5Ö3 INDEXING PROCEDURES

FOR 15 VIRUS DISEASES

OF CITRUS TREES

Agriculture Handbook No. 333

LTTB R A R Y

DEC 2 Ù 1968

R'FLT^ILE BRANCH

Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE

INDEXING PROCEDURES

FOR 15 VIRUS DISEASES

OF CITRUS TREES

Prepared by the Committee on Indexing Procedures, Diagnosis,

, and Nomenclature, Third Conference of International Organization of Citrus Virologists, Campinas and Sao Paulo, Brazil,

1963

Committee Members

J. Bové (France), E. C. Calavan (Calif., U.S.A.), L. R. Fraser

(Australia), L. C. Knorr (Fla., U.S.A.), F. Nour-Eldin (Egypt,

U.A.R.), A. A. Salibe (Brazil), Shoichi Tanaka (Japan),

L. G. Weathers (Calif., U.S.A.), and J. F. L. Childs (Fla.,

U.S.A.), chairman

Agriculture Handbook No. 333

Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE

Washington, D.C. Issued February 1968

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402 - Price 65 cents.
Contents

Page

Introduction 1

Psorosis A, blind pocket, concave gum, crinkly leaf, and

infectious variegation 5

Cachexia (xyloporosis) 16

Tristeza and seedling yellows 20

Exocortis 28

Stubborn 35

Vein enation and woody gall 44

Gummy bark of sweet orange 50

Yellow vein 54

Satsuma dwarf 56

Impietratura .'. 60

Likubin 63

Tatter leaf 68

Leaf curl 74

Podagra 77

Indexing for tatter leaf with cowpea 80

Color test for exocortis 83

Greening disease 87

Classification of Citrus species 91

Glossary 96

Italic numbers in parentheses refer to Literature Cited at

the end of each section.

The opinions expressed in this publication are those of the

authors and do not necessarily constitute an endorsement by

the U.S. Department of Agriculture.

References are given and the artwork is reproduced essentially as submitted.
Indexing Procedures For 15

Virus Diseases Of Citrus Trees

INTRODUCTION

By J. F. L. CHILDS

Recognized virus diseases of citrus trees are so numerous and

the literature is so voluminous that selecting an appropriate indexing procedure is often a problem. Information on the preferred test

plant for a certain virus, age of the test plant for best expression

of symptoms, inoculation methods, incubation period, and the

effect of environment and of other viruses can be of critical importance in testing for citrus viruses. For example. Rangpur

lime is a good test plant for exocortis virus in Brazil and California but poor in Florida. Psorosis leaf symptoms are difficult

to obtain in the State of Bahia, Brazil. In Surinam, tristeza symptoms can be obtained reliably on Key lime test plants only in an

air-conditioned greenhouse. Because of this situation, publication

of a handbook on indexing procedures for virus diseases of citrus

was recommended by the Committee on Indexing Procedures,

Diagnosis, and Nomenclature of the International Organization

of Citrus Virologists at the Third Conference held at Campinas

and Sao Paulo, Brazil, in 1963.

The Committee agreed that 15 viruses (the number depends

on whether certain closely related viruses such as psorosis A and

concave gum are grouped together) are sufficiently well established that procedures for indexing them should be presented.

Committees of from one to four people were selected to describe

the procedures required for the various viruses. Some changes

have occurred in these committees. Gaetano Ruggieri died shortly

after preparing a brief note on impietratura. A. P. D. McClean

retired from active research and declined to prepare a formal

paper on vein enation and woody gall for lack of time. We hoped

to have a paper by H. Schneider explaining his histological method

of diagnosing tristeza infection of sour orange (Citrus aurantium

L.) rooted citrus trees, but because of other commitments he was

unable to prepare one. L. C. Knorr, who was unable to attend the

Conference, suggested that podagra should be included, but that

index tests for fovea were as yet without conclusive results.

Citrus viruses occur throughout the citrus-growing districts of

the world and most often in old-line varieties propagated vegeta-
¿ AGRICULTURE HANDBOOK '¿WW, U.S. DEPT. OF AGRICULTURE

tively for many years. As a result, citrus budwood programs

were set up to supply virus-free budwood to nurserymen and

growers—first, the psorosis-free budwood program of California

and later, one in Texas. Programs to supply budwood free of

several viruses were subsequently started in Florida, California,

Egypt, Brazil, and elsewhere. It is now widely recognized that

most old-line citrus trees carry several viruses and that trees

with a single virus are as rare as those without any. Thus, the

most promising sources of virus-free citrus budwood are nucellar

seedlings of the old-line varieties. Since psorosis virus was found

to be seed transmitted by Carrizo citrange, one cannot assume

that all citrus seedlings are automatically virus free. They must

be tested for viruses at the same time that they are tested for

trueness to type, productiveness, and vigor.

A minimum indexing program that covers the bare essentials

is outlined here as an aid to those undertaking such a project

for the first time. Seedling plants are usually employed for citrus

virus indexing and seeds of the required varieties must be obtained locally or imported. Ordering large quantities of seed that

cannot be used within a year is wasteful and a hardship to others

who require seeds. A few hundred seeds of each of the four or five

index varieties are adequate for the first year of operation. Additional seeds may be ordered as the program warrants it. Kept in

a humid atmosphere at around 10° C, citrus seeds survive for

12 to 15 months with little loss in germination, but fresh seeds

should be obtained each year if possible.

The seeds required are as follows: (1) West Indian lime (Citrus

aurantifolia (Christm.) Swing.), also known as Mexican lime.

Key lime, and baladi lime, for detecting tristeza virus; (2) Labsweet or Pineapple sweet orange (C. sinensis (L.) Osbeck),

bitter sweet sour orange (C. aurantium L.), and Willowleaf

mandarin (C. reticulata Blanco); or King mandarin (C. sinensis

X C. reticulata) for detecting psorosis A, concave gum, and blind

pocket forms of psorosis; (3) Arizona 861 or U.S.D.C.S. 60 — 13

selections of Etrog citron (C. medica L. var. ethrog Engl.) and

Rangpur lime (C. reticulata var. austera hybrid) for exocortis

virus; and (4) Orlando tángelo (C. paradisi Macf. x C. reticulata

Blanco) for cachexia (xyloporosis) virus.

Pure cultures of tristeza, psorosis, exocortis, and cachexia (xyloporosis) should be available in the greenhouse for comparison.

The tristeza isolate should be an insect-transmitted strain and the

psorosis isolate should be a seed-transmitted strain. With exocortis

and cachexia (xyloporosis), the best budwood is from trees that

were indexed and found negative for other viruses. Such budwood

may be obtained through the California and Florida citrus budwood programs and possibly elsewhere.

When indexing bearing seedling trees for the purpose of selecting virus-free individuals for budwood, it is of course impractical

to index for all the known citrus viruses. However, if seedling

trees are indexed and found free of tristeza, psorosis, exocortis,

and cachexia, one can usually assume that other viruses are likewise absent. In tristeza-infested areas tristeza-free trees may be
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS ö

rare or nonexistent. In that case the only choice is to select trees

infected with a mild strain of tristeza.

Virus-free budwood programs should be planned to fit the conditions of the area involved; namely, the citrus varieties grown and

the rootstocks adapted to local soil and water conditions. For

example, trifoliate orange (Poncirus trifoliata (L.) Raf.), an

excellent rootstock, is not suited to alkaline or salty soils. The

basic indexing procedures are presented here and can be selected

or rejected according to the program chosen.

Including several uninoculated plants in an experiment on indexing is not entirely adequate as a check or control. In testing a

candidate tree for the presence of a virus, one should duplicate

the indexing procedure with a pure culture of the suspected virus,

and inoculation of test plants with the pure culture should be run

concurrently with tests of the candidate plant. This aspect of

indexing is most important because the symptoms developed

under local conditions of light and temperature may not be exactly

like those described from other localities and the test plants used

may not be exactly like those used by the original investigator.

The nomenclature of citrus species is of considerable importance to the investigator attempting to repeat the experiments

of others. Several systems have been proposed, principally those

of W. T. Swingle, T. Tanaka, and R. W. Hodgson. Swingle recognized 16 species of citrus, Tanaka 150, and Hodgson 39. Some

virologists use one system and others another. This leads to confusion, because it is not always apparent which system is being

used. Also an author may use one name from one system and

another name from another system in the same publication. In an

attempt to establish a degree of order under these conditions, a list

of species names has been included at the end of this handbook.

Prepared by P. C. Reece, it is essentially a reference list correlating the names used by Swingle and Tanaka.

Contributors to this handbook are as follows:

A. Bental, plant pathologist, Volcani Institute of Agricultural

Research, Rehovot, Israel

J. Bové, biochemist. Institute Française de Recherches Fruitières, Station Centrale de Physiologie Végétale, Versailles, France

H. C. Burnett, plant pathologist. Division of Plant Industry,

Florida Department of Agriculture, Winter Haven, Fla., U.S.A.

E. C. Calavan, plant pathologist, Citrus Research Center, University of California, Riverside, Calif., U.S.A.

J. B. Carpenter, plant pathologist, U.S. Department of Agriculture, Indio, Calif., U.S.A.

J. Cassin, horticulturist. Station de Recherches Agrumicoles,

San Giuliano, Cervione, Corsica

Henri Chapot, geneticist. Chef du Service de THorticulture,

Rabat, Morocco

J. F. L. Childs, plant pathologist, U.S. Department of Agriculture, Orlando, Fla., U.S.A.
4 AGRICULTURE HANDBOOK 3 33, U.S. DEPT. OF AGRICULTURE

Lillian R. Fraser, plant pathologist, Director of Science Services,

Department of Agriculture, Rydalmere, Australia

S. M. Garnsey, plant pathologist, U.S. Department of Agriculture, Orlando, Fla., U.S.A.

T. J. Grant, ^ former plant pathologist, U.S. Department of

Agriculture, Orlando, Fla., U.S.A.

L. C. Knorr, plant pathologist. Citrus Experiment Station,

Lake Alfred, Fla., U.S.A.

T. T. Lo, senior plant pathologist. Joint Commission on Rural

Reconstruction, Taiwan, China

A. P. D. McClean,^ former Chief of Plant Protection Research

Institute, Pretoria, South Africa

T. Matsumoto,^ former plant pathologist. National Taiwan University, Taipei, Taiwan

S. Moreira,^ former horticulturist. Instituto Agronómico,

Campinas, Brazil

Farid Nour-Eldin, plant pathologist, Ministry of Agriculture,

Cairo, Egypt, U.A.R.

E. 0. Olson, plant pathologist, U.S. Department of Agriculture,

Indio, Calif., U.S.A.

Philip C. Reece, horticulturist, U.S. Department of Agriculture,

Orlando, Fla., U.S.A.

Victoria Rossetti, plant pathologist, Chefe de Seccao de Fitopatologia Geral, Instituto Biológico, Sao Paulo, Brazil

Gaetano Ruggieri,-' former Director, Stazione Sperimentale di

Agrumicoltura e Frutticoltura, Acireale, Italy

Ary A. Salibe, citriculturist. Instituto Agronómico, Limeira

Experiment Station, Limeira, Brazil

H. Schneider, plant pathologist, Citrus Research Center, University of Cahfornia, Riverside, Calif., U.S.A.

Ralph E. Schwarz, citrus virologist, Research Institute for

Citrus and Subtropical Plants, Nelspruit, Republic of South

Africa

H. J. Su, associate professor, Joint Commission on Rural Reconstruction, Taiwan, China

Shoichi Tanaka,! former Director, National Tokai-Kinki Agricultural Experiment Station, Okitsu, Shizuoka-ken, Japan (present address: Tamagawa University, Machida City, Tokyo, Japan)

J. M. Wallace,^ former plant pathologist, Citrus Research Center, University of California, Riverside, Calif., U.S.A.

L. G. Weathers, associate plant pathologist, Citrus Research

Center, University of Cahfornia, Riverside, Calif., U.S.A.

Shunichi Yamada, plant pathologist, Okitsu Horticultural Research Station, Okitsu, Shimizu, Japan

1 Retired.

- Deceased.
PSOROSIS A, BLIND POCKET, CONCAVE GUM,

CRINKLY LEAF, AND INFECTIOUS VARIEGATION

By J. M. WALLACE i

The diseases psorosis A, blind pocket, concave gum, crinkly

leaf, and infectious variegation are considered here as a group,

because they have certain common or overlapping characteristics.

Infectious variegation was formerly regarded as directly related

to the psorosis group, but the fact that it has been mechanically

transmitted and the others have not raises a question regarding

such a close relationship. However, for brevity they are discussed

here as a group.

For the purposes of certifying virus-free sources of budwood, it

is not essential to identify the particular virus of the psorosis

group that may be present in the tree under test. Thus, short-term

indexing on seedling trees will often identify concave gum, crinkly

leaf, and infectious variegation, but in order to distinguish between psorosis A and blind pocket, the indicator plants must be

grown for seyeral years until bark lesions and concavities, respectively, develop.

Studies by C. N. Roistacher " disclosed what appear to be mild

strains of concave gum virus, which rarely if ever cause zonate

leaf patterns on sweet orange. Also, some investigations in

Australia by Lilian R. Fraser and in California by the author

have revealed sources or strains of crinkly leaf virus that do not

cause typical psorosis young-leaf flecking except possibly on certain citrus varieties or selections and only under certain environmental conditions. These findings are taken into consideration in

the indexing procedures recommended in this handbook for this

group of diseases.

Indexing should be done in glasshouses or at least in screenhouses where the plants can be maintained in pots on benches.

This facilitates care of plants, inoculation, and examination for

symptoms. Symptoms may not develop well during the hot season

when the temperature cannot be partially controlled. The individual investigator should determine the times of the year when

symptom expression is good.

^ This section was reviewed by E. C. Calavan, J. Bové, Lilian R. Fraser, and

T. J. Grant.

2 University of California, Riverside, Calif., U.S.A.
6 AGRICULTURE HANDBOOK 33 3, U.S. DEPT. OF AGRICULTURE

INDICATORS

Indexing for the viruses that cause these five diseases may be

done with seedhngs of sweet orange {Citrus sinensis (L.) Osbeck),

lemon (C limon (L.) Burm. f.), and mandarin (C reticulata

Blanco). Studies in California by Roistacher and Nauer ^ showed

that some sweet orange varieties were better indicators of psorosis

A and ordinary concave gum than others. Among the named sweet

orange selections that proved to be good indicators and that are

fairly well known are Pineapple, Madam Vinous, Parson Brown,

Bessie, Indian River, and Koethen. *

Other sweet oranges will serve as indicators of psorosis A, blind

pocket, and concave gum. In locations where the above selections

are not available, local varieties should be tested to select a useful

indicator from which seeds can be obtained.

In California the sour lemon varieties, Eureka and Lisbon, are

used as indicators of crinkly leaf and infectious variegation.

Other kinds of lemon may serve equally as well. If lemon seeds

are not available, sour orange (C. aurantium L.) can be substituted, since this species develops fairly good symptoms of crinkly

leaf and very good symptoms of infectious variegation.

Mandarin varieties or mandarin hybrids should be included,

because they react to a virus (possibly an avirulent strain of concave gum virus) that causes faint oak leaf or zonate patterns on

mandarin but not on sweet orange seedlings. Roistacher found this

symptom much more often on Kara and Dancy mandarins and

Dweet tangor (Dancy (C. reticulata Blanco) x Mediterranean

sweet orange) than on other mandarin varieties, such as Batangas,

Honey, Kinnow, Ponkan, and Willowleaf. King mandarin was a

good indicator of this virus if the inoculated plants were provided

with supplemental lighting. During the winter 5 hours of additional artificial light improved symptom expression also on Kara,

Dancy, and Dweet.

SYMPTOMS

It has been assumed that vein flecking on young leaves is a

common symptom of these five diseases, and this was the basis for

their original classification as members of the psorosis group by

H. S. Fawcett.^ There still seems insufficient reason to change

this classification, although recent studies have disclosed strains

of these viruses that do not produce the young-leaf flecking symptom as originally reported. Studies in California indicate that

this young-leaf symptom occurs regularly on trees infected separately with the viruses of psorosis A, blind pocket, or concave gum.

Roistacher found that mild strains of concave gum virus caused

some flecking or vein clearing on sweet orange, even though they

produced no oak leaf pattern on this species.

3 See footnote 2.

4 "Lab-sweet," a pineapple-type sweet orange, has given excellent leaf symptoms of psorosis under greenhouse conditions in Florida (J. F. L. Childs).

5 Deceased, formerly at University of California, Riverside, Calif., U.S.A.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS <

The vein flecking and vein clearing typical of psorosis on sweet

orange and the oak leaf pattern of concave gum are shown in

figure 1. Actually the flecking symptom is a slight vein banding,

\

BN-28374

FIGURE 1.—Young-leaf symptoms of psorosis A and concave gum on sweet

orange: Left, two leaves with vein flecking of psorosis A; right, two leaves

with zonate oak leaf patterns along midrib and with clearing and banding

of veins on other parts.

since the lighter colored area involves some of the leaf tissues

closely adjacent to the veins. The clearing is not so distinct or so

narrowly restricted to the veins as that caused by tristeza. Flecking may be general over leaves or only on part of the leaf blade.

Also, the flecks may coalesce so that individual flecks are not

apparent and the entire area of the leaf is light in color. Thus,

there may be a range of leaf efi'ects on sweet orange, and on lemon

particularly there may be circular spots or irregularly shaped

chlorotic areas. The young-leaf symptoms disappear as the leaves

mature and harden.

If the common strains of concave gum virus are present in the

inoculum, some of the leaves of sweet orange should show the oak

leaf pattern. These patterns may be visible along the sides of the

midrib, over the entire length of the leaves, or only in localized

areas. Other parts of the leaves show the characteristic vein

flecking of psorosis. Under greenhouse conditions the patterns may

not be present during all growth flushes. In California, concave

gum-infected field trees display strong oak leaf patterns during

the spring growth flush, but for the most part these trees show
8 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

only the psorosis A type of leaf flecking during the summer and

fall flushes. If the suspected mild concave gum virus is present,

it should be revealed on the mandarin indicators already mentioned. Zonate or oak leaf patterns also tend to disappear as the

leaves mature, but sometime faint traces of this symptom can

be seen on old leaves.

With certain sources of virus, the ñrst visible effect after inoculation is a shock reaction. Newly formed leaves become chlorotic

and drop off, and the shoots that develop after the seedlings are

decapitated become necrotic (fig. 2). The new leaves are usually

BN-:iS37.-|

FIGURE 2.—Psorosis shock effect on sweet orange seedlings: A and B, Shedding of newly formed leaves and terminal necrosis of soft stems; C, healthy,

noninoculated check plant.

shed before they can develop vein flecking. Later a new cycle of

growth appears with young leaves, some of which usually display

psorosis leaf symptoms. Indications are that the psorosis A, blind

pocket, and infectious variegation viruses can induce the shock

symptoms, but there may be strains of these viruses that do not

cause this symptom. Concave gum and crinkly leaf viruses apparently do not induce the shock reaction, although a combination

of either or both of these two with any of the other three will

cause shock.

The reaction of seedlings infected with blind pocket virus is

much like those infected with psorosis A. Usually there is a severe

shock effect on the first cycle of growth after infection, followed
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS V

by the appearance of vein flecking on the young leaves of succeeding flushes of growth.

Unmixed with other psorosis viruses, crinkly leaf and infectious

variegation viruses seldom induced strong vein flecking. On inoculated seedlings, the first flush of growth shows slight leaf distortion and blotching of leaves and usually there is leaf shedding

and shock symptoms from infectious variegation. Leaves formed

later commonly show pinpoint spotting, as illustrated in figure 3.

BN-28365

FIGURE 3.—Pinpoint spotting of crinkly leaf and of infectious variegation

on Eureka lemon leaves.

This spotting develops on immature leaves and persists. The

crinkle efi^ect develops as the leaves mature and forms a persistent

symptom, as shown in figure 4. A similar type of crinkle, often

associated with infectious variegation, also causes severe leaf

distortion accompanied sometimes by yellowish or whitish blotchy

patterns. These patterns are called the variegation eñ'ect (fig. 5).

Cross-protection reactions and the existence of sources or

strains of virus that induce symptom efi'ects intermediate between

crinkly leaf and severe infectious variegation suggest a close

relationship between these two viruses. Plants with infectious

variegation sometimes produce recovery shoots that show only

crinkly leaf and pinpoint spotting.

INOCULATION METHODS

In growing seedlings for virus indexing, the practice in California is to germinate the seeds in flats and to transplant the small
10 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

BN-28372

FIGURE 4.—Persistent symptoms of crinkly leaf on mature leaves of Eureka

lemon.

seedlings to pots or cans, one or two plants per container, depending on its size. Seedlings transplanted when 4 to 6 inches tall

become established faster than larger transplants. A good soil

mix that encourages vigorous growth should be used. The use of

sterilized or fumigated soil avoids trouble from root-rotting

fungi. "

Tissue-graft inoculations are made by means of small twig side

grafts, leaf patches, bark patches, or T-buds. All types of tissue

give good results, but twig grafting has three advantages. First,

side grafts can be made on seedlings when they are very small.

Second, one can readily determine whether the inoculum piece

united with the seedling and survived, without removing the grafting tape. A third possible advantage is that more virus-bearing

tissue is used. At inoculation time, the seedlings are cut back at a

point two or three buds above the inoculum. When leaves are present on the stem of the seedlings, it is advisable to leave them

because they offset some of the effects of topping.

Leaf patches consist of narrow rectangular pieces of the leaf

blade, and bark patches are similarly shaped pieces of tissue taken

preferably from small limbs or twigs with moderately thin bark.

It is a good practice to clean the leaves and twigs from which the

inoculum tissue is to be taken. Normally, washing and wiping are

sufficient, but should one consistently find a considerable amount

of decay of these tissues on the inoculated seedling, the sources of

inoculum should be cleaned with alcohol or mercuric chloride

(1-1,000), washed in water, and wiped dry before the patches are

° In Florida we find vermiculite (expanded mica chips) superior for germinating seeds, and witli our organic type soil, sterilizing often results in poorer

plant growth than unsterilized soil (J. F. L. Childs).
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 11

BN-28364

FIGURE 5.—Chlorosis and leaf distortion on Eureka lemon infected with virus

of infectious variegation.

cut out. The patches are then inserted under bark flaps made by

peehng the bark downward from one horizontal and two vertical

cuts and then wrapped in place with suitable grafting tape '

or with rubberbands.

When buds are used as inoculum, they are inserted in T-cuts

as in propagating citrus. If the index seedling is large enough, it

is advisable to put in two pieces of inoculum. These may be the

same or diiferent; for example, one twig graft and one leaf patch.

The number of test plants to be included will depend somewhat

on availability of plants and the number of indicator varieties to

be used. If a single indicator variety is used, sweet orange for

example, at least eight plants should be inoculated and there

should be two or more noninoculated controls. If plants of sweet

orange, mandarin, and sour lemon are included, four inoculated

and two controls of each variety should be sufficient. If the test

plants are large enough, using two pieces of inoculum taken from

different positions on the source tree will improve the test.

READING SYMPTOMS

If inoculation causes the shock reaction, the first symptoms are

twisting and bending of the newly developing axillary shoots.

We have found vinyl plastic tape of 0.0035-inch (3.5 mil) thickness

superior to other tapes (J. F. L. Childs).
12 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

These symptoms are followed by leaf shedding and varying

degrees of necrosis of the soft stems.

If new growth develops without the shock reaction, the leaves

should be examined regularly for vein flecking or other effects.

The leaves should be viewed while shaded and with the light passing through them. Faint leaf flecks may not be visible except

under certain angles of lighting. Examinations should be made

at 3- to 4-day intervals at least. If no symptoms are found within

6 weeks after inoculation, the plants should be cut back again and

examined throughout a second cycle of leaf growth. The absence

of symptoms from all indicator varieties throughout two growth

flushes is reasonably good assurance that the source of inoculum

was free of the viruses in question. If facilities are available and

it is thought desirable, the plants can be overwintered in a screenhouse and then cut back in the spring for further study. This

procedure sometimes reveals some concave gum-type symptoms

that do not appear on plants maintained at all times in greenhouses.

Plants infected with crinkly leaf virus may show slight or no

symptoms on the young leaves, but occasionally some faint vein

flecking is present in one or more leaves of sweet orange and

certain other kinds of citrus. This symptom may sometimes be

evident for only 2 to 3 days. As the leaves mature, the crinkling

develops on some leaves. On lemon the leaves commonly show

pinpoint spotting (fig. 3). If the infectious variegation virus is

present with the crinkly leaf virus, there is shock. Leaves that do

not drop are chlorotic or blotched and distorted. Later growth is

severely affected, showing the symptoms already described for

infectious variegation.

The presence of other commonly occurring citrus viruses in

mixture with the psorosis viruses seems to have no appreciable

effect on the symptoms of the latter, although it is not known if

this is always the case. Ideally, for budwood selection, all sources

of budwood should be rejected if inoculations from them give any

indication of virus infection, even though the virus or viruses

present cannot be specifically diagnosed. However, in some countries the tristeza virus may be present in all available citrus

budwood. Likewise, where the vein enation (woody gall) virus

spreads naturally, it may be of little value to start with citrus

trees that are free of this virus.

Regardless of whether or not virus-free budwood is used for

propagation of new trees, diseases that spread naturally must be

controlled by using tolerant or resistant rootstocks. Obviously it

is wise to avoid propagation from trees carrying viruses such as

tristeza and vein enation if the resulting trees are to be planted

in areas where these viruses are not present.

NONINFECTIOUS PSOROSISLIKE LEAF EFFECTS

In a study of citrus virus diseases in California, some noninfectious leaf patterns have been encountered that should not
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 13

be confused with symptoms of psorosis. It seems advisable to

describe the patterns so that investigators will be aware oí them.

Seedlings of Eureka lemon grown in the greenhouse sometimes

display the leaf-spotting effects shown in figure 6 Whether this

symptom is caused by genetic or environmental factors is not

known, but at times it closely resembles psorosis leaf spotting

BN-2837(»

FIGURE 6.—Psorosis and noninfectious psorosislike symptoms on young leaves

of Eureka lemon: Left, psorosis; center, false psorosis; right, normal.

on lemons and could easily be interpreted as a psorosis symptom.

Another condition closely resembling leaf symptoms of psorosis

that has been recorded occasionally as psorosis on field trees is

shown in figure 7. Extensive studies of this leaf abnormality have

been made in California. It is noninfectious and appears to be

a genetic abnormality. Seedlings derived from trees showing these

leaf patterns also display the same pattern. It has been observed

by the author on Cutter and Olinda selections of Valencia orange

in California and on Tarocco orange in Sicily.

A third noninfectious leaf abnormality (fig. 8) has been observed in California on lemon trees after severe frost damage.

This is a variegation, with some leaves showing chimeral sectors.

These symptoms developed on the spring growth of trees on which

there had been some freezing back of the small hmbs. They were
14 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

BN-283Ö3

FIGURE 7.—Psorosislike genetic leaf pattern on sweet orange: Left, two leaves

show pattern; right, normal leaf.

most striking on leaf clusters that developed from buds in close

proximity to dead stem tissue. These symptoms were found on

virus-free seedlings in nurseries as well as on larger field trees.

Individual leaves sometimes were only spotted or stippled and

some of them resembled leaves with some eflPects of psorosis.

Leaves of later growth flushes were normal.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 15

BN-28362

FIGURE 8.—Variegation and other effects on leaves of lemon shoots that

developed near tissues killed by cold.
CACHEXIA (XYLOPOROSIS)

By J. F. L. CHILDS i

Opinions differ regarding the relationship of cachexia (described from Orlando tángelo (Citrus paradisi, Macf. X C. reticulata Blanco) ) to xyloporosis (described from Palestine sweet lime

(C aurantifolia (Christm.) Swing.)). The available evidence

indicates that both diseases are caused by the same virus, and

that differences in their respective symptoms are in reality differences in the reaction of different hosts to the same virus (figs.

land 2).

FIGURE 1.—Orlando tángelo on Rusk citrange rootstock 30 months after budding. Note severe symptoms of cachexia on Orlando tángelo.

L. C. Knorr reported a disease of Murcott orange (probably a

tangor (C. sinensis (L.) Osbeck x C. reticulata Blanco)) in Florida and named it fovea. The symptoms are pitting and pegging

1 This section was reviewed by E. O. Olson and A. Bental.

16
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 17

FicrRK 2.—Hamlin sweet orange on sweet lime stock 10 years after budding.

Note mild symptoms of cachexia on sweet lime stock.

of the cambial surface of the bark and wood and starvation symptoms on the foliage, which are much the same as in cachexia.

Gum is not commonly associated with the pits and peg-s, a symptom more like xvloporosis. Knorr observed that affected trees

usually die 1 to 2 years after the first appearance of symptoms.

However, his index tests with Orlando tángelo, sweet lime, and

Clementine mandarin (C. reticidata Blanco) have been inconclusive.

Cachexia virus occurs widely in commercial varieties of citrus,

but causes primary symptoms only on certain mandarin (C.

reticnlaia Blanco) varieties, mandarin hybrids, sweet lime, and

lime hybrids. Severity of symptoms on susceptible varieties ranges

from most severe on Orlando tángelo and Parson's Special mandarin to none on Webber tángelo (C. ¡mmdisl Macf. X C. reticulutu

Blanco). Other species and varieties of citrus, such as sweet

orange (C. siuensis (L.) Osbeck), sour orange (C. unr(i)itiuin

L.), and grapefruit (C. ¡mradisi Macf.), may carry the virus

without expressing recognizable symptoms.

SYMPTOMS

The symptoms may be divided roughly into (1) primary

damage to tissues by the virus—gum impregnation of the phloem

tissues and pitting "of the cambial face of the wood and pegging

of the bai-k, followed by necrosis and cankers of the bark that

may cause partial girdling and (2) secondary damage—sparse

chlorotic foliage, yellow midrib, and retarded growth, which are

mainly starvation'symptoms resulting from girdling. When tolerant varieties such as sweet orange or grapefruit are grown on susceptible rootstocks, cachexia may cause severe starvation symptoms and stunting of the top through injury to the rootstock.
18 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

INDEXING PROCEDURE

Several test plants—Rangpur lime (C. reticulata var. austera

hybrid), Palestine sweet lime, and certain tángelo varieties—have

been widely used as indicators for cachexia virus infection. None

appear more satisfactory than Orlando tángelo (fig. 3). Rangpur

FIGURE 3.—Marsh grapefruit on Orlando tángelo rootstock with early symptoms of cachexia. (Courtesy of E. C. Calavan.)

lime is also susceptible to exocortis virus. Consequently, the diagnosis of cachexia is not always as clear as it should be when

both viruses are present. Palestine and Columbia sweet lime (C.

aurantifoUa (Christm.) Swing.) both react more slowly than

Orlando. Wekiwa tángelo (C. paradisi Macf. X C. reticulata

Blanco) gives strong symptoms but grows less vigorously than

Orlando, at least in Florida.

Experimental studies indicate that cachexia virus is not seed

transmitted. Nevertheless as a precaution, seed for test plants

should be obtained from cachexia-free parent trees if possible.

Seeds are most easily germinated in vermiculite (expanded mica

chips) in flats. Seedlings may be transferred to small pots or

cans when 15 cm. high.

The test seedlings can be budded or grafted with tissue from

candidate trees when 20 to 30 cm. high. After the bud or graft

is established and has made some growth, the seedling top may be

cut back, the bud or graft allowed to grow, and the plant transferred to the field for observation. Symptoms appear sooner in the

field than in the greenhouse. Plants transferred from pots to the

field grow well and require a minimum of care. At least three tests

should be made for each candidate tree, although some workers

use as many as eight.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 19

In test plants consisting of a top grown from a bud or graft

of the candidate tree on Orlando tángelo seedling rootstock, the

first symptom will be small spots of gum in the phloem or on the

cambial face of the bark at the bud union. Pitting of the Orlando

tángelo wood, pegging of the bark, and gum impregnation of the

bark develop later. On inoculated seedling trees growing on their

own roots but not cut back to force the candidate bud, bark symptoms may appear anywhere on the trunk, although they occur most

often near the point of inoculation and below large branches.

The Orlando tángelo grows poorly on its own roots in Brazil,

according to Ary A. Salibe. Under such circumstances, he recommends budding Orlando on a vigorous stock such as Rangpur

lime that was previously inoculated from the candidate tree. If

possible, control plants inoculated with cachexia should be grown

simultaneously as a guide to the length of the incubation period

and the severity of symptoms under local conditions.

INTERPRETATION OF RESULTS

Symptoms as described normally appear in 6 to 36 months in

Florida. An occasional plant may require 5 years. If no symptoms

have appeared on any of the Orlando test plants in 5 years, the

candidate can be considered free of cachexia. Appearance of

symptoms may be delayed under certain environmental conditions. At such times inoculated control plants are highly important

for comparison in the evaluation of results.
TRISTEZA AND SEEDLING YELLOWS

By J. M. WALLACE ^

Tristeza virus infection of intolerant combinations of top and

rootstock can be diagnosed by histológica! examination with considerable accuracy. However, with tolerant combinations and

with mild strains of the virus, it is necessary to index in order

to avoid tristeza-infected sources of budwood. The lime test provides a reliable and relatively quick index test and is widely used.

However, under certain conditions interpretation of the results

may be facilitated by testing with susceptible budded trees. Most

combinations of sweet orange (Citrus sinensis (L.) Osbeck) on

sour orange (C. aumntium L.) are susceptible. For greenhouse

experimental studies, the author has found that navel or Valencia orange on Eureka lemon (C. Umon (L.) Burm. f.) is a very

susceptible combination.

Indexing for the seedling yellows strain requires citrus species

that will give the seedling yellows reaction. West Indian lime (C.

auranUfolm (Christm.) Swing.) reacts to tristeza virus and to

the seedling yellows strain, but does not distinguish between them.

LIME REACTION

Mexican lime (C. aurantifoUa (Christm.) Swing.), also known

as West Indian lime. Key lime, and baladi lime, has been adopted

as the best indicator of tristeza virus infection. Leaves of this lime

develop distinctive vein clearing when infected, which seems to be

specific for the tristeza virus. Other types of lime as well as other

kinds of citrus develop similar vein clearing, but none is better

than Mexican lime.

Limes infected with tristeza virus also develo]) stem pitting,

which per se cannot be considered specific for tristeza, but substantiates the diagnosis when combined with the leaf symptoms.

Infected lime plants may be slightly, moderately, or severely

stunted. However, the degree of vein clearing, stem pitting, and

stunting on hme varies widely with different strains of tristeza

virus No other citrus virus has been found to produce this set of

symptoms on lime.

SEEDLING YELLOWS REACTION

Other indicator varieties are necessary to indicate the presence

Grant.

20

This section was reviewed by Lilian R. Fraser, H. Schneider, and T. J.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 21

of the seedling yellows strain. Lemon (C. limon (L.) Burm. f.),

sour orange (C. aurantium L.), and grapefruit (C. paradisi

Macf.) are good indicators for that purpose. On the basis of

extensive studies of strains of seedling yellows virus, the author

prefers Eureka lemon. However, the scarcity of seeds in Eureka

lemon fruits and the lack of uniformity among the seedlings introduce some problems not found with sour orange and certain

varieties of grapefruit. In some regions sour orange and grapefruit prove satisfactory, but in California Eureka lemon is preferred, because a strain of seedling yellows found there causes

no symptoms on standard sour orange.

The seedling yellows virus is known to spread naturally in

Australia, South Africa, South America, and some other countries,

but it has never been recovered from any commercial citrus

plantings in California. Most Meyer lemon trees in California

were positive for tristeza when tested on lime. Testing on Eureka

lemon demonstrated that all that gave the lime reaction also gave

the yellows reaction. Several strains of seedling yellows virus are

known, and two or more strains may be present in a given tree.

INDEXING PROCEDURE

Seeds of the indicator varieties should be collected, treated,

and planted as previously described (see pp. 9-10). Transplanting

the seedlings when 4 to 6 inches high encourages establishment

and rapid growth. The number of seedlings placed in each pot

depends on the size of available containers. A 6-inch pot or 1-

gallon can will support four lime or two lemon seedlings until

completion of the indexing. Mexican lime and Eureka lemon

seedlings are not all alike and offtype plants should be eliminated

by careful roguing at time of transplanting. Weak seedlings as

well as the large-leaved extra vigorous plants should be eliminated.^

For uniformity, it is often necessary to discard about one-half of

the lime seedlings.

Inoculation from the tree being tested can be made with bark

or leaf patches, buds, or grafts. On very small seedlings side

grafts are most satisfactory. In this method, part of the scion is

exposed and can be examined for survival or *'take'' without

unwrapping or cutting into the plant tissue. Side grafts involve

the use of more tissue than is the case with buds or leaf and bark

patches, and it is believed that they provide more virus for infection. In the author's studies the total percentages of infection

were not significantly different with various kinds of inoculum

when the **take" was equally good, but symptoms generally appeared sooner on the seedlings infected from grafts.

Test seedlings should be cut back at the time of inoculation to

leave two or three buds above the inoculation site for develoj)-

ment of shoots. Growth from twig grafts should be su])pressed

so as not to interfere with the growth of the seedling. Suitable

indicators of seedling yellows should be inoculated at the same

time if one wishes to know whether the seedling yellows virus is

carried by the tree being tested. Four seedlings of lime and four
22 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

of a seedling- yellows indicator normally provide a reliable test.

It IS well to include at least two noninoculated plants of each

indicator variety as controls.

SYMPTOMS

With most sources of tristeza and with the tristeza-seedlingyellows complex, the first observable symptoms on infected lime

seedlmg-s are chlorosis and cupping of terminal leaves of the

shoots that develop after the plants are inoculated and cut back

(fig. 1). In the glasshouse these symptoms may be noticeable 3 to

FiGliRi: 1.—Early symptoms of tristeza on Mexican lime: Left, stunting

4 weeks after inoculation. Young chlorotic leaves may show distinct but slight vein clearing as early as 4 weeks, but usuallv 5

weeks or longer are required before vein clearing becomes conspicuous (fig. 2 A). The amount of vein clearing and its distribution over the i)lant vary considerablv. This is governed to

sonie extent by the strain of virus jiresent, and i)robablv lime

seedlings vary somewhat in their response to infection '

Vem-clearing symptoms usually i)ersist long after the leaves

mature. Close examination of the undersurface of affected leaves

reveals a slight water-soaked api)earance of those pai-ts of the

veins that ai)pear cleai-ed when viewed by transmitted light (fio-

¿, /^).. Ihe author has not found this water-soaking svmptom

associated with any other citi'us virus.

_ Leaf symptoms nearly always a])|)ear within 3 months of the

inoculation time but when all plants of the same groui» remain

negative they should be observed for a ])eriod of « months Test

plants should be ])runed after 3 months and new shoots forced
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 23

FiGURp; 2.—Symptoms of tristeza on Mexican lime leaves: A, Vein clearing;

B, water-soaked appearance of veins on undersurface of affected leaves;

affected parts of veins correspond to vein clearings.

from buds near the inoculation site. If no vein clearing' appears

on this growth within the second 3-month period, the plants

should be cut off at that time and examined for stem pitting by

stripping the bark from the main trunk and several small

branches (fig. 3, A). Pitting appears on the base of small, green,

hardened shoots at times when it cannot be found on the older

stems. At other times pitting can be detected without removal of

the bark (fig. 3, Z> ). If none of a particular group of lime seedlings

show vein clearing or stem pitting within 6 months, it can be

concluded that the test is negative, i.e., that the tree under test

was free of tristeza virus when the inoculum was taken.

If seedling yellows virus has been introduced into lime test

plants, it commonly causes an early and fairly severe reaction,

but some strains of tristeza cause symptoms equally as severe.

Therefore, lemon seedlings, or another suitable indicator of yellows, must be used to determine whether the inoculum contains

seedling yellows virus. With some sources of virus. Eureka lemon

seedlings develop symptoms of yellows before lime plants in the

same test show any symptoms. If lemon seedlings are infected

with seedling yellows virus, growth is retarded and terminal

leaves of newly developing shoots are chlorotic and smaller than

normal (fig. 4). Later the symptoms of seedling yellows advance

rather rapidly. Some strains of the virus cause severe stunting

and almost a complete cessation of growth (fig. 5). There is a

wide range of severity dependent on the source or strain of virus.
24 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

BN-28371

FIGURE 3.—Tristeza stem pitting on Mexican lime: A, Twigs with bark removed showing characteristic wood pitting; B, older stems on which pitting

can be seen without removing bark.

After showing good yellows symptoms, seedlings of lemon, sour

orange, and grapefruit may recover by the renewal of growth

with little or no disease effects. However, in California some

strains of seedling yellows virus have been found from which

lemons failed to recover. The rate of recovery seems to be governed by the virulence of the virus strain. In some instances, the

affected plant may show strong symptoms in the terminal part

of the stem for a relatively short time and then produce growth

that gradually becomes nearly normal. In other cases, several

months may elapse before any recovery is initiated, and this may

come about slowly by the production of mildly affected or symptomless shoots from below the affected terminal of the plant.

Studies have shown that after plants of lemon, sour orange,

and grapefruit recover and no longer display seedling yellows

symptoms, the virus retained by them will cause the tristeza lime

reaction and decline of tristeza-susceptible budded trees, but will

not cause seedling yellows. The disappearance of the yellows virus

takes place gradually during plant recovery, and sometimes several months elapse before this component of the virus complex is

no longer present.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 25

BN-28380

FIGURE 4.—Seedling yellows symptoms on Eureka lemon: A, Healthy control;

B, plant 36 days after graft inoculation.
26 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

J'

BN-2836(>

FIGURE 5.—Seedling yellows-affected Eureka lemon 1 year after inoculation

with virulent strain of seedling yellows virus from which lemon seedlings

seldom recover.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 27

EFFECT OF OTHER VIRUSES

Mixtures of tristeza and other viruses occur rather commonly.

However, a thorough or complete experimental study has not been

made of the effect of other viruses on tristeza symptoms. Most of

the other viruses seem not to affect the symptoms of tristeza and

seedling yellows on indicator seedlings. Two exceptions are psorosis and greening disease viruses.

If psorosis virus is also present, its symptoms usually appear

first and may mask or prevent early development of distinct vein

clearing on lime, especially if the strain of psorosis is one that

causes shock symptoms. However, typical vein clearing will be

evident in later flushes of leaves if tristeza virus is present m

the inoculum. The presence of psorosis virus seems to have no

effect on subsequent development of stem pitting.

The reactions of sour orange. Eureka lemon, and grapefruit

seedlings will not differentiate between seedling yellows and

greening disease viruses because both produce severe chlorosis on

these varieties. However, if a positive reaction for greening disease (a chlorosis similar to that of iron and zinc deficiencies) is

obtained on sweet orange, the presence or absence of seedling

yellows must be established by transmission of the virus with the

brown citrus aphid (Toxoptera citricida (Kirkaldy)). Greening

disease virus is believed to be transmitted by psyllids and not by

aphids.
EXOCORTIS

By E. C. CALAVAN i

Exocortis is primarily a disease of citrus trees grown on rootstocks of trifoliate orange (Poncirus trifoliata (L.) Raf.), citrange (P. trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck),

and Rangpur lime (C. reticulata var. austera hybrid). Generally

bark scaling on the rootstock and stunting of the top are noted.

Sometimes scaling of the stock occurs without noticeable stunting

of the top and vice versa. Several strains of exocortis virus exist

and host reactions to exocortis infection are greatly affected by

environment.

SYMPTOMS

Exocortis virus causes vertical cracking and scaling of the

bark (fig. 1), yellow blotching of twigs, and considerable stunting

of trifoliate orange, certain mandarin limes (C. aurantifolia

(Christm.) Swing.), and some citranges (3, 5, 6, 8, 12), It causes

crackmg of the bark and stunting in Cuban shaddock (C. grandis

(L.) Osbeck), some lemons (C. limon (L.) Burm. f.), and sweet

limes {C. aurantifolia (Christm.) Swing.) {15).

On certain seedhng clones (U.S.D.C.S. 60-13 and Arizona 861)

of Etrog citron (C. medica L. var. ethrog Engl.) exocortis virus

mduces cracking of the abaxial surface of the midrib, stem and

leaf epmasty, yellow blotching of the stem, corky lesions and

vertical^ cracks on the stem, and dwarfing (i, 2, 7). The base of

the petiole and lower side of principal veins of younger leaves

may be darkened abnormally. Mature leaves of some citron shoots

may die and drop. The virus has been transmitted from citrus to

V^turvm {Petunia hybrida Vilm.) by dodder {Cuscuta subinclusa

Dur. & Hilg.) {li).

Exocortis has caused severe losses in areas where trifoliate

orange, citrange, or Rangpur lime was used as a rootstock. Many

kinds of citrus, including sweet orange (C. sinensis (L.) Osbeck)

and mandarin orange (C. reticulata Blanco), may be stunted

slightly by exocortis infection, but they usually fail to develop

specific symptoms of exocortis.

Dissemination of exocortis virus occurs principally through

propagation of infected budwood. Natural spread in California

orchards appears to have been slight, but considerable natural

1 This section was reviewed by Ary A. Salibe, H. C. Burnett, and E. O

Olson. '

28
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 29

I

,,,'.' , / .' •' ' , ; *-^f •-= ^L"' '-"

KN' 12s:i70

FIGURE 1.—Exocortis on 12-year-old trifoliate orange rootstock, partially

excavated to show lesions below soil level. Top variety is Temple orange

(probably hybrid (C. sinensis (L.) Osbeck X C. reticulata Blanco)).

spread in greenhouses has been noted (2).- Seed transmission of

exocortis virus has been suspected in Brazil (13), but has not

been found in Austraha (6) or California.

INDEX TESTS

Exocortis indexing on indicator plants of citron, such as

- Natural spread, so called, probably results from mechanical inoculation by

contaminated budding knives, according to the results of S. M. Garnsey

(J. F. L. Childs).
30 AGRICULTURE HANDBOOK 3 33, U.S. DEPT. OF AGRICULTURE

U.S.D.C.S. 60-13 or Arizona 861, requires only a few months.

The citron test (2) is the most rapid and sensitive index for

exocortis. Citron clones vary tremendously in their reaction to

exocortis infection (1, 7), and only the most sensitive are useful

as indicators. Tvv^o widely used and practical methods of inoculation are illustrated (fig. 2).

BN-288G7

-biGURE 2.—Two methods of using citron in rapid indexing for exocortis* A

Propagation of citron (C) on citrus seedling inoculated with exocortisiniected buds (AA ) ; B, splice grafting of exocortis-infected shoot (EX)

on citron cutting (C), which is then rooted and forced to develop a shoot
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 31

PREFERRED METHOD

Two buds from the candidate tree are grafted into each of

several seedlings of a suitable exocortis host, such as Rough lemon

(C limon (L.) Burm. f.) or Ponderosa lemon (probably a hybrid,

C. limon (L.) Burm. f. X C. medica L.), but are not permitted to

develop into shoots. Bark patches may be used. A bud from a

healthy plant of a sensitive exocortis-free citron clone, such as

CRC. 60-13 or Arizona 861,'^ is then inserted m each seedling

(fig 2, A). The citron bud may be inserted on the same day as

the candidate bud or up to 2 weeks after the candidate bud. Too

early infection of a seedling may inhibit growth of the citron bud.

Growth of the citron shoot is encouraged by cutting back or

bending the seedlings. The citron scions should be grown as

single shoots in a favorable environment in either the greenhouse

or the field and pests must be rigorously controlled. Iniected

plants usually develop symptoms within 1 to 3 months and betöre

the shoots are 2 feet high. Taller symptomless citron shoots may

be cut off about 15 cm. above the bud union and a new citron shoot

grown for about 3 months. . i jTo avoid overlooking mild strains of exocortis virus, each candidate should be indexed on at least five indicator plants until symptoms appear or until 6 months after inoculation. Nonmoculated

control plants should be included with the citron index plants to

detect possible natural spread of exocortis (2), Inspection ot

citron budwood plants once a week is advisable for the same

reason.

The 'first and most characteristic symptoms of exocortis infection on citron shoots are epinasty of the leaves (fig. 3, A) and

cracking of the lower side of the midrib (2) (fig. 3, B). Severely

affected leaves curl downward from the tip, and the blades twist

to varying degrees from the normal plane. Other symptoms include dwarfing, stem epinasty, yellow blotching of the stem, small

corky lesions, and vertical cracking of the stem (fig. 3, C). The

last symptoms provide corroborative evidence and are especially

valuable if one is forced to index on one of the less sensitive

strains of Etrog citron. j_ - n

An alternative method consists of splice grafting a twig trom

the candidate tree onto exocortis-free citron cuttings (2, 7). A 2-

to 3-inch section of candidate shoot is splice grafted onto a citron

cutting (fig. 2, B). The cutting is then rooted and allowed to grow

a shoot, but shoots from the candidate graft are removed. This

method gives excellent results in the greenhouse and requires no

rootstock seedlings.

OTHER METHODS

Rangpur lime seedlings or budded plants have been used extensively as exocortis indicators in Brazil (9, 11), California (2, 3),

''Another citron useful for exocortis indexing is the Beerhalter strain of

Mortimer Cohen (Indian River Field Laboratory, Ft. Pierce, Fla., U.S.A.).

Most other citron selections are worthless for exocortis indexing. (J. F. L.

Childs)
32 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 33

and Texas (10). The principal methods are (1) inoculation of

seedhngs with buds from the candidate tree and (2) topworking

healthy Rangpur lime into one or more branches of the candidate

tree. Five or more indicator plants or shoots should be used for

each candidate. The use of seedling indicator plants avoids the

possibility of accidental inoculation of a candidate tree from grafts

used for topworking.

Vigorous Rangpur lime shoots often react to exocortis infection

in 4 to 15 months. Symptoms of exocortis in Rangpur lime shoots

are yellow bark blotches of variable size and shape (fig. 3, D)

usually located near thorns, vertical cracks in or near these

blotches, limited necrosis, and sometimes scaling around the

cracks. Rangpur lime is useful as an indicator in the greenhouse

or field. Although Rangpur lime usually reacts to infection with

severe strains of exocortis virus, it is less sensitive to infection

by milder strains than certain citron clones (2).'^

Long-term indexing of exocortis, by growing a scion of the

candidate tree on exocortis-sensitive rootstocks, such as trifoliate

orange or Rangpur lime, has been widely used. However, this

method is slow and expensive compared with the more rapid

citron method. A further disadvantage is that failure of the rootstock to develop symptoms after several years does not insure

freedom from exocortis infection (2).

The presence of exocortis symptoms in exocortis-sensitive

rootstocks of candidate trees or their progeny, determined by

direct examination or by the phloroglucinol test (Í), makes

indexing unnecessary.

Incidence of exocortis in susceptible trees can be minimized by

propagating from parent trees that are exocortis-negative when

indexed on sensitive citron plants.

LITERATURE CITED

(1) ALLEN, R. M., and ODEN, W. R.

1964. VARIABILITY OF SEEDLING CLONES OF CITRON AS INDICATORS OF

EXOCORTIS VIRUS OF CITRUS. Phytopathology 54: 1431.

(2) GALAVAN, E. C, FROLICH, E. F., CARPENTER, J. B., and others.

1964. RAPID INDEXING FOR EXOCORTIS OF CITRUS. Phytopathology

54: 1359-1362. (See also Calif. Agr. 19 (1): 8-10 and

Calif. Citrograph 50: 185-188, 190.)

(3) and WEATHERS, L. G.

1961. EVIDENCE FOR STRAIN DIFFERENCES AND STUNTING WITH EXOCORTIS VIRUS. In Price, W. G. (ed.), 2d Gonf. Internatl.

Organ. Citrus Virol. Proc. 1960, pp. 26-33.

(4) CHILDS, J. F. L., NORMAN, G., and EICHHORN, J. L.

1958. A COLOR TEST FOR EXOCORTIS INFECTION IN PONCIRUS TRIFOLIATA. Phytopathology 48: 426-432.

' In Florida, Rangpur limes, both Florida and Brazilian strains, required

2 to 3 years to develop exocortis symptoms in the greenhouse (J. F. L. Childs).
34 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

(5) FAWCETT, H. S., and KLOTZ, L. J.

1948. EXOCORTis OF TRIFOLIATE ORANGE. Citrus Leaves 28 (4) : 8.

(See also Calif. Citrograph 83: 230.)

(6) FRASER, L. R., LEVITT, E. C, and Cox, J.

1961. RELATIONSHIP BETWEEN EXOCORTIS AND STUNTING OF CITRUS

VARIETIES ON PONCIRUS TRIFOLIATA ROOTSTOCK. In Price,

W. C. (ed.), 2d Conf. Internatl. Organ. Citrus Virol. Proc.

1960, pp. 34-39.

(7) FROLICH, E. F.. CALAVAN, E. C, CARPENTER, J. B., and others.

1965. DIFFERENCES IN RESPONSE OF CITRON SELECTIONS TO EXOCORTIS

VIRUS INFECTION. In Price, W. C. (ed.), 3d Conf. Internatl.

Organ. Citrus Virol. Proc. 1963, pp. 113-118.

(8) MOREIRA, S.

1959. RANGPUR LIME DISEASE AND ITS RELATIONSHIP TO EXOCORTIS. In

Wallace, J. M. (ed.). Citrus Virus Diseases, 1957, pp. 135-

140. Univ. Calif. Div. Agr. Sei., Berkeley.

(9)

1961. A QUICK FIELD TEST FOR EXOCORTIS. In Price, W. C. (ed.), 2d

Conf. Internatl. Organ. Citrus Virol. Proc. 1960, pp. 40-42.

(10) OLSON, E O., SLEETH, B., and SHULL, A. V.

1958. PREVALENCE OF VIRUSES CAUSING XYLOPOROSIS (CACHEXIA) AND

EXOCORTIS (RANGPUR LIME DISEASE) IN APPARENTLY HEALTHY

CITRUS TREES IN TEXAS. Rio Grande Val. Hort. Soc. Jour.

12: 35-43.

(11) ROSSETTI, V.

1961. TESTING FOR EXOCORTIS. In Price, W. C. (ed.), 2d Conf. Internatl. Organ. Citrus Virol. Proc. 1960, pp. 43-49.

(12) SALIBE, A. A.

1961. CONTRIBUCAO AO ESTUDO DA DOENCA EXOCORTE DOS CITROS. Thesis, Escola Superior de Agricultura "Luiz de Queiroz," Univ.

de Sao Paulo, 71 pp.

(13) and MoREiRA, S.

1965. SEED TRANSMISSION OF EXOCORTIS VIRUS. In Price, W. C. (ed.),

3d Conf. Internatl. Organ. Citrus Virol. Proc. 1963, pp. 139-

142.

(14) WEATHERS, L. G.

1965. TRANSMISSION OF EXOCORTIS VIRUS OF CITRUS BY CUSCUTA SUBINCLUSA. U.S. Agr. Res. Serv. Plant Dis. Rptr. 49: 189-

190.

(15) and CALAVAN, E. C.

1961. ADDITIONAL INDICATOR PLANTS FOR EXOCORTIS AND EVIDENCE FOR

STRAIN DIFFERENCES IN THE VIRUS. Phytopathology 51: 262-

264.
STUBBORN

By E. C. CALAVAN ^

SYMPTOMS

Stubborn disease affects all commercial varieties of citrus regardless of rootstock. The wide range of symptoms includes

bunchy upright growth of twigs and branches, with short internodes and excessive numbers of shoots; multiple buds; thickened

and sometimes pinholed bark; slight to severe stunting; small or

misshapen leaves or both, which frequently are mottled, dappled,

or chlorotic; excessive winter defoliation; unseasonal blossoming; low production of fruit, of which some may be very small,

lopsided, or otherwise deformed, including acorn shaped (fig.

1, B); dense or cheesy peel; stylar-end greening, or inverted development of ripe coloration (fig. 1, A) ; premature drop of fruit;

excessive number of mummified fruits; insipid, sour, or bitter

fruit flavor; and large numbers of poorly developed, discolored,

and aborted seeds (fig. 1, C) (1).

Stubborn symptoms are highly variable and frequently only a

few are expressed at one time. Trees with severe fruit symptoms

of stubborn disease may have relatively normal foliage and shoots;

trees with strong foliage and shoot symptoms may bear many

normal fruits. Trees may show symptoms on some parts, whereas

the remainder of the tree appears normal. Fruit and shoot symptoms tend to be more pronounced on the south half of the tree

in hot climates of the northern hemisphere.

INOCULATION METHOD

Stubborn disease has been repeatedly transmitted to healthy

citrus plants by tissue grafts and is believed to be caused by a

virus or virus complex. The plants reacting to experimental graft

inoculation have ranged from 100 to less than 5 percent. This variation suggests that the pathogen is distributed irregularly in the

host. Therefore, considerable replication of candidate tissues

and of index plants is needed to obtain reliable data.

A total of 20 or more tissue samples should be put into 10 or

more indicator plants. Each tissue sample used as inoculum should

be taken from a separate shoot, and shoots should be chosen to

represent all parts of the tree as far as possible. Seed for indicator and rootstock plants for index tests should be derived,

1 This section was reviewed by J. B. Carpenter and J. Cassin.

35
36 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

when possible, from disease-free parent trees. Indicator plants

should be vigorous and free from nutritional problems.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 37

INDICATORS

Seedlings of several varieties of sweet orange (Citrus sinensis

(L.) Osbeck), tángelo (C. paradisi Macf. X C. reticulata Blanco),

grapefruit (C. paradisi Macf.), and mandarin (C. reticulata

Blanco) are highly sensitive indicator plants for stubborn disease and suitable for indexing purposes. Numerous alternative

indicator plants are known, including the clonal seedling lemons

previously reported (2, 3). Many susceptible varieties may serve

as self-indicators, especially in the field.

INDEXING PROCEDURES

The indexing method, described in conjunction with inspection

of the candidate tree and its progeny, is at present the best rapid

test available for stubborn disease. However, it has not been

tested sufficiently to insure complete reliability under all conditions and may fail to detect some cases of stubborn disease.

Short-term indexing requires from 2 to 8 months. Good results have been obtained in heated glasshouses with pencil-sized

Madam Vinous or Hinckley sweet orange seedlings inoculated

with side grafts or tip grafts from 50 to 100 mm. long (figs. 2

and 3) during the spring or early summer. More than one piece

of candidate inoculum (buds or stem pieces) may be grafted

into each indicator seedling. Grafts should be protected from

drying until they have healed well. Multiple shoot growth should

be prevented. Instead, a single unbranched shoot (figs. 2 and 3)

should be forced from near the inoculation site and inspected

frequently for leaf and shoot symptoms. Several control seedlings should be grafted in exactly the same manner as the index

plants, substituting their own or other healthy seedling tissues

for candidate inocula.

Stubborn symptoms in glasshouse-grown seedlings of sweet

orange include slight to severe stunting, short internodes, and

small, cupped, somewhat chlorotic leaves (fig. 4). Marginal and

interveinal areas near the tips of nearly grown leaves may be

pale green (fig. 5, A, left). Leaf blades may be variously dappled and mottled with light and dark green (fig. 5, A, center)

or may occasionally show dark-green spotting similar to that

observed on stubborn-diseased lemon plants (2).

When seedling indicator plants are not available, buds of both

the indicator and the candidate plant may be grafted or budded

with a third plant that serves as a carrier for both.

Field indexing usually requires from 15 to 24 months after

spring or summer inoculation of pencil-sized indicator seedlings with two or more side grafts from 50 to 100 mm. long.

Inoculated seedlings usually are grown in the nursery until the

following spring, then cut back to about 60 cm. in height, and

transplanted to an index plot. Topping or bending plants a month

after inoculation forces growth of a single shoot near the inoculation sites and may accelerate the reaction. Excessive growth

from the candidate grafts should be prevented, although some

foliage is desirable.
38 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

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INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 39
40 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

„ BN-2S394

FIGURE 4.—Madam Vinous seedling- sweet orange plants: Left, healthy control, tip-grafted with healthy tissue. Right, plant 3% months after inoculation in greenhouse with tip graft from stubborn-infected tree; note short

internodes and small leaves.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 41

FIGURE 5.—Leaf symptoms of stubborn disease: A, left, pale-green marginal

and interveinal areas near tip of expanding leaf of Madam Vinous sweet

orange seedling in glasshouse; A, center, light-colored dappling and mottling in fully expanded leaf of Madam Vinous sweet orange seedling in

glasshouse; A, right, dark-green spotting in mature leaf of field-grown

Koethen sweet orange seedling; B, left, normal leaf from control plant;

l>, right, field-grown Sexton tángelo leaves showing chlorosis accompanied

by yellow streaking along veins.

Positive reactions to stubborn disease infection may develop

as follows: General stunting- with short internodes, numerous

growing points, and small, rigid, often upright, cupped leaves

(fig. 6). The leaves may display a variety of pale-green or

yellowish patterns in the form of dappling, mottling, interveinal

chlorosis, or yellow banding and streaking along the veins (fig.

5, B). Some leaves show green spotting on a pale-green to greenish-yellow or creamy-yellow background (fig. 5, A, right). Leaf

symptoms change rapidly. They often resemble patterns caused

by nutritional imbalance and are not diagnostic unless absent

in the control plants. On severely affected plants many leaves

age prematurely and drop during the winter.
42 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

Sexton tángelo, Marsh grapefruit, Parson's Special mandarin,

and Koethen sweet orange seedlings are among the varieties

that have been successfully used in California as indicator plants

in the field. Stubborn disease often can be detected by examining the fruit and the vegetative characteristics of a candidate

tree and its graft-propagated progeny. Symptoms that are masked

in large parent trees often appear well defined in progeny trees

in the nursery or young orchard. Annual inspections and accurate records of bud-progeny trees are essential in indexing for

stubborn disease.

I3N-2s;iS-l BN-2ÍJ3S3

FIGURE 6.—Sexton tángelo seedlings in long-term index block: Left, healthy

control; right, tree 2 years after inoculation is stunted, with small, upright,

chlorotic leaves.

LITERATURE CITED

(1) CALAVAN, E. C, and CARPENTER, J. B.

1965. STUBBORN DISEASE OP CITRUS TREES RETARDS GROWTH, IMPAIRS

QUALITY AND DECREASES YIELDS. Calif. Citrograph 50: 86-87,

96, 98-99.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 43

(2) and CHRISTIANSEN, D. W.

1961. STUNTING AND CHLOROSIS INDUCED IN YOUNG-LINE CITRUS

PLANTS BY INOCULATIONS FROM NAVEL ORANGE TREES HAVING SYMPTOMS OF STUBBORN DISEASE. In Price, W. C. (ed.),

2d Conf. Internatl. Organ. Citrus Virol. Proc. 1960, pp. 69-

76.

C¿) CASSIN, eJ. . -r^ .

1965. STUDIES ON STUBBORN DISEASE OF CITRUS IN MOROCCO. In Price,

W. C. (ed.), 3d Conf. Internatl. Organ. Citrus Virol. Proc.

1963, pp. 204-206.
VEIN ENATION AND WOODY GALL

By J. M. WALLACE i

Studies in California have now established that vein enation

and woody gall of citrus are caused by the same virus. This

virus has been reported from the United States (Calif.), Republic of South Africa, Australia, Peru, and Japan. Numerous

kinds of citrus are symptomless hosts of this virus; others develop only the vein enation symptoms. No injurious effects are

apparent in conjunction with the latter.

The woody gall phase of this disease is potentially damaging

to citrus trees. For example, woody gall has caused some alarm

m Peru, where many new plantings of citrus consist of sweet

orange (Citrus sinensis (L.) Osbeck) on Rough lemon (C. limon

(L.) Burm. f.) rootstock. These plantings include trees up to

10 years of age, and a high percentage of the trees in some locations have developed extensive galls on the Rough lemon rootstock.- No significant reduction in yields of affected trees is apparent, but the trunks of trees with considerable gall development are usually somewhat smaller than normal. This symptom

at least suggests that eventually aflfected trees will not perform

as well as those without galls.

The virus is transmitted by at least three species of aphids—

Toxoptera citricida (Kirkaldy), Myzus persicae (Sulzer), and

Aphis gossypii Glover. Thus, once the virus is introduced to a

citrus-growing region, it may become widely distributed within

a short time. Infections observed in nursery plantings in Peru

are probably caused by aphid vectors as well as from using infected budwood. Some of the nursery trees were stunted by the

development of galls when ready for transplanting.

SYMPTOMS AND HOST RANGE

Vein Enation

Symptoms on leaves range from small swellings to conspicuous projections on veins on the under surface of the leaves (fig.

1, ^). On the upper surface shallow indentations coincide with

the peghke structures on the opposite side (fig. 1, B). To the

touch and the unaided eye, these structures seem sharply pointed,

1 This section was reviewed by Lilian R. Fraser and A. P. D. McClean

- Vein enation virus is widespread in South Africa, although the incidence

/A ^S -K^^P^^Î^^ ^? ^^^ ^^ ^^^^ ^^^^s ^^^^ ^he incidence of galls is even lower (A. F. D. McClean).

44
INDEXING PROCEDURES FOR 16 VIRUS DISEASES OF CITRUS 45

BN'-28;!!)3

FiGURK 1.—Vein enations on leaves of Mexican lime: A, Vein swelling and

enations on under surface of leaf; B, indentations on upper surface of leaf

corresponding to enations on opposite side.

but on close examination some are found to be knoblike or cup

shai)ed. The number and size of the enations vary widely depending- on the variety of citrus affected.

Extensive host-range studies have not been made, but present

information indicates that all the commercially used citrus scion

and rootstock varieties are hosts of the virus, although sorne

develop no vein enations. Mexican lime (C. aurantifoUa

(Christm.) Swing.), some sour lime varieties, and several sour

orange {C. aurantium L.) selections develop pronounced enations in California in both the field and greenhouse. Rough lemon

and sweet orange develop some leaf enations in the greenhouse,

but show less conspicuous symptoms in the field.

Woody Gall

On small green twigs, the galls first appear as slight swellings, over which the bark becomes grayish as the galls enlarge.

On seedlings of Mexican lime and Rough lemon, galls may be
46 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

recognized on small green twigs 8 weeks after infection, but they

are inconspicuous in the early stages. Usually galls on the main

stem of seedlings develop close to thorns (fig. 2, A) and near

the graft-inoculation sites.

As their size increases, galls become irregular in shape and

the bark covering the gall tissue becomes rougher and lighter

colored than normal. Adjacent galls may coalesce as they become

larger and form "cauliflowerlike structures'' (fig. 2, B and C), as

originally described by LiHan Fraser. The gall tissue is very hard

and extends in a cone shape from the point of origin, which is

usually in or near the pith region of the stem (fig. 2, D).

Characteristically the cambial face of the gall is lightly pitted

because of numerous undeveloped or latent buds and is light green

because of chlorophyl (fig. 3, A). Shoots develop sometimes from

the latent buds on old galls. These characteristics assist in distmguishing true woody galls from other swellings near the base

of thorns and near leaf or twig scars (fig. 3, B) that superficially

resemble the early stages of woody gall. When the bark is removed from such structures, they are found to consist of a raised

portion of wood with a short, sharply pointed projection folded

over a slight crease in the wood (fig. 3, C). These structures apparently are undeveloped thorns.

When grown in soil, the roots of infected lime seedlings occasionally develop a few galls, but when maintained in water culture, great numbers of galls develop over the root system (fig.

3, D).

Information is limited on the kinds of citrus that develop woody

galls. The galls appear commonly on Rough lemon and Mexican

lime. In California studies galls failed to develop on sweet and

sour orange, Eureka lemon, C. excelsa Wester,'^ and Palestine

sweet hme (C. aurantifolia (Christm.) Swing.) even when infected plants were experimentally wounded. One seedling of Rangpur hme (C. limonia Osbeck)^ developed a few galls, but others

did not, even after experimental wounding. However, an unidentified mandarin-lime variety, recorded only as Indian lime, is very

susceptible to gall development.

INDEXING

Mexican lime and sour orange are good indicators. Test seedlings should be inoculated with a twig graft and examined later

tor leaf enations. Vein swellings or the early stages of enations or

both sometimes appear 4 to 5 weeks after inoculation by grafting.

OTHER KINDS OF GALLS ON CITRUS

Several types of galls or gall-like growths not associated with

^^ According to P. C Reece this variety is known by the common names

Le Nestour and Limon Real (J. F. L. Childs).

^ The author prefers C. limonia Osbeck over C. reticulata var. austera

hybrid for Rangpur lime. The taxonomy and nomenclature of this variety of

Indus^r^iJ F^rChilLf- ''' ^'''' ^" ''^ ^^^^"^ ^^^^^^^ '' ^^^ ^^^^^
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 47

the vein enation virus have been observed on citrus. Some appear

to be genetic, or at least not of virus origin, since they cannot

be reproduced. Several transplants of tissue from these galls resulted in enlargement of the transplanted tissue only. Inasmuch

BN-28380

FIGURE 2.—Woody galls on Rough lemon: A, Galls developing near thorns;

B, galls becoming irregular in shape and coalescing; C, cauliflower-shaped

gall on orchard tree; D, section through small gall showing V-shaped

development from point near center of stem.
48 AGRICULTURE HANDBOOK 3 33, U.S. DEPT. OF AGRICULTURE

BN-28388

FIGURE 3.—Woody gall of citrus: A, Virus-induced woody gall with bark

removed to show pitted appearance (adventitious buds) ; B, swellings near

thorns of Rough lemon resembling early stage of woody gall; C, bark

removed from swellings shown in B revealing short thornlike structures;

V, woody gall development on roots of Mexican lime grown in water culture
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 49

as some of them may closely resemble true woody galls, diagnosis

is best made by inoculation from affected trees to indicators for

vein enation virus.
GUMMY BARK OF SWEET ORANGE

By FARID NOUR-ELDIN i

Gummy bark disease of sweet orange (Citrus sinensis (L.) Osbeck) was first reported (5) to affect baladi '' sweet orange trees

grafted on sour orange (C. aurantium L.) rootstock in Egypt.

Later it was found (3) to affect several other sweet orange varieties: Valencia, navel, Sukkary, Khalili white, Egyptian blood,

and Sanguinoval. More than 50 percent of the examined trees

exhibited symptoms of this disease. Gummy bark was observed

on sweet orange trees in Saudi Arabia (Nour-Eldin, 1959, unpunished) and in the Sudan (Childs, 1964, unpublished). A mild

form of gummy bark was reported from Florida U) and Egypt

(6) on sweet orange trees grafted on Rough lemon (C. jambhiri

Lush.) (see p. 92) rootstock.

SYMPTOMS

Orchard trees affected with gummy bark disease are usually

stunted. When the bark of affected trees is scraped carefully,

circumferential streaks of reddish-brown, gum-impregnated tissue can be easily seen, particularly near the bud union (fig.l).

Occasionally the discoloration extends 24 inches or more into

sweet orange tissue above the bud union. In such severe cases,

streaks of gum-impregnated tissue may be either longitudinal

or circumferential and dark brownish. Bark discoloration has

never been observed on the sour orange rootstock of infected trees.

In addition to gum impregnation of the bark, pitting of the wood

of sweet orange is common. Two types have been observed. One

gives a grooved or channeled appearance to the cambial face of

the wood, and the other is a round conoid type of pitting (fig. 2).

The latter is the more common. Gum impregnates the inner tissue

of the pegs, projecting from the cambial face of the bark and

coinciding with the pits in the wood. Severe stem pitting does not

necessarily accompany gum impregnation of the bark. In many

cases stem pitting is mild and superficial. When severe pitting occurs on the scion, the sour orange stock occasionally exhibits very

slight wood pitting.

TRANSMISSION TESTS

Gummy bark disease was transmitted experimentally by bud in1 This section was reviewed by L. C. Knorr and J. F. L. Childs.

2 Arabic word meaning local.

50
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 51

oculations from infected trees to healthy sweet orange trees

budded on sour orange rootstock (Nour-Eldm, unpubhshed).

About 5 years after inoculation, a red-brownish circumferential

line was observed at the bud union after scraping away the corky

surface of the bark. Dark-brownish streaks can be seen m various

degrees of severity after scraping the bark on older affected trees.

Wood pitting may be mild on 5-year-old trees, but is seldom clearly

apparent until a year or two later.

Indexing of infected sweet orange trees was performed on rootstocks of Orlando tángelo (C. paradisi Macf. X C. rehculata

Blanco), sour orange. Rough lemon, Poncirus trifoliata (L.) Rat.,

citrange (P. trifoliata (L.) Raf. X C. sinensis (L.) Osbeck), citron (C. medica L.), and Rangpur lime (C. reticulata var. austera

hybrid). Results showed that gummy bark disease usually is accompanied by several other viruses, e.g., psorosis, cachexia, exoFlGURE 1.—Sweet orange on sour orange rootstock, showing gum impregnation of bark and pitting of wood above union.

cortis (Rangpur lime disease), and bud union constriction of sweet

orange trees on Rough lemon rootstock. Consequently, a test plant

highly specific for gummy bark virus is required.

In one of these tests, an indexed tree induced gummy bark symptoms on sweet orange grafted on sour orange and bud union constriction (fig. 2) of sweet orange grafted on Rough lemon rootstock. When grafts from the same source were used to inoculate

healthy sweet orange trees on Orlando tángelo rootstock, the rootstock did not react to give symptoms of cachexia disease, indicat-
52 AGRICULTURE HANDBOOK 3:í;í, U.S. DEPT. OF AGRICULTURE

ing that gummy bark disease is not caused by the cachexia virus

(1,2). This particular indexed tree was found to be carrying exocortis virus together with gummy bark disease. Nevertheless, exocortis was not found in other indexed trees that induced gummy

bark symptoms on sweet orange trees grafted on sour orange rootstock and bud union constriction of Rough lemon rootstock. This

finding indicates that exocortis (Rangpur lime disease) does not

induce the gummy bark symptoms described on sweet orange trees

grafted on sour orange or Rough lemon rootstocks.

FIGURE 2.—Sweet orange on Rough iemon rootstock, showing constriction and

pitting at union.

Previous indexing of virus-infected trees showed that Rough

lemon rootstock always developed bud union constriction when

gummy bark symptoms developed on sweet orange trees grafted on

sour orange rootstock. This suggests that gummy bark of sweet

orange on sour orange stock and bud union constriction of sweet

orange on Rough lemon stock are caused by the same agent.

Comparison of infected trees with healthy ones shows that the

former are much smaller and thinner as a result of the reduced

growth and severe leaf drop that occur during the winter. Setting

of fruit is affected as trees become old enough to bear fruit.

INDEXING PROCEDURE

Identification of this disease is based on the development of gum

deposits in the bark of sweet orange trees grafted on sour orange

rootstock. Another scion-stock combination that reacts to this

disease is sweet orange on Rough lemon rootstock. Symptoms, as

described above, develop on inoculated sweet orange trees grafted

on sour orange in about 5 years.

The symptoms described for infected sweet orange trees grafted

on Rough lemon rootstock are clearly apparent 4 years after indexing. When bark is removed from across the bud union, an irregular
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 53

and almost continuous ring of projections is present on the cambial

face of the bark at the bud union line, which fits into the crease

in the wood (fig. 2). Gum deposits are present in the bark tissue

of these projections. Mild stem pitting and bark discoloration can

be observed on both scion and rootstock. Such conspicuous symptoms make this combination of sweet orange on Rough lemon rootstock very useful as an indicator plant for gummy bark disease

of sweet orange on sour orange rootstock.

LITERATURE CITED

(i) GUILDS, J. F. L. n C^ A D

1950. THE CACHEXIA DISEASE OF ORLANDO TÁNGELO. U.b. Agi*. KeS.

Serv. Plant Dis. Rptr. 84: 295-298.

(2)

1952. CACHEXIA DISEASE, ITS BUD TRANSMISSION AND RELATION TO

XYLOPOROSis AND TO TRISTEZA. Phytopathology 42: 265-268.

(3) NOUR-ELDIN, F., and EL-HOSSEINY, N.

1956. OBSERVATIONS ON EGYPTIAN CITRUS DISEASES. Citrus Indus. 37

(10): 11-16.

(4) GRIMM, G. R., GRANT, T. J., and CHILDS, J. F. L.

1955. A BUD UNION ABNORMALITY OF ROUGH LEMON ROOTSTOCK WITH

SWEí:T ORANGE SCIONS. U.S. Agr. Res. Serv. Plant Dis.

Rptr. 39: 810-811.

(5) NOUR-ELDIN, F.

1956. PHLOEM DISCOLORATION OF SWEET ORANGE. Phytopathology 46:

238-239.

(6)

1959. CITRUS VIRUS DISEASE RESEARCH IN EGYPT. In Wallace, J. M.

(ed.). Citrus Virus Diseases, 1957, pp. 219-227. Univ. Calif.

Div. Agr. Sei., Berkeley.
YELLOW VEIN

By L. G. WEATHERS ^

SYMPTOMS

Symptoms of yellow vein are similar in different citrus species

and varieties, but are most striking in West Indian lime {Citrus

aurantifolia (Christm.) Swing.) > Rough lemon (C. limon (L.)

Burm. f.), and Etrog citron {C. 'medica L. var. ethrog Engl.).

The first symptom to appear on the developing leaves is yellowing

of the main veins. Yellowing is usually continuous along the

petiole, midrib, and main veins (fig. 1). Smaller veins may not be

uniformly aflPected, and frequently there are yellow areas separated by areas of normal green. Chlorosis is sometimes more pronounced on one half of the leaf than on the other, and it appears

on both the upper and lower surfaces of the leaf.

1 This section was reviewed by E. C. Calavan and J. B. Carpenter.

FIGURE 1.—Etrog citron leaves with yellow vein virus alone (left) and with

yellow vein and vein enation viruses (right).

54
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 55

In some cases inoculated plants fail to show typical yellow vein

symptoms and in many cases plants with mild symptoms recover;

i.e., no further symptoms develop after the initial appearance of

symptoms. Yellow vein symptoms are almost completely masked

in plants infected with both yellow vein and psorosis viruses.

However, in the presence of vein enation virus, symptoms of yellow vein are greatly enhanced and are more severe than in plants

infected with yellow vein virus alone or in plants containing both

yellow vein and psorosis viruses.

All available evidence indicates that yellow vein virus is not

widely distributed and may occur naturally only in the few limequat trees in which it was originally described in California.

INDEXING PROCEDURE

The unusual and erratic behavior of yellow vein virus when it

occurs alone and in combination with psorosis makes identification of yellow vein virus unreliable under those conditions. Yellow vein virus is diagnosed more reliably by combining it with

vein enation virus. The recommended procedure is as follows:

Inoculate young West Indian lime, Rough lemon, or citron plants

with a pure culture of vein enation virus. Any time after the

plants show well-developed enation symptoms, graft inoculate

these plants with tissue from plants suspected of having yellow

vein virus. If the yellow vein virus is present, severe yellow vein

symptoms will appear in the developing leaves of the doubly

infected plants after 2 to 6 weeks.
SATSUMA DWARF

By SHOICHI TANAKA ^

Dwarf disease of satsuma orange (Citrus reticulata Blanco) has

been recognized for more than 30 years in Japan. At the present

time the disease is known throughout Japan and is of economic

importance in several prefectures. Spread occurs mainly through

propagation of infected budwood. Affected trees deteriorate

slowly and growers usually postpone removing them. This contributes to inferior fruit production.

SYMPTOMS

From May to June, when leaves begin to elongate, is the best

time to detect symptoms of satsuma dwarf. During this period

very young leaves of even mildly affected trees curl downward

(abaxially). However, as the leaves mature, symptoms become

difficult to detect.

On severely affected trees, leaves remain abnormal even after

elongation and hardening. They show abnormal shapes of two

types—narrow boat shape and dwarfed spoon shape. Only one

type may appear on a single twig, but generally both appear together. Also, internodes are usually shortened, giving the twig

a witches'-broom appearance.

Fruits on mildly affected trees are normal or nearly so, but

when leaf symptoms are severe, the fruit is usually smaller and

of inferior quality, with the rind thickened at the stem end of the

fruit.

Symptoms occur mainly on spring growth. Summer and autumn flushes show no symptoms even though the trees are severely

diseased. When diseased scions are grafted on a healthy tree in

the spring, all the buds that come out and develop later that year

appear healthy. However, all the buds that come out during the

following spring from summer scions show abnormally shaped

leaves. When infected scions are top-grafted on 15-year-old satsuma trees, symptoms soon appear on other parts of the tree and

entire branches show symptoms several years after inoculation.

When this experiment is repeated with young nursery trees, ho\yever, all of them show symptoms by the following spring.

MECHANICAL TRANSMISSION

A virus has been transmitted mechanically from satsuma dwarf1 This section was reviewed by Shunichi Yamada and J. M. Wallace.

56
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 57

infected citrus trees to herbaceous plants. Blackeye cowpea

(Vigna sinensis (Torner) Sari), Satisfaction kidney bean {Phaseolus vulgaris L.), and white sesame (Sesamium indicum L.) gave

the strongest reactions of 35 species of legume and many nonleguminous species tested. Cross-inoculations of the virus from

satsuma dwarf-affected trees, between sesame and Blackeye cowpea or Satisfaction kidney bean, were positive. Therefore, the

virus is believed to be identical with the so-called satsuma dwarf

virus. Sesame proved to be susceptible only to satsuma dwarf

virus, but nonsusceptible to tristeza and vein enation viruses,

which are commonly carried in satsuma orange trees.

Sesame is more heat tolerant than most leguminous plants and

is useful as an indicator for this virus even in the summer. When

sesame plants were exposed to a temperature above 34"^ C. just

after inoculation, they usually showed no symptoms, but sometimes systemic infection appeared 10 days after inoculation. On

the other hand, when inoculated sesame plants were kept more

than 8 hours at 25°, they showed striking symptoms even if exposed subsequently to temperatures higher than 36"^. To date the

virus has not been transmitted from legumes or sesame to satsuma orange. When Mexican lime seedlings were inoculated from

satsuma dwarf-affected trees as well as apparently healthy trees,

tristeza symptoms invariably developed.

PROCEDURE FOR MECHANICAL TRANSMISSION

To perform the herbaceous plant test for satsuma dwarf, the

following procedure is recommended: Indicator plants—white

sesame, Blackeye cowpea, and Satisfaction kidney bean—should

be grown in small pots or cans in the greenhouse. The white

sesame seedlings are inoculated with expressed sap on the first

or second true leaves. Seedlings of cowpea or kidney bean are

inoculated on pairs of the primary leaves.

The sap should be prepared from young shoots or fruit rind

from satsuma dwarf-affected trees by grinding with an equal to

twice equal volume of 0.05 to 0.10 molar solution of potassium

acid phosphate or phosphate buffer of pH 7.0 in a small mortar.

The plants are inoculated by the usual rubbing method, using

carborundum as an abrasive. Inoculated plants should be kept

in the screenhouse at a temperature below 32° C. The optimum

temperature is about 28° for sesame and about 25° for the

legumes.

DIAGNOSIS

The standard and most reliable method of diagnosis is with

white sesame. Several days after inoculation the plants develop

local lesions, which are chlorotic spots at first and later become

necrotic spots with a yellowish halo. The systematically infected

leaves first exhibit vein clearing, malformation, and curling. In

later stages these leaves show vein necrosis. Necrosis spreads

to the adjacent mesophyll tissue causing the death of the entire

leaf. (Figs. 1 and 2.)
58 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

FIGURE 1.—Symptoms of virus from satsuma dwarf-affected trees on white

sesame: A, Local lesions and vein clearing; B, vein clearing, yellowing,

and dwarfing on systemically infected leaves; C, yellowing and necrosis in

later stage.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 59

FIGURE 2. Symptoms of virus from satsuma dwarf-affected trees on Satisfaction kidney bean: A, Necrotic streaks on petioles and stem; B, vem

clearing and mosaic.
IMPIETRATURA

By GAETANO RUGGIERI ^

Impietratura or stonelike disease of citrus fruits has been

known for many years in Sicily and more recently in several

other countries of the Mediterranean basin. It affects chiefly

orange (Citrus sinensis (L.) Osbeck), but it has been found in

grapefruit (C. paradisi Macf.), Clementine mandarin (C. reticulata Blanco), and tángelo (C. paradisi Macf. x C. reticulata

Blanco) trees. It has not been found in lemon (C limon (L.)

Burm. f.).

SYMPTOMS

The disease can be detected, especially when fruits are green

and actively growing, by noting the presence of small fruits

of one-fourth to one-third their normal size near normal-sized

fruits on the same tree. The percentage of small fruits varies

considerably, but in extreme cases trees produce only small fruits.

Three kinds of fruits may be picked from an affected tree:

Normal ones of standard shape and size, with an elastic rind

and without gum pockets in the albedo; apparently normal fruits

of standard shape and size, with an elastic rind except over gum

pockets in the albedo, and no gum pockets under the calyx; and

very small fruits of one-half to one-third normal size, pear shaped,

and very hard, with gum pockets in the albedo around the fruit

and also under the calyx. Less often the vascular bundles of the

central axis are invaded with gum. Small fruits always show

severe internal characters of the disease; normal-sized fruits have

milder symptoms or none at all. Hard, brown, gum-beaded areas

in the albedo may be seen by cutting across the fruit near the

base and in the part under the stem (fig. 1). Gum may be present

in the vascular bundles of the stem as much as several centimeters

away from the fruit.

In the most severe cases, the gum-containing tissues may advance inward toward the pulp and thus affect the central axis

(core) and the peripheral juice vesicles, which become dry and

1 This section was reviewed by Henri Chapot. Shortly before the author^s

death, he prepared a brief statement (Feb. 23, 1965) on the method he used

to transmit impietratura. The symptoms described here were taken from

Chapot, Henri, Impietratura in Mediterranean Countries, and Ruggieri, Gaetano, Observations and Research on Impietratura. In W. C. Price (ed.), 2d

Conf. Internatl. Organ. Citrus Virol. Proc. 1960, pp. 177-181 and 182-186.

1961.

60
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 61

0) .-^

D5 St:
62 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

brown, and they may advance outward toward the glandular and

interglandular tissue of the flavedo. As seen from the exterior,

these tissues appear slightly depressed and contain brown glands

that resemble the spots of petechia relata or slight patua. Sometimes the interglandular tissue may not be depressed but rather

protruding, and the affected area is well distinguished by the

brown color of the single glands as seen from the exterior. By cutting tangentially through the protruding areas, cavities with fluid

or coagulated gum up to 1 cc. in volume may be seen in the albedo

(fig. 1).

INDEX PROCEDURE ^

It is advisable to use 2- or 3-year-old grapefruit plants grafted

on sour orange (C. aurantium L.) as indicator plants. From September through October inoculate on the branches of the young

grapefruit trees with bark pieces taken from the candidate tree.

Sweet orange also may be used as an indicator plant. The test

grapefruit or sweet orange plant must be old enough to produce

fruit. A year after inoculation, fruits developing in the spring

on the inoculated branches will present characteristic symptoms

of impietratura if the candidate tree carried the virus.

- Translated by Victoria Rossetti.
LIKUBIN

By T. MATSUMüTO, H. J. Su, and T. T. Lo i

Likubin has long been known as a nonparasitic disease in Taiwan. However, experiments by the authors since 1956 have shown

that this disease is due to a virus closely related to the tristeza

virus. Likubin is now generally called huanglunpin, or yellowing

of scions, because a disease of similar characteristics was observed on the China mainland and reported by C. P. Cheng (1)

in 1943 under this name. However, direct comparison is not possible under existing circumstances.

SYMPTOMS

Although the disease syndrome differs somewhat as host varieties differ, the common symptoms are decline in vigor, yellowing

or mottling of leaves, premature defoliation, dieback of small

twigs, formation of multiple abnormal flowers, decay of feeder

rootlets and lateral roots, and wilting of leaves followed by death

of the entire plant.

The characteristic symptoms of likubin on several varieties of

citrus are as follows: On Ponkan (Citrus reticulata Blanco), the

leaves of diseased trees exhibit overall chlorosis or yellowing of

veins and of mesophyll tissues surrounding the veins (figs. 1 and

2), and less frequently yellowing of the mesophyll tissues with the

veins remaining green. This variation in symptom expression may

be associated with some environmental conditions or with mineral

deficiencies resulting from the virus infection. The vein corking

produced on diseased leaves later is apparently similar to the

symptom noticed on the Mexican lime (C. aurantifolia (Christm.)

Swing.) seedlings inoculated with this virus. Young leaves that develop after premature defoliation are usually small and pale green.

Diseased trees tend to blossom early and heavily, but flowers

are very small and abnormal, are prematurely shed, and fail to

produce fruits. Infected trees occasionally exhibit dieback of

branches, yellowing or mottling of leaves, and defoliation on one

side, whereas the other side is apparently normal in appearance

(fig. 3).

On Tankan (C reticulata Blanco), general decline symptoms

are similar to those of the diseased Ponkan, although defoliation

usually seems to be quicker for Tankan. On sweet orange (C.

sinensis (L.) Osbeck), leaf symptoms differ according to variety,

1 This section was reviewed by J. M. Wallace and L. G. Weathers.

63
64 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

*? j^ 4,,,.

FIGURE 1.—Likubin symptoms on four Ponkan leaves (left) and normal leaf

(right).

but for varieties such as Valencia, Sekkan, Washington navel, and

Luicheng, the more prominent symptoms are general yellowing

or mottling of young leaves, particularly yellowing of veins and

adjacent tissue, while the major part of the mesophyll remains

green for a considerable time. Stem pitting is observed occasionally in Washington navel.

Symptoms of likubin on the leaves of stocks grafted with infected scions are as follows: On Sunki (C. reticulata var. austera

Swing.), the leaf symptoms are not constant, differing sometimes as the source of scion differs, but the following symptoms

are noticed most frequently: The leaves usually exhibit indistinct

chlorotic areas between the main lateral veins in from 3 to 4

months, occasionally as early as 2 months after grafting. The

chlorotic areas are variable in size and shape, being elongated,

circular, or stippled and thus resulting in irregular spotting or

mottling with an indistinct margin. This chlorotic discoloration

intensifies in time, though rather slowly.

On seedlings of sour orange {C. aurantium L.) and of Mexican

lime infected by grafting with Ponkan scions, the symptoms are

as follows: Sour orange leaves become chlorotic and stunted, usually a few months after grafting, and young leaves of Mexican

lime develop dashlike chlorosis of lateral veins of young leaves

that can be seen by transmitted light. This symptom progresses

into vein clearing followed by vein corking.

The reaction of Eureka lemon (C. Umon (L.) Burm. f.) to

likubin is as follows: A mild virus strain, UO 14, from Valencia

orange and a severe virus strain, YMS 15, from Ponkan mandarin

were tested with Eureka lemon seedlings (Eraser's test). In both

cases, yellowing and stunting of leaves and some cessation of

plant growth were noticed on the Eureka lemon seedlings. The
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 65

^ -.-'i^A ^^" ^

FIGURE 2.—Twigs from likubin-infected (left)

Ponkan tree.

and from healthy (right)

symptoms on the leaves of these trees inoculated w^ith two different strains were almost the same, presumably similar to the

severe type of symptoms described by Fraser (2).

Response of stock-scion combinations to the disease is as follows: Trifoliate orange (Poncirus trifoliata (L.) Raf.), Cleopatra

mandarin (C. reticidata Blanco), Troyer citrange (P. trifoliata X

C. sinensis), Yuzu {C. ichangensis x C. reticidata var. austera),

Citrus tachibana (Makino) Tan., and tangerine (C. reticulata

Blanco) were used as rootstocks and were mostly topworked with

Ponkan scions. The Cleopatra rootstock was rather intolerant to

strains of the virus used by the authors, although this rootstock is

considered tolerant of tristeza in North America and South

America.

Trifoliate orange rootstock was more tolerant than any others

tested, which accords with reports from other countries. However,

it is noted that this rootstock is by no means absolutely tolerant

of all the virus strains existing in Taiwan. For instance, trifoliate

orange grafted with Ponkan scion has also been affected, though

not seriously, by a newly isolated severe strain YLA, which was
66 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

FIGURE 8.—Likubin-infected Ponkan tree partially revealing symptoms.

derived from a diseased Ponkan and proved positive when tested

with Mexican hme. It was also found through field surveys and

experiments that Rangpur lime (C reticukda var. austera hybrid) is highly tolerant of the virus.

TRANSMISSION BY APHIDS

The virus causing likubin was transmitted by Toxoptera citricida (Kirkaldy) as follows: Nonviruliferous aphids were fed on

diseased citrus trees for 24 hours and then transferred to Mexican lime seedlings (25-70 aphids per plant.) After feeding 24

hours, the aphids were removed and the plants were sprayed with

malathion solution (0.02 percent). The young leaves of Mexican

lime seedlings infested with viruliferous aphids developed vein

clearing in about 40 to 60 days. From these results, likubin disease
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 67

can be readily transmitted in nature by T. citricida from diseased

plants to healthy ones.

IDENTIFICATION

In experiments by the authors all the virus isolates derived

from the so-called likubin-affected citrus plants proved positive

when tested with Mexican lime. Therefore, the virus strains used

seem closely related to the tristeza virus prevalent in North America and South America. However, since these virus strains can

infect Ponkan on Cleopatra mandarin rootstock, whereas in the

aforementioned countries this rootstock is tolerant of the tristeza

virus, there is a possibility that the virus contains, besides tristeza, another component that affects Cleopatra. Another difference

between these strains and tristeza is the reaction on Sunki.

According to a report submitted to the Joint Commission on

Rural Reconstruction by J. M. Wallace (3), who visited Taiwan

in 1963, there seems to be no doubt regarding- the presence of

tristeza virus in citrus plantings there. However, he suggested

that the Sunki rootstock trees might be declining from the effects

of an unknown virus and not from tristeza virus, because in

Brazil, trees on Sunki rootstock were reported to be in good condition after many years' exposure to tristeza.

In the experiments by the authors all the isolates derived from

the so-called likubin-affected citrus trees growing in Taiwan

proved positive when tested with Mexican lime, exhibiting vein

clearing and vein corking, and the reactions of the isolates on

Eureka lemon were comparable with those of tristeza. On the other

hand, the isolates differ from the latter in their reactions against

Cleopatra and Sunki. From these data, it is tentatively concluded

that the likubin virus is identical with the tristeza virus in its

main component, but in addition it contains some other componentes).-

LITERATURE CITED

(1) CHENG, C. P.

1943. STUDIES ON 'HUANGLUNPIN' OF CHAUKAN. Jour. New Agr. 3:

142-177. [In Chinese.]

(2) FRASER, LILIAN.

1952. SEEDLING YELLOWS, AN UNREPORTED VIRUS DISEASE OF CITRUS.

Agr. Gaz. N.S. Wales 63: 125-130.

(3) WALLACE, J. M.

1963. COMMENTS ON CITRUS VIRUS DISEASE AND A REPORT ON THE ROLE

OF THESE DISEASES IN TAIWAN. Nati. Taiwan Univ. Col. Agr.

Misc. Pub. 22, pp. 52-65.

2 The other component (s) may be greening disease virus or be related to it.

C. I. Gonzales, horticulturist, Bureau of Plant Industry, Philippines, reported

that pictures received from T. Matsumoto are identical with Philippine leaf

mottle disease, which in turn seems to be related to greening disease of South

Africa. (J. F. L. Childs)
TATTER LEAF

By J. M. WALLACE and E. C. CALAVAN i

Tatter leaf was described in 1962 in California and so named

because leaves of infected plants, Citrus excelsa Wester (a thickskinned lime), are distorted and have a torn or ragged appearance^

Strong, yellowish leaf mottling and wood pitting or grooving on

Troyer citrange (C sinensis (L.) Osbeck X Poncirus trifoliata

(L.) Raf.) and on certain other trifoliate orange hybrids were attributed later to the tatter leaf virus. However, there is some question as to whether the symptoms described on C. excelsa are caused

by the same virus as that which produced the disease effects on

Troyer citrange. Two different viruses may be involved.

In California the tatter leaf virus was found occurring naturally

in Meyer lemon (C limon (L.) Burm. f.) only and was obtained

from 14 of 15 Meyer lemon trees tested. Through the citrus variety improvement program and other studies at the Citrus Research

Center, Riverside, approximately 400 citrus trees of both foreign

and domestic origin were indexed on C. excelsa seedlings. None

except Meyer lemon were carriers of tatter leaf virus. Most Meyer

lemon trees, although symptomless, are carriers of both tristeza

and seedling yellows viruses. It is of interest that three Meyer

lemon trees free of tristeza and seedling yellows viruses were infected with tatter leaf virus.

CITRUS REACTIONS

Citrus excelsa

Direct inoculations from Meyer lemon to C excelsa result in a

blotchy spotting on newly developing leaves that resembles somewhat the eifects of psorosis on leaves of lime and lemon. As new

leaves form, some are ragged or malformed (fig. 1, A). Abnormal

leaves may continue to develop generally over the plant for a considerable time, with production of symptomless shoots following

later. On other infected plants, a few leaves will show tatter leaf

symptoms, but leaves arising subsequently above the affected parts

will be normal. C, excelsa plants inoculated with pure cultures of

tristeza, seedling yellows, xyloporosis, exocortis, psorosis A,

crinkly leaf, infectious variegation, and vein enation have not developed the tatter leaf symptoms.

In spite of the fact that C. excelsa reacts to infection with seed1 This section was reviewed by L. G. Weathers and S. M. Garnsey.

68
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 69

BN-28391

FIGURE 1.—Tatter leaf symptoms: A, Blotching and distortion of two C.

excelsa leaves (left) and normal leaf (right) ; B, Mexican lime leaves with

varying degrees of vein chlorosis and blotching.
70 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

ling yellows virus from Meyer lemon trees, tatter leaf symptoms

are not suppressed. Occasionally C. excelsa and Troyer citrange

plants inoculated directly from Meyer lemon fail to develop tatter

leaf symptoms. Uneven distribution of the virus in the donor plant

or graft failures can explain this.

Lime

Key lime (C. aurantifolia (Christm.) Swing.) is not a satisfactory indicator if seedling yellows virus is present. When Meyer

lemons carrying both seedling yellows and tatter leaf viruses are

indexed on Key lime, the seedling yellows virus tends to mask

the symptoms of tatter leaf or the tatter leaf complex.

Sometimes lime seedlings develop no symptoms when inoculated

directly from a Meyer lemon that carries only the tatter leaf virus.

In other instances leaf symptoms range from slight tristezalike

vein clearing to a combination of vein clearing and strong blotching. Parts of the midrib and large veins become cleared or chlorotic and often this chlorosis extends irregularly into the adjacent

leaf tissues. These symptoms are shown in figure 1, B. Some of the

small cleared zones on the lateral veins resemble the vein clearing of tristeza.

However, tatter leaf virus obtained from the Meyer lemon plants

free of the seedling yellows virus complex causes no stem pitting

on Mexican lime and does not affect trees of sweet orange on

sour orange rootstock. Leaf distortion of lime is much less than

that of C. excelsa. Like C. excelsa, lime sometimes screens out the

component of the virus mixture that causes symptoms on

C. excelsa,

Troyer Citrange and Citremon

Troyer and Carrizo citrange and citremon (P. trifoliata (L.)

Raf. X C. limon (L.) Burm. f.) (C.E.S. 1448) react strikingly to

infection from Meyer lemons (fig. 2, A). Leaf symptoms consist

of yellowish mottling, vein banding, and failure of one side of the

leaflet to develop, which results in leaf curvature or distortion.

At times there is no leaf distortion and the spots or blotches appear

as rings. Some sources of the virus cause pronounced stunting.

Plants may fail to produce new terminal growth. Superficial

lesions that form on the bark of young stems retard the normal

development of tissue and cause the stems to grow at an angle.

Later the direction of growth changes so as to give the stems a

zigzag shape (fig. 2, B).

Wood pitting develops on Troyer citrange and citremon, with

the individual pits often ahned somewhat vertically (fig. 3). With

further growth of the trees the pits increase in size and the small

vertically arranged pits often coalesce and form continuous

grooves visible externally. These effects plus bud-union creasing

of trees on susceptible rootstocks later cause decline of the tree.

The possibility that the symptoms produced on C. excelsa and on

Troyer citrange are caused by two different viruses is based on

the observation that after one or two serial passages of the virus

from Meyer lemon through C, excelsa, the virus remaining pro-
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 71

^- JBN-28390

FIGURE 2.—Tatter leaf symptoms: A, Normal (left) and three affected

leaves (right) of citremon; B, leaf distortion, corky lesions on stems, and

irregular stem growth of Troyer citrange.

duces a reaction on Troyer citrange but no longer causes any symptoms on C. excelsa. This situation is being investigated further.

Whether or not this virus or virus complex exists anywhere in
72 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

i

)l

1

^

BN-28387

FIGURE 3.—Pitting on stems of citremon infected with tatter leaf virus.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 73

field citrus trees other than Meyer lemon is not established, but

the severe effects caused on the widely used Troyer citrange rootstock by the virus or virus mixture in Meyer lemon make it advisable to include proper indicators for it in all citrus virus indexing programs. Until the matter of virus mixture is settled, these

indicators should be C. excelsa and a susceptible citrange or citremon. Seeds of citranges are doubtless available to most workers.

Both Troyer and Carrizo citrange are good indicators.

HOST RANGE AND SYMPTOMS

Apparently the tatter leaf virus or virus complex has a wide

host range among citrus species and varieties. In limited studies

both suspected components were recovered from experimentally

inoculated sweet orange (C. sinensis (L.) Osbeck), sour orange

(C. aurantium L.), lemon (C. limon (L.) Burm. f.), and Rough

lemon (C. limon (L.) Burm. f.), although all of them remained

symptomless. Trifoliate orange (P. trifoliata (L.) Raf.) has only

rarely developed symptoms and appears to be a poor host. After

showing true tatter leaf symptoms, C, excelsa commonly recovers

by producing normal foliage. Inoculations from such plants result

in sjmiptoms on Troyer citrange, but usually fail to reproduce the

tatter leaf effects on C, excelsa.

INDEXING PROCEDURES

The usual procedures should be followed in indexing for tatter

leaf virus. Seedlings of indicator varieties should be grown and

inoculated by tissue grafts as in other citrus virus indexing. It is

advisable perhaps to include C, excelsa plus either Troyer or Carrizo citrange as indicators.^ As yet, no direct inoculations from

Meyer lemon have caused the C. excelsa reaction while failing to

affect the citranges. It is after passage through C. excelsa that

transfers to C excelsa usually fail to result in symptoms, whereas

the citranges continue to develop strong symptoms.

^ Garnsey finds many Meyer lemon trees in Florida that do not carry tatter

leaf virus, although some were indexed three times. However, tatter leafnegative trees carry tristeza virus, as Wallace observed in California. Garnsey

finds Rusk citrange (C. sinensis (L.) Osbeck X P- trifoliata (L.) Raf.) a

good indicator plant, better than Troyer and much better than Carrizo. (J. F.

L. Childs)
LEAF CURL

By ARY A. SALIBE i

A leaf-curling disease affecting some Hamlin and Pera sweet

orange trees was found in the Limeira area in Brazil. This disease

is of virus nature and inoculation tests show that it can aifect

trees of almost all commercial citrus varieties and species, independent of the rootstock. No vector is known for the leaf curl virus.

Affected trees decline and remain completely unproductive or die.

All trees with symptoms resembling those here described should

be tested and destroyed immediately if positive results are obtained. Testing takes from 2 to 5 months.

SYMPTOMS

Early symptoms of leaf curl usually appear on one to several

branches of a normal tree. The leaves of the infected branches

first show vein clearing, then turn yellow, and usually drop.

Sprouts grow abundantly from these branches. The new leaves

are small and curled in a manner suggestive of heavy infestation

by aphids (fig. 1). Later the symptoms progress to other branches

and then to the whole tree. Finally the branches die back, and

normal leaves sprouting from the trunk give the diseased tree a

gross appearance somewhat similar to that described for **blight"

in Florida. The young sprouts are weak, frequently have gum in

the wood vessels, and break off readily under slight pressure.

Blooming is abundant, but only a few flowers develop into fruits

and they are small. The seeds of these fruits produce normal seedlings (1),

The leaf curl virus affects trees of sweet orange (Citrus sinensis

(L.) Osbeck), sour orange (C. aurantium L.), tangerine (C reticulata Blanco), lemon (C. limon (L.) Burm. f.), acid and sweet

limes (C. aurantifolia (Christm.) Swing.) except Rangpur lime

(C. reticulata var. austera hybrid), grapefruit (C. paradisi

Macf.), shaddock or pummelo (C. grandis (L.) Osbeck), and citron (C. medica L.). Symptoms of the disease develop similarly in

trees of all these species, but are more striking in the sweet orange varieties. Infection with other viruses, such as tristeza, exocortis, and psorosis, does not protect the tree from the establishment of the leaf curl virus. Symptoms are expressed in young

trees from 2 to 5 months after inoculation whereas incubation in

1 This section was reviewed by S. Moreira and Victoria Rossetti.

74
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 75

FIGURE 1.—Bahianinha navel orange tree on Caipera sweet orange rootstock

affected by leaf curl virus at Limeira Experiment Station, Brazil, in 19bd.

old trees may take 1 to 2 years. Pruning hastens the appearance

of disease symptoms (2).

INDEXING PROCEDURE

Nursery seedlings of common sweet orange, 1 year old, are used

as indicator plants for the leaf curl virus. Budwood is taken from

the trees to be tested and is budded in the test seedhngs. it is

advisable to use five seedlings for each tree to be tested and to

insert many buds in each one of them. This recommendation is

based on the fact that the leaf curl virus is unevenly distributed

in the infected trees (1, 2). Twenty days later, if the inoculation

buds are still alive, the sweet orange seedlings are severely pruned

and sprouts are allowed to grow.

In the presence of the leaf curl virus, the leaves of the young

sprouts are small, curled, and show vein clearing. Sometimes the

first leaves are normal and the following ones are affected by the

virus. Symptoms usually appear within 2 to 5 months after inocu-
76 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

lation. Dieback of the young sprouts and death of the seedling both

occur frequently. Symptoms are more conspicuous from inoculations made in late autumn or spring.

If sweet orange seedlings are not available, testing can still be

carried out by using seedlings of other citrus species. The seedlings

should be similarly inoculated with buds from the tree to be tested,

and subsequently budded above the inoculation with a bud from a

healthy sweet orange that will serve as an indicator. Each test

seedling is then cut back and the sweet orange bud is allowed to

sprout. Sprouts should be examined for leaf curl virus symptoms.

Positive results should be promptly followed by eliminating the

diseased trees. Under favorable environmental conditions, normal

and vigorous sprouting of the indicator plants up to 12 months

after inoculation is strong evidence of the absence of the leaf curl

virus in the test tree.

LITERATURE CITED

(1) SALIBE, A. A.

1959. LEAF CURL—A TRANSMISSIBLE VIRUS DISEASE OF CITRUS. U.S.

Agr. Res. Serv. Plant Dis. Rptr. 43 (10) : 1081-1083.

(2)

1965. SUSCEPTIBILITY OF CITRUS VARIETIES TO LEAF-CURL VIRUS. In

Price, W. C. (ed.), 3d Conf. Internatl. Organ. Citrus Virol.

Proc. 1963, pp. 175-178.
PODAGRA

By L. C. KNORR i

Podagra is a disorder of Rough lemon (Citrus limon (L.) Burm.

f.) rootstocks, which enlarge to resemble trifoliate orange (Pondrus trifoliata (L.) Raf.) rootstocks and exhibit exocortislike

scaling of the bark (fig. 1).

Podagra is found in Florida wherever certam clones of Nagami

{Fortunella margarita (Lour.) Swing.) and Meiwa kumquats

(F. crassifolia Swing.) have been budded on Rough lemon. The

disease does not appear when the same clones are budded on

Cleopatra mandarin (C. reticulata Blanco), sweet orange (C.

sinensis (L.) Osbeck), and sour orange (C. aurantium L.) (i, 2),

Podagra's economic importance lies less in its damage to kumquat plantings than in its possible consequences to Rough lemon,

the rootstock on which approximately 75 percent of Floridas

citrus is grown.

Podagra-aifected trees in some kumquat groves on Rough lemon

rootstock amount to 50 percent. Despite such losses, growers still

prefer to grow kumquat trees on Rough lemon stocks because of

the lower solids produced in the fruits.

SYMPTOMS

The name "podagra," from the Greek pous (foot) plus agra

(seizure), refers to the swollen rootstock part of the trunk. Overgrowth is accompanied by scaling of the Rough lemon bark (fig.

1). In the spring profuse gumming often accompanies the shelling

of bark. At first these symptoms suggest that the trees are budded

on trifoliate orange aifected by exocortis. This erroneous impression is sustained by the exocortislike stunting and decline shown

in aifected tops. Whether aifected trees carry exocortis virus is

not known at the present time.

Symptoms of podagra may appear as early as 1 year after budding or as late as 10 years after setting of trees in the grove.

Speed of deterioration varies; some trees succumb in the nursery,

others decline gradually in the field.

TRANSMISSION TESTS

The possibility that podagra is a virus disease is supported by

1 This section was reviewed by J. F. L. Childs.

77
78 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

BN-28386

FIGURE 1.—Overgrowth and scaling of Rough lemon rootstock part of kumquat tree affected by podagra.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 79

preliminary results of transmission tests begun in 1957. Results

showed that budwood taken from affected trees reproduced podagra when budded into Rough lemon seedlings. On the other hand,

trees made from budwood of normal trees have not developed

podagra 8 years after budding.

Transmission tests in the field include test plants with interstocks of sweet orange, sour orange, Rangpur lime (C. reticulata

var. austera hybrid), and citron (C. medica L.) to determine

whether the hypothetical virus in kumquat tops will pass through

symptomless tissue and affect Rough lemon.

INDEXE^G PROCEDURE

No more critical procedure for indexing is known than the

inoculation of Rough lemon seedlings with buds from suspect

kumquat trees. The first symptom, formation of small pegs from

the cambial face of the wood, may be seen by peeling a patch of

bark. This occurs about 2 years after inoculation. Gumming and

scaling develop at about 4 years. Field trees commence to decline

after 4 years.

LITERATURE CITED

(1) KNORR, L. C.

1957. RE-APPRAISING CITRUS ROOTSTOCKS, WITH PARTICULAR REFERENCE

TO THEIR SUSCEPTIBILITY TO VIRUS DISEASES. III. ROUGH

LEMON (CITRUS LIMON (L.) BURM. F.). Citrus Mag. 20 (2) ?

8-10, 21, 24-25.

(2)

1959. SIMILARITIES AND DIFFERENCES BETWEEN TRISTEZA IN NORTH

AND SOUTH AMERICA. In Wallace, J. M. (ed.). Citrus Virus

Diseases, 1957, pp. 63-71. Univ. Calif. Div. Agr. Sei.,

Berkeley.
INDEXING FOR TATTER LEAF WITH COWPEA

By S. M. GARNSEY and L. G. WEATHERS

A virus present in Meyer lemon (Citrus limon (L.) Burm. f.)

has been transmitted by sap inoculation to several herbaceous

plants including cowpea (Vigna sinensis (Torner) Savi) (2, S),

It was suggested that this sap-transmissible virus was tatter leaf

virus (2), and recent transmission experiments by Weathers

(unpublished data, 1966) have confirmed this.

INDICATORS

Tests conducted in Florida show that the Early Ramshorn

variety of cowpea is a reliable and rapid indicator for tatter leaf

virus (TLV) when inoculated properly. Forty-three Meyer lemon

trees have been indexed for TLV by ( 1 ) bud-inoculating seedlings

of Rusk citrange (C sinensis (L.) Osbeck x Poncirus trifoliata

(L.) Raf.) and (2) inoculating cowpea plants with macerated

tissue of young citrus leaves made by grinding the leaves in

cold neutral phosphate buffer. The results correlated exactly.

Twenty-eight trees tested positively on both Rusk citrange and

cowpea, whereas the remaining 15 tested negatively.

TLV was also transmitted readily to cowpea from infected

seedhngs of sweet orange (C. sinensis (L.) Osbeck), Duncan

grapefruit (C paradisi Macf.), Cleopatra mandarin (C. reticulata

Blanco), Orlando tángelo (C paradisi X C. reticulata)^ Eureka

lemon (C. limon (L.) Burm. f.), C. excelsa Wester, and sour

orange (C. aurantium L.). It was readily transmitted from leaflets

of Rusk or Troyer citrange that showed symptoms of TLV infection, but not from leaflets that were symptomless. TLV was

transmitted inconsistently from Mexican lime (C. aurantifolia

(Christm.) Swing.).

SYMPTOMS

The diagnostic symptom in cowpea consists of spreading necrotic lesions on the inoculated primary leaves (fig. 1). The lesions

are chocolate brown and easily visible 4 to 6 days after inoculation,

provided the inoculated plants are held at moderate temperatures

(near 70° F.). High temperatures markedly reduce symptom

severity (fig. 1). Under certain conditions mosaic patterns appear

in the trifoliate leaves.

80
INDEXING PROCEDURES rOR 15 VIRUS DISEASES OF CITRUS 81

INDEXING PROCEDURE

Macerate young succulent leaf tissue in cold neutral potassium

phosphate buffer (0.05 molar), using a prechilled porcelain mortar

and pestle. A ratio of 0.1 gram of leaf tissue to 1 ml. of buffer is

satisfactory. Using a finger or cotton swab, gently apply the

macerate to the primary leaves of cowpea that have been dusted

with 500-mesh carborundum (passes a screen of 500 holes per

square inch). After inoculation, rinse the leaves with cool tapwater and incubate the inoculated plants under moderate light

(500 to 1,000 foot-candles) at 21° to 24° C.

.^

BN-28392

FIGURE 1.—Inoculated leaves of cowpea: Left, incubated at summer greenhouse temperatures of 70°-90° F.; right, held at 70° ±2°; light conditions

were similar. Note small lesions (arrows) on leaf at left.

Although there is no established rule on the number of plants

required, experience indicates that from 5 to 10 plants should be

used for each test. Cowpea plants are easily grown by planting

from six to eight seeds in 4-inch clay pots filled with steamsterilized potting soil. The seedlings are thinned to obtain four or

five uniform plants per pot. Inoculate when the primary leaves

have expanded, but before the trifoliate leaves appear. The plants

are usually ready to inoculate 8 to 12 days after planting depending on growing conditions.
82 AGRICULTURE HANDBOOK 333, U.S. DEPT. OF AGRICULTURE

This procedure consistently produced reliable results, even when

indexing leaf tissue collected in the field in midsummer. However,

it is well to note that a sap-inoculation technique requires higher

concentration of virus in the inoculum than is necessary for graft

transmission and to that degree is a less sensitive procedure.

Citrange test plants also should be graft inoculated from the same

inoculum source until it is established that the cowpea test gives

reliable results under local conditions. The viruses of tristeza,

exocortis, xyloporosis, psorosis, stubborn, vein enation, and yellow

vein have not produced symptoms on cowpea. Infectious variegation virus will infect several varieties of cowpea (i), but the

symptoms are easily distinguishable from those produced by TLV.

LITERATURE CITED

(1) GRANT, T. J., and CORBETT, M. K.

1964. PROPERTIES OF CITRUS VARIEGATION VIRUS. Phvtopatholoffv 54:

946-948.

(2) SEMANCIK, J. S., and WEATHERS, L. G.

1965. PARTIAL PURIFICATION OF A MECHANICALLY TRANSMISSIBLE VIRUS

ASSOCIATED WITH TATTER LEAF OF CITRUS. Phvtopatholofi-V

55: 1354-1358.

(3) YARWOOD, C. E.

1963. MECHANICAL TRANSMISSION OF A LATENT LEMON VIRUS. (Abstract) Phytopathology 53: 1145.
COLOR TEST FOR EXOCORTIS

By J. F. L. CHILDS

Several improvements in indexing for exocortis have been made

since the color test (2) was reported. The Rangpur lime (Citrus

reticulata var. austera hybrid) test (3) has given good results

under certain conditions, but it has proved slow and sometimes

erratic in Florida. The Etrog citron (C. medica L. var. ethrog

Engl.) test (i), using certain selections of citron, has given excellent and rapid results. However, occasions arise when the color

test can be used to advantage.

The use of radial-longitudinal sections of bark instead of cross

sections, as first reported, makes diagnosis by the color test more

certain and easier. Best results with the color test are obtained

with trifoliate orange (Poncirus trifoliata (L.) Raf.), but the

following citranges (P. trifoliata (L.) Raf. X C. sinensis (L.)

Osbeck and reciprocal) are satisfactory: Carrizo, Morton, Savage,

and Norton. The Rusk, Rustic, Thomasville, and Uvaldi citranges

react weakly.

The color test indicates an early stage of bark scaling that

occurs internally from 6 months to several years before scaling is

apparent to the eye. Consequently, trees on exocortis-susceptible

rootstocks can be examined at an early age by this method, or

seedlings of exocortis-susceptible varieties can be inoculated with

buds or grafts for the test.

PROCEDURE

A small piece of bark, approximately 5 by 30 mm., is cut from

the test tree anywhere between the bud union and the ground

line. In experiments at Orlando, Fla., better results were obtained

on the average with bark cut near the union. Specimens can be

examined fresh or stored in standard formaldehyde, alcohol, or

acetic acid fixing solution for examination later.

The outer surface of the bark should be washed with a brush to

remove adhering dirt, sand, or algae. Mount the specimen on a

freezing microtome so that sections can be cut in the radiallongitudinal plane and so that the blade enters the specimen on a

slight diagonal from the cambial side. This avoids dragging sand

across the section. A clinical freezing microtome is satisfactory

for this purpose. A sliding microtome with freezing attachment is

better, but more expensive.

Microtome blades must be very sharp to avoid tearing the sections, which are cut at 18 to 20fjL. Since blades are soon damaged

by sand adhering to the bark, examine the blade after cutting each

83
84 AGRICULTURE HANDBOOK 33 3, U.S. DEPT. OF AGRICULTURE

specimen of bark. If it is damaged, change to a sharp part of the

blade to cut the next specimen. After 20 or more good sections

have been cut, mount 5 to 10 of the best ones on a glass microscope

slide in rows of 5 sections each. It is suggested that 15 sections be

examined from each bark specimen.

Arrangement of the sections is facilitated if they are moved

into position on the glass slide in a film of water. Drain off excess

water and blot the remaining moisture by laying a piece of filter

paper over the sections and running a finger lightly over the paper.

A few drops of phloroglucinol (saturated solution in 18 percent

hydrochloric acid (HCl)) are allowed to run down the shde

between the sections so that all are wet by it. This solution keeps

for several months, but solutions 6 months or older may fail to

give the color test. A cover slip, 22 by 60 mm. (preferably No. 1,

0.15-mm. thickness), is placed over the sections, bubbles are

pressed out, and the edges sealed with vas-par (equal parts melted

paraffin wax and petroleum jelly) to keep HCl fumes from the

microscope. After 5 minutes the section may be examined. After

examination, the slides should be kept in an airtight container

until disposed of so as to keep HCl fumes away from instruments.

DIAGNOSIS

Examine only the medullary ray cells of the phloem region. The

advantage of radial-longitudinal sections over cross sections is

that, in the former, medullary ray cells are more easily distinguished from the sieve tubes. In the radial-longitudinal plane, the

rectangular ray cells are arranged with the long axis of the cell at

right angles to the cambium. All other cells in the phloem region

are arranged with their long axis parallel to the cambium. Also,

medullary rays are 6 to 30 cells wide in the radial-longitudinal

plane compared to the transverse plane where rays are only a few

cells wide.

Exocortis virus causes the formation of an aldehyde in some of

the ray cells. This aldehyde develops a rose red (pomegranate

purple by Ridgeway's color standards (i)) when treated with

phloroglucinol-HCl reagent (figs. 1 and 2). Any test for an aldehyde will work, but the phloroglucinol-HCl test proved most satisfactory. The color should be distributed uniformly throughout the

cytoplasm of the cell. Not all ray cells develop the color. A positive reaction can not be expected sooner than 6 months after

moculation, but may be delayed much longer if growth of the test

plant is poor. Ray cells occasionally develop a light pink (Cameo

pmk, Ridgeway U)), which has no known relation to the exocortis virus.

As an arbitrary standard, the author has held that if three

ray cells per slide develop the pomegranate purple, the exocortis

test is positive. In practice this should be confirmed by examining

at least 15 sections. Ray cells with red-stained cytoplasm are

frequently so numerous that there is no need to examine more

than five sections. To gain experience with the method and as a

basis for comparison, examination of bark known to be exocortis

infected is recommended.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 85

FIGURE 1.—Medullary ray cells oriented in horizontal rows; sieve tubes and

phloem fibers in vertical rows. Note rose-red cells in group of ray cells.

FIGURE 2.—Two stained cells in one medullary ray and one in other ray is

typical of young- P. trifoliata bark infected with exocortis.
86 AGRICULTURE HANDBOOK 3 33, U.S. DEPT. OF AGRICULTURE

LITERATURE CITED

(1) CALAVAN, E. C, FROLICH, E. F., CARPENTER, J. B., and others.

1964. RAPID INDEXING FOR ExocoRTis OF CITRUS. Phytopathology 54:

1359—1362.

(2) CHILDS, J. F. L., NORMAN, G. G., and EICHHORN, J. L.

1958. A COLOR TEST FOR EXOCORTIS INFECTION IN PONCIRUS TRIFOLIATA. Phytopathology 48: 426-432.

(3) MOREIRA, S.

1961. A QUICK FIELD TEST FOR EXOCORTIS. In Price, W. C. (ed.), 2d

Conf. Internatl. Organ. Citrus Virol. Proc. 1960, pp. 40-42.

(4) RIDGEWAY, R.

1912. COLOR STANDARDS AND COLOR NOMENCLATURE. 49 pp., 53 pi.

Washington, D.C.
GREENING DISEASE

By RALPH E. SCHWARZ

Greening disease has been known for more than 35 years in

South Africa, but it was proved to be a virus disease only recently

{1, 2), Transmission of the causal virus by the citrus psylla

Spanioza erythreae (Del Guercio) was established by McClean and

Oberholzer (1). The world distribution of this disease has not

been fully determined, but recent information indicates that greening or a closely related virus disease is common in several Asian

countries and present in the Mediterranean area. Greening disease

presents a more serious problem than any other known virus disease of citrus, because it affects the major citrus species irrespective of rootstock variety, reducing yields and making many fruits

worthless.

SYMPTOMS

A complete description of the symptoms of greening disease on

all commercially important varieties of citrus is not possible here.

The symptoms on sweet orange are described in detail, and those

on other varieties are noted when they differ significantly.

Greening alters the growth habit or shape of sweet orange trees

and causes symptoms on the leaves and fruits, but the best diagnostic symptoms appear on the foliage of seedling trees. Leaves

that develop after inoculation show chlorotic patterns similar to

those of zinc deficiency. The leaves may be completely yellow or

yellow with green veins and small green spots. Mature leaves

often show yellowing of the midrib, which becomes abnormally

prominent. This condition may extend along the lateral veins and

ultimately involve the whole leaf. Mature leaves may also show

mottling or blotching (fig. 1). Excessive blooming and out-ofseason blooming occur frequently on orchard trees and may be

followed by abnormal dropping of fruit and dieback of twigs.

Mature fruits may color only on the side exposed to the sun while

the unexposed side remains a dull olive green. Only a part or even

a single branch of an orchard tree may be affected. Furthermore,

the virus seems to be distributed unevenly in the tissues of infected

trees.

Leaf and fruit symptoms are masked under certain climatic

conditions. For example, when inoculated seedlings are kept at 21°

to 23° C., clear leaf symptoms develop, but if infected seedlings

are maintained at 26° to 28°, no symptoms appear on the new

growth (fig. 2). Although further information on the effects of

climate on symptoms is not available, other environmental effects

are suspected.

87
88 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

Equally severe symptoms are exhibited by many mandarin,

tángelo, and grapefruit varieties (fig. 3). On lemon varieties symptoms are less severe. Very mild symptoms appear on Palestine

sweet lime, West Indian lime, Rough lemon, and trifoliate orange

and its hybrids.^

INDEXING PROCEDURE

Indicator Plants.—Either sweet orange or tángelo seedlings may

be used as indicator plants. In the absence of seedling yellows

virus, grapefruit seedlings may be used. If there is a choice, it

would be Valencia, midseason orange varieties, and navel orange

in that order. For tangelos, the order would be Orlando, Minneola,

and Seminóle. Others may serve as well, but they have not been

used.

Preparation of Test Plants.—Seedlings for indexing greening

virus should be grown and inoculated as described for psorosis A

virus. Because acute symptoms of zinc deficiency are similar to

the symptoms produced by greening virus, test plants should be

adequately supplied with the minor elements, especially zinc. A

University of California type of potting mixture may be used.

1 For detailed descriptions of greening symptoms on many varieties of

citrus, refer to McClean (1) and Schwarz (2, 3).

FIGURE 1.—Greening symptoms on sweet orange leaves: Top, normal; middle

row, yellow vein ; bottom row, mottling and zinc deficiency.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 89

FIGURE 2.—Greening symptoms on sweet orange present on lower leaves kept

at low temperature, but absent from top leaves mamtamed at high

temperature.

Inoculation Methods.—Graft sticks from mature wood havmg

leaves with symptoms should be used for moculation. Sticks are

preferable to buds or bark patches, because the virus seems to be

unevenly distributed in the host and may otherwise be missed

Since the percentage of successful transmissions is often low,

about 20 test plants are recommended for each test. Citrus varieties used for indexing seedling yellows virus such as lemon and

grapefruit are also susceptible to greening disease, and the symptoms produced on such plants by the two viruses are difficult to

distinguish. It is therefore not advisable to use them as indicators

in the presence of seedling yellows virus.

Diagnosis.—Typical leaf symptoms usually show up on the new

growth 3 to 4 months after inoculation. The same range of leaf

symptoms observed in orchard trees can usually be produced on

test plants in the greenhouse. .

The presence of a specific fluorescent marker substance m the

tissue of greening-infected sweet orange, tángelo, and mandarin

trees can be used for indexing purposes. The albedo of sweet

orange fruits from infected trees fluoresces under ultraviolet light

of wavelength 365 m/x (3). This substance can be extracted from

the albedo and bark of citrus and its presence determined by

Chromatographie methods (3).
90 AGRICULTURE HANDBOOK 3 3 3, U.S. DEPT. OF AGRICULTURE

FIGURE 3.—Greening symptoms on Empress mandarin leaves.

LITERATURE CITED

(1) MCCLEAN, A. P. D., and OBERHOLZER, P. C. J.

1965. GREENING DISEASE OF THE SWEET ORANGE; EVIDENCE THAT IT IS

CAUSED BY A TRANSMISSIBLE VIRUS. So. African Jour. Agr.

Sei. 8: 253-276.

(2) SCHWARZ, R. E.

1964. AN INSECT TRANSMISSIBLE VIRUS TRAPPED IN SWEET ORANGE

SEEDLINGS IN ORCHARDS WHERE GREENING DISEASE IS COMMON.

So. African Jour. Agr. Sei. 7: 885-889.

(3)

1967. (?) INDEXING OF GREENING AND EXOCORTIS BY MEANS OF FLORESCENT MARKER SUBSTANCES. In Childs, J. F. L. (ed.), 4th Conf.

Internat!. Organ. Citrus Virol. Proc. 1966. [In press.]
CLASSIFICATION OF CITRUS SPECIES '

By PHILIP C. REECE

W. T. Swingle recognized 16 species in the genus Citrus, in

what he considered an approach toward a natural system of classification based on sexually inherited similarities. Borderline and

intermediate forms he considered hybrids and placed as hybrids

with the species they most closely resembled.

T. Tanaka recognized 150 species in a pragmatic system^ oí

classification and assigned Latin binomials to obvious hybrids,

apomictically self-perpetuating forms, as well as types that can

only be perpetuated by budding or other vegetative means oí

propagation. Even a single individual tree is given species designation. Thus, he considers the Temple orange, Meyer lemon, Tahiti

lime, and other cultivars to be valid species.

Whether one prefers either of these systems of classification or

takes R. W. Hodgson's intermediate position, which is mainly the

Swingle classification with an additional 23 species of Tanaka, the

fact remains that more than one system is in use.

In the following list are given the technical binomials of the

Tanaka systematics of the genus Citrus, the common name, and

the technical binomial equivalent used in the Swingle systematics:

Tanka Citrus

species name

alata Tan.

ambtycarpa Ochse

ampuUacea Hort.

ex Tan.

anonyma Hort.

ex Tan.

asahikan Hort.

ex Tan.

assamensis Dutta

et Bhatt.

aiirantifolia

(Christm.) Swing.

aurantium L.

aurata Risso

áurea Hort, ex

Tan.

balincolong Tan.

balo tina Poit. et

Turp.

benikoji Hort, ex

Tan.

Common name

Winged citron

Djerock limoo

Hyokan

Shirenbo

Asahikan

Assam lemon

Lime

Sour orange

Adam's apple

Kawabata-mikan

Balincolong

Balotin bergamot

Benikoji, Dai

Koji

Swingle Citrus

sjyecies equivalent

medica L.

reticulata Blanco

paradisi Macf. (?)

paradisi Macf.

paradisi Macf.

limon (L.) Burm. f.

aiirantifolia

(Christm.) Swing.

aurantium L.

limon (L.) Burm. f.

sinensis (L.) Osbeck

micrantha Wester (?)

limon (L.) Burm. f.

reticulata Blanco

1 Based on material from Swingle, W. T., and Reece, P. C, The Botany of

Citrus and Its Wild Relatives of the Orange Subfamily. In Reuther, W.,

Webber, H. J., and Batchelor, L. D. (eds.), The Citrus Industry, 2d ed., v. I,

ch. 3, Univ. Calif. Div. Agr. Sei., Berkeley. 1967.

91
92 AGRICULTURE HANDBOOK 333, U.S. DEPT.

bergamia Risso

3t Poit.

boholensis Tan.

canalicidata

Hört, ex Y. Tan.

celebica Koord.

Clementina Hort.

ex Tan.

combara Raf.

crenatifolia Lush.

davaoensis Tan.

deliciosa Ten.

depressa Hayata

el-kantara Y.

Tan.

erythrosa Hort.

ex Tan.

excelsa Wester

flavicarpa Hort.

ex Tan.

funadoko Hort.

ex Y. Tan.

genshokan Hort.

ex Tan.

glaberrima Hort.

ex Tan.

grandis (L.)

Osbeck

hainanensis Tan.

hanaju Sieb, ex

Shirai

hassaku Hort, ex

Tan.

himekitsu Hort.

ex Tan.

hiroshimana Hort.

ex Y. Tan.

hyalopulj)a Tan.

hystrix DC.

ichangensis Swing.

indica Tan.

inflata Hort, ex

Tan.

intermedia Hort.

ex Tan.

iwaikan Hort, ex

Tan.

¿2/0 Hort, ex Tan.

jambhiri Lush.

javanica Blume

./wnos Sieb, ex Tan.

karna Raf.

keraji Hort, ex

Tan.

Bergamot

Bohol papeda,

Kansi

Kiku-daidai

Celebes papeda

Clementine

Annam papeda

Kawla, Keonla

Davao lemon

Willowleaf

mandarin

Shekwasha

El-kantara

Fukushu, Kobeni

"Le Nestour,"

Limon Real

Maeda-kan

Funadoko-mikan

"like Temple"

Genshokan

Kinukawa-mikan

Pummelo, Shaddock,

Jabon, Zabon

Hainan wild orange

Hanayu, Tokoyu

Hassaku orange

Himekitsu

Natsuzabon

Sekiroku-kan

Mauritius papeda,

Porcupine orange

Ichang papeda

Indian wild orange

Kizu, Mochiyu

Yama-mikan

Iwaijabo

lyo-mikan

Rough lemon

Djerock Hunje

Yuzu

Karna, Khatta, Id

lemon

Keraji

OF AGRICULTURE

aurantium (L.)

micrantha Wester (?)

aurantium L.

celibica Koord.

reticulata Blanco

macroptera var.

annamensis Tan.

reticulata Blanco

aurantifolia

(Christm.) Swing.

reticulata Blanco

reticulata Blanco

limon (L.) Burm. f.

reticulata Blanco

aurantifolia

(Christm.) Swing.

paradisi Macf.

sinensis (L.) Osbeck

reticulata Blanco

paradisi Macf.

grandis (L.) Osbeck

reticulata Blanco

ichangensis Swing.

paradisi Macf.

paradisi Macf.

paradisi Macf.

aurantifolia

(Christm.) Swing.

hystrix DC.

ichangensis Swing.

indica Tan.

paradisi Macf.

aurantium hybrid

paradisi Macf.

sinensis (L.) Osbeck

limon (L.) Burm. f.

aurantifolia (Christm.)

Swing.

ichangensis X C.

reticulata var.

austera (?) hybrid

limon (L.) Burm. f.

reticulata Blanco
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 93

kerrii Tan.

kinokuni Hort.

ex Tan.

kotokan Hayata

latifolia Tan.

latipes (Swing.)

Tan.

leiocarpa Hort.

ex Tan.

limetta Risso

limettioides Tan.

limon (L.) Burm. f.

limonia Osbeck

Ihnonhnedia Lush.

longelimon Tan.

longispina Wester

lumia Risso

luteo-turgida Tan.

lycopersicaeformis

Hort, ex Tan.

macrophylla Wester

m,acroptera Mont.

maderaspatana

Hort, ex Tan,

madurensis Lour.

mairay Wester

medica L.

medioglobosa Hort.

ex Tan.

viegaloxicarpa Lush.

mellarosa Risso

meyerii Y. Tan.

micrantha Wester

mitsiiharii Hort.

ex Y. Tan.

montana Tan.

myrtifolia Raf.

nana Tan.

natsudaidai

Hayata

nippokoreana Tan.

Kerrys Thailand

papeda

Kinokuni, Kishu-mikan

Kotokan-tiger

head

Persian lime,

Tahiti lime

Khasi papeda

Koji

Mediterranean

sweet lemon

Sweet lime of

India

Lemon

Rangpur lime

Bajoura, Etrog

citron

"too long to be a

lemon"

Tamisan, Talamisan

Lumie, Lumia of

Italy

(unknown)

Cleopatra

Colo, Alemow

Melanesian papeda,

Cabuyao

Guntur sour

Shikikitsu,

Tokinkan,

Calamondin,

Shikinari-mikan

Mairay

Citron

Naruto orange,

Naruto-mikan

Amilbed, Keem

citron

Mellarosa of

France

Meyer lemon

Biasong, small

flowered papeda

Mitsuharu-mikan

Bilolo

Myrtle leaf

orange, Chinnoto

Small globose

citron, Limau

susu

Daidai-mikan,

Japanese summer

grapefruit

Korai-tachibana

macroptera var.

kerrii Swing.

reticulata Blanco

paradisi Macf.

aurantifolia

hybrid

latipes (Swing.)

Tan.

reticulata Blanco

limon (L.) Burm. f.

aurantifolia (Christm.)

Swing.

limon (L.) Burm. f.

reticulata var.

austera hybrid

medica L. var.

ethrog Engl.

limon (L.) Burm. f.

aurantifolia

(Christm.) Swing.

limon (L.) Burm. f.

sinensis (L.) Osbeck

reticulata Blanco

celehica Koord.

hybrid

macroptera Mont.

aurantium L.

reticulata var.

austera (?) X

Fortunella sp.

aurantium hybrid

medica L.

paradisi Macf.

limon (L.) Burm. f. (?)

limon (L.) Burm. f.

limon X sinensis

micrantha Wester

paradisi Macf.

aurantifolia

(Christm.) Swing.

aurantiuTYi var.

myrtifolia Ker.-

Gawl.

medica L.

paradisi hybrid (?)

sinensis (L.) Osbeck
94 AGRICULTURE HANDBOOK 33 3, U.S. DEPT.

nobilis Lour. King, Kunenbo

oblonga Hort, ex

Y. Tan.

obovoidea Hort,

ex Takahashi

obversa Hassk.

odorata Wester

ex Tan.

oleocarpa Hort.

ex Tan.

omikanto Hort.

ex Y. Tan.

otachibana Hort, ex

Y. Tan.

oto Hort, ex Y. Tan.

ovata Hassk.

panuban Tan.

papaya Hassk.

papülaris Blanco

paradisi Macf.

paratangerina

Hort, ex Tan.

pennivesiculata

Tan.

peretta Risso

platymamma Hort.

ex Tan.

ponki Hort, ex

Tan.

pseudoaurantium

Hort, ex Y. Tan.

pseudograndis

Hort, ex Tan.

pseudogulgul Hort.

ex Shirai

pseudolimon Tan.

pseudolimonum

Wester

pseudopapülaris

Tan.

pseudoparadisi

Hort, ex Y. Tan.

pseudosunki Hort.

ex Tan.

pyriformis Hassk.

reshni Hort, ex

Tan.

reticulata Blanco

rissoi Risso

rokugatsu Hort.

ex Y. Tan.

rugulosa Hort.

ex Tan.

sinensis Osbeck

sinograndis Hort.

ex Tan.

southwickii

Wester

Washington navel,

Daenkan

elliptic orange

Kinkoji

Djerock Balik of

Java

Tihi-tihi, flat

citron

Yu-pi-chieh

Omi-Kanto

Daikitsu, Otachibana

Oto-mikan

Djerock tanjoeng

boerrah

Panuban

Djerock papaya

Tizon

Grapefruit

Ladoo

Gajanimma, Moi

Peretta of France

Saagkam

Ponki

Henka-mikan

Uzon-kunebu

Yoa shaddock

False lemon

False lemon

Balanga orange

Uwa pumelo

Kobeni

Ponderosa lemon

Reshni, ChotaKitchli

Satsuma, mandarin

Risso's citrus

Za-daidai, June

sour orange

At-anni

Sweet orange

Oto-mikan

Southwicks papeda

OF AGRICULTURE

sinensis X reticulata

hybrid

sinensis (L.) Osbeck

paradisi Macf.

aurantifolia (Christm.)

Swing.

medica L.

reticulata Blanco

paradisi Macf.

paradisi Macf.

reticulata Blanco

aurantifolia (Christm.)

Swing.

grandis (L.) Osbeck

aurantifolia (Christm.)

Swing.

aurantium hybrid

paradisi Macf.

reticulata Blanco

limon hybrid (?)

limon (L.) Burm. f.

reticulata Blanco

reticulata Blanco

ichangensis hybrid

grandis (L.) Osbeck

grandis (L.) Osbeck

limon (L.) Burm. f.

limon (L.) Burm. f.

aurantiuyn hybrid

paradisi Macf.

reticulata Blanco

limon (L.) Burm. f.

reticulata Blanco

reticulata Blanco

limon (L.) Burm. f.

aurantium. L.

paradisi Macf.

sinensis (L.) Osbeck

paradisi Macf.

celebica var.

southwickii (Wester)

Swing.
INDEXING PROCEDURES FOR 15 VIRUS DISEASES OF CITRUS 95

suavissima Hort. Okan, Unshu- reticulata Blanco

ex Tan. mikkitsu

subcompressa Sakitsu reticulata Blanco

Hort, ex Tan.

succosa Hort. Nidonari, Jimikan reticulata Blanco

ex Tan.

sudachi Hort. Sudachi ichangensis hybrid

ex Shirai

suhuiensis Hort. Szu-ui-kom, reticulata Blanco

ex Tan. Shikaikan

suizabon Hort. Sujabo, Suizabon grandis (L.) Osbeck

ex Tan.

sulcata Hort. Sanbokan paradisi Macf.

ex Takahashi

sunki Hort, ex Sunki reticulata var.

Tan. austera Swing.

tahibana Tan. Tachibana orange tachibana (Mak.) Tar

taiwanica Tan. Nansho-daidai aurantium hybrid

et Shimada

tamurana Hort. New summer orange, sinensis (L.) Osbeck

ex Tan. Hyuga-natsumikan

tangerina Hort. Dancy reticulata Blanco

ex Tan.

tankan Tan. Honey barrel orange reticulata Blanco

tardiferax Mankitau reticulata Blanco

Hort, ex Tan.

tardiva Hort. Natsu-mikan, reticulata Blanco

ex Shirai Giri-mikan

tarogayo Hort. Tarogayo reticulata Blanco

ex Tan.

temple Hort. Temple orange reticulata hybrid

ex Y. Tan.

tengu Hort, ex ''Red-faced" paradisi Macf.

Tan. Shigetom,

Kinkuneba, Tengumikan

tosa-asahi Hort. Tosa-asahi paradisi Macf.

ex Y. Tan.

truncata Hort. Jagatara grandis (L.) Osbeck

ex Tan.

túmida Hort. Fukure-mikan reticulata Blanco

ex Tan.

ujukitsu Hort. Bushukan sinensis (L.) Osbeck

ex Tan.

unshiu Marc. Satsuma reticulata Blanco

vitensis Tan. Moli Kurikuri macroptera hybrid

webberi Wester Kalpi aurantifolia (Christn

Swing.

westeri Tan. Samuyao, small micrantha var.

fruit papeda microptera Wester

wilsonii Tan. Ichang lemon ichangensis X C.

grandis hybrid

yamabuki Hort. Yamabuki paradisi Macf.

ex Y. Tan.

yat&ushiro Hort. Yatsushiro-mikan reticulata Blanco

ex Tan.

yuge-hyokan Hort. Yuge-hyokan paradisi Macf.

ex Tan.

í/i¿fco Hort, ex Tan. "Yuko" ichangensis X C.

reticulata hybrid

zeylanica Tan. Gondh-huntra paradisi Macf.
GLOSSARY

Bark patch Piece of bark without bud that is inserted in

bark in same way as bud—sometimes called

blind bud

Blotch Chlorotic spot with diffuse, indefinite, or irregular margin

Candidate tree .... Tree to be tested for virus infection

Cheesy Consistency of cheese

Dappled Spotted as certain animals (ex. Percheron

horse)

Index Test a plant (citrus) for presence of certain

viruses

Indicator plant . . . Variety of citrus used for testing purposes by

reason of its strong reaction to virus in

question

Inverse pit Peglike outgrowth of wood that fits into similarly shaped pit in cambial face of bark

Leaf patch Small piece of leaf inserted under T- or Ushaped cut in bark

Lime test Test for tristeza using West Indian lime (C.

aurantifolia (Christm.) Swing.), also called

Key lime, Mexican lime, and baladi lime

Lopsided Unsymmetrical

Mild strain Form of virus that induces mild symptoms in

standard indicator plant

Peg Toothlike outgrowth from cambial face of

bark that fits into pit in wood

Pit (collectively. Depression in cambial face of wood, varying

pitting) from less than 1 mm. to several centimeters,

often elongated in direction of branch elongation

Take Union of bud, graft, or other tissue with

budded tree

T-bud Bud inserted into T-shaped cut in bark

(T may be inverted)

^GPO: 1967 O 263—337

96

f)