Karnal Bunt Manual
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03/2007-01 Karnal Bunt Manual TOC-1

Karnal Bunt 

Manual

Contents

Figures LOF-1

Tables LOT-1

General Information 1-1

Survey Procedures 2-1

Regulatory Procedures 3-1

Public Information 4-1

Data Management 5-1

Contacts 6-1

Appendix A

Definitions A-1

Appendix B

Safety B-1

Appendix C

Technical Application Data and Chemicals C-1

Appendix D

Laboratory Procedures D-1

Appendix E

Compliance Agreements E-1

Appendix F

Contributors F-1

Appendix G

References G-1

Appendix H

National Survey Protocol H-1

Index Index-1
Contents

TOC-2 Karnal Bunt Manual 03/2007-01
03/2007-01 Karnal Bunt Manual LOF-1

Karnal Bunt 

Manual

Figures

Figure 1-1 Karnal Bunt Life Cycle 1-6

Figure D-2 Grain Inspection Machine D-9

Figure D-3 Symptoms of Karnal Bunt (Tilletia indica) and Black 

Point D-15

Figure D-4 Karnal Bunt Kernel Analysis Worksheet D-49

Figure D-5 Karnal Bunt Quality Control (QC) Worksheet for Bunted 

Kernels D-50

Figure D-6 Quality Control (QC) Grain Labs D-51

Figure D-7 Tilletia indica Spore Analysis Worksheet D-52

Figure D-8 Tilletia indica Microscopy Log Sheet D-53

Figure E-9 Compliance Agreement for Hay Producers E-2

Figure E-10 Compliance Agreement for Mushroom Producers E-4

Figure E-11 Compliance Agreement for Rail Cars E-6

Figure E-12 Compliance Agreement for Trucks/Trailers E-8

Figure E-13 Compliance Agreement for Karnal Bunt Positive Grain 

Dealers E-13

Figure E-14 Compliance Agreement for Karnal Bunt Negative Grain Dealer/Broker E-17

Figure E-15 Compliance Agreement for Feed Processors E-21

Figure E-16 Compliance Agreement for Harvesting Equipment and 

Conveyances E-25
Figures

LOF-2 Karnal Bunt Manual 03/2007-01
03/2007-01 Karnal Bunt Manual LOT-1

Karnal Bunt 

Manual

Tables

Table 2-1 Designation of Grain Sampled Before Storage after Laboratory 

Testing 2-3

Table 2-2 Procedure for Sampling Seed During Storage 2-4

Table 2-3 Designation of Grain Sampled During Storage after Laboratory 

Testing 2-5

Table 2-4 Procedure for Sampling Seed Following Storage That Will Not 

Be Broken into Individual Lots 2-6

Table 2-5 Designation of Grain Sampled Following Storage after Laboratory Testing 2-6

Table 3-6 Grain Certification Decision Table 3-4

Table 3-7 Facility Treatments 3-7

Table 3-8 Grain from Karnal Bunt Regulated Areas 3-10

Table 3-9 Seed from Karnal Bunt Regulated Areas — Option 1 3-11

Table 3-10 Seed from Karnal Bunt Regulated Areas — Option 2 3-11

Table 3-11 Host Crop Hay Movement Decision Table 3-12

Table 3-12 Cleaning and Sanitizing Decision Table 3-12

Table 3-13 Cleaning and Sanitizing Seed Cleaning or Reconditioning 

Equipment 3-13

Table 3-14 Cleaning and Sanitizing Grain Storage Facilities and Conveyance Equipment 3-13

Table 3-15 Cleaning and Sanitizing Combines and Other Harvesting 

Equipment 3-14

Table D-16 Decontamination of Samples D-20

Table D-17 Preparation of Slide Labels D-38
Tables

LOT-2 Karnal Bunt Manual 03/2007-01
03/2007-01 Karnal Bunt Manual 1-1

Chapter

1

General Information

Contents

Purpose 1-1

Goals and Objectives 1-1

Orientation to Officers 1-2

Background Information 1-3

Causal Agent 1-3

Importance to the United States 1-3

Infection 1-3

How Karnal Bunt Spreads 1-4

Losses 1-4

Distribution 1-4

Hosts 1-5

Life Cycle 1-5

Purpose

The information contained in this document is intended for use only for Karnal 

bunt programs. Use this manual for guidance in the current National Survey 

Program and in implementing regulatory procedures and in preventing the 

spread of Karnal bunt to other locations. This manual provides technical and 

general information needed to implement any phase of a Karnal bunt 

regulatory program. Specific program action is to be based on information 

available at that time and in accordance with local methods and procedures.

Goals and Objectives

The goal of the Karnal bunt program is to retain U. S. wheat export markets 

while:

Protecting U. S. wheat production areas free of Karnal bunt

Providing the best possible options for those who are affected

Facilitating the movement of wheat into domestic and international 

markets

Promoting the flow of pertinent disease information to reassure our 

trading partners about the safety of exported U. S. wheat
General Information

Orientation to Officers

1-2 Karnal Bunt Manual 03/2007-01

Orientation to Officers

The following identifies principal activities necessary for conducting a 

regulatory program to prevent the spread of Karnal bunt disease.

1. Supervising and monitoring the cleaning and sanitizing of equipment and 

buildings that may have come in contact with infected grain.

2. Interacting with the following:

A. Farmers and ranchers

B. Storage and processing facilities

C. Commercial haulers of regulated articles

D. Farm equipment and implement dealers

E. Farm and garden supply dealers

F. Custom harvesters

G. Cooperators (county, state, and federal)

3. Monitoring to insure that infected or contaminated grain is handled in an 

approved manner as outlined in protocols.

4. Visiting processing establishments to verify that conditions listed on 

Emergency Action Notifications and Compliance Agreements are met.

5. Monitoring the movement of regulated articles.

6. Observing regulated area boundaries for movement of regulated articles.

7. Taking samples of grain, seed, and hay for testing.
General Information

Background Information

03/2007-01 Karnal Bunt Manual 1-3

Background Information

Causal Agent

Karnal bunt (synonym partial bunt) is caused by the smut fungus Tilletia indica

Mitra (synonym Neovossia indica (Mitra) Mundkur). It was first reported in 

1931, infecting wheat growing near the city of Karnal in the Indian state of 

Haryana. Since that time, the disease has been found to occur throughout all 

the major wheat growing states of India, as well as in Pakistan, Iraq, and 

Afghanistan. More recently (1970) the disease has also been observed in the 

Mexican state of Sonora. The disease has also been intercepted on grain 

samples from Lebanon and Syria, suggesting it may be established in these 

countries as well. Karnal bunt is known to affect only wheat (Triticum 

aestivum), triticale (Triticum aestivum X Secale cereale), and durum wheat 

(Triticum durum). Karnal bunt was first detected in the United States in 1996.

Importance to the United States

In the United States, concern about this disease increased with the interception 

of an infected sample of seed at the Mexican border in 1981. Although 

commercial shipments of wheat from Mexico to the United States do not 

represent a large volume of grain, they were believed to constitute a risk. In 

addition, the area surrounding Ciudad Obregon in the State of Sonora is the site 

of a number of experimental small grain winter increase programs conducted 

by U.S. breeders as well as being the center for the CIMMYT (International 

Center for Maize and Wheat Improvement) small grain breeding program. 

Plots located in this area have served as a major source of seed for many U.S. 

wheat breeders programs.

Karnal bunt has significance as an export problem because many countries 

believe it to be a quarantine pest, while the United States considers it to be a 

quality pest.

Infection

Karnal bunt affects the heads of wheat and triticale plants. Infection occurs at 

anthesis when sporidia or basidiospores come into contact with the developing 

ovary of a host plant. Once kernels become infected, the fungal mycelium may 

grow throughout the endosperm. As the infected kernel matures, part of the 

kernel will be converted to masses of dark fetid smut spores. The disease is 

often referred to as partial bunt, referring to the fact that, in most cases, only a 

portion of the kernel is converted to teliospores and typically only a few 

kernels on the spike will be affected. This is in marked contrast to common 

bunt and loose smut, where the entire kernel is converted to a mass of 

teliospores and the entire spike is affected. In most cases, bunted kernels are 

not readily visible until the wheat is harvested.
General Information

How Karnal Bunt Spreads

1-4 Karnal Bunt Manual 03/2007-01

How Karnal Bunt Spreads

This fungal disease spreads primarily by introduction of teliospores onto a 

field. Although infected seed planted in a field may or may not directly 

produce infected plants the first year, planting infected seed is the primary 

means of getting the spores into the soil. Infection by secondary sporidia, 

produced by teliospores germinating on the soil surface, occurs during the 

heading stages of the host plant. The greater threat of disease occurs the 

following years as soil is turned over, bringing these teliospores back to the 

surface.

The ideal conditions for infection are cool weather and rainfall or high 

humidity during heading. Overhead irrigation during heading time can produce 

excellent conditions for infection. When ideal conditions are not present, 

furrow or flood irrigation can produce an adequate microclimate to maintain 

low levels of the disease.

Although the spores may be carried on a variety of surfaces, spores and the 

sporidia they produce also can be windborne. Because the sporidia are fragile 

and can move only short distances, contaminated seeds are considered to be the 

major source of spread.

Losses

Losses due to Karnal bunt are attributed to the effect of the disease on grain 

quality. The fungus releases trimethyl-amine, a volatile compound with a 

characteristic fishy odor. The presence of 3% or more bunted kernels will 

result in flour having an objectionable odor, color, and taste. Flour made from 

wheat containing large numbers of heavily bunted kernels is discolored and has 

an unpleasant although harmless odor. All of the discoveries of Karnal bunt in 

the United States have resulted in infection rates of less than 1%, based on an 

average of 50,000 kernels per four pound sample.

Distribution

Karnal bunt currently occurs in Afghanistan, India, Iran, Iraq, Mexico, Nepal, 

Pakistan, and in limited areas of the United States. The pathogen has also been 

discovered in exported seed samples from Lebanon and Syria. In the United 

States, the Animal and Plant Health Inspection Service (APHIS) has 

designated parts of Arizona, California, New Mexico, and Texas as areas 

regulated for Karnal bunt. The regulated area boundaries are adjusted based on 

survey results.
General Information

Hosts

03/2007-01 Karnal Bunt Manual 1-5

Hosts

Hosts include Triticum aestivum (wheat), Triticum durum (durum wheat), and 

Triticum aestivum x Secale cereale (triticale—a hybrid of wheat and rye). 

Although Karnal bunt has been shown under greenhouse conditions and 

artificial inoculation to attack several grass species in the genera Aegilops, 

Bromus, and Lolium, as well as other species in the genus Triticum, the disease 

has never been observed on these grasses under natural conditions in the field.

Life Cycle

Teliospores are the overwintering life stage of Karnal bunt. The teliospores 

often require a dormancy period of at least 4—9 months.

Teliospores germinate at the soil surface by forming a promycelium. As many 

as 65—185 primary sporidia (infectious spores) form at the apex of the 

promycelium. Many secondary sporidia can be produced by budding from the 

primary sporidia or from mycelia.

Air currents and splashing water carry sporidia to the wheat spike. Infection 

occurs only during wheat heading and floral development. Germ tubes arising 

from the sporidia enter through stomata of the glumes and then enter the ovary 

through the ovary wall. Mycelia may grow into the endosperm of the wheat 

kernel. Many teliospores consume and replace the infected endosperm. The 

harvesting process releases the teliospores to contaminate the soil or wheat 

seed externally.

Environmental conditions directly affect the development of the disease. 

Frequent rains and high relative humidity (70% or greater) at the time of 

heading favor disease development. Irrigation induces a higher incidence of 

disease by increasing humidity. High humidity lengthens the wet period, 

creating conditions favorable for teliospore germination and disease 

development. Day temperatures of 18—22.5°C (64—72°F) and soil 

temperatures of 17—21°C (63—70°F) at spike emergence increase the 

severity of Karnal bunt.

Refer to Figure 1-1 on page 1-6 for a diagrammatic representation of the 

Karnal bunt life cycle.
General Information

Life Cycle

1-6 Karnal Bunt Manual 03/2007-01

Figure 1-1 Karnal Bunt Life Cycle
03/2007-01 Karnal Bunt Manual 2-1

Chapter

2 Survey Procedures

Contents

Protocol for Sampling Grain 2-1

Protocol for Sampling Seed Prior to Storage 2-2

Protocol for Sampling Seed During Storage 2-3

Protocol for Sampling Seed Following Storage 2-5

National Survey Plan 2-6

Protocol for Sampling Grain

Sampling

Every wheat field within the regulated area, and individually regulated fields, 

will be sampled. Karnal bunt program personnel will obtain four pound 

samples at the field. Samples will then be examined for bunted kernels at the 

nearest lab/bunted kernel machine location. The sample will be a time sensitive 

sample and results will be known prior to the grain leaving the field or before 

grain is off loaded at a facility.

The Karnal bunt project will supply to the facility handlers maps and/or lists 

identifying known field numbers of all fields within the regulated areas. This 

information will help industry insure that regulated area grain will not be off 

loaded without appropriate test results.

Protocol

Draw a four pound sample prior to or at time of harvest. A sample cut by a 

combine will contain approximately 1,000 pounds of wheat randomly 

harvested from the field being tested. Obtain the sample as safely and 

efficiently as possible. To avoid getting grain from a previous field that was 

left within the combine, let the grain discharge awhile before drawing the 

sample.

Step 1: Collect the sample.

Use a length of PVC pipe to obtain grain as the combine discharges into a 

truck. A probe may be used to draw the sample from the combine or truck. The 

sample may also be drawn from the inspection compartment, if available, or 

hopper of the combine.

Step 2: Bag the sample.

Place the sample grain into a collection bag (double bagged), labeled with one 

of the following conventions:
Survey Procedures

Protocol for Sampling Seed Prior to Storage

2-2 Karnal Bunt Manual 03/2007-01

Unique field number or identification, sampler’s name, producer’s name, 

and date

Unique identification number and accompanying sample worksheet

Properly seal the collection bag.

Step 3: Transport the sample.

Transport the sample to the nearest lab/bunted kernel machine as quickly as 

possible.

Personnel will process the samples as quickly and efficiently as possible and 

complete sample documentation. Results should be communicated as soon as 

known to the grower/harvester. Movement of grain will be based on a bunted 

kernel standard and accompanying paperwork will be completed as outlined 

under the heading Certification of Grain in Chapter 3, Regulatory Procedures.

Protocol for Sampling Seed Prior to Storage

Trucks may come from the fields and enter the storage facility in the usual 

manner. From the scale area, each truck will proceed to an assigned area for 

unloading. The objective is to obtain a representative sample and to maintain 

sanitation without cross-contamination.

Step 1: Draw sub-samples.

As the crop is off loaded, randomly draw three sub-samples from each truck.

SAFETY

 Exercise caution around combines and field machinery to avoid injury. Do not assume 

the operator can see you; large machines have blind spots and field conditions can inhibit 

visibility.

 Be careful climbing on and off of grain trucks and combines to take samples. Wear proper 

footwear.

 Be mindful of fire hazards in field conditions.

 Be timely and efficient in taking and reviewing samples and DO NOT RUSH or SACRIFICE SAFETY. Time is valuable to Custom Harvesters whose business is to compete 

with weather and harvest windows of opportunity.

NOTICE

Seed grown within the regulated area must be free of bunted kernels and teliospores. The seed is allowed to be planted within or outside the regulated area. It is 

highly recommended that all seed be tested prior to cleaning and treating.
Survey Procedures

Protocol for Sampling Seed During Storage

03/2007-01 Karnal Bunt Manual 2-3

Step 2: Mix the sub-samples.

Uniformly mix the random sub-samples from each truck (up to six trucks) in a 

sanitary bucket or bag.

Step 3: Send the samples to the laboratory.

Fields and lots identified for seed may be sampled drawing a 4 pound sample 

(refer to Protocol for Sampling Grain on page 2-1). Take one sample, one 250-

mL bottle sample (Retain the one 250-mL sample in the event more material is 

needed for replication retest,) and draw five 250-mL bottles with 50 grams 

each.

Either of two testing sequences may be used. (1) Test a 4-pound sample for 

bunted kernels according to the grain sampling protocol. If the sample is 

negative for bunted kernels, send the five 250-mL bottles to the laboratory for 

spore testing. If the samples are free of teliospores of karnal bunt, allow 

movement as seed. (2) Send the five 250-mL bottles to the laboratory for spore 

testing. If the samples are free of teliospores of karnal bunt, allow movement 

as seed. If teliospores are found the field or lot is not eligible for use as seed 

and will then be tested using the four pound sample for bunted kernels. If no 

bunted kernels are found, the field or lot can be used as grain.

Protocol for Sampling Seed During Storage

Seed sampled during storage will consist of one composite sample per lot. The 

lot size will be equal to the amount of seed stored in each individual bin or 

broken down into individual lots containing up to 150 tons (approximately 

5,000 bushels). Only one type of sampling method will be allowed for each 

storage facility or bin.

The preferred method is to collect the sample while turning the grain in 

larger units. Many on-farm storage bins do not have the ability to turn 

grain, hence the need for sampling grain for spores while still in storage.

Use probes for sampling where turning the grain is not possible. Probes 

used must be long enough to take samples of grain from all parts of the 

bin being tested.

Table 2-1 Designation of Grain Sampled Before Storage after Laboratory 

Testing

If: Then:

All five replications are NEGATIVE The entire lot moves into storage as SEED

One or more replications have one or 

more spores

Handle as GRAIN
Survey Procedures

Protocol for Sampling Seed During Storage

2-4 Karnal Bunt Manual 03/2007-01

Sample to the best method available depending upon circumstances. The 

objective is to get the most representative sample possible.

Draw sufficient sub-samples to provide enough composite seed to satisfy the 

sample size requirements. Uniformly mix all sub-samples from each bin in a 

sanitary bucket or bag to obtain a composite sample. Fill each of five or ten 

250-mL bottles (depending on the size of the bin) with 50 grams and send to 

the laboratory. The bottles represent separate replications of a single, wellmixed sample from the entire lot of wheat. Also submit one or two 250-mL 

samples.

A description of the number of sub-samples drawn and replications submitted 

for each bin follows. Draw sub-samples from each bin in such a manner that 

the final sample is representative of the entire lot or bin being sampled. The 

general procedure for sampling will be as follows:

The lab will retain the 250-mL bottle sample in the event more material is 

needed for replication retests.

The bottles represent separate replications of a single, well-mixed sample from 

the entire lot of wheat. The lab will run all replications simultaneously,

working toward a 24-hour turnaround. The following results are possible:

Table 2-2 Procedure for Sampling Seed During Storage

If there are: Then:

1-750 tons (up 

to 25,000 

bushels) in the 

bin

1. Draw one sub-sample from every 25 tons (approximately 833 bushels).

2. Mix all sub-samples thoroughly.

3. Collect five 50-gram replications, one four-pound sample, and one 

250-mL sample.

4. All tests must be negative for all stages of karnal bunt. The tests can 

be run in either of two sequences: (1) Examine the four-pound sample 

for bunted kernels. If the sample is negative for bunted kernels, send 

five 50- gram replications, and one 250-mL sample to the seed lab. (2) 

Send five 50-gram replications, one 250-mL and one four-pound sample to lab for teliospore test. If sample is teliospore positive it is not eligible for seed. Test one four-pound sample for bunted kernels. If 

Negative, permit as grain.

More than 750 

tons (25,000 

bushels) in the 

bin

1. Draw one sub-sample from every 100 tons (approximately 3300 bushels).

2. Mix all sub-samples thoroughly.

3. Collect ten 50-gram replications, one four-pound sample, and one 

250-mL sample.

4. All tests must be negative for all stages of karnal bunt. Examine the 

four-pound sample for bunted kernels. Proceed as described in (4) 

above, using ten 50-gram replications.
Survey Procedures

Protocol for Sampling Seed Following Storage

03/2007-01 Karnal Bunt Manual 2-5

Protocol for Sampling Seed Following Storage

Seed in storage will receive one composite sample per lot. The lot size will be 

equal to the amount of seed stored in each individual bin, or the producer has 

the option to break down all wheat in storage into individual lots containing up 

to 150 tons (approximately 5,000 bushels). These individual lots will be 

sampled as they are removed from storage. Maintain the identity of all 

individual lots after taking samples. Once sample results are known, lots can 

be recombined according to test results. Uniformly mix all sub-samples from 

each bin in a sanitary bucket or bag to obtain a composite sample and fill each 

of five or ten 250-mL bottles (depending on the size of the bin) with 50 grams 

and send to the laboratory. The bottles represent separate replications of a 

single, well-mixed sample from the entire lot of wheat. Also submit one or two 

250-mL bottle(s) filled with seed.

A description of the number of sub-samples drawn and replications submitted 

for each bin follows. Draw sub-samples as the seed is circulated in each bin. 

Take sub-samples from the grain stream at the intervals or quantities listed 

below. The general procedure for sampling will be as follows:

Table 2-3 Designation of Grain Sampled During Storage after Laboratory 

Testing

If: Then:

All replications are NEGATIVE The entire lot moves into storage as SEED

One or more replications have one or 

more spores

Handle as GRAIN
Survey Procedures

National Survey Plan

2-6 Karnal Bunt Manual 03/2007-01

The lab will retain the 250-mL bottle sample in the event more material is 

needed for replication retests.

The bottles represent separate replications of a single, well-mixed sample from 

the entire lot of wheat. The lab will run all replications simultaneously,

working toward a 24-hour turnaround. The following results are possible:

National Survey Plan

See Appendix H for the current National Survey Plan.

Table 2-4 Procedure for Sampling Seed Following Storage That Will Not Be 

Broken into Individual Lots

If there are: Then:

1-750 tons (up to 25,000 bushels) in the bin1. Draw one sub-sample from every 25 tons (approximately 833 bushels).

2. Mix all sub-samples thoroughly.

3. Collect five 50-gram replications, one four-pound 

sample, and one 250-mL sample.

4. Examine the four-pound sample for bunted kernels.

5. If the four-pound sample is negative for bunted kernels, send five 50-gram replications, one four pound 

sample, and one 250-mL sample to the seed lab. All 

tests must be negative for the grain to qualify as 

seed.

More than 750 tons (25,000 

bushels) in the bin

1. Draw one sub-sample from every 100 tons (approximately 3300 bushels).

2. Mix all sub-samples thoroughly.

3. Collect ten 50-gramreplications, one four-pound 

sample, and one 250-mL sample.

4. Examine the four-pound sample for bunted kernels.

5. If the four-pound sample is negative for bunted kernels, send ten 50-gram replications, one four pound 

sample, and one 250-mL sample to the seed lab. All 

tests must be negative for the grain to qualify as 

seed.

Table 2-5 Designation of Grain Sampled Following Storage after Laboratory 

Testing

If: Then:

All replications are NEGATIVE The entire lot moves into storage as SEED

One or more replications have one or 

more spores

Handle as GRAIN
03/2007-01 Karnal Bunt Manual 3-1

Chapter

3 Regulatory Procedures

Contents

Orientation/Instruction to Officers 3-1

Regulated Articles 3-2

Quarantine Actions 3-3

Certification of Grain 3-4

Handling Instructions for Seed Produced Inside the Regulated Area 3-4

Seed Storage 3-4

Handling Instructions for Seed Produced Outside the Regulated Area 3-5

Approved Regulatory Treatments for Equipment and Facilities 3-6

Mechanized Harvesting Equipment 3-6

Conveyances That Can Be Easily Inspected 3-7

Facilities 3-7

Sanitizing Options 3-7

Movement of Host Crops 3-8

Mushroom Producers 3-9

Compliance Agreement Stipulations 3-9

Decision Tables for Principal Activities 3-10

Regulatory Records 3-14

Orientation/Instruction to Officers

Regulatory actions will be initiated based on production, handling, and 

transportation practices, or when the program is modified by the agency to 

meet changing conditions. Officers must follow instructions for regulatory 

procedures when authorizing the movement of regulated articles. 

Understanding the instructions and procedures will serve as a basis for 

explaining procedures to persons interested in moving articles affected by the 

quarantine and regulations. Only authorized treatments may be used (see 

Decision Tables for Principal Activities on page 3-10). Specific working 

protocols may be tailored by individual states within these guidelines.
Regulatory Procedures

Regulated Articles

3-2 Karnal Bunt Manual 03/2007-01

Regulated Articles

The list that follows is taken from 7 CFR 301.89-2 (updated March 24, 2004):

Conveyances, including trucks, railroad cars, and other containers used to 

move host crops produced in a regulated area that have tested positive for 

Karnal bunt through the presence of bunted kernels

Grain elevators/equipment/structures used for storing and handling host 

crops produced in a regulated area that have tested positive for Karnal 

bunt through the presence of bunted kernels

Seed conditioning equipment and storage/handling equipment/structures 

that have been used in the production of wheat, durum wheat, and triticale 

found to contain the spores of Tilletia indica

Plants or plant parts (including grain, seed, and straw) and hay cut after 

reaching the dough stage of all varieties of wheat (Triticum aestivum), 

durum wheat (Triticum durum), and triticale (Triticum aestivum X Secale 

cereale) that are produced in a regulated area, except for straw/stalks/seed 

heads for decorative purposes that have been processed or manufactured 

prior to movement and are intended for use indoors

Tilletia indica (Mitra) Mundkur

Mechanized harvesting equipment that has been used in the production of 

wheat, durum wheat, or triticale that has tested positive for Karnal bunt 

through the presence of bunted kernels

Any other product, article, or means of conveyance when:

An inspector determines that it presents a risk of spreading Karnal 

bunt based on appropriate testing and the intended use of the product, 

article, or means of conveyance; and

The person in possession of the product, article, or means of 

conveyance has been notified that it is regulated under this subpart.
Regulatory Procedures

Quarantine Actions

03/2007-01 Karnal Bunt Manual 3-3

Quarantine Actions

Implement the following actions in sequence:

1. Issue an Emergency Action Notification, PPQ Form 523 (EAN) to the 

owner or operator of each property on which Karnal bunt is confirmed by 

presence of bunted kernels or which is identified as a source of or recipient 

of infected wheat, until such time as a regulated area is established. 

Recipients of EANs will be required to implement treatments and handling 

procedures. Issue EANs and/or comparable State notifications to the 

property owners or managers of all establishments that are involved in the 

handling, moving, or processing of articles capable of spreading Karnal 

bunt, when these establishments are located within the area that serves any 

infested property. You may issue a notification pending positive 

identification and/or further instruction from the Deputy Administrator. 

EANs are usually no longer issued when the regulated area is established.

2. If necessary, the Deputy Administrator will issue a letter directing PPQ 

Field offices to initiate specific emergency actions under the Plant 

Protection Act 2000 until emergency regulations can be published in the 

Federal Register.

The Plant Protection Act 2000 provides for authority for emergency 

quarantine action. This authority provides for interstate regulatory action 

only; intrastate regulatory action is provided under State authority. 

However, if the Secretary of Agriculture determines that an extraordinary 

emergency exists and that the measures taken by the state are inadequate, 

USDA can take intrastate regulatory action provided that the Governor of 

the State has been consulted and a notice has been published in the Federal 

Register.

The Plant Protection Act also provides the Federal Government, either 

independently or in cooperation with States or political subdivisions 

thereof, farmers’ associations and similar organizations, and individuals, 

the authority to carry out operations or measures to detect, eradicate, 

suppress, control, or to prevent or retard the spread of plant pests. This does 

not provide for tresspassing on private property, but relies upon State 

authority and willingness to use State right-of-entry authority.

All program technology and methodology employed are determined 

through discussion, consultation, or agreement with the cooperating State 

officials.

3. The Deputy Administrator, through the National Regional Directors, will 

notify State cooperators of the Karnal bunt detection, actions taken, and 

actions contemplated. USDA and cooperators will develop and provide a 

description of the regulated area with support documents to the national 

program planning staff or its equivalent.
Regulatory Procedures

Certification of Grain

3-4 Karnal Bunt Manual 03/2007-01

4. APHIS Regulatory Analysis and Development Staff (RADS) or its 

equivalent will publish emergency regulations under the Plant Protection 

Act.

5. After a reasonable time to determine the scope of the infection, a proposal 

to promulgate a quarantine under the Plant Protection Act will be 

published. The proposal will announce a date for submitting written 

comments, which shall be approximately 60 days after publication.

6. After receipt of written comments, a final determination specifying the 

action decided upon will be published in the Federal Register. A 

quarantine will be enacted if eradication has not been achieved under the 

Plant Protection Act.

Certification of Grain

Certify grain at the field or facility level by issuing PPQ Form 540s or local equivalent 

as approved by the PPQ Project Director.

Handling Instructions for Seed Produced Inside the Regulated Area

Any wheat, durum wheat, or triticale that originates within a regulated area 

must be tested and found free of any stage of Karnal bunt (Tilletia indica) 

before it may be used as seed within or outside a regulated area.

Seed Storage

Place seed in new bags, or bulk containers that are new or have been certified 

free of Karnal bunt.

Table 3-6 Grain Certification Decision Table

If the grain is determined to be: Then:

Bunted Kernel Negative  Use PPQ Form 540 or local equivalent as 

approved by the PPQ Project Director

Bunted Kernel Positive  Issue PPQ Form 530 (Limited Permit) or local 

equivalent as approved by the PPQ Project 

Director for each truck load of grain

 Move positive grain only to an approved facility 

for processing
Regulatory Procedures

Handling Instructions for Seed Produced Outside the Regulated Area

03/2007-01 Karnal Bunt Manual 3-5

Store all seed in Karnal bunt free facilities and transport in Karnal bunt 

free conveyances. One of the following conditions must be met for each 

lot:

Conveyances, storage facilities and planting equipment are new or 

have never been used to handle wheat, durum wheat, or triticale; or

Conveyances, storage facilities and planting equipment have been 

sanitized or cleaned with an approved treatment; or

Conveyances, storage facilities and planting equipment have never 

been used to hold or transport wheat that tested positive for Karnal 

bunt.

Ensure that the movement of seed from the storage facility is 

accompanied by a Certificate (PPQ Form 540).

Handling Instructions for Seed Produced Outside the Regulated 

Area

The following conditions apply for seed produced outside the regulated area 

and intended for planting inside the regulated area.

Testing for presence of Karnal bunt spores is not required.

Treating seed for planting inside of the regulated area is not required.

Advanced notification of movement into the regulated area is required in 

order to track and safeguard the identity of the seed.

Authorized Chemicals

The following chemicals are authorized to treat articles regulated for Karnal 

bunt:

Methyl bromide (15 lb/1,000 ft3

 For 96 hours)

One part Ultra Clorox brand regular bleach (6 percent sodium 

hypochlorite; EPA Reg. 5813-50) in 3 parts water; or one part CPPC Ultra 

Bleach 2 (6.15 percent sodium hypochlorite; EPA Reg. No. 67619-8) in 

3.1 parts water

Pentachloronitrobenzene (PCNB) with or without Carboxin-Thiram

Carboxin-Thiram
Regulatory Procedures

Approved Regulatory Treatments for Equipment and Facilities

3-6 Karnal Bunt Manual 03/2007-01

Approved Regulatory Treatments for Equipment and Facilities

Mechanized Harvesting Equipment

Mechanized harvesting equipment includes combines, grain buggies, etc. 

Equipment used in the harvesting or processing of wheat, durum wheat, or 

triticale that tests positive for bunted kernels will require cleaning. If 

disinfection is required by an inspector in addition to cleaning, the articles 

must be disinfected in accordance with 7 CFR 301.89-13 prior to movement 

from a regulated area.

Cleaning Procedure for Harvesting Machines

Use the following procedure to clean harvesting machines:1

Step 1: Remove the header.

Step 2: Open the hopper doors.

Open the hopper doors on the bottom of the combine to allow all of the 

remaining wheat in the hopper below the auger to fall out.

Step 3: Blow out the debris.

Put the combine in neutral, turn up the air pressure to maximum and run the 

combine up to full throttle to blow out all of the debris remaining in the system.

Step 4: Remove wheat from the return auger.

Open the return auger and run it at full throttle under maximum air pressure to 

remove all wheat in that portion of the system.

Step 5: Blow out remaining debris.

Use a high pressure air hose to blow any remaining debris out of the hopper 

and delivery system.

Step 6: Begin the wash.

Using a high pressure wash, start at the top of the combine and work down to 

remove dust, debris, etc. on the machine.

Step 7: Continue the wash.

Open all inspection doors and apply the high pressure wash to remove debris.

1 The entire process takes two people approximately 1.5 to 2 hours of steady work, depending on the type 

of combine used and the amount of residue left on the machinery. Under a worst case scenario 

(equipment breakdown, only one person to conduct cleaning, extremely dirty combine, etc.) the cleaning 

may take up to 3.5 hours. Small variations based on different machine types will occur. Discuss the 

cleaning required for a specific harvesting machine with the Project Director.

NOTICE

The inspector may require or recommend washing if necessary. Continue with the 

following steps for washing protocol.
Regulatory Procedures

Approved Regulatory Treatments for Equipment and Facilities

03/2007-01 Karnal Bunt Manual 3-7

Step 8: Remove side panels.

Remove side panels from the header.

Step 9: Remove all debris from header.

Step 10: Clean the header.

Clean the header with the high pressure wash.

Conveyances That Can Be Easily Inspected

Conveyances that can be easily inspected include truck trailers, bobtails, 

railcars, etc. Equipment used in the harvesting or processing of wheat, durum 

wheat, or triticale that tests positive for bunted kernels will require cleaning. If 

disinfection is required by an inspector in addition to cleaning, the articles 

must be disinfected in accordance with 7 CFR 301.89-13 prior to movement 

from a regulated area.

Facilities

Facilities include silos, bins, etc. Use Table 3-7 for facilities that have tested positive 

for Karnal bunt:

Cleaning is defined as the removal of all soil and plant debris.

Sanitizing is defined as using one of the following options:

Sanitizing Options

1. Sodium Hypochlorite Solution

Thoroughly wet the equipment until point of runoff with a solution of 1.5 

percent sodium hypochlorite—a solution of sodium hypochlorite mixed 

with water applied at the rate of (1) One part Ultra Clorox brand regular 

bleach (6 percent sodium hypochlorite; EPA Reg. No. 5813-50) in 3 parts 

water; or (2) One part CPPC Ultra Bleach 2 (6.15 percent sodium 

hypochlorite; EPA Reg. No. 67619-8) in 3.1 parts water. Leave solution in 

contact with all surfaces for a minimum of 15 minutes, then thoroughly 

rinse surfaces with water to minimize any adverse effect on equipment. 

Prepare a fresh solution of sodium hypochlorite and potable water every 

four to five hours.

Or

Table 3-7 Facility Treatments

If the facility will be used for: Then:

Host grain storage Clean the facility before storing host material

Host seed storage Clean and sanitize the facility before storing host material
Regulatory Procedures

Movement of Host Crops

3-8 Karnal Bunt Manual 03/2007-01

2. Hot Water And Detergent

Hot water and detergent drench with a pressure of at least 30 pounds per 

square inch. Apply the water to all surfaces until point of runoff including 

penetration into cracks and crevices. At point of runoff the temperature 

must be a minimum of 170°F.

Or

3. Live Steam To All Surfaces

You can use live steam (212°F) to treat various harvesting and cleaning 

equipment, as long as the steam can directly reach all surfaces that come 

into contact with grain. Apply steam directly to all surfaces until the point 

of runoff. At point of runoff the temperature must be a minimum of 170°F.

Movement of Host Crops

Host crops include hay and straw. Before moving hay or straw within or out of 

the regulated area it must be inspected by USDA or State Cooperator Karnal 

bunt personnel. The following distinctions apply:

Host crops cut prior to the development of the kernel (dough stage) will be 

considered no risk for having Karnal bunt. Host crops cut after the dough 

stage will be treated as grain.

Host crops cut with fully developed kernels will be tested for the presence 

of bunted kernels. Take samples from bales. The host crop hay/straw will 

be regulated according to the results of the testing.

Host crops testing negative can move within and out of the regulated area if 

they meet all of the following conditions (refer to Table 3-11 on page 3-12):

The identity of the cut host crop is retained.

The grower will be issued a Certificate for the number of bales inspected/

tested free of bunted kernels.

The number of bales noted on the Certificate for the location (field or 

storage area) will be allowed movement within or out of the regulated 

area. The Certificate must accompany any movement.
Regulatory Procedures

Mushroom Producers

03/2007-01 Karnal Bunt Manual 3-9

Host crop straw testing positive for bunted kernels may not be removed from 

the field. Host crop hay testing positive for bunted kernels can move within the 

regulated area under the following conditions:

A grower may move host crop hay from the field of production (land 

owned or leased by grower) to an area owned or leased by the grower for 

storage.

A grower may move host crop hay from the field of production (land 

owned/leased by grower) for the purpose of feeding to livestock.

A grower must contact USDA–State Cooperator–Karnal bunt personnel 

prior to movement of host crop hay. A Limited Permit will be issued for 

movement.

Host crop hay must be moved in a manner that safeguards the host crop 

hay and minimizes the potential for spreading Karnal bunt.

Host crop hay testing positive for bunted kernels or baled from a field that 

tested positive for bunted kernels can move out of the regulated area under the 

following conditions:

The host crop hay must be destined to an approved establishment.

Program personnel must be contacted before moving host crop hay. If 

movement is approved, a Limited Permit will be issued for movement to 

the approved establishment out of the regulated area.

Host crop hay approved for movement out of the regulated area must be 

moved in a manner that safeguards the host crop hay and minimizes the 

spread of Karnal bunt.

The establishment must be approved by USDA–State Cooperator–Karnal 

bunt personnel and under compliance to process the host crop hay in an 

approved manner to destroy all Karnal bunt life stages.

Mushroom Producers

Compliance Agreement Stipulations

See sample compliance agreement in Appendix E.

Wheat straw from the regulated area that tests positive must be introduced to a 

composting procedure that results in a pasteurized product. The composting 

procedure shall produce pasteurization temperatures throughout the mass of 

170°F for at least 1 minute.
Regulatory Procedures

Decision Tables for Principal Activities

3-10 Karnal Bunt Manual 03/2007-01

Decision Tables for Principal Activities

Use Either Table 3-9 or Table 3-10 as appropriate to your survey area.

Table 3-8 Grain from Karnal Bunt Regulated Areas

If grain is from: And: And: Then:

A field sampled 

for bunted kernels

Positive For animal 

feed

Issue a Limited Permit (PPQ 530) for 

movement to facilities with a Compliance 

Agreement1

1 Conveyances for Karnal bunt positive grain must be leak proof and covered prior to moving 

outside the regulated area.

For milling Issue a Limited Permit for movement to 

facilities with a Compliance Agreement

Going to a 

storage facility 

or to an export 

terminal facility

Issue a Limited Permit for movement to 

facilities with a Compliance Agreement1

Negative For domestic 

use or going to 

an export terminal facility2

2 Crops from previously tested positive fields are not eligible for export to countries that require 

an Additional Declaration (AD) on federal phytosanitary certificates.

Issue a Certificate to any destination for 

non-planting purposes and export destinations2

Storage facilities sampled for 

bunted kernels 

or storage facilities located outside the 

regulated area 

that are commingled with regulated grain

Positive For animal 

feed

Issue a Limited Permit for movement to 

facilities with a Compliance Agreement1

For milling Issue a Limited Permit for movement to 

facilities with a Compliance Agreement

Going to a 

storage facility 

or to an export 

terminal facility

Issue a Limited Permit for movement to 

facilities with a Compliance Agreement1

Negative For domestic 

use

Issue a Limited Permit for movement to 

any location for any purpose other than 

planting

Going to an 

export terminal facility

1. Issue a Limited Permit for movement 

to facilities with a Compliance Agreement.

2. Allow movement to countries with no 

Karnal bunt certification requirements.
Regulatory Procedures

Decision Tables for Principal Activities

03/2007-01 Karnal Bunt Manual 3-11

Table 3-9 Seed from Karnal Bunt Regulated Areas — Option 1

If the seed tests: And the seed tests: And the seed is: Then:

Negative for 

bunted kernels

Positive for spores Untreated Handle as grain

Treated with fungicideBury or dispose of seed 

with approved methods 

according to label

Negative for spores The seed is eligible for 

planting within or outside 

the regulated area

Positive for 

bunted kernels

The wheat is ineligible to 

be tested for use as seed. 

Handle as positive grain.

Table 3-10 Seed from Karnal Bunt Regulated Areas — Option 2

If the seed tests: And the seed tests: And the seed is: Then:

Negative for 

spores

The seed is eligible for 

planting within or outside 

the regulated area

Positive for 

spores

Negative for bunted 

kernels

Untreated Allow as negative tested 

grain

Treated with fungicideBury or dispose of seed 

with approved methods 

according to label

Positive for bunted 

kernels

The wheat is ineligible for 

seed. Handle as positive 

grain.
Regulatory Procedures

Decision Tables for Principal Activities

3-12 Karnal Bunt Manual 03/2007-01

Table 3-11 Host Crop Hay Movement Decision Table

If moving: And: And: Then:

Within the 

regulated area

No kernels are 

present1

1 Host crop hay cut prior to the dough stage is unregulated and does not require an inspection. 

The hay is eligible for a Certificate (PPQ 540), if requested by the producer, but the certificate 

is not a requirement or a condition of movement.

Hay cut prior to the dough stage is 

unregulated

Kernels are present 

(collect a 4-pound 

sample of grain) or

a survey has determined the field’s 

status

The field 

or kernels 

test KB 

negative

Issue a Certificate (PPQ 540 or PPQ 

527). The producer must maintain a 

movement log, if necessary, based on 

local farming practices.

The field 

or kernels 

test KB 

positive

1. Send certified notification of positive field status to the owner and 

producer of the field.

2. No movement of straw is allowed 

out of the field.

Outside the 

regulated area

No kernels are 

present1

Hay cut prior to the dough stage is 

unregulated

Kernels are present 

(collect a 4-pound 

sample of grain) or

a survey has determined the field’s 

status

The field 

or kernels 

test KB 

negative

Issue a Certificate (PPQ 540 or PPQ 

527). The producer must maintain a 

movement log, if necessary, based on 

local farming practices.

The field 

or kernels 

test KB 

positive

1. Send certified notification of positive field status to the owner and 

producer of the field.

2. Call the field manager to explain 

options.

3. Issue a conveyance Compliance 

Agreement (CA) to move host 

crop hay under Limited Permit 

(PPQ 530) to an approved processing establishment that has a 

CA.

4. No movement of KB-positive straw 

is permitted.

Table 3-12 Cleaning and Sanitizing Decision Table1

1 For handling equipment within the regulated areas of AZ, CA, NM, and TX.

If item is: Then:

Seed cleaning or reconditioning equipment GO to Table 3-13

Grain storage facilities and conveyance equipment 

(bins, elevators, bags, legs, augers, railcars, etc.)

GO to Table 3-14

Combines and other harvesting equipment (thrashers, headers, grain carts, buggies, etc.)GO to Table 3-15
Regulatory Procedures

Decision Tables for Principal Activities

03/2007-01 Karnal Bunt Manual 3-13

Table 3-13 Cleaning and Sanitizing Seed Cleaning or Reconditioning 

Equipment

If: And: Then:

Working in any 

regulated area 

and processed host 

crop material

Used in the conditioning of seed that was tested 

and found to contain spores or bunted kernels 

of Tilletia indica and being used in the conditioning of seed that has tested negative for the 

spores of Tilletia indica or being moved from a 

regulated area

Cleanining and disinfection are required in 

accordance with 7 

CFR 301.89-13

Used in the conditioning of seed that was tested 

and found negative for spores of Tilletia indica

Cleaning and sanitizing are NOT required; 

follow best management practices for that 

state

Transiting the 

regulated area

Table 3-14 Cleaning and Sanitizing Grain Storage Facilities and Conveyance 

Equipment1

1 Includes bins, elevators, bags, legs, augers, railcars, etc.

If: And: Then:

Stored or 

transported 

positive tested 

host material 

(kernels or 

spores)

Will be used in the production, harvest, transportation, or storage of 

SEED

 Cleaning and sanitizing are required

 Issue Certificate (PPQ Form 540) after 

treatment is observed by program personnel

Will only be used in the 

production, harvest, 

transportation, or storage of GRAIN

 Cleaning is required; follow best management practices for that state

 Issue Certificate (PPQ Form 540) after 

treatment is observed by program personnel

Stored or 

transported 

negative 

tested host 

material; spore 

status 

unknown

Will only be used in the 

production, harvest, 

transportation, or storage of GRAIN

 Cleaning and sanitizing are NOT required; 

follow best management practices for that 

state

Will be used in the production, harvest, transportation, or storage of 

SEED

 Cleaning and sanitizing are required

 Issue Certificate (PPQ Form 540) after 

treatment is observed by program personnel
Regulatory Procedures

Regulatory Records

3-14 Karnal Bunt Manual 03/2007-01

USDA–APHIS and State Cooperators recommend following the best 

management practices (BMPs) when cleaning any seed processing, seed/

grain storage facilities, and harvesting equipment. The best management 

practices are designed by the local Cooperative Extension Service to 

mitigate the risk of spreading Karnal bunt and the unintentional 

movement of other plant pests, diseases, and noxious weeds. Some States 

do require cleaning and sanitizing treatments when moving from state to 

state. The best management practices for your state can be obtained from 

the local Cooperative Extension Service Office.

Cleaning is defined as the removal of all host crop material (straw, heads, 

grain, etc.) from the equipment which would prevent the crosscontamination of the next field’s harvest and/or the spread of bunted 

kernels into unregulated areas, between regulated areas.

Regulatory Records

Maintain records as necessary to carry out an effective, efficient, and 

responsible regulatory program. When presented with requests for information 

(FOIA, etc.) be sure to check with Agency legal counsel. Guidance on length 

of time to retain records is available from OPM.

Table 3-15 Cleaning and Sanitizing Combines and Other Harvesting Equipment1

1 Includes thrashers, headers, grain carts, buggies, etc.

If: And: And: Then:

Transiting 

the regulated area

 Cleaning and sanitizing are NOT

required; follow best management 

practices for that state

Working in 

the regulated area

Known to 

have harvested 

positive 

tested host 

material 

(bunted 

kernels)

Moving within or 

between regulated 

areas

 Cleaning required between regulated areas, optional within regulated area; follow best management 

practices for that state

Leaving the regulated area Cleaning and if disinfection is determined to be necessary by an 

inspector, disinfected in accordance 

with 7 CFR 301.89-13

 Issue Certificate (PPQ Form 540) 

after treatment is observed by program personnel

Known to 

have harvested 

negative 

tested host 

material

Moving between 

regulated areas, 

leaving the regulated area, or crop 

from next field is 

intended to be used 

as seed

 Cleaning and sanitizing are NOT

required, but optional; follow best 

management practices for that state

 Issue Certificate (PPQ Form 540) if 

treatment is performed
03/2007-01 Karnal Bunt Manual 4-1

Chapter

4

Public Information

Contents

Introduction 4-1

Audiences 4-1

Goals 4-2

Actions Occurring Upon Detection of Karnal bunt 4-2

Media Contact 4-2

Public Meetings/Gatherings 4-2

Complaints & Concerns 4-2

Introduction

As an agency concerned about pest and disease situations that can occur or 

change rapidly, the Animal and Plant Health Inspection Service (APHIS) has a 

vital need to effectively communicate program activities to its target audiences 

using a wide variety of informational materials. During outbreak situations, 

effective and timely communication becomes even more crucial. APHIS 

provides onsite support when appropriate, serving along with state officials as 

primary liaisons with the news media to provide accurate information to 

stakeholders, industry, and the public.

Audiences

Media

State, city and county governments

Industry/stakeholders

Environmental groups

General public

Special interest groups

Trading partners

Congress

Other Federal government counterparts

Agency headquarters personnel
Public Information

Goals

4-2 Karnal Bunt Manual 03/2007-01

Goals

To provide accurate, timely information to all identified audiences.

To proactively inform and involve identified audiences about program 

activities.

To be responsive to inquiries from various audiences about program 

activities.

To create and disseminate informational materials on program activities to 

increase awareness.

To communicate information to all identified audiences about program 

risks and risk-reducing measures.

Actions Occurring Upon Detection of Karnal bunt

The agency provides support to line and staff operations through Legislative 

and Public Affairs to address public and media concerns regarding program 

activities. Communication of program activities will be through the Project 

Director, regional offices, and staff.

Media Contact

Creating a rapport with local media results in accurate coverage of a program. 

To avoid conflicting and confusing statements, all outgoing information should 

be processed through project management. All program personnel should refer 

media questions to project managers.

Public Meetings/Gatherings

Public meetings/gatherings will be coordinated through SPHDs and the 

regional office with headquarters support.

Complaints & Concerns

Complaints and concerns directed to project managers will be communicated 

to SPHDs, the regional office, and headquarters.

NOTICE

Note that whether State or federal officials take the primary responsibility for the following actions will depend on circumstances and resources at the time of the outbreak.
03/2007-01 Karnal Bunt Manual 5-1

Chapter

5 Data Management

Contents

Introduction 5-1

Karnal Bunt Information System (KBIS) 5-1

Accessing KBIS 5-2

Creating a New Karnal Bunt Program Office in KBIS 5-2

Mapping Regulated Areas 5-2

Introduction

Data, mapping, and graphics will be accomplished by local, state, or federal 

TDY personnel with the specialized skills to perform these functions. Sources 

of personnel could be the Rapid Response Team (RRT), Regional TDY rosters, 

or State Departments of Agriculture. For assistance, contact APHIS Business 

Services and/or Pest Detection and Management Programs (PDMP). Basic 

duties include but are not limited to the following:

1. Prepares maps, overlays, charts, signs, placards, and Power Points.

2. Maintains computer equipment and other technical needs of program (fax 

machine, digital camera, GPS units).

3. Prepares graphics.

4. Manages data acquisition, manipulation, and retrieval.

Karnal Bunt Information System (KBIS)

The Karnal Bunt Information System (KBIS) was developed beginning in 

April, 2002, in a combined effort with a contractor, REI Systems; 

representatives of the USDA–Karnal Bunt Program offices in Arizona, 

California, New Mexico, and Texas; the Western Regional office in Ft. Collins, 

CO; and PPQ Headquarters in Riverdale, MD. The initiative was mounted in 

order to preserve relevant program historical data and to create a unified 

information collection system for the individual existing regulatory programs 

and record data for National Karnal Bunt Grain Survey samples analyzed by 

PPQ. Also included is the ability to create a new program office KBIS system 

in the event karnal bunt is discovered in a new area. The KBIS Lotus Notesbased system is an interim step in the creation of a larger, comprehensive 

Oracle-based emergency programs database in the future.
Data Management

Accessing KBIS

5-2 Karnal Bunt Manual 03/2007-01

Accessing KBIS

If a computer does not have KBIS installed in Lotus Notes, it will be necessary 

to access the KBIS system icons on the DATA02 production server. There are 

three Lotus Notes databases required to support the KBIS system, including 

the Karnal Bunt Information System latest version, the KBIS Keyword Lookup 

database, and the KBIS Technical Support database. These can be installed as 

icons on the users Lotus Notes Desktop by clicking on File, Database, and 

Open. Then in the server dialogue box, select DATA/O2, and find the icons for 

the three KBIS databases. Clicking on them will open the databases and 

closing them will leave the icon on the Lotus Notes desktop for future use.

A KBIS User’s Manual is available from PPQ Headquaters and copies are at 

each program office and the regions.

Creating a New Karnal Bunt Program Office in KBIS

When it becomes necessary to establish a new program area office, the new 

location program manager with a Lotus Notes connection must first define the 

program office.

1. Open KBIS database.

2. Click on the Program Office button from the main screen (the screen with 

the six buttons). This opens Program Office section of the KBIS.

3. On the left Navigator of the Program Office section, there are two main 

options: Reports and Menus. Click on Administration under the Menus 

option. This opens Administration section of the KBIS.

4. On the left Navigator of the Administration section, there are three options: 

Tasks, Reports, and Menus. Click on the Add Program Office under the 

Tasks. This creates a new program office document.

5. Fill in the fields and save the document. The new program office name will 

be included in the program office list.

Mapping Regulated Areas

Currently, each program office is maintaining their own mapping information 

that is geo-referenced. Information from the mapping programs will eventually 

be linked to KBIS when it becomes an Oracle based system.
03/2007-01 Karnal Bunt Manual 6-1

Chapter

6

Contacts

Contents

Introduction 6-1

Introduction

At the outset of a Karnal bunt program the following list of various groups 

must be informed and continually updated in all operational phases of 

emergency programs. Bear in mind the success of a program is heavily 

dependent on voluntary cooperation, assistance, and understanding from other 

interrelated groups. Following is a partial list of various groups which must be 

kept informed of and/or involved in all operational phases of an emergency 

program.

Other Federal, State, County, and municipal agricultural officials

Grower groups

Universities and Agricultural Research Stations

Foreign agricultural interests

National, State, and local news media

State and local law enforcement officials

General public

Public health

Private agricultural consultants

Public and private plant breeders

State grain organizations

Bureau of Indian Affairs (BIA)
Contacts

Introduction

6-2 Karnal Bunt Manual 03/2007-01
03/2007-01 Karnal Bunt Manual A-1

Appendix

A Appendix A

Definitions

Best Management Practices (BMP). Procedures and techniques designed to 

negate or minimize the spread of Karnal bunt.

Certificate. PPQ Form 540 or local equivalent.

Cleaning. Removing all soil and plant debris.

Compliance Agreement (CA). A written agreement between APHIS and a 

person engaged in growing, handling, or moving regulated articles, in which 

the person agrees to comply with the provisions of 7 CFR 301.89 and any 

conditions imposed under that subpart.

Confirmed Detection. Positive laboratory identification of a submitted 

sample (specimen) of Karnal bunt disease (a bunted kernel of wheat).

Contaminated Seed. Seed from sources in which the Karnal bunt pathogen 

(Tilletia indica (Mitra) Mundkur) has been determined to exist by the presence 

of bunted kernels or teliospores.

Contamination. Exposure that occurs when wheat, triticale, or durum wheat 

grain or any building, equipment, or tools, come into contact with bunted 

wheat kernels.

Conveyance. Any container used to move wheat, durum wheat, triticale, or 

their products, including trucks, trailers, railroad cars, bins, and hoppers.

Cultivated Crop. Any crop where the soil is prepared annually to a friable 

state at the time of planting. No till rotation does not constitute cultivation.

Decontamination. Treatment by the application of an approved 

decontaminating solution or by methyl bromide fumigation of contaminated 

implements, material, or equipment.

Delimiting Survey. A survey conducted in the area where a confirmed 

detection of Karnal bunt has occurred to determine the extent of the outbreak.
Appendix A

Definitions

A-2 Karnal Bunt Manual 03/2007-01

Detection. Any site (1) where bunted kernels are found in the field; or (2) that 

has been the source of grain with bunted kernels; or (3) that has seed found 

positive with Tilletia indica.

Disease. Any prolonged disturbance in a plant that interferes with its normal 

structure or function.

Emergency Action Notification (EAN). A document (PPQ Form 523) giving 

notice of necessary action to mitigate pest risk within a limited time frame.

Fumigation. The application of an approved fumigant (methyl bromide) to 

infected grain or to contaminated grain, straw, soil, tools, or buildings.

Harvesting Equipment. Mechanized equipment for harvesting purposes, e.g., 

combines, reapers, and balers.

Grain. Wheat, durum wheat, or triticale for consumption or processing.

Hay. Host crops cut and dried for feeding to livestock. Hay cut after reaching 

the dough stage may contain mature kernels of the host crop.

Host. A plant which is invaded by the fungus and from which the fungus 

obtains its nutrients.

Host Crops. Plants or plant parts, including grain, seed, or hay, of wheat, 

durum wheat, and triticale.

Infection. The establishment of the fungus within a host plant.

Infestation (infected). The presence of Karnal bunt, or any identifiable stage 

of development (i.e., bunted kernels in grain, bunted kernels or teliospores in 

seed) of the fungus Tilletia indica (Mitra) Mundkur, or the existence of 

circumstances that make it reasonable to believe that Karnal bunt is present.

Inoculation. The transfer of a pathogen onto a host.

Inoculum. The pathogen or its parts that cause disease. The portions of 

individual pathogens that are brought into contact with the host.

Karnal Bunt (synonym partial bunt). A smut disease affecting the grain of 

wheat, triticale, and durum wheat caused by the fungus Tilletia indica.

Known Infested Property. The entire field where bunted kernels were 

detected.
Appendix A

Definitions

03/2007-01 Karnal Bunt Manual A-3

Limited Permit. A document (PPQ Form 530) in which an inspector affirms 

that a specified regulated article not eligible for a certificate is eligible for 

movement only to a specified destination and in accordance with conditions 

specified on the permit.

Mechanized Cultivating Equipment and Mechanized Harvesting 

Equipment. Mechanized equipment used for soil tillage, including tillage 

attachments for farm tractors — e.g., tractors, disks, plows, harrows, planters, 

and subsoilers; mechanized equipment used for harvesting purposes — e.g., 

combines, grain buggies, trucks, swathers, and hay balers.

Pathogen. Any organism that can incite a disease (T. indica is the pathogen 

that incites or causes Karnal bunt disease).

Plant. Any plant (including any plant part) for or capable of propagation.

PPQ-APHIS-USDA. Plant Protection and Quarantine, Animal and Plant 

Health Inspection Service, United States Department of Agriculture.

Property. A land unit under one owner or operator that is handled as a single 

farming or production unit.

Regulated Area. (1) An area (any definable area surrounding a three mile 

circular buffer from a known infected field) within a 3-mile radius from any 

field that has tested positive for bunted kernels. (2) Individual fields planted 

with spore positive seed. (3) Individual elevators under certain conditions.

Sanitize. To devitalize the pathogen by means outlined in 7 CFR 301.89-13.

Seed. Wheat, durum wheat, or triticale for propagation.

Smut. Any disease caused by fungi in the order Ustilaginales. Usually masses 

of dark powdery spores are present at maturity.

Sporidia. Spores produced by teliospores of smut fungi. These spores are the 

fragile and short lived basidiospores of the smut fungi.

Straw. The vegetative material left after the harvest of host crops. Straw is 

generally used as animal feed, bedding, mulch, or for erosion control.

Stubble. Standing stalks of hostplants following harvest.

Teliospore. The thick walled resting spores of smut fungi.
Appendix A

Definitions

A-4 Karnal Bunt Manual 03/2007-01

Tilletia indica (synonym Neovossia indica). The fungus that incites Karnal 

bunt disease in wheat, triticale, and durum wheat.

Tilling. The turning of a minimum of the top 6 inches of soil.
03/2007-01 Karnal Bunt Manual B-1

Appendix

B Appendix B

Safety

Contents

General Information B-1

First Aid Suggestions B-2

Managing/Monitoring Pesticide Spills B-3

Safety B-3

Cleanup B-3

General Information

Personnel and public safety must be a prime consideration at all times. Stress 

safety practices in pre-program planning. Supervisors must enforce on the job 

safety procedures.

Pesticides authorized for use vary in toxicity. When used in accordance with 

label instructions, materials do not constitute a threat to people, wildlife, bees, 

etc. Specific safety precautions for each pesticide are listed on the label. In 

addition, any special precautions listed in this or specific manuals shall be 

observed.

Keep pesticides in closed, properly labeled containers in a dry place. Store 

them where they will not contaminate food or feed and where children and 

animals cannot reach them. When handling a pesticide, follow all 

precautionary labeling.

Should there be contact through spillage or otherwise, wash immediately with 

soap and water. Should clothing become contaminated, launder before wearing 

again. Refer to PPQ Treatment Manual, Section X, for additional information.

Dispose of empty pesticide containers in an approved sanitary landfill, by 

incineration, or by other methods approved by the Federal Environmental 

Protection Agency whereby they will not present a hazard or problem. 

Arrangements for disposal of such containers should be completed and 

thoroughly understood by all parties directly involved with a program before 

the actual start of operations. Consult PPQ Regional offices and the Pest 

Detection and Management Programs (PDMP) staff for pertinent information 

in States where operations are conducted.
Appendix B

First Aid Suggestions

B-2 Karnal Bunt Manual 03/2007-01

When applying a pesticide, consider the potential impact of the pesticide on all 

components of the total environment, including humans, crops, livestock, 

wildlife, aquatic life, non-target insect species, and domesticated honey bees. 

Avoid contamination of lakes, streams, ponds, or watersheds.

You should be aware of and heed general safety considerations and those 

specific to the Karnal bunt project. This manual integrates safety procedures 

into all applicable sections. Remember that inherent hazards are involved when 

working in field conditions, when working with mechanized harvesting 

equipment, and when working with chlorine bleach.

Refer also to the APHIS Safety and Health Manual for additional information.

First Aid Suggestions

In case of accidental poisoning or as soon as any person shows symptoms of 

having been affected by any pesticide, do the following:

1. Remove the person to a place where there will be no further contact with 

the pesticide.

2. Have the person lie down and keep quiet.

3. Call a physician and provide the name and formulation of the pesticide in 

use and first aid given.

Keep the local Poison Control Center telephone number posted where 

pesticides are stored and used. The number may also be found on the inside 

front cover of the telephone directory. Call Chemtrex on toll free area code 

800- 424-9300 for another source of assistance in the event of spills, leaks, 

fires, exposures, accidents, or other chemical emergencies.
Appendix B

Managing/Monitoring Pesticide Spills

03/2007-01 Karnal Bunt Manual B-3

Managing/Monitoring Pesticide Spills

Supervisors involved in pesticide applications must be familiar with 

Guidelines for Managing and Monitoring Pesticide Spills dated March 1981. 

A useful reference is The Standard Pesticide User’s Guide, Fourth Edition, by 

Bert L. Bohmont, Prentice-Hall, Inc., 1997. In addition, the following pesticide 

spill safety and clean up equipment must be present at all job sites where 

pesticides are stored or used:

Safety

1. First Aid Kit—Bus and Truck Kit or equivalent (GSA66545-00-5312)

2. Fire extinguisher—5 lb size for Class A, B, or C fires

3. Portable eyewash kit

Cleanup

1. Shovel, square point, D-handle

2. Large heavy duty plastic bags with ties (23)

3. Rubber boots (2 pairs)

4. Disposable coveralls (4 pairs)

5. 5 gallons of water

6. Rubber gloves (4 pairs)

7. Respirators and pesticide cartridges (2 sets)

8. Broom

9. Dust pan

10. Liquid detergent (1 pint bottle); paper towels

11. Scrub brushes (2)

12. Plastic cover or tarpaulin to cover dry spills (10’ x 12’)

13. Absorbent material to absorb liquid spills (sand, sawdust, vermiculite, kitty 

litter, etc.)

14. Portable light source
Appendix B

Managing/Monitoring Pesticide Spills

B-4 Karnal Bunt Manual 03/2007-01
03/2007-01 Karnal Bunt Manual C-1

Appendix

C Appendix C

Technical Application Data and Chemicals

Contents

Methyl Bromide C-1

Chlorine Bleach C-1

PCNB (Pentachloronitrobenzene) C-1

Methyl Bromide

When fumigating contaminated elevators, storage buildings, bins, rail cars, 

equipment, and infected or contaminated grain, use methyl bromide at the rate 

of 5 pounds per 1,000 ft3 for a duration of 48 hours. Do not attempt fumigation 

when the temperature is below 70°F (21°C). Under normal conditions, 

maintain the recommended concentration of methyl bromide for 48 hours. 

Monitor the concentration of methyl bromide at regular intervals to ensure 

adequate treatment. Additional measures such as fumigant recirculation may 

be required in some cases.

Chlorine Bleach

Use chlorine bleach to decontaminate tools, equipment, etc., that may have 

come in contact with infected or contaminated grain or soil. Dilute one part 

Ultra Clorox brand regular bleach (6 percent sodium hypochlorite; EPA Reg. 

No. 5813-50) in 3 parts water; or one part CPPC Ultra Bleach 2 (6.15 percent 

sodium hypochlorite; EPA Reg. No. 67619-8) in 3.1 parts water. Since the 

working solution rapidly breaks down, prepare fresh solutions each time they 

are to be used. Use the chlorine bleach solution to thoroughly wet down all 

equipment and implements to be decontaminated. Allow the bleach solution to 

remain in contact with the equipment for at least 15 minutes.

Use one of the following formulations:

Ultra Clorox brand regular bleach (EPA Reg. No. 5813-50); 6% sodium 

hypochlorite

CPPC Ultra Bleach 2 (EPA Reg. No. 67619-8); 6.15% sodium 

hypochlorite

PCNB (Pentachloronitrobenzene)

Use PCNB with or without carboxin-thiram as a seed treatment. Treat all seed 

prior to use with one of the following:
Appendix C

PCNB (Pentachloronitrobenzene)

C-2 Karnal Bunt Manual 03/2007-01

1. 6.8 fl. oz. of carboxin-thiram (10% + 10%, 0.91 + 0.91 lb. active ingredient 

(ai/gal)) flowable liquid and 3 fl. oz. of pentachloronitrobenzene (2.23 lb. 

ai/gal) per 100 pounds of seed; or

2. 4.0 fl. oz. of carboxin-thiram (1.67 + 1.67 lb. ai/gal) flowable liquid and 3 

fl. oz. of pentachloronitrobenzene (2.23 lb. ai/gal) per 100 pounds of seed; 

or

3. 6.8 fl. oz. of carboxin-thiram (10% + 10%, 0.91 + 0.91 lb. ai/gal) flowable 

liquid per 100 pounds of seed; or

4. 4.0 fl. oz. of carboxin-thiram (1.67 + 1.67 lb. ai/gal) flowable liquid per 

100 pounds of seed; or

5. 3 fl. oz. of pentachloronitrobenzene (2.23 lb. ai/gal) per 100 pounds of 

seed.
03/2007-01 Karnal Bunt Manual D-1

Appendix

D Appendix D

Laboratory Procedures

Contents

Introduction and Background D-1

Grain Analysis Methods D-3

Background D-3

Obtaining Samples D-3

Automated Methods D-3

High Speed Optical Sorting Method D-4

Grain Inspection Machine Method D-7

Quality Control Procedures D-11

Manual Method D-12

Identifying Tilletia indica D-14

Storing Positive Kernels D-16

Decontaminating Labware and Instruments D-17

Seed Analysis Methods D-17

Background D-17

Setting Up the Laboratory D-18

Receiving Samples D-19

Extracting Samples D-21

Preparing Slides D-34

Examining Slides for Karnal Bunt D-39

Training Technicians and Verifiers D-42

Selecting and Training Bunted Kernel Verifiers D-44

Preparing New Sieves for Use D-46

Maintaining Sieves D-47

Ordering Sieves D-48

Forms D-48

Introduction and Background

The purpose of the laboratory section is to describe procedures for examining 

wheat grain samples for the presence of Tilletia indica infected grain (i.e., 

bunted kernels) and seed samples for the presence of T. indica teliospores. 

Grain analysis laboratories normally process grain samples from fields, bins, 

or national surveys for the presence of bunted kernels. Seed laboratories 

process seed samples and perform extraction procedures to determine the 

presence of T. indica teliospores.
Appendix D

Introduction and Background

D-2 Karnal Bunt Manual 03/2007-01

In program areas where bunted kernel analysis precedes seed analysis for 

spores, seed lots from regulated areas must be negative for bunted kernels prior 

to being considered for spore testing, and seed samples may also be processed 

at grain labs for the detection of bunted kernels. However, because of 

contamination potential, a different subsample of that seed lot must be 

submitted for spore analysis than the one processed for bunted kernel analysis 

and that subsample should not enter grain analysis laboratories unless it is 

sealed in nalgene bottles and is unopened. Some program areas analyze seed 

samples first for spores, then do a bunted kernel analysis on positive spore lots.

Grain and seed analysis laboratories must be physically separate and personnel 

working in grain laboratories (or field personnel) should not enter a seed 

analysis laboratory without proper decontamination safeguards.

The details of these procedures may be modified to individual laboratory 

needs; however, basic analysis methods and contamination safeguards should 

not be deviated from. Some seed analysis laboratories may wish to do the spore 

wash first and then the analysis of bunted kernels from the remaining dry 

sample afterward, and that is permissible.

Be very careful to maintain the identity of all grain and seed samples, with 

careful, accurate labeling.

The techniques for extracting teliospores by selective sieving were developed 

by Gary Peterson and Morris Bonde1

, United States Department of 

Agriculture, Agricultural Research Service (USDA, ARS), Ft. Detrick, MD. 

Plant Protection and Quarantine (PPQ) Plant Pathologist Ted Boratynski 

developed the grain inspection machine for bunted kernel analysis. Arizona 

Department of Agriculture Plant Pathologist Ron Ykema and others at the 

State Agriculture Laboratory in Phoenix, Arizona assisted in refinement of all 

the techniques described here and in the preparation of this manual. Joel Floyd, 

as PPQ Area Identifier in Nogales, AZ, wrote the teliospore and bunted kernel 

identification sections.

1 Peterson, G. L. and M. R. Bonde. 2000. Size-selective sieving for detecting teliospores of Tilletia indica in 

wheat seed samples. Plant Disease 9:999-1007.
Appendix D

Grain Analysis Methods

03/2007-01 Karnal Bunt Manual D-3

Grain Analysis Methods

Laboratories for bunted kernel detection should be in an enclosed area and 

equipped with the supplies listed below. A process for logging in samples 

received should be in place. The area designated for grain analysis must be 

physically separate from the seed (spore) analysis laboratory.

Background

This subsection describes three methods used to identify wheat kernels 

infected with Tilletia indica, the causal agent of Karnal bunt. These infected 

kernels are called bunted kernels. Included are specific procedures for 

decontamination of samples and facilities.

Obtaining Samples

The laboratory manager will specify which types and the priority of samples to 

be examined for bunted kernels. If assigned the task of detecting bunted 

kernels, do not enter any seed analysis areas.

Automated Methods

The automated methods are preferred for analyzing grain samples for bunted 

kernel detection. There are two types of automated methods in use. One 

involves optical high speed sorting technology and the other employs the use 

of a grain inspection machine. The optical scanner, where available, is used for 

both samples from the regulated area and National Survey samples. When 

grain inspection machines or high speed optical sorting machines are not 

available, the manual method can be used.

NOTICE

Seed samples tested for bunted kernels and/or spores must not utilize the same 

subsamples.
Appendix D

Grain Analysis Methods

D-4 Karnal Bunt Manual 03/2007-01

High Speed Optical Sorting Method

Supplies

Laboratory personnel will need the following supplies to examine wheat 

kernels for Karnal bunt using (Satake®) optical sorting technology:

Bags, paper

Bags, plastic

Beaker, glass or plastic, 600-mL

Bleach (6%), 1 gallon

Bottle, spray

Boxes, slide holder

Brush, fine tip applicator

Brushes, paint

Cover slips, glass

Dust masks

Fluorescent paint/dye

Forceps

Gloves, latex

Illuminators, microscope

Labels for slides

Lab towels, disposable

Ladder

Lamp, magnifying

Markers, permanent, fine-point, black

Microscopes, compound

Microscopes, dissecting

Nail polish, clear

Needle probes

Paper towels

Parafilm

Petri dish, plastic disposable

Razor blades

Reinforcements, white
Appendix D

Grain Analysis Methods

03/2007-01 Karnal Bunt Manual D-5

Shear’s mounting medium (see Preparing Shear’s Mounting Medium for 

preparation)

Slides, glass

Slides, identification aid

Tape, packing

Tape, quarantine

Tissue, toilet

Trash can, small

Tub, small

Tubs, 18-gallon/sample storage

Ultra-violet light

Vacuum cleaner, large shop type

Vacuum cleaner, small portable

Procedure

The protocol for the high speed optical sorting method (using the Satake® 

Scanmaster II) follows:

At the beginning of the day and about every four hours thereafter:

1. Make sure the machine is on for at least 30 minutes before use and that the 

valve for the compressor hose is at the “ON” position.

2. Check the light levels. Both should be at about 12.0–14.4 V (Setup 1 

Menu).

3. Select the MEDIUM Preset (Setup 3 menu). Be sure to press RECALL 

after selecting the correct product. When you go back to the Main menu, 

make sure the proper Preset is showing at the top of the screen.

4. Spike the sample with 3-13 fluorescent QC kernels. Recovery of spiked 

kernels will be done using the manual seed inspection machine.

5. Weigh the rejects. Based on a 4-lb sample, rejects will run between 180g–

320g, depending on the color of the wheat and the number of darkened 

kernels.

NOTICE

Before opening front panel ensure that the ejectors are turned off. Failure to do so 

could result in damage to the ejectors!
Appendix D

Grain Analysis Methods

D-6 Karnal Bunt Manual 03/2007-01

6. Adjust the DARK TRIP as needed to achieve the appropriate percentage of 

rejects (10-15% depending on variety). Light colored samples require 

higher trip rates. Red winter wheat trip rates are between 784 and 796. 

Durum wheat trips are between 844 and 864.

7. Open and clean the machine. Check the feeder, chute, and sorting chamber 

for kernels.

For each sample:

1. Clean the sample as needed.

2. Select the correct pre-set based on sample color and variety.

3. Run the sample.

4. Open and clean the machine. Check the feeder, chute, and sorting chamber 

for remaining kernels.

Repeat 1 through 4 for each sample.

To shut down the sorter:

1. Press the “X” in the upper right hand corner of the screen. This should get 

you back to the computer “desktop.”

2. Press “START” in the lower left hand corner.

3. Press “SHUTDOWN.”

4. A window will appear that says “What do you want the computer to do?” 

The “SHUTDOWN” command should already be selected. Press “OK.”

5. When the screen says “It is OK to turn off your computer,” press the power 

switch on the upper left side of the sorter. Do NOT shut down the sorter 

without going through this process.

6. Turn off the air supply valve.
Appendix D

Grain Analysis Methods

03/2007-01 Karnal Bunt Manual D-7

Grain Inspection Machine Method

Supplies

Laboratory personnel will need the following supplies to examine wheat 

kernels for Karnal bunt using the grain inspection machine method:

Beakers, glass, 600-mL

Bleach, 6 percent solution

Cover slips, glass (any size)

Dazzlens, glass cleaner

Dish, petri, plastic disposable, 50 x 9 mm

Dust mask or respirator

Forceps, specimen, stainless steel, large tip, small tip

Gloves, latex, small, medium, and large

Grain inspection machine

Kimwipes XL

Labels, white paper

Microscope, compound

Microscope, dissecting

Nail polish, clear, toluene/formaldehyde free

Paint brushes

Plastic bags, 12 x 12 inch, zip-lock

Shear’s mounting medium (see Preparing Shear’s Mounting Medium for 

preparation)

Slides, microscope, 3” x 1”

Tape, labeling

Tech Spray, inert dusting gas, or vacuum with hose attachment

Vials, plastic

Worksheets, Karnal bunt kernel analysis, in-house
Appendix D

Grain Analysis Methods

D-8 Karnal Bunt Manual 03/2007-01

Preparing the Workstation

Designated bunted kernel readers must wear clothing that will prevent sample 

contamination and protect them from harmful chemicals.

Step 1: Put on protective clothing.

Put on a dust mask or respirator, and latex gloves as needed. If the sample 

shows pink or violet dye, wear a dust mask when examining the grain. The dye 

shows that the grain has been treated with pesticide.

Step 2: Prepare the grain inspection machine.

Turn the feeder rheostat control knob on the grain inspection machine counter 

clockwise to the OFF position (see Figure D-2 for a diagram of a grain 

inspection machine). Turn ON the power switch located on the control panel. 

Place a clean petri dish and a pair of clean forceps on the work surface to the 

right side of the feeder trough. Open the acrylic lid above the grain hopper.

Analyzing the Sample

Step 1: Pour the sample into the hopper.

Obtain the sample to be tested and record the Karnal bunt sample number of 

the worksheet. Carefully open the sample bag(s) without tearing. Being careful 

not to over-fill, pour a portion of the sample into the grain hopper and close the 

acrylic lid.

Step 2: Activate the vibrating mechanism.

Turn ON the light switch. Turn ON the feeder rheostat control knob (vibrator 

switch). Adjust the grain hopper discharge opening and the feeder rheostat 

control until you get the desired feed rate. Only one level of grain should cover 

the bottom of the feeder trough as it vibrates, permitting observation of the 

kernels from all sides.

NOTICE

Be careful to empty the grain bin cup frequently to avoid spillage. You may wish to 

pour only a cup full of sample at a time into the grain hopper.
Appendix D

Grain Analysis Methods

03/2007-01 Karnal Bunt Manual D-9

Figure D-2 Grain Inspection Machine
Appendix D

Grain Analysis Methods

D-10 Karnal Bunt Manual 03/2007-01

Step 3: Check for bunted kernels.

When you observe a suspect bunted kernel, turn down the feed rate or the 

rheostat control and use forceps to move the kernel to the petri dish. Take the 

suspect bunted kernels to the dissecting microscope and examine for the 

presence of teliospores. Place all of the bunted kernels into a clean plastic vial 

or in a sealed petri dish and prepare a paper label to include in the vial with the 

Karnal bunt number, your initials, and the date. Using a black marker label the 

outside of the vial/petri dish. Follow identification authority verifier’s training 

procedures. If teliospores are present, prepare a microscope slide using Shear’s 

mounting medium. Seal the cover slip with nail polish. Review the slide using 

the compound microscope, verifying the characteristics of the T. indica

teliospore. Have an identifier with identification authority verify the presence 

of T. indica on the kernels found in a sample. Decontaminate the forceps with 

bleach and dispose of or disinfect the used petri dish.

Step 4: Record results.

Record your results on the appropriate worksheet. For positive results, record 

the total number of bunted kernels found in the sample. Place all positive 

samples in the storage area for positive samples. Place all negative samples in 

the storage area for negative samples, or dispose of as per project guidelines.

Step 5: Clean the inspection machine.

Clean the grain inspection machine as follows: Turn OFF the power switch and 

the light switch. Remove the seed bin cup and tilt the unit to permit any loose 

kernels to fall out through the seed bin cup area. Use inert dusting gas to blow 

any kernels from the unit. Return the unit to its upright position. Clean the 

magnifying lens with Dazzlens and Kimwipes. Periodically open the top of the 

machine and use paint brushes, or a vacuum with a hose attachment, or high 

pressure air to clean kernel pieces and dust from the inside of the machine.

Step 6: Clean the area and remove protective clothing.

When all of the samples have been examined, sweep up any spilled grain and 

dispose of it in a disposal bag in the area. Remove all protective clothing and 

place in the disposal bag.
Appendix D

Grain Analysis Methods

03/2007-01 Karnal Bunt Manual D-11

Quality Control Procedures

The following are quality control procedures for detection of Tilletia indica

bunted kernels.

As part of Quality Control (QC) procedures, samples are spiked with QC 

kernels, Tilletia indica bunted kernels, to measure efficacy of the Satake 

Optical Sorter and grain inspection machine personnel. These QC kernels are 

actual bunted kernels painted with a dye that fluoresces under UV light. 

Collected data will include the number of QC kernels added to the sample and 

the number of recovered QC kernels. The laboratory supervisor will use this 

data to keep the project director informed concerning validity of the screening 

procedures, and to provide feedback to lab personnel.

Each lab will have a vial containing 16 bunted kernels, painted with 

fluorescent paint, with which to “spike” the QC sample.

The 4-lb QC sample consists of previously tested negative grain and 3-13 

QC kernels determined by the random number generator.

Facilities Equipped with Satake Optical Sorter Technology

1. Use the random number generator to determine the number of bunted 

kernels with which to spike the QC sample.

2. Place the bunted kernels into the QC sample and run through the sorter at 

the lowest acceptable Dark Trip (784).

3. Look at the QC sample “discard container” with the UV light to ascertain 

that all of the QC kernels were ejected.

If all of the QC kernels are found, proceed to next step.

If any QC kernels are missing, look at clean portion container with the 

UV light to determine if the sorter missed any QC kernels. If it is 

determined that QC kernels were missed, raise the Dark Trip one unit 

(i.e. 784 to 788) and return the QC kernels to the QC sample and run 

again. Repeat this process until all QC kernels are ejected into the 

discard portion container. This will be the Dark Trip value to use for 

one shift (every four hours). Run a QC sample for each shift.

4. Replace all QC Kernels into discard portion. Have each lab technician 

working in the lab look at the discard using a manual inspection station 

(bunt machine) until he or she finds one bunted kernel.

5. Remove and secure all bunted kernels from the QC sample into a safe 

container and safeguarded in an appropriate location.

Appropriate worksheets will be provided for the daily recording of all 

QC results (see Figure D-4).
Appendix D

Grain Analysis Methods

D-12 Karnal Bunt Manual 03/2007-01

Field Facilities without Satake Optical Sorter Technology

1. The laboratory supervisor should spike each 4-lb QC sample with 3-13 

fluorescent QC kernels. Determine the number of kernels per sample by 

using a random number generator.

2. Inspect the sample using the manual grain inspection machine until all the 

QC kernels are found.

3. Record quality control information (see Figure D-4). If all QC kernels are 

recovered, no further action is required. If all QC kernels are not recovered, 

screen the 4-lb sample using the portable UV light to insure the indicated 

number of QC kernels were present.

Manual Method

Laboratory personnel will need the following supplies items to examine wheat 

kernels for Karnal bunt using the manual method:

Supplies

Bags, disposal, 12" x 24"

Beaker, glass, 600-mL

Bleach, 6 percent solution

Bottle, spray, 1-L, hardware store

Boxes, cardboard

Cover slips, glass (any size)

Dish, petri, plastic disposable, 50 x 9 mm

Dish pans, plastic, department store

Forceps, specimen, stainless steel, large tip

Gloves, latex, small, medium and large

Labels, white paper

Lamp, magnifying

Microscopes, dissecting and compound

Nail polish, clear, toluene/formaldehyde free

Shear’s mounting medium (see Preparing Shear’s Mounting Medium for 

preparation)

Slides, microscope, 3" x 1"

Storage units

Towels, lab

Vials, plastic

Wash basin, double sided

Worksheets, Karnal Bunt Kernel Analysis, in-house
Appendix D

Grain Analysis Methods

03/2007-01 Karnal Bunt Manual D-13

Preparing the Workstation

Designated bunted kernel readers must wear clothing that will prevent sample 

contamination and protect them from harmful chemicals.

Step 1: Put on protective articles.

Put on a dust mask or respirator, and latex gloves as needed. If the grain 

samples show pink or violet dye, wear a dust mask when examining the grain. 

The dye indicates that the grain has been treated with pesticide.

Step 2: Set up items.

Take one 600-mL beaker, one petri dish, one large forceps, and one plastic tray. 

Set these items up at your workstation and use the appropriate worksheet for 

recording data.

Analyzing the Sample

Step 1: Scoop grain into the beaker.

From the sealable plastic bag holding the grain sample, scoop approximately 

500 mL of grain and pour it into the beaker. Take the sample to your 

workstation for examination.

Step 2: Spread the sample.

Spread a portion of the sample throughout the plastic tray in approximately one 

layer of grain. Do a visual overview of the sample in the tray, looking for grain 

that shows a darkening on one end or obvious deterioration of the grain tissue.

Step 3: Check for bunted kernels.

When you observe a suspect bunted kernel, use forceps to move the kernel to 

the petri dish. Take the suspect bunted kernels to the dissecting microscope and 

examine for the presence of teliospores. Place all of the bunted kernels into a 

clean plastic vial or a sealed petri dish and prepare a paper label to include in 

the vial with the Karnal bunt number, your initials, and the date. Using a black 

marker label the outside of the vial. Follow identification authority verifier’s 

training procedures. If teliospores are present, prepare a microscope slide using 

Shear’s mounting medium. Seal the coverslip with nail polish. Review the 

slide using the compound microscope, verifying the characteristics of the T. 

indica teliospore. Have an identifier with identification authority verify the 

presence of T. indica on the kernels found in a sample. Decontaminate the 

forceps with bleach and dispose of or disinfect the used petri dish.

Step 4: Record results.

Record your results on the appropriate worksheet. For positive results, record 

the total number of bunted kernels found in the sample. Place all positive 

samples in the storage area for positive samples. Place all negative samples in 

the storage area for negative samples, or dispose of as per project guidelines.
Appendix D

Grain Analysis Methods

D-14 Karnal Bunt Manual 03/2007-01

Step 5: Clean the work area.

Clean and disinfect the work area with bleach solution.

Identifying Tilletia indica

Early stages of infection first appear at the germ end of the wheat kernel. These 

symptoms are usually not visible when the grooved side is facing down. 

Therefore, an effective technique for the manual method is to turn many seeds 

over to look for symptoms.

The first visible stage of Karnal bunt infection is a swollen area that appears 

gray or dark at the germ end forming a triangular shaped area on either side of 

the groove (see illustrations A2, B1, and B2 on the laminated insert titled 

Symptoms of Karnal Bunt (Tilletia indica) and Black Point). This is the thin 

pericarp, or outer epidermal layer, of the seed coat through which a darkening 

is visible and is evidence of a mass of teliospores beneath (the sorus). This 

swollen area of the pericarp may already be ruptured or can easily be 

punctured with a teasing needle, exposing many dark brown teliospores.
Appendix D

Grain Analysis Methods

03/2007-01 Karnal Bunt Manual D-15

0

Figure D-3 Symptoms of Karnal Bunt (Tilletia indica) and Black Point
Appendix D

Grain Analysis Methods

D-16 Karnal Bunt Manual 03/2007-01

Karnal bunt teliospores, large by fungal spore standards, are easy to see with a 

hand lens. However, to verify Karnal bunt symptoms from a seed sample, 

viewing the infected kernels under a dissecting microscope will be necessary 

to see if teliospores are present. They appear as hundreds or thousands of 

small, granulate, shiny dark brown to black spheres, resembling microscopic 

ball bearings or buckshot. They are contained within the area of the seed that 

the fungus has consumed, together forming the sorus.

Refer to the laminated insert. More advanced infection (A3 and A4) in the 

wheat kernel manifests itself as a blackening along the groove, progressively 

consuming more of the inner portions of seed tissue. A very advanced stage of 

infection results in the wheat kernel taking on the appearance of a boat or 

dugout canoe (A5).

Black point is a disease of non-quarantine importance caused by a variety of 

fungal species (Alternaria spp. and Helminthosporium spp.). The superficial 

symptoms of Karnal bunt have been confused with those of black point, but 

close examination reveals distinct differences. While both diseases give the 

germ end of the wheat a darkened appearance, black point is actually more of a 

darkening or discoloration of the pericarp (C1 and C2, laminated insert). Upon 

examination under a dissecting microscope, no spores, nor anything that 

resembles teliospores, are visible. Also, the darkening caused by black point 

organisms may extend farther from the tip of the seed and be more general in 

its occurrence on the seed, often appearing on the ventral and dorsal surfaces. 

Black point shows no obvious erosion of the seed tissue as in Karnal bunt.

Other smut diseases such as common bunt (T. laevis or T. tritici) may occur in 

the samples. Consult with a bunted kernel identifier to verify.

To make a positive identification for any spore, making a microscope slide 

will be necessary to view the spores under high magnification.

Storing Positive Kernels

Step 1: Place bunted kernels in a plastic vial or petri dish.

Place all of the bunted kernels from the same sample in a plastic vial or petri 

dish. Using a black marker, label the vial with the sample number, your initials, 

and the date on the outside of the vial, and inside the vial on a white label.

Step 2: Verify presence of Tilletia indica.

Have an identifier with identification authority for the bunted kernel verify the 

presence of T. indica on the kernels found in a sample. Return all negative 

grain to its original packaging. Also return the positive sample (minus any 

bunted kernels) to its original packaging and place it in the positive storage 

area. Verify the presence of T. indica teliospores on the kernel by making a 

slide and storing all positive slides with proper labels. Retain voucher slides for 

a project-specified time in a secure storage container.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-17

Step 3: Record findings.

Record your findings, initials, and date on the appropriate analysis worksheet 

(Figure D-4). Have the verifier initial in the appropriate space for the positive 

samples verified.

Step 4: Take care of used equipment.

Place all labware and instruments into the dirty bin for decontamination. Use 

new gloves, labware, and lab tools for each new sample. Continue with the 

next sample.

Decontaminating Labware and Instruments

Follow this procedure to decontaminate labware and instruments used in 

manual or automated methods:

Take all dirty labware and lab tools to a designated area for decontamination. 

As a precaution, keep labware “wet” until decontamination with the bleach 

solution. Soak all labware and lab tools in a 30 percent bleach solution for 15 

minutes, rinse with clear water, dry, and return to the bunt area for the next use.

After examining all the samples, or at the end of a shift, decontaminate the 

area. Decontamination includes these activities:

Bleach all workstation surfaces (hoods, chairs, tables, etc.).

Make sure no dirty labware or instruments remain in the area.

Sweep the floor and mop with 30 percent bleach solution.

When decontamination is complete, dispose of all shoe covers, hair covers, 

coveralls, and gloves into the biohazard container. Dispose of all waste during 

the examination process into the biohazard container. Turn off all hoods and 

lights when finished.

Seed Analysis Methods

Background

The purpose of seed analysis laboratories is to determine the presence or 

absence of Karnal bunt, T. indica, teliospores in submitted wheat seed samples. 

For a seed lot to be considered negative, five replications must be performed 

and all five tests must be negative for the presence of T. indica teliospores. 

Seed lot samples with only spore fragments that cannot be measured can be 

resampled and run a second time.

NOTICE

Handle all samples as if positive to help prevent contamination.
Appendix D

Seed Analysis Methods

D-18 Karnal Bunt Manual 03/2007-01

Setting Up the Laboratory

Whether laboratory personnel process a few samples per day or a hundred, the 

same minimum facility requirements are necessary. By meeting these 

requirements, each laboratory will have the flexibility to use its available 

resources, while still maintaining a national standard. The seed analysis 

laboratory must have three basic areas:

A reception area

A dirty room

A clean room

Combining functions performed in the dirty room and the clean room into one 

room is acceptable under the following conditions:

Samples for processing come from a non-Karnal bunt area.

No positive samples have been in the laboratory.

Once a suspect positive sample is encountered, these rooms and functions need 

to be separate. Also, workers should always process a negative control wheat 

sample with each group of samples once any samples entering the laboratory 

test Karnal bunt positive.

Reception Area

The reception area, a place for initial delivery of samples and decontamination 

of packaging, must be outside the laboratory facilities, whether literally outside 

or in a designated building, loading dock, or shed. This area can also be used 

for storing the remaining sample not used in the laboratory evaluation. The 

area must have access to running water.

Dirty Room

The dirty room is an area for opening dry seed samples, adding extraction 

solution, and transferring or weighing samples. The primary concern is to 

prevent dry, loose teliospores from becoming airborne and contaminating other 

samples and the external surfaces of the extraction equipment. The minimum 

equipment required is a clean air station or biological containment hood, to 

prevent direct air movement and ventilation from this room into the clean 

room. Between each sample, workers must wipe down all working surfaces 

with detergent and 30 percent bleach (6 percent sodium hypochlorite). 

Technicians must change gloves between samples and not wear contaminated 

garments into clean areas.

NOTICE

Field sample personnel or individuals entering the grain analysis laboratory are not 

allowed entry into the seed analysis laboratory without proper decontamination safeguards. Showering and changing clothes is usually sufficient.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-19

Clean Room

No sample should enter the clean room unless it is in liquid suspension. 

Workers moving from the dirty room into the clean room should have separate 

lab coats. Activities performed in the clean room are sample extraction, slide 

preparation, extraction ware decontamination, and microscopy. Laboratory 

personnel must decontaminate the extraction table surfaces between sets of 

samples. Workers must clean up any spills before they dry and spray the 

area of any spill with 30 percent bleach. After extracting samples on trays, 

remove and decontaminate the trays.

Receiving Samples

Background

This subsection describes procedures used to receive seed samples at approved 

laboratories for the analysis for Tilletia indica. Included are specific 

procedures for decontaminating the seed samples before they enter the 

laboratory, and log-in procedures.

Supplies

Laboratory personnel will need the following supplies to receive the samples:

Bleach, (sodium hypochlorite), 6 percent

Cardboard boxes

Centrifuge tubes

Chain-of-custody seals (if appropriate)

Container to hold bleach solution

Coveralls

Dish pans, plastic, department store

Garden hose with nozzle, hardware store

Karnal bunt spore analysis worksheets, in-house designed

Lab coats

Latex gloves

Plastic disposal bags, 12" x 24"

Safety glasses

Secure storage units for samples

Spray bottle, 1-liter, hardware store

Utility cart
Appendix D

Seed Analysis Methods

D-20 Karnal Bunt Manual 03/2007-01

Sample Decontamination

When samples arrive at the laboratory, make sure to receive them at a 

designated check-in/decontamination area. This area must be physically 

separate from the laboratory facility where samples are processed. A wash 

basin and hose setup is necessary at this location. Use the following procedure 

to check in and decontaminate the samples before bringing them inside the 

laboratory. Wear gloves, safety glasses, and a lab coat or coveralls during this 

procedure.

Step 1: Check samples.

Check each sample against the accompanying paper work to ensure matching 

sample number. Check all seals and samples for integrity.

Step 2: Decontaminate samples.

Decontaminate the sample containers using Table D-16 as a guide:

Step 3: Log-in samples.

Take the paperwork and samples, if necessary, into the sample receiving area 

of the laboratory for log-in.

Table D-16 Decontamination of Samples

If the samples (or 

subsamples) are in: Then:

Plastic bottles 1. REMOVE the bottles from the sample bag.

2. TIGHTEN the lids to ensure a water tight seal.

3. SUBMERGE each bottle in a 30 percent bleach solution prepared fresh daily.

4. After 15 minutes, RINSE the bottles with tap water from a garden 

hose.

5. GO to Step 3.

Plastic bags 1. MAKE sure the bags are sealed tightly.

2. FILL a spray bottle with 30 percent bleach solution, prepared 

fresh daily.

3. SPRAY the bags with the bleach solution and ALLOW the solution to remain on the bags for 15 minutes.

4. After 15 minutes, RINSE the bags with tap water from a garden 

hose.

5. GO to Step 3.

NOTICE

Never take any samples into the laboratory before decontaminating them.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-21

Sample Log-In

Accurate sample log-in is very important. Be very careful to maintain each 

sample’s identity.

Step 1: List the sample number on the worksheet.

List the assigned sample number on the analysis worksheet, and label sample 

containers with the assigned laboratory number. Place samples for spore 

analysis on a utility cart after logging-in and labeling them. Enter a “Quality 

Assurance (QA) Negative” laboratory control on the worksheet as well. Make 

sure to include on the cart a label for the control (for assembly in the weighing 

room). Use positive laboratory controls occasionally to prove the laboratory’s 

ability to detect and identify positive samples.

Step 2: Label the centrifuge tubes.

Label centrifuge tubes with laboratory sample numbers for each sample in 

each set. Place the tubes in a disposable tube rack supplied with the tubes, and 

label the rack with the set number. Place the samples, worksheet, and 

centrifuge tubes on a utility cart and deliver these items to the weighing area or 

the solution addition area.

Reserve Sample Storage

Using approved sample journals, label excess sample bags and bottles with 

correct laboratory sample numbers. Sign and date any broken seals. Reseal the 

samples and dispose of used gloves in a biohazard bag at the decontamination 

area. Group samples by box number and place them in cardboard boxes in a 

secure storage area. Identify the boxes appropriately.

Extracting Samples

Background

This section describes procedures used at approved laboratories to extract 

teliospores of Tilletia indica from wheat or other crop samples. Included are 

specific procedures for preparation of extraction solution and preparation of 

Shear’s mounting medium. Also included are procedures used to prepare 

extraction containers and measure out samples, and an alternative method for 

adding extraction solution to premeasured bottled samples. This section also 

details the extraction technique itself.

Supplies

Laboratory personnel will need the following supplies to process the samples:

Aprons, rubber, 45" x 35"

Bags, biohazard, 38" x 48"

Bags, biohazard, 12" x 24"
Appendix D

Seed Analysis Methods

D-22 Karnal Bunt Manual 03/2007-01

Balance, analytical

Beakers, glass, 2-L and 1-L

Beakers, 600 mL

Bleach (6 percent sodium hypochlorite)

Bottle, glass, 1-L with ground glass stopper

Bottle, spray, plastic, 1-L, hardware store

Bottles, HDPE wide mouth, 250-mL, plastic

Bottles, wash, 500-mL

Box, broken glass disposal

Bulb, pipette

Carboy, 20-L with spigot

Cart, glassware

Cart, utility, stainless steel

Centrifuge, minimum 1,000 r/min required

Citric acid monohydrate (C6H8O7.1H2O)

Cups, disposable, paper, 5-oz

Cups, disposable, paper, 3-oz

Cups, portion, paper, 1-oz

Cylinders, graduated, 25-mL, 250-mL, 500-mL, 1-L

Deionized water

Detergent

Dissecting microscope, linen tester, or 10X magnifying glass

Dust masks

Ethyl alcohol, 95 percent, anhydrous

Flasks, Erlenmeyer, graduated, 500-mL

Funnels, glass, 60-mm ID top x 13-mm OD stem

Funnels, glass, 100-mm ID top, 18-mm OD stem

Glasses, safety

Gloves, gauntlet, sizes 9, 10, and 11

Gloves, latex, surgical

Gloves, nitrile, sizes 8, 9, 10, and 11

Glycerol

Hood, biosafety with HEPA filter
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-23

Hoods, fume

Inverted light microscope, equipped with ocular micrometer

Isopropyl alcohol, 70 percent

Karnal Bunt Sieve Log

Karnal Bunt Sieve Usage Log

Labels, 1" x 2", 3 up x 10, Avery, 5160

Liner, bench (substitute for plastic trays)

Magnifying glass, illuminated

Mixer, vortex, thermolyne

Parafilm

Pipette tips, 200-1000 μL, for Eppendorf adjustable pipettor

Pipette tips, repeating

Pipettes, Mohr, 1-mL and 10-mL

Pipettor, adjustable, Eppendorf, 100-1000 μL

Pitcher, 3000-mL

Potassium acetate (KC2H3O2)

Rack, test tube

Scoop, lab, stainless steel

Shaker, orbital, thermolyne

Sieve, nylon, 20 m pore with square openings, 3" ID x 50 mm high, 

USDA2

Sieve, nylon, 53 m pore with square openings, 3" ID x 50 mm high, 

USDA2

Silicone sealant

Sodium phosphate, dibasic, anhydrous (Na2HPO4)

Stage micrometer

Stir bars, magnetic, 11⁄2" x 1⁄2"

Stirrer, magnetic

Syringes, 10 ml

Tape, lab, blue, 1"

Tape, lab, red, 1⁄2"

Tape, yellow stretchy, lab safety

2 See attachment for construction of the sieves.
Appendix D

Seed Analysis Methods

D-24 Karnal Bunt Manual 03/2007-01

Terri-wipes

Timers

Trays, plastic, 18" x 12" x 1⁄2" deep (substitute for bench liners)

Tubes, centrifuge, disposable, plastic, conical, 15-mL

Tubs, disinfectant

Tubs, self-draining and holding

Tween 20 or wetting agent

Preparing the Extraction Solution

Prepare the extraction solution in a separate, clean room. Never bring grain 

samples into this room.

Step 1: Fill a carboy with deionized water.

Wearing safety glasses and latex gloves, fill a 20-L carboy with 18 L of 

deionized water.

Step 2: Add detergent.

Using a graduated cylinder, add 12 mL Tween 20 detergent or wetting agent to 

the carboy. Rinse the cylinder several times with deionized water into the 

carboy.

Step 3: Label the carboy.

Place a strip of red lab tape at the 18-L mark and label as Tween 20 solution. 

Cap tightly and roll the carboy on a utility cart to make sure that the Tween is 

well dispersed in the water.

Preparing Shear’s Mounting Medium

After centrifuging aqueous extracts of the sample and decanting the 

supernatant, re-suspending the remaining pellet in mounting fluid is necessary. 

Shear’s mounting medium, the standard mounting medium for smut fungi, 

provides excellent buffering capacity. The fluid maintains the structural 

integrity of the Karnal bunt spores, while limiting evaporation. Additionally, 

the glycerol in the Shear’s fluid maintains a refractive index of 1.47 at 27°C, 

which allows the effective transmission of light by the compound microscope. 

Shear’s mounting fluid preserves sample extracts for future microscopic 

evaluation. You can use the procedure for preparation below, or purchase the 

mounting fluid from a commercial source.

Preparing 1 L of 0.2 M Na2HPO4 solution

Step 1: Weigh anhydrous Na2HPO4.

Wearing safety glasses and latex gloves, weigh 38.39 g of anhydrous Na2HPO4

into a tared 2-L beaker on a 3 decimal balance.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-25

Step 2: Add deionized water.

Using a graduated cylinder, add 1 L of deionized water to the beaker and stir 

with a magnetic stirring bar until all granules are dissolved.

Step 3: Transfer the solution.

Using a funnel, transfer the solution to a 1-L glass stoppered bottle and label as 

0.2 M Na2HPO4 with the date of preparation and your initials.

Preparing 1 L of 0.1 M citric acid solution

Step 1: Weigh citric acid monohydrate.

Weigh 21.01 g of citric acid monohydrate into a tared 2-L beaker on a 3 

decimal balance.

Step 2: Add deionized water.

Using a graduated cylinder, add 1 L of deionized water to the beaker and stir 

until all granules are dissolved.

Step 3: Transfer the solution.

Using a funnel, transfer the solution to a 1-L glass stoppered bottle and label as 

0.1 M citric acid solution with the date of preparation and your initials.

Preparing 600 mL of Shear’s Mounting Medium

Step 1: Transfer Na2HPO4.

Using a pipette, transfer 19.45 mL of 0.2 M Na2HPO4 into a 1-L beaker.

Step 2: Transfer citric acid.

Using a 1-mL pipette, transfer 0.55 mL of 0.1 M citric acid solution into the 

same beaker.

Step 3: Add potassium acetate and deionized water.

Add 6 g of potassium acetate and 280 mL of deionized water to the beaker.

Step 4: Stir to dissolve solids.

Stir with a magnetic stirring bar until all solids are dissolved and the solution is 

uniform in appearance.

Step 5: Add glycerol and ethyl alcohol.

Continue stirring and add 120 mL of glycerol and 180 mL of 95% ethyl 

alcohol to the beaker.

Step 6: Transfer the solution.

After the solution is uniform in appearance, use a funnel to transfer to a 1-L 

glass stoppered bottle. Label as Shear’s mounting medium, and include the 

date of preparation and your initials.
Appendix D

Seed Analysis Methods

D-26 Karnal Bunt Manual 03/2007-01

Step 7: Clean glassware.

Clean all glassware with lab detergent and rinse with tap water followed by 

deionized water. Place pipettes in the pipette washer with tips up.

Optional Method for Preparing Extraction Flasks

Prepare and label an Erlenmeyer flask containing extraction solution for each 

sample that requires weighing or measuring out before extraction. Carry out 

the following procedure in the room designated for preparing extraction 

solution:

Step 1: Fill flasks with extraction solution.

Wearing safety glasses and latex gloves, fill 500-mL flasks to the 100 mL mark 

with extraction solution directly from the 20-L carboy. Place two 3-oz 

disposable paper cups over the mouth of each flask immediately after adding 

the solution.

Step 2: Label flasks.

Label one flask for each sample in the set to be extracted. Place the flasks for 

that set on the top shelf of a utility cart in the order listed on the worksheet and 

move it to the sample weighing area.

Step 3: Place worksheet and tubes on cart.

Place the spore analysis worksheet and labeled centrifuge tubes on the top 

shelf of the cart. Place the samples in order from left to right on the second 

shelf of the cart and take the cart to the room designated for measuring out the 

samples.

Weighing and Measuring the Samples

It is preferable to have 50-g pre-measured samples arrive at the laboratory in 

sealed nalgene bottles that are disinfected with chlorine solution before 

entering the laboratory. Use these procedures when larger, unmeasured, or 

dirty samples arrive at the seed testing laboratory.

Take samples that require weighing to the sample weighing area. It is 

recommended that this procedure be carried out in a biosafety hood fitted with 

a HEPA filter. Generally, the only samples of this type are bin sweepings or 

other “dirty” samples that contain mostly dirt and chaff and very little grain. 

Measure most other samples by volume rather than by weight. Consult 

your supervisor if weighing or measuring unusual sample matrices or sample 

types is necessary. Wear safety glasses, latex gloves, and a lab coat or 

overalls.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-27

Sanitizing the hood. Before weighing or measuring any samples, first sanitize 

the hood thoroughly.

Step 1: Turn on power.

Turn on power to the hood, light, and balance.

Step 2: Sanitize the hood.

Thoroughly sanitize the interior of the hood (including walls, glass door, and 

ceiling) by spraying with 30 percent bleach solution. This also includes the 

balance and the area under the balance. Let the bleach stand for 15 minutes.

Step 3: Spray hood again.

Spray the hood again with the bleach solution and wipe dry with terry wipes. 

Dispose of used terry wipes in the biohazard bag.

Step 4: Clean bleach residues from hood.

Spray the entire hood with 70 percent isopropyl alcohol and wipe dry. Doing 

this will help to clean bleach residues from hood surfaces, particularly the glass 

window of the hood. Dispose of used terry wipes in the biohazard bag.

Weighing the samples. For samples that are very dirty and contain very little 

sample by weight, measure them out by weighing at least a 2.0 g portion of 

sample, up to 50 g, on an analytical balance. Wear a dust mask when handling 

dirty and dusty samples.

Step 1: Take a sample from the cart.

From the cart containing the sample set to be weighed, take a sample and make 

sure that the sample number and the number on the container of extraction 

solution are identical. Place the sample and the corresponding container in the 

hood. Pull down the hood door to allow only enough space to move sample 

containers in and out of the hood.

Step 2: Put on gloves.

Put on a new pair of latex gloves; place one of the two paper cups on the 

balance and tare it.

Step 3: Mix the sample.

Mix the sample well by inverting and rolling thoroughly. Open the sample 

container and use one paper cup to scoop out 2.0 g ± 0.1 g of sample into the 

cup on the balance. Transfer excess sample that has not touched the hood or 

balance back to the sample container.

Step 4: Transfer the contents.

Transfer the contents of the paper cup to the sample container and seal the 

opening with parafilm.
Appendix D

Seed Analysis Methods

D-28 Karnal Bunt Manual 03/2007-01

Step 5: Close the sample container.

Close the sample container and place the cups in the palm of one gloved hand 

and fold that glove over them. Now place this glove in the palm of the other 

hand and repeat. Dispose of the used materials in the biohazard bag.

Step 6: Remove the sample from the hood.

Remove the sample and the container from the hood and place the container 

back on the cart. Return the sample to the storage area.

Step 7: Repeat procedure for additional samples.

For additional samples, repeat Step 1 through Step 6 using new gloves for each 

sample. Deliver the cart to the extraction lab after weighing all samples. Return 

sample bags to the storage area.

Step 8: Maintain cleanliness.

Always maintain cleanliness of the weighing area. If any sample falls on the 

balance or the hood floor, do not place in any sample container. Dispose of the 

sample with the gloves and cups at the end of the weighing cycle. If any 

sample falls on the hood or balance, sanitize the area of the spill before 

weighing the next sample. Wipe up the spilled sample and dispose of it in a 

biohazard bag. Spray the area where the sample fell and the surrounding area 

with the bleach solution. Wipe up the bleach solution and dispose of the dirty 

gloves and terry wipes in the biohazard bag.

Step 9: Resanitize the hood and balance.

Resanitize the hood and balance after each sample.

Measuring the samples. Measuring the samples by volume is preferable to 

measuring by weight. Measure by volume these types of samples:

Samples that do not arrive premeasured in plastic bottles

Samples that require additional analyses

Measure these samples into clean glass or new plastic sample containers.

Step 1: Measure the samples.

The procedure is the same as for weighing samples in the previous section, except 

that this procedure involves measuring the samples by scooping three 1-oz 

cupfuls into a container (may be different for other types of crops). Use the paper 

portion cups for this purpose and then dispose of them in the biohazard bag.

Step 2: Take plastic bottles by cart.

When measuring the samples into plastic bottles, take them by cart to add 

extraction solution before taking them to the extraction lab. See Adding 

Extraction Solution on page D-29.
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03/2007-01 Karnal Bunt Manual D-29

Step 3: Measure control wheat.

Also, measure control wheat into containers using the above procedure. Seal 

these containers of control wheat and store them in the solution addition room 

for later use in extraction sets as needed.

Step 4: Resanitize the hood.

When finished weighing or measuring samples for the day or at the end of a 

shift, resanitize the hood.

Adding Extraction Solution

When an extraction set consists of premeasured samples in plastic bottles, add 

the extraction solution to the bottles in the hood in the solution addition room. 

Take only decontaminated samples into this room. Wear safety glasses and 

latex gloves throughout this procedure.

Step 1: Turn on power and sanitize the hood.

Turn on power to the hood and sanitize the hood as described in the subsection 

Sanitizing the hood. Pull down the hood door to allow only enough space to 

move sample bottles in and out of the hood.

Step 2: Check samples.

Before adding solution to any of the bottles, first check to make sure that each 

sample on the utility cart has a matching sample number listed on the 

worksheet. The bottles should also appear to contain approximately 50 g of 

sample for wheat; other crops may be different. If any bottle appears to contain 

too much or too little sample, call it to the attention of your supervisor before 

measuring a new sample from the bag of grain retained in storage.

Step 3: Add extraction solution.

For each sample, place bottle(s) in the hood and put on a new pair of latex 

gloves. Add extraction solution from the carboy on the counter outside the 

hood to a clean, disposable paper cup. Fill to a level equal to the mark on the 

reference cup next to the carboy. This should be approximately 100 mL of 

solution. Place the cup of solution in the hood and open the sample bottle. Add 

the solution to the bottled sample. Tighten the bottle cap and place the sample 

back on the cart. After completing a sample, place the cup(s) in the palm of one 

gloved hand and fold that glove over it. Now place this glove in the palm of the 

other hand and repeat. Dispose of the gloves and cup in the biohazard bag. 

Repeat this procedure for each sample on the cart, using a new pair of gloves 

and cup for each sample. Do not reuse paper cups. Once extraction fluid has 

been added to the sample containers, samples must be extracted within 30 

minutes.
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D-30 Karnal Bunt Manual 03/2007-01

Step 4: Resanitize the hood.

When finished weighing or measuring samples for the day or at the end of a 

shift, resanitize the hood.

Extracting the Samples

The clean room must have sufficient counter space (approximately 15 ft) to 

carry out the extractions. The lab must also have a shaker table, a chemical 

fume hood for bleach baths, and sinks to rinse glassware. A separate counter 

top is necessary to support the centrifuge station.

Extract the samples in a clean room, always wearing safety glasses. As an 

added precaution, wear protective clothing such as a rubber apron and heavy 

rubber gloves when decontaminating glassware with bleach solution. Wear 

latex gloves when extracting samples and change the gloves between samples.

Setting up for extraction. 

Step 1: Position bench liner or plastic trays.

Place the laboratory bench liner on the bench top with the plastic side down. To 

prevent confusion when processing multiple samples, use lab tape to mark out 

lanes approximately 30 cm wide. Another approved method is to use 

individual plastic trays (18" x 12" x 1⁄2") for each sample. Plastic trays will 

contain spills, and tubes cannot roll into the wrong lane. The trays also work 

well at smaller labs that do not have room to set up multiple lanes.

Step 2: Lay out beaker and sieve sets.

Place one 600-mL beaker toward the rear of each lane and set a 20 μm pore 

nylon sieve on top of it. Place another 600-mL beaker in front of the first 

beaker and set a 53-μm pore nylon sieve on top of that beaker. Lay out as many 

beaker and sieve sets as there are samples to extract, and place centrifuge racks 

between each set. Place small glass funnels (60 mm ID) in the center of the 

bench, along with pitchers and wash bottles containing tap water. When using 

the plastic tray option, keep all the extraction equipment for one sample in that 

sample’s tray. Assure that flasks, beakers, and centrifuge tubes are properly 

labeled with corresponding sample numbers.

Step 3: Prepare bleach baths.

Prepare bleach baths by filling large disinfectant tubs inside fume hoods (or in 

a separate room) with 30 percent bleach solution. The bleach solution baths 

need to contain a sufficient volume of solution to completely submerge the 

glassware being decontaminated.
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03/2007-01 Karnal Bunt Manual D-31

Extracting the samples. 

Step 1: Shake samples.

Place sample bottles or flasks containing extraction solution on an orbital 

shaker. Secure the containers in the shaker so that the fit is appropriate for the 

type of container used. Shake samples for 5 minutes at 200 r/min. Do not allow 

samples to remain in the extraction solution for longer than 30 minutes due to 

release of starch granules. While samples are shaking, remove the centrifuge 

tubes that arrived with the samples from the disposable rack and place one per 

lane on the bench top. Place the disposable centrifuge tube rack and worksheet 

in the center of the bench. Sanitize the utility cart with 30 percent bleach 

solution before returning it to the sample receiving area.

Step 2: Place samples with tubes.

After shaking the samples, place each in the lane/tray with the tube that 

corresponds to that sample. Double check to make absolutely sure that tubes 

and sample bottles have matching sample numbers.

Step 3: Pour sample into 53-μm sieve3.

For each sample, invert the bottle or swirl the flask to re-suspend the grain and 

debris. Immediately pour the contents into the 53-μm sieve, collecting the 

filtrate in the 600-mL beaker. Add an additional 100 mL of water to the 

container, swirl or recap and shake the bottle, and then pour into the sieve to 

rinse the grain. Take care to avoid splashing the sample solution during sieving 

procedures. Reextract samples if cross-contamination occurs. Rinse a 

minimum of twice, preferably three times, tilting the sieve on top of the beaker 

to aid draining. Place the empty extraction container on the glassware cart.

Step 4: Pour filtrate into 20-μm sieve3.

After the sieve has drained, discard the grain into a small disposal bag, and 

place the used sieve on a glassware cart. Pour the filtrate from the beaker into 

the 20-μm sieve, collecting the filtrate in the second 600-mL beaker. Add an 

additional 100 mL of water to the first beaker, swirl, and pour through the 

sieve. Use a wash bottle to rinse down the side walls of the beaker, and pour 

this rinsing into the sieve as well. Rinse a minimum of twice, preferably three 

times. Place the used beaker on the glassware cart.

3 Follow quality assurance checks for sieves.

NOTICE

The sieves are manufactured with a ring to make them stackable as an option for 

using them together on one beaker.
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D-32 Karnal Bunt Manual 03/2007-01

Step 5: Rinse the nylon screen.

Once the 20-μm sieve has drained, tilt the sieve on top of the beaker and use 

the wash bottle to rinse down the sides and upper portion of the nylon screen. 

Tip the sieve over the 50-mL beaker at a 45° angle and use a wash bottle with a 

steady stream to rinse debris into the beaker. Repeat this rinse twice. Debris 

will pool in the lower portion of the sieve.

Step 6: Rinse debris into the centrifuge tube.

Open the centrifuge tube and place it upright in the centrifuge tube rack. Use a 

wash bottle to rinse debris from the 20-μm sieve into the tube. You may also 

use a small glass funnel to facilitate this transfer. To assure that all tubes have 

the same amount of liquid, fill the tubes with liquid to just below the screw cap 

threads. Recap the tube and return to the disposable centrifuge rack. Pour the 

extraction filtrate into a 20-L carboy containing 1⁄2 gallon of bleach. Place 

filtrate from samples treated with fungicide in a separate carboy. Place used 

glassware and sieve on the glassware cart. As an added precaution against 

contamination by airborne spores, soak all used labware in a water bath when 

not in use, until decontamination.

Step 7: Repeat procedure for each sample.

Repeat Step 3 through Step 6 for each sample in the extraction set. If extracting 

multiple samples becomes necessary, processing all samples through one step 

before proceeding to the next step is most efficient. Be sure to remain 

undistracted while doing this. Take care not to mix samples or confuse the 

extraction steps.

Step 8: Notify your supervisor in case of spills.

In case of a sample spill, notify your supervisor to decide if a second extraction 

of the sample is necessary. If so, discard the sample and show the result for that 

sample as “L” on the worksheet with your initials and date. Record all lab 

accidents in the lab notebook kept at the centrifuge station. If a sample spills on 

the bench liner, replace it with a clean liner. If any sample contacts the bench 

surface, spray a 30 percent bleach solution on the area to decontaminate it, and 

wipe with a clean towel.

Step 9: Transfer glassware and sieves to bleach bath.

Immediately transfer all used glassware and sieves from the glassware cart to a 

bleach bath and soak them for 15 minutes. Do not allow sieves to remain in the 

bleach bath for more than 15 minutes, as this will rapidly degrade the nylon 

screens. Rinse glassware and sieves thoroughly with hot tap water. Hang 

glassware on a rack to drain and place sieves in self-draining plastic tubs. 

Discard plastic sample bottles after decontaminating them in the bleach bath.
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03/2007-01 Karnal Bunt Manual D-33

Step 10: Centrifuge the tubes.

After preparing all of the tubes for the set, take them to the centrifuge station 

and centrifuge at 1,000 r/min for 5 minutes. Pour off the supernatant and resuspend the pellet by pipetting 500 μl of Shear’s mounting medium into the 

tube. Recap and vortex a few seconds to mix the pellet with the Shear’s fluid. 

Record the set information in the centrifuge log book before delivering the 

centrifuge tubes and worksheet to the microscope area.

Cleaning Up the Extraction Lab

Clean up the clean room by following these steps:

Step 1: Remove the bench liner.

At the end of each extraction series or as necessary, remove the bench liner and 

discard in a large biohazard bag along with the small bags of extracted grain 

and other potentially contaminated materials. Tie these bags shut with lab tape. 

Place in red biohazard containers in a designated hazardous waste area. When 

using plastic trays, removing and discarding bench liners is not necessary.

Step 2: Dispose of the filtrate.

Pour the bleached sample filtrate from the carboy down a sink drain as 

necessary. After filling any carboys containing filtrate from seed treated with 

pesticide, pour the contents into a labeled hazardous waste container.

Step 3: Sanitize surfaces.

Spray all bench tops, carts, and other potentially contaminated surfaces with 30 

percent bleach solution. Wipe all these surfaces down at the end of each 

extraction series. Sweep and mop the floor in the clean room with bleach water 

at the end of each extraction series as well.

Step 4: Sanitize pitchers and wash bottles.

Soak all pitchers and wash bottles in 30 percent bleach solution for at least 15 

minutes at the end of each shift. Take care to rinse these items thoroughly 

before using them again.

! CAUTION

Fumes from chlorine bleach solutions can be hazardous if inhaled. Make sure the 

area is properly ventilated and consider having personnel use a respirator during disinfection.
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D-34 Karnal Bunt Manual 03/2007-01

Preparing Slides

Background

This section describes procedures used to prepare sample slides for 

microscopic examination for Tilletia indica teliospores. Included are 

specifications for lab garments and set-up of the working area. The section also 

identifies priorities in the processing of samples. Topics include receiving 

sample tubes, preparing slides, filling out slide labels and data sheets, 

preparing quality assurance slides, handling spills, and cleaning up. Keep the 

slide preparation area separate from other sample processing areas.

Supplies

Laboratory personnel will need the following supplies to prepare the slides:

Applicators, cotton-tipped

Bags, biohazard 38" x 48"

Bags, biohazard 12" x 24"

Beakers, 150-mL, 250-mL, and 400-mL

Bleach (6 percent sodium hypochlorite)

Bottles, plastic, narrow mouth

Bottles, plastic, spray

Box, microscope slide

Boxes, glass disposal, floor

Bulbs, rubber pipette

Cabinet, storage

Caps, bouffant, 21"

Centrifuge, minimum 1,000 r/min required

Cleanser, skin, antimicrobial

Coats, lab, polyethylene, medium, large, and extra large

Cover slips, microscope, 22 x 50 mm

Cytoseal 60, low viscosity

Flasks, Erlenmeyer, 500-mL and 1,000-mL

Fume absorber

Gloves, latex, medium and large

Graduate, transfer pipettes, polyethylene, 5-mL

Labels, multi purpose, white

Petroleum jelly (100 percent)
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03/2007-01 Karnal Bunt Manual D-35

Pipettes, Pasteur, disposable, 9-inch

Pipettes, transfer, Padl-Pet 0.5-mL

Scissors

Shear’s mounting medium

Slide warmer

Slides, microscope, 3" x 1"

Teri Reinforced Towels

Tray, slide, aluminum or plastic

Store all supplies in a designated Karnal bunt cabinet. Designate a clean area 

specifically for slide preparation. Clean all surfaces in this area with a 30 

percent bleach solution before setting up the work area.

Setting-Up the Working Area

Prepare the slides under a fume extractor or canopied laminar flow hood to 

minimize exposure to chemicals. Keep a supply of Shear’s solution on hand. 

During the process of slide making, Shear’s solution is necessary to dilute 

dense samples or to add liquid under the cover slip as needed.

Step 1: Disinfect surfaces.

Before making any slides, disinfect surfaces with 30 percent bleach solution 

from a spray bottle.

Step 2: Fill plastic bottles.

Fill separate plastic bottles with Cytoseal and Shear’s solution. Label the 

bottles.

Step 3: Fill a container with bleach.

Fill a container half full with 30 percent bleach solution for disposal of Pasteur 

pipettes.

Step 4: Add bleach to a plastic bottle.

Label a large plastic screw-cap bottle with “Quarantine Material.” Add 200 mL 

of 30 percent bleach. Place the bottle by the centrifuge. Use this bottle to 

decant supernatant from centrifuged samples.

Step 5: Use new supplies for each sample.

Use new slides, cover slips, and pipettes for each sample. Check supplies and 

restock if needed. Place plastic pipettes, Pasteur pipettes, and padl-pets in 

separate, clean beakers.
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D-36 Karnal Bunt Manual 03/2007-01

Step 6: Turn on the slide warmer.

Under a hood place one layer of reinforced towel. On top of the towel, place a 

colored sheet of paper (throw away between samples). Turn on the slide 

warmer to approximately 58°C.

Identifying Sample Priority

Sample processing priority will vary by region. Laboratory managers will 

establish sample priorities based on program needs. Contact the laboratory 

manager if sample priority is in question.

Processing Sample Tubes

Count the tubes and data entries on the sheet. Make sure that the sample 

number matches the data sheet number, along with the set number. Report 

discrepancies to the laboratory manager.

Preparing the Slides

Step 1: Extract the pellet.

Use a new piece of paper, pipettes, gloves, slide(s) and cover slip(s) for each 

sample. Use clean pipette bulbs for each sample if not using cotton-plugged 

Pasteur pipettes. Move the sample tube to be prepared to the front of the rack. 

Put on new gloves. Use a Pasteur pipette with a pipette bulb to extract the 

whole pellet at the bottom of the tube. Squeeze the pipette bulb before inserting 

into the tube. Slowly release the pressure on the pipette bulb to extract the 

pellet. Avoid extracting excess supernatant to reduce the number of slides 

required per sample pellet. However, transferring the entire pellet to slides 

is important.

Step 2: Homogenize dense samples.

When processing dense, muddy, or thick samples, introduce the Pasteur pipette 

into the sample tube and homogenize the sample by pressing the pipette bulb 

several times. Homogenize the sample between each slide. Maintain pressure 

on the pipette bulb and remove the pipette from the tube. Hold the pipette at 

approximately a 45° angle above the slide and expel two or three small drops 

onto the slide depending on the sample density. For dense samples, dispense 

two drops of sample onto the slide and using a plastic pipette, place a drop of 

Shear’s mounting medium in the middle. Doing this will help produce a 

nonlayered, uniform slide.

Step 3: Spread the sample.

Use the end of the Pasteur pipette to spread the sample to a small rectangle. 

Avoid placing excess sample on the slide to eliminate seepage, or preparing a 

slide that will be too dense to read easily. Use more slides if necessary.
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03/2007-01 Karnal Bunt Manual D-37

Step 4: Place a cover slip on the slide.

Return the tube with Pasteur pipette to the sample rack or pipette bucket. Place 

a cover slip over the drops on the slide. Do this by holding the cover slip above 

the slide at approximately a 45° angle and then touching the drop nearest the 

edge of the slide with the edge of the cover slip. A padl-pet pipette may help to 

move the opposite side of the cover slip down slowly until it touches the slide. 

This may help to reduce air under the cover slip. Remove air bubbles only by 

tapping lightly on the cover slip. Do not lift the cover slip to remove air 

bubbles.

Step 5: Position the cover slip.

Leave a 1⁄4 inch margin between the cover slip and the slide edge opposite the 

label. Place the Pasteur pipette in 30 percent bleach solution. Cap the sample 

tube and discard the tube only after making the label.

Step 6: Seal the cover slip.

Seal the slides if you are unable to read them immediately. All positive voucher 

slides must be sealed for storage.

Dispose of the padl-pet pipette in a disposal bag. Seal the cover slip to the slide 

after preparing all of the slide(s) from a sample. Using a plastic pipette, extract 

the needed amount of Cytoseal. Seal the long sides of the cover slip, then the 

narrow sides to the slide. Release the Cytoseal (or clear nail polish) slowly, 

running the pipette along the edge of the cover slip. Avoid making a pass over 

an area more than once, but inspect the perimeter of the cover slip for missed 

or poorly sealed areas. Touch up with Cytoseal as needed. Discard excess 

Cytoseal from the plastic pipette into a plastic screw cap bottle for disposal. 

Keep containers of Cytoseal and Shear’s mounting medium closed when not in 

use. Remove gloves and discard along with sample tubes, colored paper, and 

plastic pipettes into the biohazard bag.

Making Slide Labels and Filling Out the Data Sheet

Make slide labels only for the current sample. Never use correction fluid on 

slide labels; make a new label.

Step 1: Prepare slide labels.

Prepare the label from top to bottom using a permanent ink pen as instructed in 

Table D-17 on page D-38.
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D-38 Karnal Bunt Manual 03/2007-01

Step 2: Label the slides.

Affix the label(s) to the slide(s). Record the number of slide(s) prepared for the 

sample in the comment section of the data sheet.

Step 3: Warm the slides.

Keeping the slide horizontal, place it on the warmer for 5 to 15 minutes. 

Transfer slides to a slide tray. Repeat the slide preparation for each sample in 

the rack.

Step 4: Complete data sheet.

After completing a data sheet of samples, count the number of slides prepared 

and compare it with the total listed on the data sheet. Initial “Slide Preparer” 

section on the data sheet.

Step 5: Place tray and data sheet on counter.

Place the tray and data sheet on the counter marked for reading. After readers 

read the slides and record the results on the data sheet, they should return each 

slide to its appropriate sequential location in the slide tray.

Handling Spills

Cover all spills of supernatant or sample with 30 percent bleach solution and a 

reinforced towel. Let soak for 15 minutes. Wipe up the bleach and rinse with 

water. Discard towels in a biohazard bag.

Cleaning Up

Discard all used disposables, clean slide-making area with 30 percent bleach 

solution, and replace supplies. Turn off the extraction hood and slide warmer.

Table D-17 Preparation of Slide Labels

Description Slide Label (Example)

Sample Code CG-96-172

Number of slides prepared 1/3, 2/3, 3/3, etc.

Set Number Set 3052

Person who read the slide Reader Initials:

Authorized person who verified the positive finding of 

Tilletia indica or person who completed negative reread

Ver. Initials:

Date the sample was read Date:
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03/2007-01 Karnal Bunt Manual D-39

Examining Slides for Karnal Bunt

Background

This section describes procedures used for the microscopic examination for 

and identification of teliospores that may be Tilletia indica. A compound 

microscope is necessary to examine the slides. Included are procedures for 

selecting, examining, and identifying Karnal bunt. The section also includes 

procedures for handling positive slides, negative slides, and record keeping. 

Microscopic identification requires a room with sufficient counter top and 

chair space to hold multiple microscope stations.

Selecting a Slide

Select a slide based on the priority established by the laboratory manager. 

Before taking a slide, place your initials in the “Slide Taken By” box and 

record the slide number in the “Slide” box of the Tilletia indica Spore Analysis 

Worksheet (Figure D-7).

Examining the Slide

Examine the slide using the 10X objective and 10X ocular. Begin at one corner 

of the cover slip, including the area on the edge of the cover slip containing 

slide sealant. Scan the entire slide, either vertically or horizontally, left to right 

or right to left. Be consistent in your scanning procedure and be aware that the 

image is reversed. Take care to remember your direction if interrupted during a 

scan. A simple method is to place a pen or pencil on the table pointed in the 

direction of the scan at the time of interruption.

Identifying Karnal Bunt Teliospores

After carefully examining the Tilletia indica reference slides, and recognizing 

the variability in mature and immature spores, look for similar objects. Refer 

also to the laminated color illustration titled Karnal Bunt Teliospores, Tilletia 

indica Mitra. Upon finding a suspect spore, change the objective to 40X and 

look for the following characteristics:

Mature Teliospores. 

Spherical shape with an average diameter of 39 m and a range of 24–47 

m. Atypical spores that are broken and/or irregularly shaped may be 

present.

Color light brown to near black

Smooth clear or yellowish sheath enveloping the spore that is visible 

when focusing on cross section of the spore

Distinctive truncate projections that are visible on the circumference of 

the spore when focusing through a cross section and dark brown spots in 

the middle of the spore when focusing on the surface of the spore
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D-40 Karnal Bunt Manual 03/2007-01

Immature Teliospores Combined with Mature Teliospores. 

Similar shape but often smaller than mature teliospores

Color yellow to lighter brown

More distinct, finer ornamentation than mature teliospores

Any of the above characteristics will suggest a suspect find of Tilletia indica

teliospores.

Verifying Teliospores

Refer suspect teliospores (intact, broken, or fragmented) to the appropriate 

authority for verification.

If identified as T. indica, the verifier will do the following:

1. Circle the position of the verified teliospore using a permanent marker.

2. Print his or her initials on the slide label and in the “Verified By” column of 

the Tilletia indica Spore Analysis Worksheet.

If the teliospores are indicative of T. indica, but are undersized or comprised of 

fragments less than 50 percent, the verifier will follow the steps above and in 

addition will do the following:

1. Record an asterisk (*) in the “# of Spores” column.

2. Add the statement “undersized spores” or “fragments” to the “Comments” 

column on the Tilletia indica Spore Analysis Worksheet (Figure D-7).

Counting Teliospores as Positive

A seed sample will be considered positive for T. indica if at least one spore is 

found equal to or exceeding 33 in diameter and possessing morphological 

characteristics consistent with T. indica and not other Tilletia species (i.e., T. 

walkeri, T. horrida syn. T. barclayana). All teliospores used to make a positive 

determination for a sample must be verified by an identifier with identification 

authority for the T. indica teliospore in absence of the host.

Keeping Records

Maintaining accurate records of samples is extremely important. Always 

record information in ink. NEVER use correction fluid on laboratory 

documents, slide labels, or sample labels. If a correction is necessary, draw a 

line through the incorrect entry, enter the correct entry, and place your initials 

and date beside the correct entry.

Tilletia indica Microscopy Log Sheet. Keep your own record of the samples and 

their counts using the Tilletia indica Microscopy Log Sheet (Figure D-8). 
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03/2007-01 Karnal Bunt Manual D-41

Record the sample #, set #, and 1st/2nd reads. Use the Sample # section to 

record slide number, if there are multiple slides. For example, “1 of 2 slides.” 

Record spore coordinates in the Comments section.

Slide labels. After completing a slide, place your initials and the date on the 

slide label.

Tilletia indica Spore Analysis Worksheet. Return the slide to the appropriate slide 

holder. Record the following data on the Tilletia indica Spore Analysis 

Worksheet (Figure D-7):

Your initials

The date

Have the verifier who confirmed the first T. indica teliospore record his or her 

initials under the Confirmed By section.

Interpreting Laboratory Results

A laboratory result, whether positive or negative, stands on its own. Repeating 

a test does not necessarily invalidate or validate the previous test unless the 

results are the same.

Rating on the Karnal Bunt Spore Analysis Worksheet. After a slide reader reads the 

slides and documents the results on the worksheet, the laboratory manager will 

rate the results using the following codes (only one rating is necessary per 

sample):

P for positive. Used when at least one spore of T. indica is confirmed for 

the sample.

N for negative. Used when no T. indica spores are confirmed for the 

sample.

X for retest. Used when the verifier cannot make a positive identification.

L for laboratory accident. Used when a sample is spilled, mislabeled, 

etc. Retesting is required.

R for redundant. Used when there are multiple test results for the same 

sample, if a rating of P, X, or L is used. One test is rated and the others are 

rated with an R.
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D-42 Karnal Bunt Manual 03/2007-01

Quality Control

The following are recommended minimum procedures for quality control 

purposes:

Have a different reader do a second examination of every 5th negative 

slide for beginners. Experienced readers should have every 10th negative 

slide re-read.

Include slides of known negative and positive controls to test readers and 

verifiers.

Training Technicians and Verifiers

Background

To assure accuracy and efficiency when laboratory personnel are processing 

samples, technicians need to be appropriately trained and successfully 

complete documented procedures. Technicians should complete the training 

program before actually processing real samples.

Bunted kernel verifiers should receive appropriate training, including 

identification and equipment usage. Spore verifiers, as well, should have 

training in examining isolates of Tilletia indica, and should meet certain 

educational and experience requirements. Slide readers should receive all 

laboratory procedure training, including gross teliospore identification and 

slide preparation. Written training records for all analysts covering all 

procedures will be maintained at the laboratory.

Suggested training content for all laboratory personnel. 

Presenting general orientation about Karnal bunt

Identifying and explaining the purpose of processing areas

Setting up the reception (sample receiving) area

Setting up the dirty rooms

Setting up the clean rooms

Receiving samples

Maintaining supplies

Decontaminating samples

Logging-in samples

Storing reserve samples

NOTICE

Do not allow technicians involved in bunted kernel analysis or field personnel to 

enter the laboratory areas where seed analysis is taking place.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-43

Extracting samples

Maintaining supplies

Preparing Shear’s mounting fluid

Preparing sample containers

Weighing and measuring samples

Adding extraction solution

Extracting the samples

Cleaning up the extraction lab

Preparing slides

Maintaining supplies

Setting up the working area

Establishing priority and processing sample tubes

Preparing the slides

Making labels and data sheets

Handling spills and cleanup

Examining slides for Karnal bunt

Selecting and examining slides

Identifying Karnal bunt teliospores

Verifying teliospores (for spore verifiers)

Counting teliospores, if appropriate

Reading positive slides

Keeping records and interpreting laboratory results

Examining wheat kernels for Karnal bunt (automated and manual 

methods)

Maintaining supplies

Processing samples

Preparing the workstations

Identifying Tilletia indica from bunted kernels

Storing samples

Decontaminating equipment and cleaning up
Appendix D

Seed Analysis Methods

D-44 Karnal Bunt Manual 03/2007-01

Selecting and Training Bunted Kernel Verifiers

Background

The Karnal bunt bunted kernel verifier must be able to positively identify 

Tilletia indica Mitra, the causal agent of Karnal bunt, in or on the wheat grain 

kernel to the exclusion of other fungi. The bunted kernel verifier serves as the 

final authority for the identification of Karnal bunt from grain samples.

Requirements

The bunted kernel verifier should have work experience in microscopy, or 

other work experience in an agricultural or biological field. The verifier must 

pass the bunted kernel identification course given by a PPQ Identifier.

Job Description

The incumbent is responsible for verifying the identification of T. indica 

bunted kernels found during Karnal bunt screening to the exclusion of other 

fungi. This identification will be based on a visual examination of infected 

grain. Symptoms must be verified with a microscopic examination of Karnal 

bunt teliospore gross morphological characteristics.

Training

Kernel verifiers need to complete the following training activities:

Receive introduction and background information on Karnal bunt and 

training and testing from a PPQ Plant Pathology Identifier.

Learn about Tilletia indica, specifically:

Life cycle

Initial infection

Morphology of symptoms at various stages of infection

Teliospore morphology

Recognize other fungi that infect wheat kernels:

Black point symptoms (Helminthosporium sativum, Alternaria sp.)

Common bunt (T. laevis, T. tritici) kernel infection/teliospore 

morphology

Dwarf bunt (T. controversa) kernel infection/teliospore morphology

Learn slide making and record keeping procedures.

Observe grain examination machine demonstration and practice runs with 

known infected samples.

Examine specimens.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-45

Identification Authority

After the incumbent completes the required training and shows the ability, 

through testing or correct identifications, to identify T. indica bunted kernels in 

Karnal Bunt Project samples, designated USDA/APHIS personnel (PPQ Plant 

Pathology Identifiers) will grant identification authority for use on the Karnal 

Bunt Project.

Selecting and Training Spore Verifiers

The Karnal bunt teliospore verifier must be able to positively identify Tilletia 

indica Mitra, the causal agent of Karnal bunt, to the exclusion of other fungi. 

The verifier serves as the final authority for identification of Karnal bunt.

Requirements

The verifier should hold a college degree in mycology, plant pathology, or 

other related fields. The verifier should have work experience with 

microscopy, fungal taxonomy, and morphology, or course work in plant 

pathology and mycology and work related experience in plant pathology and 

mycology. Knowledge or experience in plant pathology is desirable but not a 

requirement. For those without a degree in plant pathology or a related field, 

the requirement should be a foundation in mycology or plant pathology with 

recent work experience in that area (for example, PPQ Port Identifier or 

backup, in plant pathology).

Job Description (the incumbent is responsible for these duties)

1. Verifies the identification of T. indica teliospores found by microscopists 

during Karnal bunt screening to the exclusion of other fungi.

2. Distinguishes by microscopic examination other species of Tilletia that 

could be encountered (for example, T. walkeri and T. horrida).

3. Using established guidelines on spore size, makes determinations of when 

to send “suspect spores” to National Mycologist for final determination.

4. Confirms wheat kernels infected by T. indica.

Training

Training will consist of microscopic examination of several isolates of T. 

indica. Depending on their age and condition, teliospores will exhibit 

significant variation in size, shape, and color. Verifiers will frequently find 

immature and fragmented spores.

Karnal bunt teliospore verifiers will complete the following training activities:

Analyze slides of T. indica, T. horrida (rice smut), and T. walkeri.

Review microscope slides of actual samples to familiarize themselves 

with the other fungi commonly found associated with wheat and pollen 

grains frequently encountered.
Appendix D

Seed Analysis Methods

D-46 Karnal Bunt Manual 03/2007-01

Scan a minimum of 10 training slides of actual wheat samples, with 

known numbers of teliospores. At least one of these slides will be a 

negative control. The purpose of this training slide exercise is to 

familiarize the verifiers with the microscopist’s tasks, not for them to 

become skilled microscopists. For the verifier to qualify as a reader, the 

verifier must meet the requirements for readers described in the previous 

subsection, Training Technicians.

Examine wheat kernels macroscopically and learn how to positively 

confirm a bunted kernel caused by T. indica.

Retesting

A system of retesting verifiers should be in place. This should include 

examining slides with various wheat smuts, particularly variations of Tilletia

species.

Identification Authority

After the candidate completes the required training and shows the ability to 

identify T. indica teliospores and bunted kernels in Karnal Bunt Project 

samples, designated USDA personnel (PPQ National Mycologist or designee) 

will grant identification authority for use on the Karnal Bunt Project.

Preparing New Sieves for Use

Background

New sieves generally arrive at the laboratory by mail from the USDA 

manufacturer. These sieves should arrive pre-labeled with the mesh size (20 or 

53), month and year of manufacture, and unique serial number. Each size 

should be uniquely color coded. Upon receipt of newly manufactured sieves, it 

is the laboratory’s responsibility to ensure the quality of each sieve prior to use.

Step 1: Log the date received.

For each new sieve, log the date received at the laboratory and that sieve’s 

unique serial number into the Karnal Bunt Sieve Log.

NOTICE

Because sieves have a limited life, quality control measures require replacement at 

prescribed intervals. It is important to anticipate future needs for sieve replacement 

well in advance of when new sieves are required.
Appendix D

Seed Analysis Methods

03/2007-01 Karnal Bunt Manual D-47

Step 2: Examine the sieve.

Using a microscope equipped with an ocular micrometer, verify the quality and 

mesh size of each sieve.

Examine the entire mesh under the microscope to look for tears, holes, or 

gaps in the mesh (recommended at 40x).

Measure the mesh pore size with an ocular micrometer at any location 

where a gap in the mesh appears, and additionally at five or more separate 

random locations across the mesh (recommended at 200x).

Examine the silicone seals. If the silicone seal is imperfect and can be 

repaired, touch up with silicone; otherwise it is unusable. After a silicone 

application, do not place the sieve into service for 24 hours.

If you find no damage, the mesh size is correct, and the silicone seal is intact, 

the sieve can go into service following step 3 below. If the sieve is unusable 

due to damage, improper mesh size, or an unsalvageable silicone seal, the sieve 

cannot be used, and must be destroyed. The person performing the verification 

enters the results into the Karnal Bunt Sieve Log. When a sieve is destroyed, 

log the date and your initials into the Karnal Bunt Sieve Log sheet.

Step 3: Place the sieve into use.

Each sieve that passes the examination can be placed into use. Log the date 

into use on the Karnal Bunt Sieve Log. Then soak the sieve in soapy water for 

15 minutes and rinse with clear water. After rinsing, soak the sieve in 30 

percent bleach solution for an additional15 minutes and rinse once more in 

clear water. Log the serial numbers of the sieves that are now ready for use into 

the spaces across the top row of the Karnal Bunt Sieve Usage Log. These 

sieves can now be used for sample extraction.

Maintaining Sieves

Background

To ensure that intact sieves are used to extract samples, inspect the sieves 

before each use.

Step 1: Record the sieve’s use.

Record every use of each sieve into the Karnal Bunt Sieve Usage Log. In the 

column below each sieve’s serial number on the log sheet, enter the sample/

sub-sample number that the sieve is used to extract. Before performing an 

extraction, visually inspect every sieve to be used for any tear in the mesh or 

other imperfection, such as damaged silicone. If you find a sieve to be 

unusable, destroy it. Record the date, your initials, and the reason destroyed on 

Karnal Bunt Sieve Log sheet. Also record your initials and the date destroyed 

at the bottom of the column for that sieve’s serial number on the Karnal Bunt 

Sieve Usage Log.
Appendix D

Forms

D-48 Karnal Bunt Manual 03/2007-01

Step 2: Recheck the sieve.

After ten uses, recheck each sieve for quality as in Step 2 above. Record your 

initials and the date of damage check on the Karnal Bunt Sieve Usage Log in 

the space below the 10th extraction set entry. If you find the sieve to be usable, 

soak it in warm soapy water for 15 minutes and rinse in clear water before 

further use. If you destroy the sieve at this point, record the action in both the 

Karnal Bunt Sieve Log and Sieve Usage Log. Perform these rechecks after the 

tenth and twentieth uses. Take each sieve out of service and destroy it after 

30 uses, regardless of the sieve’s condition at that point.

Ordering Sieves

Sieves are manufactured at the USDA, APHIS, PPQ Aircraft and Equipment 

Operations Center in Mission, Texas. If you need replacement sieves, fax a 

request including the number of sets required, the requested date of delivery, 

and delivery information.

Contact the Operations Center at the following numbers:

Fax: (956) 580-7276

Phone: (956) 580-7270

Forms

Each laboratory location will develop forms used for recording results of 

bunted kernel and teliospore analyses. Assure that the appropriate levels of 

information collection are included on each data collection form. Refer to 

samples of such forms on the following pages.
Appendix D

Forms

03/2007-01 Karnal Bunt Manual D-49

Figure D-4 Karnal Bunt Kernel Analysis Worksheet
Appendix D

Forms

D-50 Karnal Bunt Manual 03/2007-01

Figure D-5 Karnal Bunt Quality Control (QC) Worksheet for Bunted Kernels
Appendix D

Forms

03/2007-01 Karnal Bunt Manual D-51

Figure D-6 Quality Control (QC) Grain Labs
Appendix D

Forms

D-52 Karnal Bunt Manual 03/2007-01

Figure D-7 Tilletia indica Spore Analysis Worksheet
Appendix D

Forms

03/2007-01 Karnal Bunt Manual D-53

Figure D-8 Tilletia indica Microscopy Log Sheet
Appendix D

Forms

D-54 Karnal Bunt Manual 03/2007-01
03/2007-01 Karnal Bunt Manual E-1

Appendix

E Appendix E

Compliance Agreements

Contents

Examples E-1

Hay Producers E-2

Mushroom Producers E-4

Rail Cars E-6

Trucks/Trailers E-8

Karnal Bunt Positive Grain Dealers E-13

Karnal Bunt Negative Grain Dealer/Brokers E-17

Feed Processors E-21

Harvesting Equipment and Conveyances E-25

Examples

The following examples serve as guides. Stipulations may be altered to meet 

local needs and/or farming practices.
Appendix E

Hay Producers

E-2 Karnal Bunt Manual 03/2007-01

Hay Producers

Figure E-9 Compliance Agreement for Hay Producers

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Hay Producers

03/2007-01 Karnal Bunt Manual E-3

HAY PRODUCERS

Compliance Agreement Stipulations

Before hay may be moved within and/or out of the regulated area it must be inspected by Karnal bunt 

personnel. The following distinctions will be made:

 Hay cut prior to the development of the kernel will be considered no risk for having Karnal bunt.

 Hay cut with fully developed kernels will be tested for the presence of bunted kernels. Samples can be 

taken from bales or from standing wheat around the edges of the field from which the hay was cut. The 

hay will be regulated according to the results of the testing.

 Hay cut from previously tested fields will be regulated according to the results of that previous test.

Hay testing NEGATIVE or hay cut PRIOR TO DEVELOPMENT OF KERNEL will be allowed 

movement under the following conditions:

Movement of hay within and out of the regulated area:

 Hay must be inspected/tested free of bunted kernels prior to movement from the production area.

 If hay has already been moved to a storage area inspection/testing will be done at the storage area.

 Grower will be issued a Certificate for the number of bales and/or weight inspected/tested free of bunted 

kernels.

 The number of bales noted on the Certificate for the location (field or storage area) will be allowed 

movement within or out of the regulated area. The Certificate must accompany any movement.

Hay testing POSITIVE for bunted kernels will be allowed movement under the following conditions:

Movement of hay within the regulated area:

 A grower may move his/her hay from the field of production (land owned/leased by grower) to an area 

owned by the grower for storage.

 A grower may move his/her hay from the field of production (land owned/leased by grower) for the 

purpose of feeding to livestock.

 A grower must contact Karnal bunt personnel prior to movement of hay. A limited permit will be issued 

for movement.

 Hay must be moved in a manner that safeguards the hay and minimizes the potential for spreading 

Karnal bunt.

Movement of hay out of the regulated area:

 Hay testing positive for bunted kernel or cut from a field that tested positive for bunted kernels can only 

move out of the regulated area if destined to an approved establishment.

 The establishment must be approved by Karnal bunt personnel and under compliance to process the hay 

in an approved manner so as to destroy all Karnal bunt teliospores.

 Program personnel must be contacted prior to movement of hay. If movement is approved, a limited 

permit will be issued for movement to the approved establishment out of the regulated area.

 Hay approved for movement out of the regulated area must be moved in a manner that safeguards the 

hay and minimizes the spread of Karnal bunt.
Appendix E

Mushroom Producers

E-4 Karnal Bunt Manual 03/2007-01

Mushroom Producers

Figure E-10 Compliance Agreement for Mushroom Producers

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Mushroom Producers

03/2007-01 Karnal Bunt Manual E-5

MUSHROOM PRODUCERS

Compliance Agreement Stipulations

5. I/We agree to the following:

 Wheat straw from the regulated area must be introduced to a composting procedure that results in a 

pasteurized product. The composting procedure shall produce pasteurization temperatures of 170°F for 

at least 1 minute.
Appendix E

Rail Cars

E-6 Karnal Bunt Manual 03/2007-01

Rail Cars

Figure E-11 Compliance Agreement for Rail Cars

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Rail Cars

03/2007-01 Karnal Bunt Manual E-7

RAIL CARS TRANSPORTING BUNTED KERNEL POSITIVE GRAIN, 

MILLFEED, AND/OR ASSOCIATED ARTICLES

Compliance Agreement Stipulations

5. I/We agree to the following:

 All rail cars designated for use must pass a pre-use inspection performed by a Karnal Bunt Program 

inspector. Cars can be used only if he/she determines that the car is leak-proof and is enclosed or 

adequately covered. Cars having passed the initial inspection need not be reinspected unless the car is 

damaged in some way which would require an additional inspection.

 To transport regulated articles only when authorized to move under Limited Permit (PPQ Form 530).

 To present the PPQ Form 530 to the appropriate consignee representative at destination.

 To ensure that rail cars carrying regulated articles move to their approved destination by the most direct 

route prior to the void date on the PPQ Form 530.

 To notify the Karnal Bunt Office 24 hours in advance of any changes in movement as described in the 

PPQ Form 530. Approval must be obtained prior to any change movement of regulated articles.

 To immediately contact the Karnal Bunt Office in the event of a spillage or accident.

 To obtain approval in advance from the Karnal Bunt Office, if transferring of regulated grain from one 

rail car to another becomes necessary.

 To ensure rail cars removed from service on this project are properly cleaned in accordance with USDA 

protocols before being placed in service for other purposes.

 To perform treatments in accordance with the Karnal Bunt treatment protocols.
Appendix E

Trucks/Trailers

E-8 Karnal Bunt Manual 03/2007-01

Trucks/Trailers

Figure E-12 Compliance Agreement for Trucks/Trailers

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Trucks/Trailers

03/2007-01 Karnal Bunt Manual E-9

TRUCKS/TRAILERS TRANSPORTING BUNTED KERNEL POSITIVE 

GRAIN, MILLFEED, AND/OR ASSOCIATED ARTICLES

Compliance Agreement Stipulations

5. I/We agree to the following:

Transporting Regulated Grain

 To transport regulated articles only when authorized to move under Limited Permit (PPQ Form 530).

The PPQ Form 530 will be issued by the inspector or designee under compliance after:

 Trucks/trailers transporting positive grain have been determined to be leak proof and properly 

covered to avoid spillage of grain.

 All debris has been removed from the exterior of the truck/trailer after loading.

 To perform treatments in accordance with the Karnal bunt treatment protocols. Refer to Cleaning and 

Sanitizing Protocols for approved treatments.

 To immediately contact the USDA/State Department of Agriculture Karnal bunt office in the event of a 

spillage or accident. Spills can be picked up and returned to the truck hauling the positive grain.

 To obtain approval in advance from the USDA/State Department of Agriculture Karnal bunt office, if 

transferring of regulated grain from one truck to another becomes necessary.

 To notify the USDA/State Department of Agriculture Karnal bunt office 24 hours in advance of any 

changes in movement as described in the PPQ Form 530. Approval must be obtained prior to any 

movement of regulated articles.

 To ensure that trucks/trailers carrying regulated articles move to their approved destination by the most 

direct route prior to the void date on the PPQ Form 530.

Discharging Regulated Grain

 To ensure that trucks/trailers are cleaned following discharge and that small quantities of debris resulting 

from this cleaning are:

 Store back in the bin with bunted kernel positive grain, or

 Buried in an approved landfill, or

 Treated with an approved treatment.

 To clean vehicles dedicated to transporting bunted kernel positive grain (not back hauling) in accordance 

with attached information upon completion of transporting bunted kernel positive grain. All other 
Appendix E

Trucks/Trailers

E-10 Karnal Bunt Manual 03/2007-01

vehicles transporting Karnal bunt positive grain and back hauling will be cleaned in accordance with 

attached information prior to loading any agricultural products.

 To provide a copy of this compliance agreement to the facilities where the trucks/trailers will be loading.

Approved Conveyances

The following license plates belong to conveyances that have been inspected and approved by the USDA/State 

Department of Agriculture Karnal bunt project.

____________________ ____________________ ____________________ ____________________

____________________ ____________________ ____________________ ____________________

____________________ ____________________ ____________________ ____________________

Cleaning and Sanitizing Protocols for

Grain Bins, Elevators, Seed Plants, and Associated Equipment

Seed equipment

Grain bins, elevators, seed plants, conveyances, and associated equipment used in the storage, transportation 

and/or processing of wheat, durum wheat or triticale seed which had tested positive for Karnal bunt spores or 

kernels will require cleaning and sanitizing under the following guidelines:

 “Cleaning and sanitizing” requirements are defined as the removal of all host material followed by:

A. SODIUM HYPOCHLORITE SOLUTION

The equipment should be thoroughly wetted until point of runoff with a solution of 1.5 percent 

sodium hypochlorite—e.g., with a solution of sodium hypochlorite mixed with water applied at the 

rate of 1 gallon of household chlorine bleach (5.2 percent sodium hypochlorite) mixed with 2.5 

gallons of water. Leave solution in contact with all surfaces for a minimum of 15 minutes then 

thoroughly rinse surfaces with water to minimize any adverse effect on equipment. Prepare a fresh 

solution of sodium hypochlorite and potable water every four to five hours.

NOTE: EPA approved bleach products are as follows:

 Ultra Clorox brand bleach (EPA Reg. No. 5813-50); 6% Sodium Hypochlorite

 CPPC Ultra Bleach 2 (EPA Reg. No. 67619-8); 6.15% Sodium Hypochlorite

These are the only two products that have EPA approval to be used for the sanitization of equipment.

Or

B. HOT WATER AND DETERGENT
Appendix E

Trucks/Trailers

03/2007-01 Karnal Bunt Manual E-11

Hot water and detergent drench with a pressure of at least 30 pounds per square inch. The water 

must be applied to all surfaces until point of runoff including penetration into cracks and crevices. 

At point of runoff the temperature must be a minimum of 170°F.

Or

C. LIVE STEAM TO ALL SURFACES

Live steam (212°F) can be used to treat various harvesting and cleaning equipment, as long as all 

surfaces that come into contact with grain can be directly contacted with the live steam. Apply steam 

directly to all surfaces until the point of runoff. At point of runoff the temperature must be a 

minimum 170°F.

Or
Appendix E

Trucks/Trailers

E-12 Karnal Bunt Manual 03/2007-01

D. FUMIGATION

The storage area can be fumigated with methyl bromide at the dosage of 15 pounds/1,000 ft3

 for 96 

hours.

The USDA Karnal Bunt office must be notified 24 hours in advance of the sanitizing to allow 

monitoring of the treatment by a PPQ officer or State inspector.

Grain-only equipment

A facility, portions of a facility, associated equipment, and transportation conveyances that do not 

handle seed, only grain, may be approved to be cleaned and disinfected by the following manner:

Grain handling equipment (elevator legs, etc.) used to move grain between bins must be cleaned, but 

sanitizing is not required. The bins and other equipment must be cleaned of all kernels and 

particulate matter. The purpose is to clean to the point of freedom of bunted kernels, not spores. This 

is not approved for anything that may come into contact with seed.

Certification

 A PPQ Form 540, Certificate, or equivalent documentation will be provided stating that the treatment 

has met the requirements according to Karnal Bunt Project Protocol.
Appendix E

Karnal Bunt Positive Grain Dealers

03/2007-01 Karnal Bunt Manual E-13

Karnal Bunt Positive Grain Dealers

Figure E-13 Compliance Agreement for Karnal Bunt Positive Grain Dealers

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Karnal Bunt Positive Grain Dealers

E-14 Karnal Bunt Manual 03/2007-01

KARNAL BUNT POSITIVE GRAIN DEALER

Compliance Agreement Stipulations

5. I/We Agree To The Following:

Certificates/Permits

 To receive and safeguard PPQ Form 530 (Limited Permits).1

 These forms are accountable and must be 

maintained in a secure location. Only employees designated by the signatory of the Compliance 

Agreement are approved to handle and complete these documents. The signatory of the Compliance 

Agreement shall provide a list of designees to the USDA/State Department of Agriculture Karnal Bunt 

Office. Documents shall be completed by following the instructions that are included as an attachment to 

this Compliance Agreement.

Receiving Bunted Kernel Positive Grain

 To handle bunted kernel positive wheat grain from the regulated area only if it has been authorized to 

move under PPQ Form 530 (Limited Permit).

 To verify, upon receipt of the shipment, the identity and quantities stated on PPQ Form 530.

Storage of Grain

 To notify the USDA/State Department of Agriculture Karnal Bunt Project Office 24 hours in advance of 

turning/moving of stored grain.

Shipping Grain

 To notify the Consignee purchasing the grain that it is strictly prohibited to utilize this grain for the 

purpose of planting.

 To retain original copies of PPQ Form 530 (Limited Permit) and make them available at the request of 

the USDA/State Department of Agriculture Karnal Bunt Program Office, and to maintain supporting 

records of grain movement associated with the permit.

 To contact the USDA/State Department of Agriculture Karnal Bunt Office 24 hours in advance of each 

planned movement of grain to allow for the possible inspection of loads by an inspector.

 To ensure that the truck/trailer that will be loaded is covered by a compliance agreement to transport 

positive grain.

 To inspect each conveyance to ensure that it is leak-proof and is enclosed or adequately covered with a 

tarp to prevent the loss of grain in transport. Seal each truck with PPQ issued wire seals and record seal 

numbers on the PPQ Form 530. Attach a copy of the weight ticket for each load to the associated PPQ 

Form 530 - white copy to serve as a verification of the grain movement. Present the PPQ Form 530 to 

the appropriate consignee representative at destination.

 To immediately contact the USDA/State Department of Agriculture Karnal Bunt Office in the event of a 

significant spillage or accident.

 To obtain approval in advance from the USDA/State Department of Agriculture Karnal Bunt Office if 

transferring of regulated grain from one conveyance to another becomes necessary.

1 For guidance, see Instructions for filling out PPQ Form 530 (Limited Permit).
Appendix E

Karnal Bunt Positive Grain Dealers

03/2007-01 Karnal Bunt Manual E-15

 To ensure that all wheat kernels are removed from the conveyance following discharge and that small 

quantities of debris resulting from this cleaning are:

 Collected and placed in the conveyance/bin with the positive grain, or

 Buried under two feet of soil in an area not to be cultivated or in an approved landfill2

, or

 Treated with an approved treatment.

 To remove all volunteer plants resulting from debris by cultivation or by using an herbicide.

 To retain copies of permits and other related documentation related to the transport of said regulated 

grain and to ensure that all documentation of the transport of the regulated wheat is made available upon 

request to an inspector.

 To contact the USDA/State Department of Agriculture Karnal Bunt Office prior to breaking the seals or 

immediately after discovering that a seal has been broken from the top loading doors of the railcars that 

are returned from discharge destination. This notification will allow for the monitoring of the 

compliance agreement by the USDA/State Department of Agriculture Karnal Bunt Project.

2 Contact the USDA/State Department of Agriculture Karnal Bunt Office for information on approved establishments/facilities.
Appendix E

Karnal Bunt Positive Grain Dealers

E-16 Karnal Bunt Manual 03/2007-01

 Instructions for filling out PPQ Form 530 (Limited Permit)

Block #1: Enter the date in the following format: (October 12, 2001).

Block #2: Enter the Void Date in the following format: (October 20,2001). Note: Allow a sufficient amount of 

time for the shipment to arrive directly to the destination to include any possible delays.

Block # 3: Enter your name/company that is selling the commodity.

Block #4: Enter the location from where the grain is being shipped.

Block #5: Enter name and address of the person/company that is purchasing the grain.

Block #6: Enter license plate number of the trailer and the issuing state.

Block #7: Enter rail car number.

Block #8:

A. Enter the quantity in pounds.

B. Enter Positive KB Wheat and enter the seal numbers.

C. Enter the following statement: “To designated address only.”

Block #9: The signatory and designees authorized by the signatory of the Compliance Agreement sign in this 

block (Shipping Point Signatory).

Block#10: Enter the date that the shipment arrives at its destination in the following format: (October 12, 

2001).

Block #11: The signatory and designees authorized by the signatory of the Compliance Agreement sign in this 

block (Receiving Point Signatory).

Distribution of copies issued by party under compliance:

 PINK COPY:

 Goes with shipment

 Held by consignee at destination

 YELLOW COPY:

 Goes with shipment

 Destination PPQ Officer picks up copy, or

 Consignee that is under compliance maintains copy until requested by PPQ

 WHITE COPY:

 Consignor maintains until requested by PPQ
Appendix E

Karnal Bunt Negative Grain Dealer/Brokers

03/2007-01 Karnal Bunt Manual E-17

Karnal Bunt Negative Grain Dealer/Brokers

Figure E-14 Compliance Agreement for Karnal Bunt Negative Grain Dealer/Broker

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Karnal Bunt Negative Grain Dealer/Brokers

E-18 Karnal Bunt Manual 03/2007-01

KARNAL BUNT NEGATIVE GRAIN DEALER/BROKER

Compliance Agreement Stipulations

FOR REGULATED ARTICLES TESTING NEGATIVE FOR KARNAL BUNT

5. I/We Agree To The Following:

Certificates/Permits

 To receive and safeguard PPQ Form 530 (Limited Permits) and PPQ Form 540 (Certificates). These forms 

are accountable and must be maintained in a secure location. Only employees designated by the signatory 

of the Compliance Agreement are approved to handle and complete these documents. The signatory of the 

Compliance Agreement shall provide a list of designees to the USDA/State Department of Agriculture 

Karnal Bunt Office. Documents shall be completed by following the instructions that are included as 

attachments to this Compliance Agreement.

Receiving Grain

 To handle wheat from the regulated area only if it has been authorized to move under PPQ Form 540 

(Certificate) or PPQ Form 530 (Limited Permit).

 To verify, upon receipt of the shipment, the identity and quantities stated on PPQ Form 540 or PPQ Form 

530.

Storage of Grain

 To notify the USDA/State Department of Agriculture Karnal Bunt Project Office 24 hours in advance of 

turning/moving of stored grain.

Shipping Grain

 To notify the Consignee purchasing the grain that it is strictly prohibited to utilize this grain for the 

purpose of planting outside the regulated area. This grain can only be utilized for planting inside the 

regulated area after it has tested negative for karnal bunt spores and has been treated with an approved 

fungicide.

 To issue PPQ Form 530 (Limited Permit) or PPQ Form 540 (Certificate) on each conveyance of grain 

moving to another location. With this permit, grain may be moved to locations within the regulated area or 

to destinations outside the regulated area. This grain has been certified as free of Karnal bunt (bunted 

kernel free) and may be moved to any potential market (except for planting) both domestic and/or export. 

Note: many foreign countries requiring an additional declaration on the Phytosanitary Certificate (PPQ 

Form 577) prohibit wheat grain originating within Karnal bunt regulated areas and/or grain that is 

commingled with grain from regulated areas from fields that have not been tested. We advise before 

contracts for the exportation of grain are signed you contact the USDA/State office for the importing 

country’s Karnal bunt export certification requirements.

 To retain original copies of PPQ Form 530 (Limited Permit) or PPQ Form 540 (Certificate) and make 

them available at the request of the USDA/State Department of Agriculture Karnal Bunt Program Office, 

and to maintain supporting records of grain movement associated with the permit or certificate.

 To obtain all Phytosanitary Certificates, for the purpose of exporting grain, from the USDA/State 

Department of Agriculture Karnal Bunt Project Office.

NOTICE

Bin-tested negative grain is eligible for a Limited Permit (PPQ Form 530).

See Instructions for Filling out PPQ Form 530 (Limited Permit).

Field-tested negative grain is eligible for a Certificate (PPQ Form 540).

See Instructions for filling out PPQ Form 540 (Certificate).
Appendix E

Karnal Bunt Negative Grain Dealer/Brokers

03/2007-01 Karnal Bunt Manual E-19

Instructions for Filling out PPQ Form 530 (Limited Permit)

Block #1: Enter the date in the following format: (October 12, 2001).

Block #2: Enter the Void Date in the following format: (October 20,2001). Note: Allow a sufficient amount of 

time for the shipment to arrive directly to the destination to include any possible delays.

Block # 3: Enter your name/company that is selling the commodity.

Block #4: Enter the location from where the grain is being shipped.

Block #5: Enter name and address of the person/company that is purchasing the grain.

Block #6: Enter license plate number of the trailer and the issuing state.

Block #7: Enter rail car number.

Block #8:

A. Enter the quantity in pounds.

B. Enter Wheat Grain.

C. Enter “Tested negative for KB.” Stamp the center of the bottom two lines of Block #8 with the 

stamp that reads as folows: “Regulated wheat grain. Tested negative for Karnal bunt. Nonpropagative domestic use or limited export only.”

Block #9: The signatory and designees authorized by the signatory of the Compliance Agreement sign in this 

block (Shipping Point Signatory).

Block#10: Enter the date that the shipment arrives at its destination in the following format: (October 12, 

2001).

Block #11: The signatory and designees authorized by the signatory of the Compliance Agreement sign in this 

block (Receiving Point Signatory).

Distribution of copies issued by party under compliance:

 PINK COPY:

 Goes with shipment

 Held by consignee at destination

 YELLOW COPY:

 Goes with shipment

 Destination PPQ Officer picks up copy, or

 Consignee that is under compliance maintains copy until requested by PPQ

 WHITE COPY:

 Consignor maintains until requested by PPQ
Appendix E

Karnal Bunt Negative Grain Dealer/Brokers

E-20 Karnal Bunt Manual 03/2007-01

Instructions for filling out PPQ Form 540 (Certificate)

Block #1: Enter the date in the following format: (October 12, 2001).

Block #2: Enter the Void Date in the following format: (October 20,2001). Note: Allow a sufficient amount of 

time for the shipment to arrive directly to the destination to include any possible delays.

Block # 3: Enter your name/company that is selling the commodity.

Block #4: Enter the location from where the grain is being shipped.

Block #5: Enter name and address of the person/company that is purchasing the grain.

Block #6: Enter license plate number of the trailer and the issuing state.

Block #7: Enter Railcar number.

Block #8:

A. Enter the quantity in pounds.

B. Enter the Item name that is being issued a certificate.

C. Enter KB tested negative. Note: In the bottom two lines of block # 8 enter the following statement: 

“Produced in an area where Karnal bunt is not known to occur. For non-propagative use only.”

Block #9: The signatory and designees authorized by the signatory of the Compliance Agreement sign in this 

block (Shipping Point Signatory).

Distribution of copies issued by party under compliance:

 WHITE COPY (PART 1): Goes with shipment to consignee

 GREEN COPY (PART 2): Consignor’s Copy

 WHITE COPY (PART 3): Consignor maintains until requested by PPQ
Appendix E

Feed Processors

03/2007-01 Karnal Bunt Manual E-21

Feed Processors

Figure E-15 Compliance Agreement for Feed Processors

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Feed Processors

E-22 Karnal Bunt Manual 03/2007-01

FEED PROCESSOR

Compliance Agreement Stipulations

5. I/We agree to the following:

Receiving Bunted Kernel Positive Grain

 To handle regulated mill feed and/or bunted kernel positive wheat grain from the regulated area or an 

approved facility and only if it has been authorized to move under PPQ Form 530 (Limited Permit).3

 To verify, upon receipt of the shipment, the identity and quantities stated on PPQ Form 530, (lines 1-8).

 To receive and safeguard PPQ Form 530 (Limited Permits). These forms are accountable and must be 

maintained in a secure location.

 To retain original copies of PPQ Form 530 (Limited Permit) and make them available at the request of 

the USDA/State Department of Agriculture Karnal Bunt Program Office, and to maintain supporting 

records of grain movement associated with the permit.

 To immediately notify the USDA/State Department of Agriculture Karnal Bunt Project Office if any 

discrepancies occur in the shipment, if the shipment arrives without a PPQ Form 530, if seals are 

broken, or if it is received after the void date or in case of emergency (no offloading of shipments with 

discrepancies, broken seals, or shipments arriving after void date is permitted until approved by the 

USDA/State Department of Agriculture Karnal Bunt Project Office).

 To ensure that conveyances (trucks/trailers) delivering contaminated wheat grain are cleaned of all grain 

and/or millfeed contaminants prior to leaving the facility.

 To ensure that any material resulting from cleaning or processing bunted kernel positive wheat grain is 

destroyed by:

 Burial under two feet of soil in an area not to be cultivated or in an approved landfill4

, or

 Collection of all debris and placement in the conveyance/bin with the positive grain, or

 Sanitizing with an approved treatment.

 To remove all volunteer plants resulting from debris by cultivation or by using an herbicide.

Storage of Bunted Kernel Positive Grain

 To ensure that regulated millfeed and/or bunted kernel positive wheat grain is not subsequently moved 

from the initial processing facility to another facility without advance approval from the USDA/State 

Department of Agriculture Karnal Bunt Project Office.

 To notify the USDA/State Department of Agriculture Karnal Bunt Project Office 24 hours in advance of 

turning/moving of stored grain.

3 For guidance, see Instructions for filling out PPQ Form 530 (Limited Permit).

4 Contact the USDA/State Department of Agriculture Karnal Bunt Office for information on approved establishments/facilities.
Appendix E

Feed Processors

03/2007-01 Karnal Bunt Manual E-23

Processing Bunted Kernel Positive Grain

 To ensure that no changes occur in processing (changes that would not meet specified minimum moist 

heat treatment requirements) unless approved by the USDA/State Department of Agriculture Karnal 

Bunt Project Office.

 To ensure that all regulated millfeed, by-products, and/or positive wheat grain that is received is 

processed and treated with a moist heat treatment at a minimum of 170°F for at least one minute.

All entities that are under compliance are subject to 7 CFR 301.89.

Copies of the Code of Federal Regulations are available from the following Web site:

http://www.gpoaccess.gov/cfr/index.html

Penalty may be assessed for misuse of the agreement (7 USC 163).
Appendix E

Feed Processors

E-24 Karnal Bunt Manual 03/2007-01

 Instructions for filling out PPQ Form 530 (Limited Permit)

Block #1: Enter the date in the following format: (October 12, 2001).

Block #2: Enter the Void Date in the following format: (October 20,2001). Note: Allow a sufficient amount of 

time for the shipment to arrive directly to the destination to include any possible delays.

Block # 3: Enter your name/company that is selling the commodity.

Block #4: Enter the location from where the grain is being shipped.

Block #5: Enter name and address of the person/company that is purchasing the grain.

Block #6: Enter license plate number of the trailer and the issuing state.

Block #7: Enter Railcar number.

Block #8:

A. Enter the quantity in pounds.

B. Enter Positive KB Wheat and enter the seal numbers.

C. Enter the following statement: “To designated address only.”

Block #9: The signatory and designees authorized by the signatory of the Compliance Agreement sign in this 

block (Shipping Point Signatory).

Block#10: Enter the date that the shipment arrives at its destination in the following format: (October 12, 

2001).

Block #11: The signatory and designees authorized by the signatory of the Compliance Agreement sign in this 

block (Receiving Point Signatory).

Distribution of copies issued by party under compliance:

 PINK COPY:

 Goes with shipment

 Held by consignee at destination

 YELLOW COPY:

 Goes with shipment

 Destination PPQ Officer picks up copy, or

 Consignee that is under compliance maintains copy until requested by PPQ

 WHITE COPY:

 Consignor maintains until requested by PPQ
Appendix E

Harvesting Equipment and Conveyances

03/2007-01 Karnal Bunt Manual E-25

Harvesting Equipment and Conveyances

Figure E-16 Compliance Agreement for Harvesting Equipment and Conveyances

According to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information 

unless it displays a valid OMB control number. The valid OMB control numbers for this information collection are 0579-0054, 0088, 0129, 0198, 0257, 0310, 0317, 

0322, 0337, 0346, 0363, and 0369. The time required to complete this information collection is estimated to average 1.25 hours per response, including the time

for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

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Appendix E

Harvesting Equipment and Conveyances

E-26 Karnal Bunt Manual 03/2007-01

HARVESTING EQUIPMENT AND CONVEYANCES

Compliance Agreement Stipulations

I/We agree to the following:

 Fields within the regulated area will be sampled and results known prior to the grain leaving the field or 

before grain is off loaded at a facility.

 Each load of negative grain from fields within the regulated area will be accompanied by the 

appropriate certificate, PPQ Form 540 or local equivalent.

Positive grain:

 To transport equipment, conveyances and/or grain from a known positive field only when authorized to 

move under Limited Permit (PPQ Form 530). The PPQ Form 530 will be issued by the inspector or 

designee under compliance after:

 Trucks/trailers transporting positive grain have been determined to be leak proof and properly 

covered to avoid spillage of grain.

 All debris has been removed from the exterior of the truck/trailer after loading.

 To perform treatments in accordance with the Karnal bunt treatment protocols. Refer to cleaning 

Protocols for approved treatments.

 To immediately contact the USDA/State Department of Agriculture Karnal bunt office in the event of a 

spillage or accident. Spills can be picked up and returned to the truck hauling the positive grain.

 To obtain approval in advance from the USDA/State Department of Agriculture Karnal bunt office, if 

transferring of regulated grain from one truck to another becomes necessary.

 To notify the USDA/State Department of Agriculture Karnal bunt office 24 hours in advance of any 

changes in movement as described in the PPQ Form 530. Approval must be obtained prior to any 

movement of regulated articles.

 To ensure that trucks/trailers carrying regulated articles move to their approved destination by the most 

direct route prior to the void date on the PPQ Form 530.

 To ensure that trucks/trailers are cleaned following discharge and that small quantities of debris resulting 

from this cleaning are:

 Stored back in the bin with bunted kernel positive grain, or 

 Buried in an approved landfill, or 

 Treated with an approved treatment

 To clean vehicles dedicated to transporting bunted kernel positive grain (not back hauling) in accordance 

with attached information upon completion. All harvesting equipment associated with Karnal Bunt 

positive grain must be cleaned prior to leaving the regulated area (see Cleaning Protocol)

 To provide a copy of this compliance agreement to the facilities where the trucks/trailers will be loading 

upon request.

INITIAL: ________________.
03/2007-01 Karnal Bunt Manual F-1

Appendix

F Appendix F

Contributors

State Regulatory Officials

A. Pfender, Laboratory Manager, State Agriculture Laboratory, Arizona 

Department of Agriculture, Phoenix, Arizona

T. Sim IV, Plant Pathologist, Entomology Division, Kansas State Board of 

Agriculture, Topeka, Kansas

C. Umbdenstock, Program Manager, Arizona Department of Agriculture, 

Phoenix, Arizona

R. Ykema, Plant Pathologist, State Agriculture Laboratory, Arizona 

Department of Agriculture, Phoenix, Arizona

USDA Agencies

G. Adams, State Plant Health Director (Montana), Plant Protection and 

Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Helena, Montana

R. Berry, Supervisory Biological Technician, Invasive Pest Management 

Laboratory, Plant Protection and Quarantine, Animal and Plant Health 

Inspection Service, U. S. Department of Agriculture, Niles, Michigan

M. R. Bonde, Plant Pathologist, Foreign Disease—Weed Science 

Research Unit, Agricultural Research Service, U. S. Department of 

Agriculture, Ft. Detrick, Maryland

T. Boratynski, Plant Pathologist, Plant Protection and Quarantine, Animal 

and Plant Health Inspection Service, U. S. Department of Agriculture, El 

Centro, California

K. L. Bryan, State Plant Health Director (New Mexico), Plant Protection 

and Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Albuquerque, New Mexico

N. Diaz, Botanist, Plant Protection and Quarantine, Animal and Plant 

Health Inspection Service, U. S. Department of Agriculture, Houston, 

Texas

F. E. Dowell, Research Leader, Grain Marketing and Production Research 

Center, Agricultural Research Service, U. S. Department of Agriculture, 

Manhattan, Kansas
Appendix F

USDA Agencies

F-2 Karnal Bunt Manual 03/2007-01

W. M. Dowler, Plant Pathologist, Plant Disease Research, Northeastern 

Region, Agricultural Research Service, U. S. Department of Agriculture, 

Frederick, Maryland

T. Ely, State Operational Support Officer (Arizona), Plant Protection and 

Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Phoenix, Arizona

J. Floyd, Emergency Programs Coordinator, Plant Protection and 

Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Riverdale, Maryland

D. Grooms, Biological Technician, Invasive Pest Management 

Laboratory, Plant Protection and Quarantine, Animal and Plant Health 

Inspection Service, U. S. Department of Agriculture, Niles, Michigan

B. Guerra, Assistant Program Manager, Plant Protection and Quarantine, 

Animal and Plant Health Inspection Service, U. S. Department of 

Agriculture, Olney, Texas

M. Hennessey, Program Manager, Plant Protection and Quarantine, 

Animal and Plant Health Inspection Service, U. S. Department of 

Agriculture, Blythe, California

J. Hoffman, Plant Pathologist, Crops Laboratory, Western Region, 

Agricultural Research Service, U. S. Department of Agriculture, Logan, 

Utah

J. Levitt, State Plant Health Director, Plant Protection and Quarantine, 

Animal and Plant Health Inspection Service, U. S. Department of 

Agriculture, Phoenix, Arizona

C. Maneely, PPQ Technician, Plant Protection and Quarantine, Animal 

and Plant Health Inspection Service, U. S. Department of Agriculture, 

Olney, Texas

R. McKinney, State Plant Health Director (Kansas), Plant Protection and 

Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Topeka, Kansas

T. McNary, Regional Program Manager, Western Region, Plant Protection 

and Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Ft. Collins, Colorado

P. McPherren, State Plant Health Director (Colorado), Plant Protection 

and Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Aurora, Colorado

G. H. Nash, State Operational Support Officer (Texas), Plant Protection 

and Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Austin, Texas

J. Pace, PPQ Technician, Plant Protection and Quarantine, Animal and 

Plant Health Inspection Service, U. S. Department of Agriculture, Olney, 

Texas
Appendix F

USDA Agencies

03/2007-01 Karnal Bunt Manual F-3

G. Peterson, Research Scientist, Foreign Disease—Weed Science 

Research Unit, Agricultural Research Service, U. S. Department of 

Agriculture, Ft. Detrick, Maryland

S. R. Poe, Emergency Programs Pathologist, National and Emergency 

Programs, Plant Protection and Quarantine, Animal and Plant Health 

Inspection Service, U. S. Department of Agriculture, Riverdale, 

Maryland

C. Randal, PPQ Technician, Plant Protection and Quarantine, Animal and 

Plant Health Inspection Service, U. S. Department of Agriculture, Olney, 

Texas

L. Richardson, PPQ Technician, Plant Protection and Quarantine, Animal 

and Plant Health Inspection Service, U. S. Department of Agriculture, 

Olney, Texas

M. Royer, Acting Senior Program Advisor, Pest Detection and 

Management Programs, Plant Protection and Quarantine, Animal and 

Plant Health Inspection Service, U. S. Department of Agriculture, 

Riverdale, Maryland

J. Schoenholz, Senior Program Manager, Eastern Region, Plant Protection 

and Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Raleigh, North Carolina

B. Shambaugh, Regional Program Manager, Western Region, Plant 

Protection and Quarantine, Animal and Plant Health Inspection Service, 

U. S. Department of Agriculture, Ft. Collins, Colorado

T. Sheikh, Safeguarding Specialist, Plant Protection and Quarantine, 

Animal and Plant Health Inspection Service, U. S. Department of 

Agriculture, Olney, Texas

B. Smith, Program Manager, Plant Protection and Quarantine, Animal and 

Plant Health Inspection Service, U. S. Department of Agriculture, Olney, 

Texas

B. Spaide, National Karnal Bunt Program Manager, Plant Protection and 

Quarantine, Animal and Plant Health Inspection Service, U. S. 

Department of Agriculture, Riverdale, Maryland

T. Weir, PPQ Officer, Plant Protection and Quarantine, Animal and Plant 

Health Inspection Service, U. S. Department of Agriculture, Aurora, 

Colorado

The aforementioned individuals were major contributors to the development, 

preparation, and review of the Karnal Bunt Manual. Other contributors and/or 

reviewers were research scientists of Agricultural Research Service and 

regional and staff personnel of Plant Protection and Quarantine.
Appendix F

USDA Agencies

F-4 Karnal Bunt Manual 03/2007-01
mm/yyyy-ee Manual Title Here G-1

Appendix

G Appendix G

References

Aujla, S. S., Sharma, Y. R., Chand, K., and Sawney, S. S. 1977. Influence of 

weather factors on the incidence and epidemiology of Karnal bunt disease of 

wheat on the Punjab. Indian J. Of Ecol. 4:71-74.

Bonde, M. R., Peterson, G. L., Schaad, N. W., and Smilanick, J. L., Karnal bunt 

of wheat. Plant Disease (St. Paul, Minn. American Phytopathological Society) 

Dec. 1997, v. 81 (12) p. 1370-1377.

Dowell, F. E., Boratynski, T., Ykema, R., Dowdy, A. K., and Staten, R. 2002. 

Use of optical sorting to detect wheat kernels infected with Tilletia indica. 

Plant Dis. 86:1011–1013.

Fuentes, S. F., Torres, E. T., and Dominquez, C. G., 1982. Estudios sobre el 

control del carbon pareial del trigo. Paper presented at the Associan 

Colombiana de Fitopathologica, Cali, Colombia.

Joshi, L. M., Singh, D. V., Srivastava, K. D., and Wilcockson, R. D. 1983. 

Karnal bunt—a major disease that is now a new threat to wheat. Botanical 

Review (accepted for publication, December 1983).

Malik, V. S., and Mathre, D. E. 1998. Bunts and smuts of wheat: an 

international symposium. North American Plant Protection Organization, 

Ottawa (proceedings of symposium held in Raleigh, North Carolina, August 

17-20, 1997).

Peterson, G. L., Bonde, M. R., and Phillips, J. G., Size-selective sieving for 

detecting teliospores of Tilletia indica in wheat seed samples. Plant Disease 

(St. Paul, Minn. American Phytopathological Society) Sept. 2000, v. 84 (9) p. 

999-1007.

Singh, A., and Prasad, R. 1980. Karnal bunt of wheat in India. Unpublished 

manuscript.

Smilanick, J. L., Dupler, M., Goates, B. J., Hoffman, J. A., Clark, D., Dobson, 

G., Germination of teliospores of karnal, dwarf, and common bunt fungi after 

ingestion by animals. Plant Disease (St. Paul, Minn. American 

Phytopathological Society) March, 1986, v.70 (3) p. 242-244.

Torres, E. T. 1982. Information on Neovossia indica (Mitra) Mundkur, the 

causal agent of partial bunt in wheat. Unpublished manuscript (CIMMYT).
Appendix G

G-2 Manual Title Here mm/yyyy-ee
03/2007-01 Karnal Bunt Manual H-1

Appendix

H Appendix H

National Survey Protocol

The National Survey for Karnal bunt is conducted in each state with technical 

support provided to SPHDs/SPROs from the National Survey Coordinator of 

Pest Detection and Management Programs (PDMT). Numbers of samples 

taken vary depending on the amount of wheat produced within a state. The 

Karnal bunt protocol is available from the National Survey Coordinator for 

Karnal bunt on the PDMP Staff in Riverdale Headquarters. You can also 

download the survey plan located on the Karnal bunt Web site by clicking on 

the following link:

http://www.aphis.usda.gov/ppq/ep/kb/KB_Survey_Plan_FY2003.pdf
Appendix H

H-2 Karnal Bunt Manual 03/2007-01
mm/yyyy-ee Karnal Bunt Index-1

Karnal Bunt

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

Index

A

absorbent material B-3

accidental poisoning B-2

accidents B-2

acrylic lid D-8

adding extraction solution D-43

Aegilops 1-5

Afghanistan 1-3, 1-4

Agency legal counsel 3-14

agricultural consultants 6-1

Agricultural Research Service (ARS) D-2, F-1

Agricultural Research Stations 6-1

air hose, high pressure 3-6

air pressure 3-6

airborne spores D-32

Aircraft and Equipment Operations Center D-48

alcohol

ethyl D-22, D-25

isopropyl D-23, D-27

Alternaria D-16, D-44

analysis

bunted kernel D-2

spore D-2

worksheet D-21

analytical balance D-27

Animal and Plant Health Inspection Service

(APHIS) 1-4, 3-14, 4-1

anthesis 1-3

APHIS (Animal and Plant Health Inspection Service) 1-4, 3-14, 4-1

APHIS Business Services 5-1

APHIS Safety and Health Manual B-2

applicators D-34

approved establishment 3-9

approved laboratories D-21

aprons, rubber D-21

aquatic life B-2

area

decontamination D-20, D-21

hazardous waste D-33

microscope D-33

processing D-42

sample processing D-34

sample receiving D-31, D-42

sample weighing D-26

secure storage D-21

slide preparation D-34

slide-making D-38

solution addition D-21

storage D-28

weighing D-21, D-28

work D-35

Arizona 1-4

Arizona Department of Agriculture D-2, F-1

ARS (Agricultural Research Service) D-2, F-1

augers 3-6, 3-12, 3-13

automated method D-3

B

bags

biohazard D-21, D-27–D-29, D-33, D-34, D37, D-38

disposable D-37

grain storage 3-12, 3-13

plastic D-20

sanitary 2-3, 2-4

balance

3 decimal D-24, D-25

analytical D-22, D-27

bales 3-8

basidiospores 1-3

beakers D-25

150-mL D-34

1-L D-22, D-25

250-mL D-34

2-L D-22, D-24, D-25

400-mL D-34

50-mL D-32

600-mL D-7, D-12, D-13, D-22, D-30

bees B-1

bench liners D-23, D-30, D-32, D-33

best management practices (BMPs) 3-14, A-1

BIA (Bureau of Indian Affairs) 6-1

bin sweepings D-26

bins 3-7, 3-12, 3-13, D-1

biohazard bags D-21, D-27–D-29, D-33, D-34, D37, D-38

biohazard containers D-17, D-33

biological containment hood D-18

biosafety hood D-22, D-26

black point D-16, D-44

bleach

baths D-30, D-32

decontaminating forceps with D-10, D-13

decontaminating labware and instruments

with D-17

household D-7, D-12, D-19, D-22, D-34

in carboy for holding extraction filtrate D-32

residues D-27

sanitizing the hood with D-27

solution D-14, D-27, D-28, D-30–D-33, D35, D-37, D-38, D-47

wiping down working surfaces with D-18

BMPs (best management practices) 3-14, A-1

bobtails 3-7
Index

Index-2 Karnal Bunt mm/yyyy-ee

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

Bohmont, Bert L. B-3

Bonde, Morris D-2

boots B-3

Boratynski, Ted D-2

bottles

250-mL 2-4

glass D-22

HDPE wide mouth D-22

nalgene D-26

plastic D-20, D-28, D-29, D-34, D-35

sample D-31, D-32

screw-cap D-35

spray, plastic D-22

wash D-22, D-30, D-31, D-32, D-33

boxes

cardboard D-12, D-19, D-21

glass disposal D-34

microscope slide D-34

Bromus 1-5

broom B-3

brushes B-3, D-7

bucket, sanitary 2-3

bulbs, pipette D-22, D-34

bunt

common D-44

dwarf D-44

bunted kernels

alternate term for infected kernels D-3

analysis D-2, D-42

as an indicator of infected grain D-1

checking for D-10, D-13

equipment testing positive for 3-7

examining samples for D-3

fields sampled for 3-10

host crops free of 3-8

identification authority for D-16, D-45, D-46

identification course D-44

identification of D-44

identifier D-16

machine 2-2

movement of host crops testing positive for 3-

9

readers D-8, D-13

recording number of D-10, D-13

seed samples tested for D-3

seed testing negative for 3-11

spread of 3-14

storing D-16

testing host crops for 3-8

verifiers D-42, D-44

Bureau of Indian Affairs (BIA) 6-1

C

CA (Compliance Agreement), definition of A-1

cabinet, storage D-34

California 1-4

caps D-34

carboxin-thiram 3-5, C-2

carboy D-22, D-24, D-26, D-29, D-32, D-33

cardboard boxes D-19, D-21

cart

glassware D-22, D-31, D-32

utility D-22

centrifuge

log book D-33

racks D-30, D-32

required r/min D-22, D-34

station D-30, D-32, D-33

tube rack D-31, D-32

tubes D-19, D-21, D-24, D-26, D-30, D-31, D-32, D-33

certificate

definition of A-1

for grain 3-10

for hay/straw movement 3-12

for host crops testing negative 3-8

chain-of-custody seals D-19

chairs D-17

charts 5-1

check-in area D-20

chemical emergencies B-2

chemical fume hood D-30

chemicals authorized to treat regulated articles 3-5

Chemtrex B-2

chlorine bleach B-2, C-1

CIMMYT (International Center for Maize and Wheat

Improvement) 1-3

citric acid

monohydrate D-22, D-25

solution D-25

Ciudad Obregon 1-3

clean air station D-18

clean room D-18, D-19, D-24, D-30, D-42

cleaning

definition of 3-7, 3-14, A-1

extraction lab D-43

of mechanized harvesting equipment 3-6

treatments 3-14

cleanser, skin D-34

coats, lab D-34

collection bag 2-1

combines 3-6

commercial haulers 1-2

common bunt 1-3, D-44

complaints 4-2

compliance agreements

definition of A-1

for grain 3-10

for processing establishments 1-2

composite sample 2-3, 2-4

composting procedure 3-9

compound microscope D-7, D-10, D-13, D-39

concerns 4-2

conditions for infection 1-4

confirmed detection, definition of A-1

Congress 4-1

consultants 6-1

contacts 6-1

containers

as a regulated article 3-2

biohazard D-33

hazardous waste D-33

contaminated materials D-33

contamination, definition of A-1

control wheat D-29

conveyances
Index

mm/yyyy-ee Karnal Bunt Index-3

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

as regulated articles 3-2

definition of A-1

equipment 3-12, 3-13

for seed produced inside the regulated area 3-

5

Cooperative Extension Service 3-14

cooperators 1-2

correction fluid D-37, D-40

counting teliospores D-43

cover slips

adding liquid under D-35

beginning examination at the corner of D-39

for examining wheat kernels D-7, D-12

new for each sample D-35, D-36

positioning D-37

sealing D-10, D-13, D-37

size D-34

coveralls B-3, D-17, D-19, D-20

CPPC Ultra Bleach 2 C-1

crops B-2

cross-contamination 2-2, 3-14

cups

disposable D-22

portion D-22

custom harvesters 1-2

cylinders, graduated D-22, D-24

Cytoseal D-34, D-35, D-37

D

data

acquisition 5-1

entries D-36

management 5-1

sheets D-34, D-38

dealers

farm and garden supply 1-2

farm equipment and implement 1-2

debris 3-6

decontaminating equipment D-43

decontaminating samples D-42

decontamination

area D-20, D-21

definition of A-1

of extraction ware D-19

of labware D-17

of samples D-3

safeguards D-2, D-18

deionized water D-22, D-24, D-25, D-26

delimiting survey, definition of A-1

delivery system 3-6

Deputy Administrator 3-3

designated building D-18

detection, definition of A-2

detergent B-3, D-18, D-22, D-24, D-26

digital camera 5-1

dirty room D-18, D-42

disease

definition of A-2

unintentional movement of 3-14

dish pans D-12, D-19

disinfectant tubs D-24, D-30

disposable centrifuge rack D-32

disposable coveralls B-3

disposable rack D-31

disposable tube rack D-21

disposal bag D-10, D-37

disposal of pesticide containers B-1

dissecting microscope D-7, D-10, D-13, D-16, D22

distribution 1-4

dough stage 3-8, 3-12

dry spills B-3

durum wheat

affected by Karnal bunt 1-3

as a host of Karnal bunt 1-5

equipment used in harvesting or processing of

3-7

seed storage 3-5

treatments for equipment used in harvesting or

processing of 3-6

dust 3-6

dust mask D-7, D-8, D-13, D-22, D-27

dust pan B-3

dusting gas D-7, D-10

dwarf bunt D-44

dye D-8, D-13

E

EAN (Emergency Action Notification) 1-2, 3-3, A-2

elevators 3-12, 3-13

emergencies, chemical B-2

Emergency Action Notification (EAN) 1-2, 3-3, A-2

emergency quarantine action 3-3

endosperm 1-3, 1-5

environment, impact of pesticides on B-2

environmental groups 4-1

Environmental Protection Agency B-1

epidermal layer D-14

Eppendorf adjustable pipettor D-23

equipment

as a regulated article 3-2

cleaning 3-8

decontaminating D-43

harvesting 3-8

mechanized harvesting 3-6, B-2

reconditioning 3-12, 3-13

seed cleaning 3-12, 3-13

Erlenmeyer flasks D-22, D-26, D-34

establishing priority D-43

ethyl alcohol D-22, D-25

examining slides D-43

examining wheat kernels D-43

export terminal facility 3-10

exposures, pesticide B-2

extracting samples D-21, D-43

extraction

container D-31

equipment D-18

filtrate D-32

fluid D-29

hood D-38

lab D-28

procedures D-1

series D-33

set D-32

solution D-18, D-21, D-24, D-26, D-27, D-
Index

Index-4 Karnal Bunt mm/yyyy-ee

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

28, D-29, D-31

extraordinary emergency 3-3

eyewash kit B-3

F

facilities 3-7

farmers 1-2

fax machine 5-1

Federal Environmental Protection Agency B-1

Federal Register 3-3, 3-4

feeder trough D-8

field number 2-2

fields D-1

filter, HEPA D-26

filtrate D-31, D-32, D-33

fire extinguisher B-3

fires B-2

first aid B-2

first aid kit B-3

flasks D-30, D-31

flasks, Erlenmeyer D-22, D-26, D-34

Floyd, Joel D-2

forceps D-7, D-8, D-10, D-12, D-13

Ft. Detrick, MD D-2

fume

absorber D-34

extractor D-35

hood D-23, D-30

fumigant recirculation C-1

fumigation A-2, C-1

fungal morphology D-45

fungal taxonomy D-45

fungicide 3-11, D-32

funnels D-22, D-25, D-30, D-32

G

garden hose D-19

general public 4-1, 6-1

germ tubes 1-5

glass bottle D-22

glass cleaner D-7

glasses, safety D-19, D-20, D-22

glassware D-26, D-30, D-32

glassware cart D-31, D-32

glassware cart, D-31

glassware, D-30

gloves

gauntlet D-22

latex D-7, D-12, D-17, D-18, D-19, D-20, D22, D-34, D-36

nitrile D-22

rubber B-3

glumes 1-5

glycerol D-22, D-24, D-25

governments 4-1

GPS units 5-1

graduate, transfer pipettes D-34

graduated cylinder D-24, D-25

grain

analysis D-3

analysis laboratories D-1, D-2, D-18

buggies 3-6

certification of 3-4

definition of A-2

elevators 3-2

examination machine D-44

host crop material 3-14

inspection machine D-2, D-3, D-7, D-8, D-9, D-10

organizations 6-1

protocol for sampling 2-1

samples 1-2, D-1, D-2, D-3, D-44

storage 3-7

storage facilities 3-12, 3-14

graphics 5-1

grasses, as hosts of KB 1-5

grower groups 6-1

Guidelines for Managing and Monitoring Pesticide

Spills B-3

H

hair covers D-17

hand lens D-16

handling spills D-43

harvesters, custom 1-2

harvesting and cleaning equipment 3-8

harvesting equipment 3-6, 3-14, A-2, B-2

harvesting machines 3-6

Haryana, Indian state of 1-3

hay

definition of A-2

movement decision table 3-12

movement of host crop 3-8

movement of KB positive 3-9

movement of KB positive, 3-9

samples of 1-2

hazardous waste

area D-33

container D-33

header 3-6, 3-7

heading, of wheat plants 1-4

headquarters 4-2

heads 3-14

Helminthosporium

sativum D-44

spp. D-16

HEPA filter D-26

high pressure air D-10

honey bees B-2

hood

biosafety D-22, D-26, D-28, D-29, D-30

extraction D-38

fume D-23

in solution addition room D-29

laminar flow D-35

hood, D-29

hood, sanitizing D-29

hopper 3-6, D-8

hose, garden D-19

host

definition of A-2

material 3-7

host crop

material 3-13, 3-14

movement of 3-8

hosts of Karnal bunt 1-5

hot water and detergent drench 3-8
Index

mm/yyyy-ee Karnal Bunt Index-5

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

humidity, relative 1-5

I

identification authority D-13, D-45, D-46

identifier D-13

identifying teliospores D-43

incineration B-1

India 1-3, 1-4

industry 4-1

inert dusting gas D-7

infection

definition of A-2

occurrence of 1-3

stages of D-14

infectious spores 1-5

inoculation A-2

inoculum A-2

International Center for Maize and Wheat Improvement (CIMMYT) 1-3

interpreting laboratory results D-43

interstate regulatory action 3-3

intrastate regulatory action 3-3

inverted light microscope D-23

Iran 1-4

Iraq 1-3, 1-4

irrigation 1-4, 1-5

isopropyl alcohol D-23, D-27

K

Kansas State Board of Agriculture F-1

Karnal bunt

definition of A-2

sieve log D-23, D-46, D-47

sieve usage log D-23, D-47, D-48

Karnal, city of 1-3

keeping records D-43

kernel verifiers D-44

Kimwipes D-7

kitty litter B-3

known infested property, definition of A-2

L

lab coats D-19, D-20, D-26

lab tape D-24, D-30, D-33

lab tools D-17

label instructions B-1

labeling D-2

labeling tape D-7

labels D-7, D-12, D-23, D-34

laboratories

grain analysis D-1, D-2

seed D-1

seed analysis D-2

laboratory

control D-21

documents D-40

grain analysis D-18

manager D-36, D-39, D-41

number D-21

personnel D-19, D-21, D-42

procedures D-1

sample numbers D-21

seed analysis D-17, D-18

labware D-17

lakes B-2

laminar flow hood D-35

lamp, magnifying D-12

landfill B-1

latex gloves D-8, D-13, D-19, D-24, D-26, D-27, D-29, D-30, D-34

law enforcement officials 6-1

leaks, pesticide B-2

Lebanon 1-3, 1-4

Legislative and Public Affairs 4-2

legs 3-12, 3-13

life cycle 1-5

light source B-3

light switch D-8, D-10

Limited Permit 3-9, 3-10, A-3

linen tester D-22

liner, bench D-23

liquid detergent B-3

liquid spills B-3

liquid suspension D-19

live steam 3-8

livestock B-2

loading dock D-18

logging-in samples D-42

log-in procedures D-19

Lolium 1-5

loose smut 1-3

M

machine, bunted kernel 2-2

magnetic stirring bar D-25

magnifying glass D-22, D-23

magnifying lamp D-12

maintaining supplies D-42, D-43

making labels D-43

manual method D-12

mapping 5-1

maps 5-1

marker D-10, D-13, D-16

mechanized harvesting equipment 3-2, 3-6, B-2

media 4-1

methyl bromide

application rate C-1

approved fumigant A-2

authorized chemical for treating regulated articles 3-5

Mexico 1-3, 1-4

microclimate 1-4

micrometer

ocular D-23

stage D-23

microscope

area D-33

compound D-7, D-10, D-12, D-13, D-39

dissecting D-7, D-10, D-12, D-13, D-16, D22

inverted light D-23

slides D-7, D-10, D-12, D-13, D-16, D-35, D-45

stations, D-39

microscopic evaluation D-24

microscopic examination D-34, D-39, D-44, D-45

microscopic identification D-39

microscopy D-19, D-45
Index

Index-6 Karnal Bunt mm/yyyy-ee

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

Mission, Texas D-48

mixer, vortex D-23

mycelia 1-5

mycelium 1-3

mycology D-45

N

nail polish D-7, D-10, D-12, D-37

nalgene bottles D-2, D-26

National Mycologist D-45

National Program Planning Staff 3-3

National Regional Directors 3-3

national standard D-18

National Survey Coordinator H-1

National Survey Plan 2-6

national surveys D-1, H-1

negative control D-46

negative samples D-10, D-13

negative slides D-39

Neovossia indica 1-3, A-4

Nepal 1-4

New Mexico 1-4

news media 6-1

Nogales, AZ D-2

non-target insect species B-2

noxious weeds 3-14

nylon screens D-32

nylon sieve D-30

O

ocular micrometer D-23, D-47

Office of Personnel Management (OPM) 3-14

officials

federal 4-2

state 4-2

OPM (Office of Personnel Management) 3-14

options, sanitizing 3-7

orbital shaker D-31

outbreak situations 4-1

ovary 1-3, 1-5

overalls D-26

overlays 5-1

P

packaging D-18

padl-pets D-35, D-37

paint brushes D-7, D-10

Pakistan 1-3, 1-4

paper cups D-26, D-27, D-28, D-29

paper label D-10

parafilm D-23, D-27

partial bunt 1-3, A-2

Pasteur pipettes D-35, D-36, D-37

pasteurized product 3-9

pathogen, definition of A-3

PCNB (pentachloronitrobenzene) 3-5, C-1

PDMP (Pest Detection and Management Programs) 5-1, B-1

pentachloronitrobenzene (PCNB) 3-5, C-1

pericarp D-14, D-16

Pest Detection and Management Programs (PDMP) 5-1, B-1

pesticides B-1

application B-2, B-3

authorized B-1

cartridges B-3

containers B-1

seed treated with D-8, D-13, D-33

spillage B-1

Peterson, Gary D-2

petri dish D-7, D-8, D-10, D-12, D-13, D-16

petroleum jelly D-34

Phoenix, Arizona D-2

pipette

bulbs D-36

bulbs, D-36

Mohr D-23, D-26

Pasteur D-35, D-36, D-37

Pasteur, D-35, D-36

plastic D-25, D-35, D-36, D-37

tips D-23

transfer D-35, D-36

washer D-26

pipettor D-23

pitchers D-23, D-30, D-33

placards 5-1

plant breeders 6-1

plant pathology D-45

plant pests 3-14

Plant Protection Act 2000 3-3

Plant Protection and Quarantine (PPQ) D-2

planting equipment 3-5

plastic bags B-3, D-7, D-13, D-20

plastic bottles D-20, D-28, D-29, D-35

plastic cover B-3

plastic disposal bags D-19

plastic pipettes D-35, D-36, D-37

plastic trays D-13, D-24, D-30, D-33

plastic tubs D-32

plastic vials D-7, D-10, D-13, D-16

Poison Control Center B-2

poisoning B-2

pollen grains D-45

ponds B-2

portable eyewash kit B-3

portable light source B-3

positive samples D-10, D-13, D-18, D-21

positive slides D-39

potassium acetate D-23, D-25

Power Points 5-1

PPQ (Plant Protection and Quarantine) D-2

PPQ Field offices 3-3

PPQ Form 523 3-3, A-2

PPQ Form 530 3-10, A-3

PPQ Form 540 3-4, 3-12, A-1

PPQ Identifier D-44

PPQ National Mycologist D-46

PPQ Plant Pathology Identifiers D-45

PPQ Port Identifier D-45

PPQ Project Director 3-4

PPQ Regional Offices B-1

PPQ Treatment Manual B-1

PPQ-APHIS-USDA, definition of A-3

precautionary labeling B-1

preparing sample containers D-43

preparing Shear’s mounting fluid D-43

preparing slides D-34, D-43

preparing the workstations D-43
Index

mm/yyyy-ee Karnal Bunt Index-7

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

primary sporidia 1-5

private agricultural consultants 6-1

probes 2-3

processing areas D-42

processing samples D-43

program activities 4-2

program risks 4-2

Project Director 4-2

project guidelines D-10, D-13

project managers 4-2

promycelium 1-5

property, definition of A-3

protective clothing D-8, D-10, D-30

public health 6-1

public meetings/gatherings 4-2

PVC pipe 2-1

Q

quality assurance D-21

quality assurance slides D-34

quality control measures D-46

R

rack, test tube D-23

RADS (Regulatory Analysis and Development

Staff) 3-4

railcars 3-7, 3-12, 3-13

railroad cars

as a regulated article 3-2

ranchers 1-2

Rapid Response Team (RRT) 5-1

reading positive slides D-43

receiving samples D-42

reception area D-18

reception area, D-18

record keeping D-39

records 3-14

reference slides D-39

references G-1

regional offices 4-2

regulated area

boundaries 1-2

definition of A-3

equipment leaving the 3-13

hay/straw leaving the 3-9

hay/straw movement within the 3-12

spread of bunted kernels from 3-14

regulated articles 1-2

Regulatory Analysis and Development Staff

(RADS) 3-4

regulatory program 3-14

relative humidity 1-5

replacement sieves D-48

replications D-17

respirator B-3, D-7, D-8, D-13

return auger 3-6

rheostat D-8, D-10

right-of-entry authority 3-3

risk-reducing measures 4-2

RRT (Rapid Response Team) 5-1

rubber aprons D-21, D-30

rubber boots B-3

rubber gloves B-3, D-30

running water D-18

S

safety B-1

safety considerations B-2

safety glasses D-19, D-20, D-22, D-24, D-26, D29, D-30

sample bags D-21

sample bottles D-31, D-32

sample containers D-21, D-27, D-28, D-29

sample contamination D-13

sample extraction D-19, D-47

sample journals D-21

sample log-in D-21

sample priorities D-36

sample receiving area D-20, D-31, D-42

sample slides D-34

sample tubes D-34

sample weighing area D-26

samples

analyzing D-8

centrifuged D-35

delivery of D-18

dry grain D-18

seed D-19

sand B-3

sanitary bucket 2-3, 2-4

sanitary landfill B-1

sanitize, definition of A-3

sanitizing 3-7, 3-14

sawdust B-3

scale area 2-2

scissors D-35

scoop, lab D-23

screen, nylon D-32

screw-cap bottle D-35

scrub brushes B-3

sealant, silicone D-23

Secale cereale 1-5

secondary sporidia 1-4, 1-5

Secretary of Agriculture 3-3

secure storage area D-21

secure storage units D-19

seed

analysis D-3, D-42

analysis laboratories D-2, D-17, D-18

bin cup D-10

coat D-14

conditioning equipment 3-2

definition of A-3

processing facilities 3-14

produced outside the regulated area 3-5

protocol for sampling during storage 2-3

protocol for sampling following storage 2-5

protocol for sampling prior to storage 2-2

samples 1-2, D-1, D-2, D-16

storage 3-4, 3-7

storage facilities 3-14

testing laboratory D-26

selecting slides D-43

selective sieving D-2

serial number D-46, D-47

set number D-21

setting up the working area D-43
Index

Index-8 Karnal Bunt mm/yyyy-ee

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

shaker table D-30

shaker, orbital D-23

Shear’s mounting medium

containers of D-37

labeling D-25

laboratory supply D-7, D-12

laboratory supply, D-35

mixing pellets with D-33

placing onto slides D-36

preparation of D-21, D-24, D-25

preparing microscope slides with D-10, D-13

Shear’s solution D-35

shed D-18

shoe covers D-17

shovel B-3

side panels 3-7

sieve log D-23

sieve usage log D-23

sieves

20-μm D-23, D-31, D-32, D-46, D-47

53-μm D-23, D-46, D-47

53-μm, D-31

signs 5-1

silicone seal D-47

silicone sealant D-23

silos 3-7

sinks D-30

slide

labels D-37, D-40, D-41

making area D-38

microscope D-7, D-12, D-35

preparation D-19, D-35, D-42

preparation area D-34

readers D-42

sealant D-39

tray D-38

warmer D-35, D-36, D-38

smut

definition of A-3

fungi D-24

spores 1-3

sodium

hypochlorite 3-7, D-18, D-22

phosphate D-23

solution addition

area D-21

room D-29

Sonora, Mexican state of 1-3

sorus D-16

special interest groups 4-1

special precautions B-1

SPHDs 4-2

spike 1-3

spills D-19

spills, pesticide B-1, B-2

spore

analysis D-2, D-3, D-21

analysis worksheets D-19, D-26

black point D-16

fragments D-17

infectious 1-5

Karnal bunt 1-4

seed positive 3-11

verifiers D-42

sporidia

contact with ovary, 1-3

definition of, A-3

primary 1-5

secondary 1-4, 1-5

spread of 1-4

spray bottle D-12, D-19, D-22

stage micrometer D-23

stakeholders 4-1

Standard Pesticide User’s Guide, the B-3

starch granules D-31

State Agriculture Laboratory D-2, F-1

State Cooperators 3-3, 3-14

State Departments of Agriculture 5-1

State regulatory officials F-1

steam 3-8

stir bars, magnetic D-23

stirrer, magnetic D-23

stirring bar D-25

stomata 1-5

stoppered bottle D-25

storage

and processing facilities 1-2

area D-21, D-28

bins 2-3

container D-16

facilities 2-2, 3-5, 3-10

units D-12, D-19

storing samples D-42, D-43

straw

definition of A-3

movement of 3-8

removal of 3-14

streams B-2

stubble, definition of A-3

sub-samples 2-2, 2-5

supernatant D-24, D-33, D-35, D-36, D-38

suspect spores D-45

Syria 1-3, 1-4

syringes D-23

T

T. barclayana D-40

T. controversa D-44

T. horrida D-40, D-45

T. laevis D-16, D-44

T. tritici D-16, D-44

T. walkeri D-40, D-45

table surfaces D-19

tables D-17

tap water D-26, D-30, D-32

tape D-7, D-23

tarpaulin B-3

TDY personnel 5-1

TDY rosters 5-1

teasing needle D-14

technicians D-42

teliospore

analyses D-48

contamination D-18

definition of A-3

examination D-16, D-34
Index

mm/yyyy-ee Karnal Bunt Index-9

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

extraction D-2, D-21

gross morphological characteristics D-44

identification D-39, D-42, D-45

introduction of 1-4

kernel conversion 1-3

mass D-14

morphology D-44

overwintering life stage 1-5

positive determination D-40

presence of D-1, D-10, D-13, D-16, D-17

verification D-16, D-40

verifiers D-45

terry wipes D-24, D-27, D-28

test tube rack D-23

Texas 1-4

Tilletia indica

analysis for D-19

as a regulated article 3-2

as causal agent of Karnal bunt 1-3

definition of A-4

determining presence of D-17

examination for D-34

examining isolates of D-42

identification D-14, D-43

identifying wheat kernels infected with D-3

initial infection D-44

life cycle D-44

microscopy log sheet D-40, D-53

morphology D-44

reference slides D-39

spore analysis worksheet D-39, D-40, D-41, D-52

teliospores of D-21, D-39, D-40

verifying presence of D-16

verifying samples for the presence of D-1

timers D-24

towels D-12, D-35

toxicity of pesticides B-1

trading partners 4-1

training

activities D-44

content D-42

procedures D-13

program D-42

records D-42

slides D-46

tray, slide D-35

trays D-19, D-24

Treatment Manual B-1

treatments, approved regulatory 3-6

trimethyl-amine 1-4

triticale 1-3, 1-5, 3-5, 3-6, 3-7

Triticum 1-5

Triticum aestivum 1-3, 1-5

Triticum aestivum X Secale cereale 1-3, 1-5

Triticum durum 1-3, 1-5

truck trailers 3-7

trucks 3-2

tube rack D-21

tubes

centrifuge D-24, D-26, D-31, D-32, D-33

sample D-34

tubs

disinfectant D-24, D-30

self-draining and holding D-24

Tween 20 D-24

U

ultra chlorine bleach 3-5

Ultra Clorox brand regular bleach C-1

United States 1-3, 1-4

United States Department of Agriculture (USDA) 3-

14, D-2

universities 6-1

unregulated areas 3-14

USDA (United States Department of Agriculture) 3-

14, D-2

USDA agencies F-1

utility cart D-19, D-21, D-24, D-26, D-29

V

vacuum D-10

verifying teliospores D-43

vermiculite B-3

vials D-7, D-10, D-12, D-16

vibrating mechanism D-8

visual examination of infected grain D-44

voucher slides D-16, D-37

W

wash basin D-12, D-20

wash bottles D-30, D-31, D-32, D-33

wash, high pressure 3-6

water

deionized D-22, D-24, D-25, D-26

deionized, D-24, D-25

tap D-26, D-30, D-32

watersheds B-2

weighing and measuring samples D-43

weighing area D-21, D-28

wetting agent D-24

wheat

affected by Karnal bunt 1-3

as a host of Karnal bunt 1-5

as a regulated article 3-2

equipment used in harvesting or processing of

3-6, 3-7

floral development 1-5

grain samples D-1

heading 1-5

samples D-46

seed storage 3-5

spike 1-5

spike emergence 1-5

straw 3-9

wildlife B-1, B-2

work area D-35

worksheets D-7, D-8, D-10, D-12, D-13

Y

Ykema, Ron D-2
Index

Index-10 Karnal Bunt mm/yyyy-ee

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z