Greening Disease of Citrus in the Deccan Trap Country and its Relationship with the Vector
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Stubborn, Greening, and Related Diseases
Greening Disease of Citrus in the Deccan Trap
Country and its Relationship with the Vector,
Diaphorina citri Kuwayama
S. P. C,apoor,D. G. Rao, and S. M. Viswanath
Citrus greening was observed for the LaflPche and BovC (10) observed myco-
first time in India in 1960 (3, 6, 7 ) .T h e plasmalike bodies in sieve tubes of
disease was again observed at Modlimb, greenhouse-grown sweet orange seed-
on Kagzi lime trees, in 1963. Thereafter lings affected with greening, and con-
it was commonly encountered in or- cluded that it is most probably caused
chards in which trees were being in- by a mycoplasmalike organism.
dexed for virus-free budwood. T h e ori- T h e data in this paper pertain to the
ental citrus psyllid, Diaphorina citri Deccan T r a p Country, and include stu-
Kuwayama, was established as the vec- dies of etiology, spread, host range, and
tor of greening pathogen in India (4). relationship with the psyllid vector.
MATERIALS AND METHODS
Greening disease was maintained in Inoculations were made by the leaf-
seedlings of sweet orange previously rubbing method, with 600-mesh carbo-
infected by psylla in the glasshouse. rundum. Test plants were predarkened
T h e disease was identified from leaf before inoculation, and maintained at
and fruit symptoms, from the general 22 to 2 4 O C after inoculation.
growth of the trees ( I I ) , and by index- Acquisition and transmission feeding
ing budwood from affected trees on OF the insect species for insect transmis-
seedlings of sweet orange, Kagzi lime, sion tests was the same as used previ-
and grapefruit (4). Later, only sweet ously (4). As a check, the Psyllids were
orange was used because it consistently also used to transmit the greening path-
gave a typical reaction to the greening ogen from source plants used in these
disease. Seedlings were raised inside a experiments. Test plants were sweet
glasshouse, and selected nucellar seed- orange seedlings. Newly emerged adults
lings were transplanted individually in from a colony of Diaphorina citri
15-cm earthen pots. Testing was done (mixed population) were used in the
by budding, shoot grafting, or by leaf- experiments unless stated otherwise.
patch grafting (15). After test-feeding, psyllids were killed
Bark extracts of several plants were by spraying with 0.02 per cent Folidol-
also examined for fluorescent-marker E 605.
substance by the method described by I n surveys for greening, particular
Schwarz (12,13). attention was given to the nurseries
For sap inoculation, young, diseased from which the citrus trees in orchards
leaves of sweet orange were homog- had originated.
enized with 10 per cent sucrose in 0.1 A survey for the disease in the Dec-
M phosphate buffer at p H 7.6 ( 5 ) . can T r a p Country, carried out from
Leaves were frozen for four hours be- 1965 to 1970, revealed that, but for a
fore grinding with 50 mg of activated few areas, the greening disease was pres-
charcoal to every 10 gm of leaves. In- ent in most of the localities of the re-
oculum was kept cool i n crushed ice gion, and trees of sweet orange, man-
until the inoculations were completed. darin, grapefruit, lime and lemon wereSixth ZOCV Conference
affected. Trees showing dieback symp- for greening were found free of tristeza
toms invariably proved positive for virus, but mixed infections of tristeza
greening. Several trees that were positive and greening were prevalent.
EXPERIMENTS AND RESULTS
Symptomatology. Infected trees show
stunted growth, sparsely foliated
branches, unseasonal bloom, leaf and
fruit drop, and twig dieback (fig. 1).
Young leaves are chlorotic, with green
banding along the major veins. Mature
leaves have yellowish-green patches be-
tween veins, and midribs are yellow. I n
severe cases, leaves become almost chlo-
rotic, with scattered spots of green
(fig. 2).
Fruits on greened trees are small,
generally lopsided, underdeveloped,
and unevenly colored. Sides exposed to
the sun develop prominent, deep or-
ange spots, while the rest of the fruit
remains a dull orange-green. Fruits are
hard, and poor in juice. T h e columella
was almost always curved in sweet or-
ange fruits, and appears to be the most
reliable diagnostic symptom of green-
Fig. 2. Chlorotic leaf of. sweet orange with
green patches on narrow lamina.
ing. Some seeds in diseased fruits de-
velop normally, but most are small and
dark colored.
Transmission by tissue grafts. A high
percentage of transmission was ob-
tained by bark or leaf-patch grafting.
Shoot grafts almost always transmitted
greening. Budding was less successful
because of failure of bud-take. Symp-
toms appeared within eight weeks in
summer (March to June) and 10 weeks
in other months, in Poona. Sometimes
test seedlings developed symptoms after
two years or more.
Transmission by mechanical means.
Fifteen plants each of Phaseolus vul-
garis, Vigna sinensis, Nicotiana ta-
Fig. 1. Greening-affected Mosambi sweet
orange tree showing sparsely foliated bacum cvs White Burley and Xanthi-nc,
branches, twig dieback, and poor yield. N . sylvestris, Chenopodium quinoa, C.Stubborn, Greening, and Related Diseases
v u s cv. Long Green (fig. 3) and C . v u l -
garis cv. fistulosus. Subsequent leaves,
however, appeared normal. Back inocu-
lation from cucumber to citrus was un-
successful. Vigor of cucumber plants was
greatly reduced, and leaf size was about
half that of normal leaves. Schwarz (14)
reported similar results with the Na-
tional Pickling variety of cucumber in-
fected with South African greening dis-
ease.
Seed transmission. Twenty-four hun-
dred seeds of Mosambi sweet orange
and 850 seeds of grapefruit extracted
from greened fruits were sown for a test
of seed transmission. Normal-looking
seeds and brown and abortive seeds
were planted. Of these, 2,025 seeds of
sweet orange and 704 of grapefruit ger-
minated and produced normal plants.
No evidence was found of seed trans-
mission of greening in seeds of sweet
Fig. 3. Leaves of Cucumis satims cv. Long orange and grapefruit.
Green, showing characteristic symptoms fol- Transmission by insects. Although
lowing mechanical inoculation with extract Diaphorina citri was established as the
from leaves of greened sweet orange.
Healthy leaf at top.
vector of c i t k s greening in India ( 4 ,
attempts were made to transmit green-
ing by means of other insects collected
amaranticolor, Carica papaya, C u c u m i s on greened sweet orange shoots in or-
sativus cvs. Long Green and Khira chards in Poona.
Local, C. m e l o , and Citrullus vulgaris Insect species tested were: T o x o p t e r a
cv. fistulosus were inoculated mechan- citricidus (Kirk.); T. aurantii (B. de F.);
ically. Four plants of each species were Phyllocnistis citrella (Staint); Dialeu-
kept as uninoculated controls. Yellow- rodes citri (Ash.); Papilio demoleus L.;
ing, puckering, and marginal distortion and an unidentified leafhopper, Eupoc-
at the apical region of the first and the retis sp. Greening appeared only in test
second true leaves appeared in C. sati- seedlings exposed to psyllids.
RELATIONSHIPS O F
T H E GREENING PATHOGEN
W I T H I T S VECTOR
I n preliminary experiments on trans- of the insect. Percentage of transmis-
mission of greening, adult female psyl- sion of disease in these tests was uni-
lids were collected from greened trees, formly high. These observations indi-
and liberated directly on test seedlings. cated a biological relationship between
Results were erratic, and percentage of greening and the psyllids.
transmission was low. Subsequently, Effect of numbers of psyllids on
psyllids were fed o n a diseased plant in transmission. Adult psyllids were main-
the insectary for 24 hours and then lib- tained on diseased sweet orange seed-
erated on to the test plants for the life lings and liberated on test seedlings for46 Sixth ZOCV Conference
TABLE 1
INCUBATION PERIOD OF GREENING PATHOGEN IN DlAPHORlNA ClTRl
ON SWEET ORANGE SEEDLINGS
Greening symptoms in a series of test seedlings exposed to
Groups feeding of psyllids on successive days after access feeding*
of
psyllids 4 6 8 9 10 11 12 13 14 15 16
First........
Second - - - + + + + + + t +
Third......
Fourth....
Fifth.......
Sixth......
1 -- -
-
-
- +
-
+
-
+
-
+
-
+
- +
+
+
+
+
+
+
+
Seventh.. - - - - - - + + + +
Eighth....
Ninth......
} -- -
- - - - - + + + + + +
Tenth...... - - - - - + + + + + + +
* t = positive transmission of greening pathogen; -= no transmission of greening.
a period of 24 hours. Sixty test seedlings Incubation period of pathogen in
were exposed to varying hopulations of psyllids. T o determine the latent period
psyllids. Forty-one of 60 plants exposed in the insect, groups of five freshly
to a single psylla were infected with emerged, pathogen-free female psyllids
greening. Groups of five or more psyl- were fed on diseased plants for 24 hours,
lids infected all 60 plants. and transferred serially to healthy test
~inimurn-acquisitionfeeding period. seedlings of sweet orange every second
Adult ~svllidsmaintained on healthy
L J
day for the first 8 days and every day
sweet orange seedlings were fasted for for the second 8 days. Data in table 1
2 hours before access feeding on dis- show that a waiting period of from 8
eased plants. T h e psyllids were then to 12 days is required before the psyllid
transferred, in groups of five, to test can transmit the pathogen following
plants and allowed to feed for a min- acquisition feeding. Only 10 per cent
imum period of 12 days (4). T h e psyl- of the psyllids could transmit the dis-
lids transmitted the disease to 45 of 50 ease on the eighth day, while all trans-
test plants after access feeding of 15 mitted after 12 days.
minutes, and to all the plants after ac- I n similar experiments with 4th- and
cess feeding of 30 minutes or longer. 5th-instar nymphs, it was observed that
Minimum-infection feeding period. the incubation period in the adult was
Freshly emerged adult psyllids were reduced by several days.
caged on diseased sweet orange seed- Retention by psylla. Freshly emerged
lings for a period of 12 days. Psyllids female psyllids were fed on diseased
from these colonies were transferred. in sweet orange seedlings for 24 hours, and
groups of five, to each of the test seed- transferred to a series of test seedlings.
lings for periods ranging from 5 min- T h e first transfer was made at 2 days
utes to 24 hours. Psyllids transmitted and the others at intervals of 8 days
greening in a minimum infection feed- until the insects died. Under the con-
ing of 15 minutes, but the percentage ditions of the experiment, the psyllids
of transmission was low. One hundred lived for 70 to 86 days, although the
per cent infection was obtained when life span of disease-free psyllids is about
the psyllids fed for 1 hour or more. 150 days at Poona. T h e test was re-Stubborn, Greening, and Related Diseases
TABLE 2
RETENTION OF GREENING PATHOGEN BY ADULTS OF DlAPHORlNA ClTRl
ON SWEET ORANGE SEEDLINGS
Test plants serially exposed to psyllids on
Experiment successive transfer at intervals (days)*
no.
2 10 18 26 34 42 50 58 66 74 76 78 86
* + = positive transmission of greening pathogen; - = no transmission of greening.
t Psyllids collected from orchard trees.
$ Psyllids died.
peated 10 times, starting with a group the 4th- and 5th-instars, however, did
of five adults for each test. Three of acquire it and transmitted it to a high
the 10 tests in table 2 were carried out percentage of test seedlings of both
with freshly emerged adult psyllids col- series (193/200 plants). Symptoms ap-
lected from orchard trees of sweet peared in 110 to 150 days.
orange. Results of these experiments Effect of number of nymphs on trans-
(table 2) clearly show that the psyllids mission. Fourth- and 5th-instar nymphs
retained infectivity throughout their
life span.
TABLE 3
Transmission by nymphs. T h e vector
TRANSMISSION OF GREENING
Dinphol-ina citl-i has five nymphal in-
PATHOGEN BY NYMPHS OF
stars in addition to the egg and the DlAPHORlNA ClTRl ON SWEET
adult (8). Nymphs of all stages readily ORANGE SEEDLINGS
settle down to feed if young
, - and suc-
culent flush is available. Nymphs of Number of test plants
each instar were collected and fed for infected/number exposed
Nymphs
24 hours on diseased sweet orange i n 1st stage* 2nd staget
the insectary and then transferred, in
First instar ........ 0/50 0/50
groups of 20, to each test plant and
allowed to feed for 15 to 20 days until Second instar .. 0/50 0/50
they emerged into adults. T h e freshly Third instar........ 0/50 0/50
emerged adults were transferred to an- Fourth instar .... 43/50 50/50
other test plant for the rest of their life Fifth instar ........ 50/50 50/50
span. I n each series, 50 test plants were
exposed to nymphs of each instar. T h e * Indicates first series of test plants on which
nymphs were liberated for test feeding for first 15
data in table 3 show that the 1st-, 2nd-, days.
and 3rd-instar nymphs were unable to t Indicates series of test plants on which nymphs
were transferred for life after 15 days of feeding on
acquire the pathogen. T h e nymphs of first series of test plants.48 S i x t h ZOCV Conference
fed for 24 hours on diseased sweet or- had copulated. These were then trans-
ange were transferred, singly and in ferred to healthy, luxuriantly growing
groups of 3, 5, 10, 25, and 50, to each seedlings of Mosambi sweet orange, on
of the 20 test seedlings. Twenty-five or which they laid eggs. Nymphs that
more nymphs per test plant gave 100 hatched were carefully transferred, in
per cent transmission, whereas only 15 groups of 5, to test seedlings, where
per cent of the plants became infected they spent their entire lives. None of
when only 1 nymph per plant was used. the 304 test seedlings that were col-
Absence of transovarial transmission. onized with 1,520 nymphs over a period
Freshly emerged adults of Diaphorina of 3 years developed greening. These
citri were colonized on diseased plants data prove that greening is not carried
of sweet orange. Close watch was main- over to the next generation through the
tained in order to pick u p females that eggs of infective females of D. citri.
DISCUSSION AND CONCLUSIONS
T h e greening disease of citrus in the thrips, the vector of tomato spotted-wilt
Deccan T r a p Country in India appears virus, which must feed on infected
similar to the Soutli African greening plants in the larval stage in order to
and Philippines leaf mottling diseases become viruliferous (1).
since all three induce identical symp- T h e significance of these findings is
toms in certain citrus species, and are that psyllids continue to serially infect
transmitted by psyllids. Since mycoplas- healthy test plants over a long period,
malike organisms are found associated and remain infective after molting.
with these diseases of citrus ( l o ) , and This strongly indicates that the causal
are not found in healthy citrus tissues, agents of greening multiply in the body
it has been concluded that they are the of the psyllid. T h e greening pathogen
probable cause of greening or citrus may be classed as propagative as well
decline in India, greening in South Af- as circulative (2). Since there is no trans-
rica, and leaf mottling in the Philip- ovarial transmission, however, it would
P':nes. be necessary to demonstrate multipli-
T h e relationship existing between cation of greening pathogen in psyllids
the greening disease pathogen and the in order to class it finally as propaga-
vector, Diaphorina citri, is clearly tive. T h e only other disease known to
shown to be a circulative type ( 2 ) . T h e be transmitted by a psyllid, pear de-
psyllid requires an incubation period cline, vectored by Psylla pyricola Foer-
of about 21 days in which to transmit ster, is caused by a circulative pathogen
the pathogen, which it retains for life (9).
following a short access feeding on a I n South Africa the vector of the
diseased plant. Apparently most of the greening pathogen, Trioza erytreae
psyllids in orchards usually acquire the (Del Guercio), does not require an in-
pathogen as 4th- and 5th-instar nymphs, cubation period, and becomes infective
and become vectors for life after going after 24 hours o n diseased tissues. T h e
through a latent period of 5 to 9 days. greening pathogen persists for at least
It is unnecessary for adult psyllids aris- 2 to 3 weeks in adults, but nymphs do
ing from infectious nymphs to have ac- not appear to transmit it, and there is
cess feeding on diseased shoots in order no evidence for transovarial transmis-
to become vectors. Nor is it essential sion (Dr. H . D. Catling, personal com-
that the psyllids have access feeding as munication). How Philippines leaf mot-
nymphs in order to become infectious tling disease pathogen is related to its
in the adult stage-a requirement with vector, D i a p h o ~ i n ac i t ~ i is
, not known.Stubborn, Greening, and Related Diseases 49
Of several herbaceous hosts mechan- cumber. It is believed that the disease
ically inoculated with extracts of symptoms in cucumber were induced
greened sweet orange leaves, only Cu- by a vivotoxin produced by mycoplas-
cumis sativus, C. melo, and Citrullus malike organisms present in greened
vulgaris cv. fistulosus developed disease trees, and not by the organisms them-
symptoms. These could not be trans- selves. Experiments are being carried
mitted back either to citrus or to cu- out to check this point further.
LITERATURE CITED
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13. SCHWARZ, R. E.
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Conf. Intern. Organ. Citrus Virol. (J. F. L. Childs, ed.) Gainesville: Univ. Florida Press,
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