THE GOLDEN NEMATODE HANDBOOK 15

NONINFESTED FIELD
263 BUSHELS

Ficure 10.—Yields of Green Mountain potatoes per acre, based on a 5-year average.

ture, Cornell University, Department of Plant
Pathology, maintains a laboratory and green-
house facilities at Farmingdale. Approximate-
ly 8 acres of land is available here for field
tests (fig. 10).

Research and Methods

The laboratory at Farmingdale investigates
many phases of the golden nematode problem.
But its primary concern involves (1) investi-
gations into rotation and resistance, biotype
buildup, race determination, and breeding of
potato varieties resistant to the golden nem-
atode, and (2) investigations into chemical
control, which include screening of nema-
tocides, evaluation of promising new nemato-
cides, and determination of techniques and
conditions for optimum nematocide effective-
ness.

The Plant Pest Control Division’s Methods
Improvement Operations works closely with
Cornell scientists in their research program.
The Methods Improvement personnel develop
and improve machinery for applying nemato-
cides to the soil, ways of field-scale testing of
candidate nematocides that show promise for
use in a control program, and ways of field-
seale testing of fumigants under tarpaulin
seal; they also develop regulatory treatments
to free of infestation nursery stock, farm ma-
chinery, burlap bags, and other commodities
so that they may be safely moved.

Research has gone forward with very signi-
ficant progress in various fields. A chemical
treatment program and commercially accept-
able varieties of potatoes resistant to the golden

HEAVILY
INFESTED FIELD
64 BUSHELS

BN-28195

nematode have been the two most valuable
findings in making a breakthrough in solving
this pest problem.

Survey Results

During the first 10 years of the program
the golden nematode seemed to be confined to
western Long Island, mainly Nassau and west-
ern Suffolk Counties. The vast potato acreage
of eastern Long Island remained free of the
nematode until 1950, when several infested
properties in Bridgehampton were found (fig.

. With establishment of systematic surveys
in 1946, the number of properties known to
be infested rose sharply. During the period
1946-52, the number of properties found in-
fested averaged 34 annually, comprising 1,362
acres. Beginning in 1947 real estate interests
bought up much of Nassau County’s agricul-
tural land for houses and by 1956 few potato
fields remained. As farmland became scarce in
Nassau County, builders began to purchase
potato fields in western Suffolk County. Dur-
ing the period 1954-67, the average number of
properties found infested each year dropped
to 7, and the average acreage dropped to about
361 (fig. 12).

Since the golden nematode program began,
387 properties consisting of 17,151 acres, have
been found infested (table 1 and fig. 12). Of
this total about 11,413 acres of infested agri-
cultural land have been permanently removed
from agriculture for real estate development
(table 2). Of the remaining infested acres,
4,389 have received soil treatment and have


32 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

BN-80007

Ficure 29.—Floating material is transferred from
beaker to 80-mesh sieve.

Figure 30.—Flotsam is transferred to each section of
the dish, and water is added for microscopic
examination.

The Golden Nematode Act

In_1948, the Federal Government, through
the Congress of the United States, announced
the Government’s policy for the protection of
the potato and the tomato industries from the
golden nematode should further action become
necessary. The Government’s policy was set

forth in the Golden Nematode Act, Title 7,
U.S. Code, Sec. 150-150g. The act stated, “It
is the policy of the Government of the United
States independently or in cooperation with
State or local governmental agencies and other
public and private organizations, associations
and individuals to eradicate, suppress, control,
and to prevent the spread of the pest.” The
Secretary of Agriculture is empowered, either
independently or in cooperation with States
and other agencies, to make inspections, apply
suppressive measures, enforce quarantine, en-
force restrictions on planting tomatoes and
potatoes, destroy tomatoes and potatoes grow-
ing in soil found infested with or- exposed to
infestation of the golden nematode, and com-
pensate growers in areas infested with or ex-
posed to infestation of the golden nematode
for not planting tomatoes and potatoes or for
losses resulting from destruction of crops. The
mandatory restrictions on planting or destruc-
tion of crops must be supported by similar
State authorizing legislation.

Quarantine

The following are the basic provisions of the

New York State Golden Nematode Quarantine:

1. All lands within the boundary of Nassau

and Suffolk Counties on Long Island,

the towns of Prattsburg and Wheeler

in Steuben County, and the town of

Italy in Yates County are placed under
quarantine.

2. All potatoes grown within the designated
area on “clean” land shall be packaged in
an approved paper bag or other approved
container for movement within the con-
ian United States and Canada (fig.

3. Potatoes grown on lands which become
part of a regulated area after planting
shall be packaged in paper bags or other
approved containers, restricted in move-
ment to approved outlets and moved only
under permit, or washed in approved
manner under supervision, packaged in
paper bags or other approved containers
and shall move only under permit, or shall
be subjected to such procedures and safe-
guards which may be prescribed and
shall move only under permit.

4. No potatoes shall be grown on any land
known to be infested with the golden
nematode or dangerously exposed to in-
festation except where such lands have
received a prescribed treatment and have
been declared safe, following soil sampl-
ing, for growing potatoes.


THE GOLDEN NEMATODE

Background shows normal potato plant
Galt) and oun exposed to heavy rmmatnde
atta

Greatly magnified portion of infested root:

A, Foul fart lcvakiog through root

Byer develeoel
partly developed cyst.

GD, al Ie propreanve cole clianges if
cysts,

F, cyst in the soil.

Gy creas teetion of eyht hiowing eggs and
hatching larvae leaving cyst to enter roots.

)
.

-

Fioure 5



THE GOLDEN NEMATODE HANDBOOK 19

festation was detected by Plant Pest Control
Division personnel who had conducted surveys
in western New York potato-growing counties
in October and November.

Survey Procedures

The undramatic nature of the pest in its
early stages is an aid to its survival. It is very
important that the organism be recognized be-
fore crop damage becomes apparent; there-
fore, it is necessary that a constant alert be
maintained for its presence throughout the
potato- and tomato-producing areas of the
country. Small or isolated infestations may
exist unnoticed, since plant symptoms are not
reliable in the detection of the golden nema-
tode. In the early stages, buildup of populations
is slow, and crop damage may not be notice-
able for several years.

The most reliable method for the detection
of the golden nematode is the collection of
soil samples and the microscopic examination
of the washed residue. Yield losses are quite
evident when the concentration of cysts reach
approximately one billion per acre. However,
the presence of the golden nematode may be
detected before the concentration of the cysts
in the soil reach the level where they will
cause crop loss.

From the records of more than 100 fields of
the many hundreds surveyed on Long Island,
it has been determined that, with the survey
procedures herein outlined, cysts of the golden
nematode may be found on the average when
the concentration is approximately one mil-
lion per acre. Assuming 10 viable cysts were
initially introduced into a field and there was
a tenfold increase per year, it would take ap-
proximately 6 years for a cyst population to
build up to the discovery level. Crop damage
may not become apparent for another 3 years,
during which time the cyst population may
build up to a billion per acre (fig. 8).

From this evidence it can easily be under-
stood that a large number of cysts may exist
in a potato field in which no symptoms are
noticeable on the tops or the tubers. For this
reason nematodes may be found in a field that
has been surveyed with negative results for
several years. This is also the reason that sur-
veys must be conducted continuously and sys-
tematically.

Survey Crew

Soil survey crews normally consist of three
or four men, one of whom is designated as the
crew leader. The number of men assigned to
survey a given property or area will vary with

the district in which they operate and the

scope of such operations. One man can survey

about 25 acres per day; therefore, the crew

may be varied from one to several men, de-

pea upon the size of fields to be surveyed
fig.

Equipment

Each crew is provided with a map of the
area to be surveyed, a supply of No. 12 wet-
strength, fungusproof, double-wall paper bags,
a felt marker or wax marking crayon, a
long-handled (28-inch) pointing trowel for
each member of the crew, wire staplers or tape
for sealing the bags, a bristle brush for clean-
ing shoes and equipment, and forms on which
to record field survey operations (fig. 14).

Field Soil Sampling

Upon arrival at the premises to be surveyed,
the crew leader should look over the property
and determine the boundaries, size, and shape
of the field, and plan how the field will be
surveyed. If the owner or operator has not
been previously contacted, the crew leader
should do so before beginning work.

It is advisable to divide the field into several
small subdivisions or working units. With such
subdivisions, it will be possible to return to a
given unit to pinpoint an infestation should
cysts be found in the sample. They also permit
a systematic survey of the field and give the
laboratory soil samples of proper size for pro-
cessing.

In the initial survey, the field is divided in-
to working units of approximately three-
fourths to one acre each. The survey is usually
conducted in a grid pattern; about a tablespoon
of soil is collected on the end of the trowel
about every eight paces. This procedure is

BN-29989
Ficure 13.—Survey crew collecting soil samples.


Introduction
History and origin of the golden
nematode
Origin
Discovery i in the United States ___

The Genus Heterodera -.-

Key to the mature cysts of the
more common species of
Heterodera __

Type host plants -

Biology
Life history -
Symptoms of infestation _.__-___-
Host plants
Rate of population increase.

The potato and tomato industries __
The potato industry
The tomato industry

Control of the golden nematode in the

United States -_...__.
Research and methods -
Survey results -__-.__--- -
Survey procedures

Survey crew __-

Equipment ----

Field soil sampling

Plant-root examination _

Labeling and recording

Sanitation __- _
Laboratory procedures. -

Soil processing -_-.__...-

Examination of soil samples ____.

Checking specimens for viability -
Regulatory

The golden nematode act _

Quarantine

Approved regulatory treatments

Compensation ---
Chemical control -._._. -

Chemicals to be used -

Equipment needed

Calibration of fumigant meter-

ing systems __ - -

Field operations for shank in-

jection equipment

Decontamination of equipment

Precautions _____
Resistant potato varieties -

The international problem of control - _

vi

Page

e

® cpp

World distribution of the golden
nematode _...-...--.._--_---
Algeria -.--______
Argentina
Austria -
Belgium _-
Bolivia
Canada -
Canary Islands... -
Chile
Czechoslovakia
Denmark _
England and Wales --.-..-.--_--_-
Faeroe Islands -
Finland
France __-
Germany (East).
Germany (West)
Greece __._.
Guernsey __
Iceland
India _-
Ireland .___-
Israel ___-
Italy
Jersey - -
Luxembourg _-.----__-____
Netherlands
Northern Ireland .._.-._..-
Norway -- Woe
Panama -
Peru
Poland __.___.- --
Portugal __- ween eee
Saarland _-.___-
Scotland _-
Spain _ a
Sweden ._-....__-
Switzerland _.___-
Union of Soviet Socialist Republics |
United States
Yugoslavia ____-
Literature cited ____
Appendix I—World potato produc tion
Appendix II—Intensity of infestation _
Appendix III—Soil sampling for
potato root eelworm _-
The United Kingdom method
The Dutch method -
Comparison of the two methods -



30 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

level must be examined more thoroughly to
determine whether treatment was successful.

The viability of the cysts is determined by
placing them in a drop of water on a glass
slide. With the aid of a 90-power dissecting
stereo microscope and a surgical lance or an
iris knife, the cysts are cut open and the eggs
and larvae removed. At times it is necessary
to rupture the eggs to free the larvae. This is
accomplished by placing a cover glass over the
drop of water containing the eggs and apply-
ing just enough pressure on the cover glass
with the eraser end of a pencil to free the
larvae.

In those fields where eradication treatments
have not been applied, the specimens are con-
sidered viable if the internal structure of one
or more larvae is intact and there is no indica-
tion of any breakdown. In fields where eradi-
cation treatments have been applied but the
larval specimen(s) is intact and there is no
indication of breakdown, further checks must
be made to determine that the larva is capable

(N-88212
Ficure 26.—The machine and all equipment is thoroughly washed after processing the last sample.

of movement. As the golden nematode is ex-
ceptionally sluggish, it may at times take from
1 to 24 hours before any movement is noted.

Regulatory

Soon after the golden nematode was discov-
ered in the United States, the New York State
Department of Agriculture and Markets, un-
der the broad coverage of the State’s “Agri-
culture and Markets Law” relating to insect
pests and plant diseases, took action designed
to prevent the spread of the golden nematode.
In 1944, the State enacted a specific quarantine
against the golden nematode. The quarantine
has been modified and amended with changing
conditions, receipt of new information, and
development of treatments (28).

The State’s quarantine was drafted in con-
sultation with USDA’s regulatory officials. The
Plant Pest Control Division has been a full
partner with the State and has cooperated in
the administration and the enforcement of the
quarantine.


Brookville!
ie

ons 50 miles

Commack @St. James
°

Huntington Suffolk County
Station

\SLAND

@ Mt. Sinai

Northville

@ Manorville

LONG ISLAND

[CUINFESTED AREA
[QUARANTINED AREA

Ficure 11

9T

GYNLINOWSV JO LNAWLYVddd ‘s’n “Eos MOOMGNVH AYNALINOSV


Dy} AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

collected, survey pattern—that is, 8 x 8, and so
forth—name of farm operator, mail address,
field location including map designation (if
any), State, county, and the names of the
inspectors that collected the samples (fig. 18).
On the reverse of the survey form a sketeh
should be made of the field, showing the prop-
erty location, definite landmarks, and the loca-
tion in the field from which each sample was
collected.

Sanitation

Every reasonable precaution should be taken
to prevent the spread of this organism. Vehicles
assigned to a survey should not be permitted
to enter any property. They must remain on
highways or thoroughfares, and must be kept
clean at all times (fig. 19). Trowels must be
free of soil collecting recesses and grooves and
should be cleaned when taken from a field.
The inspector should clean his shoes with a

N-88191
Ficure 17.—A sketch should be made of the field to show the property location, land marks, and location in the
field from which each sample was collected.


18

AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

TABLE 1.—New infestation of golden nematode in Nassau, Suffolk, and Steuben Counties, by
years, 1941-67

Nassau County | Suffolk County | Steuben County]
Year [Prop- | Acreage| Prop- [Acreage| Prop-|Acreage] Total | Gross Remarks
erties | infested | erties | infested | erties | infested [properties acres *
1941. 2 115.66] 0 0 0 0 2 116 | First discovered in U.S.
1942 9 541.86 0 0 0 0 9 542 Plant pulling or irregular pat-
1943 8 437.36 | 0 0 0 0 8 437 tern of soil sampling, 1941-45.
1944 5 142.98] 0 0 0 0 5 143
1945 ___ 5 165.88 0 0 0 0 5 166
1946 41 | 1,656.50) 0 0 0 0 41 1,656 | Golden nematode project estab-
lished—systematic ‘soil sam-
pling began.
52 | 2,793.28) 1 30.00 | 0 0 53 | 2,828 ‘| First Suffolk Gounty.
27 | 103466; 6 | 21695] 0 0 33 1,252 | Commack, N.Y.
22 663.00 | 7 350.15 | 0 0 29 1,018
22 660.56 | 6 32:88] 0 0 28 898 | Bridgehampton, N. Y.
16 544.75 | 10 302.80 | 0 0 26 848 | Cutchogue, N.Y.
13 261.12 | 12 790.61 0 0 25 1,052 Mt. Sinai, N. Y.
8 167.43 | 18 989.20 | 0 0 26 1157
3 143.24| 5 266.00 | 0 0 8 409
2 130.00| 3 85.30} 0 0 5 215 | St. James, N. Y.
0 0 3 153.00] 0 0 3 153 | None in Nassau County.
1 7.92) 7 263.70 | 0 0 8 272
2 181.00 4 322.94 0 0 6 504
1 72.00| 11 629.75 | 0 i) 12 702
0 0 14 | 629:39| 0 0 14 629
0 0 io | 827.63| 0 0 10 828
0 0 2 113.00 | 0 0 2 113
0 0 6 304.95 0 0 6 805,
0 0 7 339.62 | 0 0 7 340
1 30.00 4 116.85. 0 0 5 147 Manorville, N. Y.
0 0 10 382.36 | 0 0 10 382
0 0 ne 1 BB 1 55 | First infestation found off Long
Island.
Total | 240 9,749.20 | 146 7,347.08 1 5B 387 17,151

+ Rounded to nearest acre.

TABLE 2.—Infested land at beginning of each year, land found infested during year, land removed
from agriculture by real estate development, land treated and released for potato production,
and agricultural land remaining to be treated, Long Island, 1955-67

Infested land Land found Land removed Land treated | Agricultural land
Year at beginning | infested during from agriculture| and released for remaining to
of each year year by realestate | potato production be treated
development
Gross acres Gross acres Gross acres Gross acres Gross acres
1955 | *12,722.17 6,832.18 0 5,889.99
1956 — 12,722.17 153,00 796.96 10.00 5,236.08
1957 - “| 121875.17 271.62 103.37 0 5,404.28
1958 | 13,146.79 503.94 0 10.61 5,897.61
1959 13,650.73 687.30 691.47 67.32 5,826.12
1960 14,388.03 643.84 876.57 656.31 4,951.53
1961 14/996.32 827.63 107.20 1,275.68 4;381.88
1952 15,809.50 113,00 624,17 783.11 3,087.60
1963 __ 15,922.50 304.95 451.06 511.50 2,429.99
1964 16,227.45 339,62 517.45 360.04 1,892.12
1965 - 16,567.07 146.85 252.00 240.87 1,546.10
1966 | a 16,713.92 382.36 29.00 429.14 1,470.82
1967. 17,096.28 55.00 181.10 44.20 1,350.02
1968 _. 817,151.28 | a ee ne

* Cumulative fig

‘ures for 1941-55. See table 1.
* Includes infestation found in Steuben Co., N. Y.


THE GOLDEN NEMATODE HANDBOOK 31

N-38214

‘
4 —

<4.
oe

N-88215
FicuRE 28.—Laboratory technician with equipment needed for detecting nematode cysts.


PRODUCTION
SEASON 1000 CWTS.
WINTER G 430 16

EARLY SPRING HH 3900 is

Uae spRING [) 24,400 92
EARLY SUMMER 14,000 53
LATE SUMMER MI 34,900 131
FALL BD 194,500 3
TOTAL 286,000

Figures under state nemen indicate the production
of tha date in thevsand hundredeighs and the
percentage of the U. S. crop (1958-62 averoge)

\ak \

NEBRASKA
2438

as7e t
=
°°
SOUTH DAKOTA
x6
on
‘on

Figure 9

MOOHANVH SGOLVWAN N&d’105 AHL

&I


THE GOLDEN NEMATODE HANDBOOK

FicurRE 2.—Numerous immature swollen females on the roots of a potato plant.


28 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

(A) BN-28191, (8) BN-28190, (c) BN-28189, (D) BN-28192

Ficure 24.—(A) Soil sample being placed into soil processing machine. (B) Surface debris is directed to over-
flow spout by hand spray. (C) Top sieves are sprayed directly with hand spray to wash cysts through to the
screen below. (D) Flotation tank is cleaned by turning it upside down and opening the fast overflow valve.



THE GOLDEN NEMATODE HANDBOOK 25

N-38209

FIGURE 21—Storage racks containing soil samples for processing.

Soil Processing

The identity of every soil sample must be
maintained throughout the washing procedure.
It is important that the beaker bears the same
number as the sample and that the technician
makes certain that he is washing the sample
into the proper beaker (fig. 22).

Before a sample is introduced into the soil
processing machine (fig. 23), the slow over-
flow valve (No. 5) is opened. This keeps the
apertures through which the water enters the
flotation tank from being contaminated and
clogged with soil. By the time the paper bag
containing the soil is opened, enough water is in
the flotation tank so that the fast acting over-

flow valve (No. 6) may be opened and the
soil introduced (fig. 24A). The fast valve
serves to roil and stir the soil. This action is
best obtained if the soil is poured in gradually.
The fast valve is closed as soon as material
starts to overflow into the top sieve.
Overflowing is allowed to continue until the
water runs fairly clear or until no floating
material car be seen on the surface. Material
clinging to the sides of the tank is dislodged
and directed through the overflow spout with
the hand spray (No. 14). The spray should be
directed laterally along the surface of the
water so it will not submerge floating particles
(fig. 24B). Material in the top sieve should be
sprayed thoroughly with the hand spray to


2 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

acres. In the United States and Canada, the use
of nematocides has been adopted on a large scale
in an effort to eliminate the golden nematode.
Continuing research will undoubtedly refine
present methods of control. Basic information
being gathered by nematologists will develop

HISTORY AND ORIGIN OF

During Germany’s campaign against sugar-
beet nematodes in 1881, Julius Kuhn observed a
cyst-forming nematode attacking potatoes. He
recorded it as a curiosity—a possible subrace of
Heterodera schachtii (48).

That the nematode was actually a serious
parasite of potatoes was not established, how-
ever, until 19138, when Massee (Scotland) and
Zimmerman (Germany, 1914) proved Kuhn’s
“oddity” to be the real cause of what was then
known as “soil sickness of potatoes” (9).

Within the following decade, the existence of
the pest was discovered in England (1917),
Sweden (1922), and Ireland (1922). Still
thought to be a new species of Heterodera
schachtii, the pest gained the common English
name of “potato root eelworm” (48).

In 1923, however, Wollenweber observed that
“potato larvae are slightly shorter than those
of the sugarbeet nematode, and that their cysts
are pear-shaped rather than lemon-shaped.” De-
claring that this was an entirely different spe-
cies from that attacking sugarbeets, he proposed
the name Heterodera rostochiensis since his
specimens came from Rostock, Germany (48).

Despite Wollenweber’s findings, scientific
journals of the day continued to refer to the
nematode as a race of the sugarbeet nema-
tode (15). It was not until the excellent work
of Franklin, years later, that Heterodera ros-
tochiensis was given general recognition as a
valid species.

The impact of the pest on the world’s potato
industry ? has been spectacular since infested
seed had been spread throughout much of the
world long before Heterodera rostochiensis was
named. Infestations have been discovered in
such widely separated nations as Peru (1952),
aan (1943), India (1961), and Panama

967).

‘Italie numbers in parentheses refer to Literature
Cited, p. 77.

*For information on world potato production, see
Appendix I.

more efficient control measures. Screening of
chemicals for control or eradication is being
stepped up. As more effective and less expensive
nematocides are developed, together with more
efficient methods of applying them, the use of
chemicals for nematode control will undoubtedly
increase.

THE GOLDEN NEMATODE

Origin

For many years it was thought that the
golden nematode originated in Europe. Some
workers suggested that the nematode’s sud-
den appearance was by mutation from a
species of Heterodera, originally a parasite of
some European garden weeds or ornamentals.
Some advanced the theory that it was trans-
ported from an isolated area where the species
may have lived on a sporadically occurring
Solanacaea.

On April 3, 1951, a ship from Peru arrived
at the port of Seattle, Wash. Plant quarantine
inspectors of the USDA making routine in-
spection of the ship for plant pests collected
soil from potatoes in the ship’s stores and
found the golden nematode. On September 25,
1951, a ship from Peru in the New York har-
bor was inspected and golden nematode cysts
were found in the ship’s stores in two different
collections. These specimens were examined by
USDA_nematologists and were confirmed as
being Heterodera rostochiensis. Since this pest
had not been previously reported in Peru, a
careful check was made of the ship’s log to
determine if the ship had taken on stores in
other ports. All evidence pointed to the fact
that the contaminated potatoes had originated
in Peru.

A. L. Taylor of the Nematology Section,
Crops Research Division, ARS, wrote to
C. Bazan de Segura, Centro Nacional de
Investigacion y Experimentacion Agricola de
la Molina, Peru, advising of the finding of the
golden nematode on ships from Peru and sug-
gesting that a search be made in the country
for the presence of the pest. As a result of this
communication the nematode was found in
February 1952 on potatoes from Tarma. A
scientist from ARS, who was in Peru at the
time the discovery was made, brought speci-
mens back to Mr. Taylor in May 1952. Mr.
Taylor confirmed identification as Heterodera
rostochiensis.

The first account of this discovery was pub-
lished in 1952 (36). The author, C. Bazan de
Segura, stated that it was her opinion the
Heterodera rostochiensis is indigenous to the


THE GOLDEN NEMATODE HANDBOOK

Survey, Laboratory, Control,
and Quarantine Procedures

By
Joseph F. Spears, Assistant Director,
Plant Pest Control Division, Agricultural Research Service

INTRODUCTION

The golden nematode is undoubtedly the most
serious pest threatening the American potato
industry. It is recognized throughout the tem-
perate regions of the world as one of the most
difficult of all crop pests to control. It also at-
tacks tomatoes, but it is not a major threat to
this crop.

The golden nematode is the subject of strin-
gent quarantine regulations in most countries
where it occurs. Countries not known to be in-
fested have rigid regulations governing the im-
portation of potatoes or other products that
might carry the pest. Agricultural officials in
countries where the nematode occurs agree that
potatoes cannot be grown economically on land
containing large numbers of the nematode.
Tuber yields of less than seed planted have been
reported from fields heavily infested.

The golden nematode poses many problems
in its control. It has a remarkable ability to sur-
vive under unfavorable conditions. It can re-
main dormant in the soil in the absence of host
plants for many years because the unhatched
nematode is protected by both the shell of the
eggs and the durable cyst wall, which acts as a
protection against adverse conditions. The lack
of any one aboveground symptom to betray its
presence is an aid to its survival. Thus, light or
scattered infestations of the nematode repro-
duce and increase in the soil undetected. During
this time of buildup, infestations may spread to
new fields, and because the populations are low
they will not be detected by the best survey
techniques. This type of spread and slow build-
up of populations is undramatic, and low levels
of infestations are always one step ahead of
survey. Crop reduction as a result is negligible.

The buildup may require 5 to 7 years or more
after introduction of the pest into a field before
noticeable crop injury and reduction in yields
occur. However, buildup of nematode popula-
tion increases like compound interest until the
crop is destroyed.

As the damage caused by plant nematodes is
becoming more widely recognized, general in-
terest in the problem is increasing and a mount-
ing volume of questions are being asked by
growers. Most countries now have nematolo-
gists devoting their efforts to problems caused
by plant nematodes. More and more surveys are
being undertaken in many countries to deter-
mine distribution and identification of plant
nematodes.

Control of the golden nematode in most areas
of the world still depends on crop rotation. How-
ever, scientists have been devoting more and
more attention to the development of resistant
varieties. Resistance-breaking biotypes of the
golden nematode have now been reported from
several countries, and investigators are devot-
ing much attention to the influence this has on
the problem.

The use of nematocides to control the golden
nematode is also being investigated by workers
in several countries. Some European countries
are making limited use of these chemicals. How-
ever, the high cost of chemicals has been a fac-
tor in preventing large-scale use.

In the United States the use of nematocides
is increasing annually for nematode control on
many crops. Over 100 million pounds of nema-
tocides are used annually in this country, prin-
cipally on tobacco, pineapple, and vegetable
soils. Nematocides are applied to about 1 million

1


14 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

Bulgaria, Brazil, Japan,
Mexico, and Argentina.

Greece, Rumania,

The Potato Industry

Potatoes are grown commercially in every
State (fig. 9), and there is not a single month
in the year when potatoes are not being
planted or harvested somewhere in the country
(49). Potato yields per acre have risen from
a national average of 80 hundredweight per
acre in 1940 to 210 cwt. in 1966. It has been
predicted that the national average per-acre
yield may rise to as much as 250 ewt. by 1975.
The total acreage decreased from 2.8 million
in 1940 to about 1.5 million in 1966.

Potatoes have a market value of about $600
to $800 million. When transportation, packag-
ing, and marketing facilities are included,
potatoes are a $3 billion industry. The 10
leading potato-producing States are Idaho,
Maine, California, New York, Washington,
North Dakota, Minnesota, Wisconsin, Colorado,
and Oregon.

As a potato-producing area, Suffolk County,
Long Island, is outranked in the United States
only by Aroostook County, Maine, and Bing-
ham County, Idaho. The potato acreage on the
Island was rapidly expanded during World
War II. It may have been during this expan-
sion that the golden nematode spread from the
original field in Nassau County to eastern
Suffolk County—a distance of over 100 miles.
Potato production on Long Island reached its

peak in 1946, when well over 62,000 acres were
produced. It decreased to about 40,000 acres in
the midsixties.

The yield per acre, which rose rapidly dur-
ing and following World War II, continues to
increase but at a slower rate. In recent years,
yields have averaged 250 to 260 cwt. per acre.
Of this yield, about 235 to 240 cwt. may be
sold; the rest is offgrade or used for home con-
sumption.

Hicksville—once the center of potato pro-
duction in Nassau County—is now the center
of a large suburban area of New York City.
The population of Nassau County has grown
from about 400,000 in 1941 to more than
1,400,000 in 1967. Almost all of the infested
potato fields in Nassau County have become
house lots or industrial sites. The golden nem-
atode, however, continues to threaten the po-
tato industry in Suffolk County.

The Tomato Industry

Tomatoes are widely grown in the United
States for both processing and fresh market.
Most of the tomato crop is field grown. About
275,000 acres of tomatoes are grown for pro-
cessing; the crop value is about $160 million.
Tomatoes for the fresh market are grown on
170,000 acres; the annual value is about $190
million.

The 10 leading tomato-producing States are
California, Ohio, Florida, New Jersey, Indi-
ana, Illinois, Pennsylvania, Maryland, Michi-
gan, and New York.

CONTROL OF THE GOLDEN NEMATODE IN THE UNITED STATES

When the golden nematode was discovered
on Long Island in 1941, Cornell University
and the U.S. Department of Agriculture un-
dertook a cooperative research program, and
the New York State Department of Agricul-
ture and Markets and the USDA conducted
surveys. With the involvement of the United
States in World War II, however, the amount
of attention that could be devoted to the pro-
blem was limited.

By 1944, it had become obvious that the
nematode was much more widespread on Long
Island than it had been thought at first. In that
year, the State invoked a quarantine (33), and
the Bureau of Entomology and Plant Quaran-
tine, USDA, began a survey in 19 potato-pro-
ducing States east of the Mississippi River to
see if the nematode had spread outside Long
Island (10).

Following World War II, a cooperative Fed-
eral-State Golden Nematode Control Project

was established. Research facilities were great-
ly expanded, and the staff to operate the pro-
gram was also expanded. The program was
divided into four main branches: Survey, lab-
oratory processing of samples, quarantine en-
forcement, and research. Systematic soil sur-
veys were begun of all fields on Long Island,
and surveys were expanded to include all po-
tato-producing areas of the United States (fig.
9). Quarantine regulations were strengthened.

The following offices and laboratories have
been set up for the golden nematode program
on Long Island:

(1) A headquarters building at Hicksville
houses the program offices of both the USDA’s
Plant Pest Control Division and the New York
State Department of Agriculture and Markets,
a laboratory, and a storage garage.

(2) A work unit office is maintained at
Riverhead, Long Island.

(3) New York State College of Agricul-


4 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

soil analysis or other explanation of his crop
injury (5).

In July of 1941, Orson S. Cannon, a gradu-
ate student of the Department of Plant
Pathology, Cornell University, attached to the
Nassau County Farm Bureau, Long Island,
visited the potato field and observed that the
stunted and off-color appearance of the pota-
toes was similar to spots in sugarbeet fields
infested with Heterodera schachtii, with
which he was familiar (4). Cannon examined
potato roots from the field and found numer-
ous swollen female nematodes on the roots.
Suspecting that this was the causative organ-
ism, he took specimens to the USDA Bulb
Laboratory in Babylon, Long Island, where
B. G. Chitwood, USDA nematologist, identi-
fied the specimens as Heterodera rostochiensis
(28). Subsequent examination of the property
showed that the entire field was infested and
crop losses were as high as 70 percent; in an
adjoining field, marked injury was apparent

11).

Available information on the distribution of

the pest indicates that all Long Island infesta-
tions may have originated from this 40-acre
field. The reuse of burlap bags for picking
potatoes and the movement of farm machinery
by renters were major factors in the local
spread of the disease. More than 30 additional
fields farmed by the operator of the original
infested field have since been found infested.
How and when the nematode gained entrance
into the United States is not known. However,
it could have been brought on military equip-
ment being returned to Long Island military
camps after World War I. From the extent of
the infestation in 1941 and the history of the
field, the nematode had probably been present
for 30 years before it was identified (13).

H. rostochiensis is usually called the potato
root eelworm in most European countries.
However, to avoid confusion with other potato
nematodes in the United States, Chitwood
gave the pest its popular name of “golden
nematode” (7) because the flask-shaped imma-
ture females attached to the roots become
golden in color about the time the plants begin
to bloom (fig. 1).

THE GENUS HETERODERA’

The golden nematode (Heterodera rostochi-
ensis Wollenweber, 1923) (51) belongs in the
“rostochiensis group” of the genus Heterodera
Schmidt, 1871 (35). This group is typified par-
ticularly by the globular to ovate shape of the
white females and cysts, and the character of
the cyst wall pattern (fig. 2). Specific identi-
fication is based primarily on vulva-anal size
and relationship, and to some extent, on
morphological characters of the females and
males, if these are available (fig. 3).

When H. rostochiensis nematode was first
discovered in this country about 25 years ago,
there was in the group only one other known
and described species, H. punctata Thorne,
1928 (46). Since then several very closely re-
lated species have been found, and their accu-
rate identification requires the efforts of
someone thoroughly familiar with all the
species.

In 1958, Cobb and Taylor (16) described
H. leptonepia found in potatoes obtained from
a ship’s stores at Callao, Peru. This form is
distinctive especially because of the strikingly
slender larvae. Shortly after that, another
species, H. tabacum Lownsbery and Lowns-
bery, 1954 (25), from tobacco in Connecticut
was described. In 1959, a Heterodera, still un-
described and intermediate between H. rosto-

*Prepared by M. Golden, nematologist, Crops Re-
search Division, ARS.

chiensis and H. tabacum was found on horse-
nettle, Solanum carolinense, in Virginia; it is
known to occur in other areas also. In 1961,
another Heterodera in the rostochiensis group
was found on tobacco in Virginia. These forms
have been referred to, respectively, as the
“horsenettle-cyst nematode” and the “Osborne
cyst nematode” (29). The latter is closely re-
lated, and possibly identical, to H. tabacum.

Knowing of the above closely related nema-
todes and keeping in mind the very probable
existence of still undiscovered new species in
the “rostochiensis group,” one should be very
cautious and thorough before concluding that
any globular cysts that might be associated
with potatoes are actually the golden nema-
tode.

Key to the Mature Cysts of the More
Common Species of Heterodera‘

This key is designed to facilitate identifica-
tion of the more common species of Heterodera
that might be encountered in soil samples col-
lected during a survey. Only characters of the
mature cysts and their contents—that is, eggs
with second stage larvae—are used. Certain
characters used in the key may not be visible
on other than fully mature cysts.

“Prepared by A. L. Taylor, nematologist, Crops Re-
search Division, ARS. (Retired in 1967.)


20 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

commonly referred to as the 8 x 8 block
method. By this method, one composite sample
weighing 4 to 6 pounds will be collected from
every three-fourths to one acre of soil inspected.
If a more intensive survey is desired, soil is
collected on a 4 x 4 pace interval. By this
system, four times the volume of soil will be
collected as by the 8 x 8 pace method.

SPIRAL
NOTEBOOK

3 TROWELS

For a still more intensive survey, soil may
be collected on a 2 Xx 2 pace interval. By this
procedure, 16 times the volume of soil will be
collected as ‘by the 8 x 8 pace method. The
4 x 4 and 2 x 2 pace methods of survey are
generally used for pinpointing an infestation
following the initial discovery by the 8 x 8
pace method or for collecting samples for vi-
ability purposes following field soil fumigation.

3 FELT
MARKERS

CLIPBOARD

Figure 14.—Soil survey equipment: Notebook; pointing trowels; bristle brush for cleaning shoes and equip-
ment; tape for sealing bags; felt markers; map of area to be surveyed; No. 12 wet-strength fungusproof,

double-wall bags; and record forms,


THE GOLDEN NEMATODE HANDBOOK
Survey, Laboratory, Control,
and Quarantine Procedures

By

Joseph F. Spears, Assistant Director,
Plant Pest Control Division, Agricultural Research Service

eer
BBug 5)

SMOG WINS

AU VHaTT

>
Sunt uOIR,

MSY
TAS 39 tag NOUN
an)

Agriculture Handbook No. 353

UNITED STATES DEPARTMENT OF AGRICULTURE
Agricultural Research Service

Washington, D.C. Issued September 1968

For sale by the Superintendent of Documents, U.S. Government Printing Office,
Washington, D.C, 20402—Price $1.25


THE GOLDEN NEMATODE HANDBOOK qT

Measurements of larvae are from Fenwick
and Franklin (18) for most species, from Jones
(22) for H. carotae, from Ichinohe (21)
for H. glycines, and from Kirjanova (23) for
H, fici.

1. Body of cyst, ovoid to globular, that is,
with posterior portion rounded and
vulva not located on a distinct protub-
erance

Heterodera rostochiensis group 4

Body of cyst, lemon-shaped, that is, with
vulva located on a distinct protuber-
ance

2. Basic element of pattern of outer layer of
cyst wall at middle portion of cyst,
short zigzag lines with little or no trace
of regular transverse arrangement;
sometimes modified to appear as net-
work ___ 3

Basie element of | pattern at outer layer of
cyst wall at middle portion of cyst,
straight or wavy lines at right angles
to axis of cyst; sometimes broken by
short oblique or vertical lines; outer
layer of cyst may have grainy appear-
ance ______ Heterodera cacti group 7

8. Mature cysts with dark bodies (brown
knobs) and often sheaf-shaped object
(lining of vagina) at posterior end. On
immature cysts, these are seldom visi-
ble, and then do not appear dark _____

Heterodera schachtii group 8

Mature cysts without brown knobs or
sheaf-shaped object at posterior end

Heterodera gottingiana group 11

4, H. rostochiensis group. Cyst often ovoid,
anus located at a transparent spot on
cyst so that anal and vulvar openings
appear to be about the same size when
seen by transmitted light. Hyaline por-
tion of larval tail much longer than
stylet ____-.--- Heterodera punctata

Cyst ovoid to globular; anal opening ap-
pears much smaller than vulva opening.
Hyaline portion of larval tail about the
same length as stylet _--____..-___- 5

5. Larvae very slender; length about 39
times greatest width; orifice of dorsal
oesophageal gland about two-thirds
stylet length posterior to stylet knobs

Heterodera leptonepia.

Length of larvae about 22 times greatest
width; orifice of dorsal oesophageal
gland about one-fourth stylet Tength
posterior to stylet

6. Distance between vulva and anus rreen
two and one-half times diameter of
vulva ---___ Heterodera rostochiensis

Distance between anus and vulva about
one and one-half times diameter of
vulva _----- Heterodera tabacum

7. H. cacti group. Hyaline portion of larval
tail about as long as stylet; stylet knobs
concave anteriorly __Heterodera weissi

Hyaline portion of larval tail usually
shorter than stylet; stylet knobs convex
anteriorly _________. Heterodera cacti

8. H. schachtii group. Cysts always with dis-
tinct, coarse punctation consisting of
dots of uniform size but not in rows;
brown knobs closely clustered around
vulva. Hyaline portion of larval tail at
least one and one half times longer than
stylet ______.___._Heterodera’ avenue

Cyst with or without punctation, fine and
mostly in rows; brown knobs not closely
clustered around vulva. Hyaline portion
of larval tail about as long as stylet _.9

9. Average length of larvae:
480 microns _
about 460 micr

10. Average length of larvae:
484 microns _.____Heterodera glycines
502 microns Heterodera trifolii
518 microns _
Heterodera schachtii galeopsidis.

11. H. gottingiana group.
Average length of larvae:
414 microns ___Heterodera cruciferae
454 microns ___..._Heterodera carotae
474 microns _. Heterodera gottingiana
405 microns _ __Heterodera_humuli
406 microns ___ Heterodera fici

Type Host Plants °

Many lists of host plants of Heterodera
species have been published, but it seems pro-
bable that many of these are inaccurate in that
they include plants that are not hosts of the
species discussed. This is true especially of the
older lists and of those based on information
compiled from the literature rather than from
host tests. To avoid this particular error, the
following list of host plants includes only the
type host of each species and an indication of
the other plants that it attacks, so far as
there is general agreement or information on
host tests available. That is, the list is not in-
tended to be complete, but I believe it is ac-
curate so far as what is included is concerned.

° Prepared by A. L. Taylor, nematologist, Crops Re-
search Division, ARS, (Retired in 1967)


6 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

BN 28041

FiGuRE 3.—The golden nematode of potatoes, Heterodera rostochiensis Wollenweber, 1923: (A) Infective larva,
hatched from egg; (B) head of infective larva: (C) adult male; (D) head of adult male; (E) young adult
female; (F) section of cyst wall (7).


THE GOLDEN NEMATODE HANDBOOK 27

SOIL WASHER ASSEMBLY

TOP VIEW

SINK

REMOVABLE WASHER
FLOTATION TANK

SPOUT

SLOW OVERFLOW, VALVE 1
FAST OVERFLOW, VALVE 2
ADJUSTING VALVE
20-MESH SIEVE

60-MESH SIEVE

DRAINAGE BASIN AND SPOUT
HOSE FITTING

SPLASH SHIELD

SIEVE RECEPTACLE

HAND SPRAY

FPENROWSNOATPWN >

LEFT SIDE VIEW FRONT VIEW

Ficure 23.—Soil washing machine.


26 AGRICULTURE HANDBOOK 353, U.S, DEPARTMENT OF AGRICULTURE

N-B8210

Ficure 22.—Soil samples are washed and residues
placed in beakers, Identity of every soil sample must
be maintained throughout the laboratory processing.

wash cysts through to the screen below (fig.
24C). Tests have shown that cysts tend to be-
come trapped in the coarse material in the
top sieve.

When the flotation tank is free of floating
material, it is emptied and cleaned by turning
it upside down and opening the fast overflow
valve (fig. 24D). The tank is then allowed to
pivot back to its normal position and the spout
is washed outward with the hand spray. Be
sure the tank is washed clean.

Again, the material in the top screen is
washed, using full force on the hand spray.
The screen is then held over the sink and all
surfaces washed, inside and out, with the hand
spray. Using medium force from the hand
spray, the material in the bottom sieve is
washed and then transferred to a 600-ml.
beaker until the beaker is about three-fourths
full (fig. 25A). This sieve is washed and
sprayed in the same manner as the top sieve
(fig. 25B). After each screen is washed, its
holding receptacle should be sprayed clean. The
shelf holding the beaker should also be sprayed
clean after each transfer of material to a
beaker.

The machine is now ready for the next
sample (fig. 26).

Examination of Soil Samples

The soil samples are examined to recover,
for identification, nematode cysts that may be
present (fig. 27). The equipment needed in-
cludes a microscope (binocular with 15 x
magnification), small 80-mesh sieve (about the
size of a muffin tin), examining dishes, scalpel,
forceps, needle, 50-ml. bulb syringes, 600- and
250-ml. beakers, 500-ml. metric bottles, and a
lamp (fig. 28).

Floating material, which has been transfer-
red into 600-ml. beakers by the washers, is
poured into the small 80-mesh sieve (fig. 29).
While the material is being poured, the
beaker is rotated to clear the sides of adher-
ing material. A scalpel is used for transfer-
ring this material into the examining dishes.
The correct amount of material per dish is
about one-half teaspoonful. Using the bulb sy-
ringe, particles adhering to the scalpel and
sieve are rinsed into one of the dishes. Suf-
ficient water is added to float the material
near the brim of each dish. A probing needle
should be held in the free hand for use in
spreading clumps of material and for closer
inspection of cystlike objects.

A standard Syracuse dish may be used for
examining screened flotsam; however, a spe-
cial plastic dish developed by personnel at the
golden nematode laboratory is better (fig. 30).
The dish is divided into three sections. Each
section of the dish is the width of the view
through the examiner’s binocular microscope.

Upon completion of the examination, all
equipment is thoroughly washed to avoid con-
taminating the next sample. Suspect cysts are
transferred to frosted dishes or vials and ident-
ified with collection and sample numbers.
Cysts, or objects suspected of being cysts,
should be given to the supervisor. All 80-mesh
sieves that have contained positive material
must be examined under the microscope before
they are used for another sample (fig. 31).

Checking Specimens for Viability

It is necessary to determine whether golden
nematode cysts were viable at the time of col-
lection, since quarantine and control action
may depend upon such determination. Except
for specimens originating in fields fumigated
at the level recommended for eradication, the
viability of the cysts is based on the appearance
of the larvae within. Cysts recovered from
areas that were fumigated at the approved


THE GOLDEN NEMATODE HANDBOOK 3

Andes, Later investigations conducted by
Peruvian scientists determined that the nema-
tode was widely distributed and was of an
ancient origin.

Later, the nematode was discovered in the
highlands of Bolivia and Argentina, where it
is believed to be indigenous also.

The theory basing Heterodera rostochiensis
origin in Peru is accepted by most nematol-
ogists today. Also, most botanists agree that
the potato originated in Peru. Thus, one might
logically ask why the golden nematode did not
become a problem to European potato grow-
ers much earlier.

Potato tubers from South America had been
well-distributed in Europe by early 1600. No
doubt cysts of Heterodera rostochiensis were
also distributed with these tubers, but under
primitive cultural methods buildup was slow.
By 1700 the potato had become a vital part of
the basic food supply in Ireland, Scotland,
caeaaly, and other western European coun-

ies.

However, little was done by agricultural
scientists to improve the potato. Their interest
was generally for its botanical characteristics.
These earlier horticulturists did not seriously
influence potato production or improvement
of the few varieties that existed during this
period. There was no serious attempt to de-
velop new potato varieties until after the crisis
in Ireland caused by late blight (1845). The
potato famine that resulted gave the necessary
stimulus to a new era of potato breeding,
which led to commercial potato production.

From the mid-1800's to the end of that cen-
tury there were revolutionary changes in po-
tato growing. Plant breeders for the first time
were guided by scientific principles of hered-
ity and immunity in crop improvement.
Between 1856 and 1876, large numbers of dis-
tinct varieties were produced and consumption
of the potato in the people’s everyday diet
increased. It was probably during this period
of intensified growing of potatoes and the dis-
tribution of seed for the establishment of
commercial potato production that Heterodera
rostochiensis was able to gain a foothold in
Europe's potato fields.

Discovery in the United States

A farmer near Hicksville, Long Island, N.Y.,
was little concerned in 1934 when he noticed a
few isolated spots in his potato field where
vines were stunted and off color. Within 4
years, however, the spots had multiplied and
commercial losses to his potato crop were be-
ing sustained. The farmer made repeated re-
quests to his county farm bureau agent for

Ficure 1.—Mature females attached to the roots be-
come golden in color, hence the popular name
“golden nematode” in the United States.


THE GOLDEN NEMATODE HANDBOOK 17

been released for agricultural use, thus leav-
ing 1,850 acres of agricultural land to be
treated.

The following tabulation shows the status
on January 1, 1968, of the infested land re-
maining to be treated:

Land use Acres
Vegetable crops ...-.--._------- BTT.TA
Potato land (Steuben County) 55.00
Cultivated sod 281.74
Pasture 108.87
Pending development (for real estate
purposes) —_- wee eens 326.67
Total 1,350.02

Early regulatory action and the strict en-
forcement of the State quarantine has played
a large part in restraining the spread of the
golden nematode. The withholding of potatoes
and tomatoes from infested land has been the

most important single thing done to control
the spread. Fortunately, the infested fields
had low nematode populations, and the fields
were on an island.

However, administrators of the quarantine
had recognized, before discovery of the golden
nematode on Long Island or the enactment of
strict quarantine regulations, that the pest
could be transported to other potato-growing
areas,

During the early 1940’s a few Long Island
farmers either moved to western New York or
operated farms both in western New York and
on Long Island. Because of the operational ex-
posure of these farms, surveys are made an-
nually in western New York.

On December 2, 1967, the golden nematode
was found on a 55-aere field in Steuben County,
N.Y., near the town of Prattsburg. The in-

ACRES INFESTED PROPERTIES
3,000 60
i Nassau County 1
2,000 H 1 *“—— Acres — 40
1
r Ne Properties 4

1,000

Suffolk County

— 20

Lilo

1940 1950

1970

Ficure 12.—Results of Long Island nematode survey. Number of properties and gross acreage infested, Nassau
and Suffolk Counties, 1941-66.


34

10.

AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

Top soil and sod shall be moved under
permit only to designated locations in
accordance with an agreement and under
permit.

Used farm equipment, construction,
equipment, used containers or any other
articles that may be contaminated shall
not be moved from the area until they
have been disinfested in accordance with
prescribed procedures and a permit is-
sued.

FicurE 32.—All potatoes on Long Island are shipped in nonreusable paper bags.

Approved Regulatory Treatments

Portable steam generators mounted on pick-
up trucks are used for cleaning farm machin-
ery. Methyl bromide may be used to fumigate
equipment, burlap bags, tools, crates, or other
contaminated materials. The following treat-
ment schedules have been worked out for free-
ing materials and commodities from the golden
nematode.

st 617-4


THE GOLDEN NEMATODE HANDBOOK

35

Temperature Exposure

Type, of enclosure or CF.) of
material to be treated enclosure or
material

Chamber or tarpaulins _... 60° or above

Chamber or tarpaulins -.- 60° or above

Vacuum chamber (27 inches
sustained vacuum).

Gastight chamber (less than
10 cubie feet).

Gastight chamber (100
cubic feet or more).

Gastight chamber (100
cubic feet or more).

60° or above
60° or above
60° or above

60° or above

Dry oven (preheat) - 221°
Dry oven (preheat) ...... 287°
Greenhouse benches or 180°

large containers of soil.

The following dips have been developed for
treatment of bulbs and corms.

Commodity

Gladiolus corms, hy-
acinth, iris, or tulip
bulbs, and narcissus
bulbs.

Lily-of-the-valley pips

Treatment

L-hour dip in L-pet. aqueous
solution of Aaventa 46N or
0.5 pet. for 3 hr. or 0.25 pet.
for 6 hr.!

Separate bundles one from an-
other just before treatment
begins. Without preliminary
warmup, submerse in hot
water at 118° F. for 30 min.;
follow with 5-min. drain, an
finish with 5-min, cooling dip
or hosing in tapwater.

Wash free of soil, then sub-
merge for at least 5 min. in
water maintained at a tem-
perature of not less than

Potato tubers

1 Request for registration has not been presented in U.S.A,

Compensation

To aid growers whose income was drastical-
ly cut because infestations were found on their
farms, the State of New York enacted legisla-
tion in 1946 to compensate them for withhold-
ing their lands from potato and tomato pro-
duction.

Funds for this purpose were provided solely
by the State of New York until 1948 when the
Golden Nematode Act was passed by the 80th
Congress. This act authorized Federal parti-
cipation in an owner-operator compensation
program. In 1948, Federal and State funds
were made available for payments to owner-

period
(Ar.)
Treatment
16 28 Ib. methyl bromide per 1,000
cubic feet.
2 46 lb. methyl bromide per 1,000
cubic feet.
16 8 Ib. methyl bromide per 1,000
cubic feet.
3 1 Ib, methyl bromide per 1,000
cubic feet.
3 4.6 Ib. methyl bromide per 100
cubic feet.
16 2.3 Ib. methyl bromide per 100
cubic feet.
1 Dry heat.
% Dry heat.
1 Live steam injected through-

out soil mass,

operators at the rate of $150 per acre, each
cooperator paying $75 per acre. Under an
agreement, the State assumed full payment to
renters on the basis of a fair rental value as
determined by the Commissioner of the New
York State Department of Agriculture and
Markets.

In 1950, payments to owner-operators were
reduced to $120 per acre, each cooperator pay-
ing $60. In 1951, payments were reduced to $80
per acre, each cooperator paying $40. In 1952
and 1953, the payments were $60 per acre,
Federal and State Governments each paying
$80. In 1958, the State of New York and the
U.S. Department of Agriculture agreed that
the Federal Government would withdraw from
the compensation program but would increase
its participation in surveys and,certain regula-
tory activities. Beginning in 1954, the State
of New York assumed the total cost of the
compensation program, paying owner-opera-
tors of infested land $60 per acre.

The compensation program provided a very
useful means whereby farmers were able to
adjust farm operations. Growers were en-
couraged to plant their infested potato land
to pasture, small grain, or field corn. The grow-
ing of these crops entitled the grower to re-
ceive compensation; however, if he elected to
grow high-value crops, such as vegetables,
compensation payments were not made.

With the advent of the field soil fumigation
program in 1955 and the returning of potato
land to production, the need to compensate
growers no longer existed and the amount of
compensation paid to growers decreased to a
very low level.


Pre gaye

U.S. Department of Agriculture A.

.R.S. Plant Pest Control Division

00 NOT WRITE IN THIS SPACE

CYST NEMATODE SURVEY

couwes [2] sowseC—] omen

bare (3/6, suse xo. 232 B |
aap pestonatron $2- W- 7 Far operator LO4U POE
cousrnon wo, 2AL__77E [oon inne 10 WEST STORCH,

Mz, COB WEST, 57, CEUEAL AVE)
ACRES SURVEYED 25 FIELD LOCATION LUKALTON.
NO. SAMPLES COLLECTED 4 stare LA” county ASSAY
SURVEY PATTERN Bx inspectors EMMETT - JONES

Ini tialf7Confirmatory] delimit:

ine]

prant [__] nursery [__]  cropranp [__]

KIND OF SURVEY

SOIL GREENHOUSE OTHER

Foartive | W,.0F | GENTIFIGATION NO OF
save wa. Srsr" | QSSES SViacs LED

Posirive | NO, OF | 1DENTIFICATION NO.OF
SAMPLE No.| cysts. | SLIDES @ VIALS FILED

3 l lA M720 H. Ros.
aia SS e¥878 - Vide 12

Ale

SAUPLES ARE 4BK72

PACES ACART UULESS
OTHECUL8E SHOWN

E

LUCAL Ai

[SgSa5" 8 5 OO

OUNERS
RESIDES
# FARM BUILDINGS

ap. fa.
petermineo py “C7 Geely

DATE _/Z, CHA

Ficure 18.—(Left) Front of Plant Pest Control form PPC 3-12 used for recordkeeping in nematode survey. (Right) Back of form PPC 3-12,

ype op cnor(s) on propery __25 ACKES OF POTATOES

ESTIMATED ACREAGE OF EACH

STATUS OF FIELD AT TIME OF SURVEY.
REMARKS

HARVESTED

showing how form is used to identify samples collected from a farm.

MOOMGNVH AGOLVWON Ndd'109 GHL

&@


PREFACE

This handbook has been prepared to meet the needs of agricultural workers
interested in the protection of crops from nematode outbreaks. Most of the
procedures set forth in this handbook are the outgrowth of research conduct-
ed by the New York State College of Agriculture, Cornell University, and the
Agricultural Research Service (ARS) of the U.S. Department of Agriculture
(USDA). These methods and procedures have been successfully used for sev-
eral years. Through the efforts of the scientists working on this problem these
methods are constantly being improved.

Many people have contributed in various ways to make this handbook pos-
sible. The author is sincerely grateful to all of them. Special acknowledgment
is made to personnel of the New York State Department of Agriculture and
Markets; the Department of Plant Pathology, Cornell University; the Nema-
tology Section of the Crops Research Division and the Plant Pest Control Di-
vision, ARS, USDA; and the Foreign Agricultural Service, USDA.


10 AGRICULTURE HANDBOOK 353, U.S, DEPARTMENT OF AGRICULTURE

Symptoms of Infestation

Soil populations of the golden nematode suf-
ficiently high to cause plant damage are pro-
duced only after buildup on several potato
crops. At first there is no aboveground evi-
dence of plant damage. This lack of distinguish-
able symptom of the early stages is an aid to
the organism. After host crops have been _re-
peatedly grown on infested land, then distinct
aboveground signs of damage begin to appear
(40) (fig. 6). .

The first sign is poor growth of plants in
small spots in the field. These spots are similar
to that of plants in low spots in wet years and
high spots during dry years. No one plant
symptom alone can be relied upon as evidence
of the disease. If the infestation grows, the
spots become larger and additional spots ap-
pear (27).

Aboveground symptoms appear only in the
presence of large numbers of nematodes in the
soil. Heavy infestations cause considerable
wilting of plants, especially during midday,
stunting of growth, poor root development, and
poor plant growth. Before enough nematodes
develop in the soil to cause aboveground symp-
toms, immature females may be seen on the
roots. By carefully removing the plants and
roots at blossom time, the white, yellow, or

BN 30006
Ficure 6,—Aboveground symptoms of golden nematode damage.

golden flask-shaped females attached to the
root may be seen with the unaided eye (40).

Populations of the golden nematode on Long
Island are so low that aboveground symptoms
seldom occur except in the experimental area
of the golden nematode laboratory. In this area
symptoms of heavily infested plants are gen-
erally evident the latter part of May and early
June. Extensive soil surveys detect infestations
as soon as they reach the discovery level.

The effect of the golden nematode on toma-
toes is similar to that on potatoes. Growth of
plants is retarded and wilting is particularly
noticeable (fig. 7). Leaves of heavily infested
plants are purplish, instead of yellowing as in
potatoes, and the lower leaves may die (38,
p. 178).

The nematode in the early stages of the at-
tack on tomatoes causes slight swelling of the
roots, which may be confused with galling
caused by the root-knot nematode (38, p 178).

Host Plants

The only commercial crops known to be at-
tacked by the golden nematode are Solanum
tuberosum (potatoes), Lycopersicon esculentum
(tomatoes), and S, melongena (eggplant)
(27).


12
VIABLE CYSTS PER ACRE

AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

10,000,000,000
CONTINUAL CROP FAILURES
1,000,000,000 +
CROP SEVERELY DAMAGED
100,000,000 +
DAMAGE APPEARING IN SPOTS
10,000,000
[_ DETECTABLE LEVEL BY
1,000,000 SOIL SAMPLING yf
|
100,000 |- VIRTUALLY _ @ |
INDETECTABLE Y | |
10,000 | e | |
| 1
1,000 |- e | /
! 4
al NO PLANT SYMPTOMS
' 4
Poe
0- @ | I
| 1
1 Lae I { ! !
0 ou 2 3 4 5 6 7 8 9 10
YEAR

Ficure 8.—Increase in golden nematode populations in a continuous potato culture, assuming a tenfold increase.

formed. Research workers in the Netherlands
have, in general, agreed with USDA findings.
In the Government-enforced rotation program,
the Netherlands has assumed, for practical pur-
poses, a tenfold increase of the nematode on a

crop of potatoes and a decrease of 50 percent
in the absence of a potato crop.

Figure 8 shows graphically an increase of
golden nematode infestation in a continuous
potato culture assuming a tenfold increase (44).

THE POTATO AND TOMATO INDUSTRIES

The economic potential of the golden nema-
tode, if left uncontrolled, can have serious ef-
fects on the potato industry. Potatoes are a
major food crop in many countries.

Potato production in 1966 in the principal
producing countries, estimated at 5,100 million
hundredweight (100 Ib.), was 1 percent above
the 1965 crop but 2 percent below the 1960-64
average. Total acreage, estimated at 44 million
acres, continued its gradual downward trend,
whereas yields in general increased. Higher
yields more than offset the effect of decreased
acreage.

Tomatoes are not as widely grown as pota-
toes; however, they are an important crop for
both the fresh market and for processing.
Tomato production in 1965 in the principal
producing countries of the world is estimated
at 420 million hundredweight produced on
about 2.5 million acres.

Europe leads the world in tomato produc-
tion, followed by North America, the Near
East, Latin America, the Far East, Africa,
and Oceania. The 10 leading tomato-producing
countries are United States, Italy, Spain,


Trade names are used in this publication solely to provide specific information. Mention
of a trade name does not constitute a guarantee or warranty of the product by the U.S.
Department of Agriculture or an endorsement by the Department over other products not
mentioned.


THE GOLDEN NEMATODE HANDBOOK 33

BN-28899

Figure 31.—A microscopic view of golden nematode cysts in flotsam.

The golden nematode in any state of de-
velopment may not be moved or trans-
ported except as authorized.

There shall be no movement of hay,
straw, or plant litter from regulated area
except in accordance with an agreement
and under permit.

Vegetable root crops, transplants, nurs-

ery stock, bulbs, corms, and tubers are
all subject to appropriate sanitation reg-
ulations or treatment when grown on
land infested or dangerously exposed to
infestation.

Potato grading stations shall be operated

* under permit, which provides for the safe

disposal of grader dirt and other waste.


THE GOLDEN NEMATODE HANDBOOK 21

N-9204
Ficure 15.—Plant root examination for nematode cysts.

Plant-Root Examination

The following method of examining potato
roots for nematodes may be used to advantage
under certain conditions. The best time is
while the nematodes are in the white or orange
stage. On Long Island this is about the last 2
weeks in June. The cysts are generally in this
stage when the potatoes begin to blossom.
Fields are looked over carefully, and patches
showing plants with weak, spindly stems and
stunted tops are selected. Plant-root examina-
tion should also be made around buildings or
where grading debris has been disposed of. The
plant is carefully removed from the ground
with a pointing trowel. The roots are separ-
ated from the soil, but no attempt is made to
remove soil that is clinging to individual roots,
as unnecessary handling causes the nematodes
to fall from the roots (fig. 15). With the aid of
a hand lens, the root system is examined care-
fully for female cysts. Specimens collected in
this manner are placed in vials containing 15
percent formalin or equal parts of rubbing
alcohol and water, labeled, and sent to the
laboratory for determination.

Labeling and Recording

It is very important that samples be pro-
perly labeled and adequate records prepared
to clearly identify the samples from each pro-
perty inspected. Each sample should be la-
beled in such a manner that all information is
visible after the bag has been sealed (fig. 16).

The inspector’s collection number, which con-
sists of his initials followed by a number, is
recorded on the top of the sealed bag. For ex-
ample, the first collection made on a survey in
the calendar year is ‘No. 1. Each collection
thereafter, regardless of the State or county,
is numbered in a series for the same calendar
year. Thus, the inspector’s first collection ap-
pears as “RAL-1,” his second collection “RAL—
2,” and so on throughout the year. The name
of the farmer, the map designation, or the
field number is placed on the second line. The
date is recorded in the lower right-hand corner
of the bag. As the samples or bags of soil are
collected in a given field, they are also num-
bered in a series beginning with No. 1. On the
last sample in a series from a particular farm,
the notation “End” is made following the
sample number.

The collected soil sample is only as good as
the records kept of its origin. Thus, in addi-
tion to labeling the collected sample another
important step in any survey is good record-
keeping (fig. 17). If the collected sample is
found in the laboratory to contain golden nem-
atode cysts, one should be able to return to
the location in the field from which the
sample came. A field survey form should be
kept and it should include all necessary details,
such as the date sample was collected, collection
number, acres surveyed, number of samples

N-88185
FIGURE 16.—An important job of the survey crew is
to see that bags are properly labeled.


FOREWORD

The history of nematology in the United States is characterized by its rapid
growth and by general recognition by growers, industry, and agricultural
workers of the importance of nematodes in limiting crop production.

The first regulatory activity against nematodes was in 1926 when the U.S.
Department of Agriculture’s Bureau of Entomology and Plant Quarantine
imposed regulations against the bulb and stem nematode. Today, several hun-
dred Federal and State people are engaged in regulatory nematology and are
spending about $4.5 million a year on surveys, enforcement of quarantine reg-
ulations, and the application of nematocides.

Before World War II, about 35 people were engaged in nematological re-
search, and the annual appropriation was less than $150,000. Today, at least
175 persons are working full or part-time on nematological research, and the
annual expenditure is about $4 million.

The discovery of the nematocidal properties of dichloropropene-dichloropro-
pane in 1943 marked the beginning of the present soil fumigation industry.
For the first time farmers and nurserymen had a practical and economical
means of controlling nematodes. About the time of this discovery the golden
nematode was found on Long Island (1941). Although a handful of nematolo-
gists in the United States had been working on the nematode problem for a
number of years, the discovery of the golden nematode brought the problem
forcibly to public attention.

In 1958, nematologists discovered that the burrowing nematode, Radopholus
similis, caused spreading decline of citrus, and in 1954, they discovered that
the soybean cyst nematode, Heterodera glycines, was causing economic losses
to soybeans.

After the discoveries in 1941, 1953, and 1954, survey, regulatory, and con-
trol programs were initiated and special methods were developed for’ large-
scale control programs.

Regulatory measures and the laboratory and field techniques used to carry
out the program against the golden nematode are described in this hand-
book. Most of the methods are suitable for use on a large scale and are appli-
cable to most other cyst nematodes. No other publication includes such a com-
prehensive treatment of a nematode control program. The information will
be valuable not only to those conducting regulatory and control programs in-
volving the golden nematode, but also to agricultural administrators, plant
protection officials, and students in many other areas of agriculture.

Joseph F. Spears is well qualified to write this handbook. He was one of the
organizers of the program. He pioneered in the development of many of the
program operations and has been associated with the work in some capacity
for more than 20 years. He initiated the present chemical control program. In
addition, he has had broad experience with large-scale plant protection pro-
grams involving other nematodes and other organisms, such as insects and
fungi.

William F. Mai, Ph.D.
Professor

Department of Plant Pathology
Cornell University

Ithaca, New York

ms
=:
=


(A) BN-28194, (B) BN-28193
Figure 25.—-(A) The washed residue is transferred to a beaker. (B) Each sieve
is carefully washed after transfer of flotsam.

MOOMGNVH AGOLVWAN NadTOD AHL

62


24 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

N-88189

Ficure 19.—All vehicles must remain on highways or
thoroughfares.

brush when he leaves fields or storage houses
(fig. 20).

Laboratory Procedures

Procedures outlined for recovering nematode
cysts from soil samples are based on the fact
that dry cysts will float in water. A machine
has been developed by ARS, USDA, to float
out these cysts and separate them from most
of the extraneous material. To prevent con-
tamination, equipment is thoroughly washed
after the processing of soil samples from each
field; shelves and racks bearing samples are
brushed and washed before being reloaded with
new samples (fig. 21); when infested material
is found, sieves in use at the time are replaced
until they have been thoroughly cleaned and
inspected.

Testing sieves currently in use are made
from medium-gage tin and brass screening by
tinsmiths. The sieves are 4 inches deep. The
bottom diameter is 1014 inches. For this pur-
pose such a sieve is superior to the factory-
made nesting type of testing sieves, because
it has a greater capacity and is more rugged.

'N-38190
Ficure 20.—Survey crews should clean shoes and equipment thoroughly before proceeding to the next property.


8 AGRICULTURE HANDBOOK 353, U.S. DEPARTMENT OF AGRICULTURE

The species of Heterodera and their princi-
pal hosts are as follows:

H. schachtii. Type host, sugarbeet (Beta
vulgaris L). Also other Chenopodiaceae, many
species of Cruciferae and various species of
other plant families (47). It seems possible
that H. schachtii attacks a wider variety of
plants than any other known species of Hetero-
dera.

H. gottingiana. Type host, garden peas
(Pisum sativum L.) and other Leguminosae.
But according to Oostenbrink (31), this species
does not attack beans (Phaseolus vulgaris L.),
clover (Trifolium spp.), alfalfa (Medicago
sativa L.), or soybeans (Soya maz Piper).

H. trifollii. Type host, red clover (Trifolium
pratense L.) and other Leguminosae, includ-
ing beans (Phaseolus vulgaris L.) but not peas
(Pisum sativum L.) alfalfa, or soybeans. _

H. glycines. Type host, soybean (Glycine
max L.) and snap bean (Phaseolus vulgaris
L.), Adzuki bean (P. angularis), vetch (Vicia
sp.), annual lespedeza (Lespedeza stipulacea
Maxim.), henbit (Lamium sp.).

H. major. Type host, oats (Avena sativa
L.) and other Gramineae.

H. cruciferae. Type host, cabbage (Bras-
sica oleracea L.) and other Cruciferae.

H. carotae. Type host, carrot (Daucus caro-
tae L.). Wild carrot (Daucus carotae) is the
only other known host (22).

H. humuli. Type host, hops (Humulus lupu-
lus L.) and other Urticaceae.

H. galeopsidis. Type host, hemp nettle
(Galeopsis tetrahit L.). Also other Labiatae
and some species of Chenopodiaceae and Caro-
phyllaceae (22).

H. fici. Type host, rubber plant (Ficus sp.).

H. weissi. Type host, knotweed (Polygonum
pensylvanicum L.). No other hosts known.

H. cacti. Type host, Phyllocactus (Epiphyl-
lum ackermanni) and other Cactaceae.

H. rostochiensis. Type host, potato (Solanum
tuberosum L.). Also tomato (Lycopersicon
esculentum Mill.) and a few other species of
Solanaceae (30) but not tobacco (Nicotiana
tabacum L.) (45).

H. tabacum. Type host, tobacco (Nicotiana
tabacum L.) and tomato.

H. punctata. Type host, wheat (Triticum
vulgare Vill.) and other Gramineae.

BIOLOGY

Life History

The golden nematode passes through the egg,
larval, and adult stages in its development.
From 38 to 48 days are required for the egg-
to-adult cycle (9).

The eggs hatch within the dead, swollen
bodies of fertilized females, which are called
cysts. Cysts are flask shaped and are much
smaller than a pinhead. Each cyst may con-
tain up to 500 eggs. The cyst is a protective
covering for the eggs and is resistant to chemi-
cals, to drying, and to some soil organisms
(fig. 4).

In the spring when the soil temperature is
around 60° F. a chemical given off by potato
or tomato roots stimulates larvae to hatch from
the eggs, leave the cyst through an opening in
the wall, and migrate to host plants. Larvae
enter the roots where they feed. At tempera-
tures below 55° F. there is little activity of the
larvae (10).

In the roots, golden nematodes take up a
position near the plant’s vascular system and
then undergo a series of changes. The females
become stationary; they swell and break
through the roots, to which they remain at-
tached by a thin neck. The males retain their
slender eellike form and mate with the females.
After fertilization, several hundred eggs de-

velop within the female whereupon the female
dies.

The golden nematode cyst is visible to the
unaided eye. At first, it is pearly white. Later,
it turns golden, orange, and finally brown.
Cysts become detached from the roots and re-
main in the soil after the crops have been
harvested (40) (fig. 5).

BN 30081

Ficure 4.—Golden nematode females washed from
potato soil. Each cyst may contain up to 500 eggs.


THE GOLDEN NEMATODE HANDBOOK ll

Among other plants that may also become
infected, thereby helping to propagate this pest,
are Solanum dulcamara (bitter nightshade),
S. rostratum (buffalo bur), S. triflorum (cut-
leaf nightshade), S. elaeagnifolium. (silverleaf
nightshade), S. blodgettii, S. xanti (purple
nightshade), and S. integrifolium (tomato
eggplant) (15).

Plant breeders have found about 90 species
of the Solanum to be host of the golden nem-
atode. Although most of these plants are wild
species not occurring in the United States,
most do occur in South America (38 p. 176).

Rate of Population Increase

Damage by the golden nematode to host
crops is caused by mass invasion on the roots
by nematodes that kill or damage the root sys-
tem, depriving the plant of the necessary food.

The relative increase in the number of nem-
atodes in the soil from the growing of a potato
crop is greatest when the number of nematodes
is low (27). This is because the plant is able
to produce a vigorous root system, and the
nematodes have an abundant root system on
which to feed. The nematodes attacking the
root are less concentrated, and thus few roots
are killed. However, as the number of nema-
todes increases in the soil, the annual increase
of nematodes from the continual growing of
potatoes is less because more roots are killed
and both the plant and the nematodes are de-
prived of an adequate food supply.

When the concentration of nematodes
reaches a level to cause total failure of the
potato crop, the number of living nematodes
in the soil drops substantially (27). This, of
course, is due chiefly to the lack of food to
sustain the population. The following year it

BN 29988
FicurE 7.—(Left) Healthy tomato plants. (Right) Tomato plants stunted by the golden nematode.

may be possible to grow a fair potato crop
because enough nematodes have died so the
plant can produce a root system to sustain it-
self. At these high degrees of infestation, the
crop will always be irregular and always be
poor.

In the absence of host crops, the rate of de-
crease in viable cysts may be as much as 50
to 80 percent the first year; however, the rate
of decrease continues at a smaller rate with
some viable cysts remaining for many years.
Grainger (19) of the West Scotland Agricul-
tural College reported that the golden nema-
tode wastes away at a fairly standard percent
annually in the absence of host plants, because
of normal aging and ultimate death of the
nematode and attack by other organisms.

Normal aging appears to be logarithmically
dependent on temperature, being greater in
warm climates than in cooler climates. Infesta-
tions of the golden nematode never assume
very serious proportions in warm countries,
where natural wastage is so high that even re-
latively short crop rotations would reduce popu-
lations to very low levels and longer rotations
would eliminate them entirely. In cool areas,
like Scotland, however, the annual wastage
could be as low as 18 percent. Grainger also
says that a moderate infestation of nematodes,
still in a viable state, could be expected to
cause serious disease “up to 30 years after
the last potato crop.” This expectation, accord-
ing to Grainger, has been confirmed by care-
ful checking of actual instances, which in-
volved persistence of 27 to 28 years (19).

United States Department of Agriculture re-
search workers Chitwood and Feldmesser (14)
found the increase on one potato crop on Long
Island to be about tenfold to twentyfold until
approximately 50,000 cysts per plant were