Abstracts of Papers Accepted for Presentation at the Seventh Annual
i Meeting of the Philippine Phytopathological Society in Conjunc-
Stion with the First National Pest Control Conference, Iloilo City,
S5-8 M ay 1970 .......................................... 1
Histopathology of Mango Fruits Infected by Diplodia natalensis
P.M Halos and G. G. Divinagracia .......................... 14
Cultural, Physiological, and Biochemical Properties of Isolates of Philip-
SEldon I. Zehr ......................... ............. 29
Strains of Pseudomonas solanacearum in the Philippines as Deter-
mined by Cross-inoculation of Hosts at Different Temperatures
lldon 1. Zehr ....................................... 44
: A Bacterium Causing Vein Necrosis of Rambutan
A. N. Pordesimo and F. C. Barredo ....................... 55
T-.i':; issue Incompatibility, the Likely Cause of Bud union Crease of Cala-
Smondin Grafted to Certain Citrus Varieties
A. L. Martinez ................................ .......... a62
S Further Study on.the Assessment of Yield Losses Due to Rice Blast
. R. Exconde and A. D. Raymundo ............ ....... 66
5 effect of Exposure Period in Moist Chamber on Gercospora fuligena
Spore Germination, Penetration, Infection and Sporulation on
T. R. Magda and F. C. Quebral ............ ........... 75
A' PHYTOPATHOLOGICAL NOTES
.. :: Local Diseases of Sorghum :
Asuncion Karganilla. and F.A. Elasegui .................. 83 ; :
;. Diseases of Corn Unreported in the Philippines .
S. C. Dalmacio and 0. R. Exconde ..................... 89
Official Organ of
THE PHILIPPINE PHYTOPATHOLOGICAL SOCIETY
R'.: Entered as second class mail matter at the Manila Post Office on 22 September 1970.
GIL G. DIVINACACIA
CEAIM our in t ha pl C iAlOLCOG
U:PC A CDLLE6E/ LAGUNA
vt'-.. T R a d nd F .Q e ra . . .. . . . 5 "*ii
STreasurer, T. REYES, UPCA, College, Laguna
REditor-inrChie, A. N. P QODESIMO, UPCA, College, Laguna
Vie-wsletter Editole, G. DAVINDE, UPCA, CColege, Laguna
SBusiness Manager, C. A. BANIQUED, Bureau of Plant Industry, San Andres, Manili
cAuditor, F. L. RNUQUE, Plant Protection Division, IRRI, College, Laguna
F, T.. C, QUEBRAL, UPCA, College, Laguna (Luzon)
i... R RVERA, Philippine Sugar Inst., Bacolod City (Visayas)
A. L. ELOJA, Bureau of Plant Industry, Davao City (Mindanao)
.'Bayer Chemicals, Inc., 622 Shaw Blvd., Mandaluyong, Rizal
:Canlubang Sugar Estate, Canlubang, Calamba, Laguna
'iCdtral Azucarera de Bais, Bais, Negros Oriental
CIBA-GEIGY, LTD., Switzerland
Dole Philippines, Inc., Madrigal Bldg., Makati, Rizal
Filipinas Pest Control & Filipinas Agricultural Trading, E. de los Santos Ave., Cubao
FMC Corporation, Niagara Chemical Division, Middleport, New York, U.S.A.
HOECHST PHILIPPINES, INC., Corer Pioneer & Reliance Sts., Mandaluyong, Rize
MERCK SHARP & DOHME (PHIL.) INC., 113 United Nations Ave., Manila
Philippine Sugar Institute, Diliman, Quezon City
Tarlac Development Corporation, Hacienda Luisita, San Miguel, Tarlac
''Union Carbide (Philippines) Inc., Makati, Rizal
Velsicol Chemical Co., Makati, Rizal
Vintnrina Millina Cr'n In Virtnrie Nearn fotidelanal
Official Organ of the Philippine Phytopathological Society
A. N. PORDESIMO, Editor-in-Chief, Department of Plant Pathology
University of the Philippines, College, Laguna
C. A. CALICA, Bureau of Plant Industry, San Andres, Manila
B. P. GABRIEL, University of the Philippines, College, Laguna
M. E. LOPEZ, Philippine Sugar Institute, Quezon City
A. L. MARTINEZ, Bureau of Plant Industry, Lipa City
DOLORES RAMIREZ, University of the Philippines, College, Laguna
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ABSTRACTS OF PAPERS ACCEPTED FOR PRESENTATION AT
THE SEVENTH ANNUAL MEETING OF THE PHILIPPINE
PHYTOPATHOLOGICAL SOCIETY IN CONJUNCTION
WITH THE FIRST NATIONAL PEST CONTROL
CONFERENCE, ILOILO CITY, 5-8 MAY 1970
(Arranged alphabetically according to first author's surname)
The build-up of Thielaviopsis in pineapple trash and its effect on
crop plants. C. A. Alaban. Thielaviopsis paradoxa, a wound parasite of
pineapple, is a primary agent in the decomposition of pineapple trash.
Quantity of pineapple trash undergoing decomposition was found to be
related to mortality rate of newly-set plants as well as growth condition
of survivors. Large amounts of decomposing trash caused poor stands
and uneven growth of plants. Evidence to demonstrate the pathogenicity
of Thielaviopsis was obtained.
Response of improved mungo varieties to four diseases.- R. V. Corta-
do. -Seven improved varieties of mungo (MG50-10A, MG55-3, MG59-6,
MG60, MG61-2, CES 14 and BPI Glabrous) were tested for resistance to 4
most common diseases in Cagayan Valley, viz., Cercospora leaf spot, pow-
dery mildew, mosaic, and root knot. Except in root knot, the test plants
were subjected to natural inoculation in the field. Disease ratings re-
vealed that none of the 7 improved varieties of mungo are resistant to
Cercospora leaf spot, root knot, and powdery mildew. These varieties,
however, reacted differently to mosaic. Based on infection percentage, all
the MG varieties showed higher degree of mosaic resistance than BPI
Glabrous. Except MG55-3, the tested MGs were more resistant than CES
14. No significant differences in mosaic resistance among the MG varie-
ties were obtained. CES 14 was more resistant to mosaic than BPI Gla-
brous; the latter was the most susceptible.
Distribution of citrus leaf mottling and its vector in the Philippines. -
R. E. Cortez and C. S. Celino. Leaf mottling is a citrus disease which
was described by several investigators as the principal factor responsible
for the decline of citrus in Batangas. Studies on its etiology proved that
the disease is of virus nature, with the causal agent determined to be
distinct from tristeza virus. The disease was likewise found to be vectored
by the citrus psylla, Diaphorina citri Kuway. An extensive indexing of
citrus trees grown in various parts of the country was pursued by the
BPI Lipa Experiment Station to determine the distribution of the disease.
r'H ILIPPINE F'HYTOPATHOI.OGV
The activity of the insect vector was likewise assessed. Indexing was
performed by collecting budsticks from trees exhibiting certain aspects
of the disease syndrome and then subsequently inoculating suitable in-
dicator seedlings maintained in the screenhouse. A minimum of 5 trees
from each orchard was indexed on the indicator varieties which included
the Madame Vinous Sweet Orange, Sexton Tangelo, and Rough Lemon.
The insect vector was surveyed by taking 100 young shoots from citrus
trees selected at random from each orchard. Insect population was ex-
pressed as percentage of young shoots infested.
On the basis of incidence and natural spread of leaf mottling, the
citrus growing areas of the Philippines can be divided into 3 zones, viz.,
Zone I areas with high incidence of the disease and high psylla popula-
tion, where the rate of spread of leaf mottling appears to be extremely
rapid. Nucellar virus-free materials planted in the field never prosper
as trees die before reaching bearing age. The entire province of Batangas
which is the center and origin of the citrus industry in the Philippines
belongs to this zone. Zone II areas with intermediate incidence of the
disease and the psylla population ranges from low to high. The decline
of trees in this zone is noted only in comparatively young groves and
the rate of spread increases every year. The provinces of Laguna and
Cavite, the towns of Pili, Tigaon, and Isarog of Camarines Sur, General
Santos City in Cotabato, and Mandawi City in Cebu belong to this zone.
Nucellar plantings started in some of these places have attained consider-
able progress within the last 3 to 5 years of observation. Zone III -areas
wherein there is no natural spread of leaf mottling because the psyllid
vector or the source of inoculum, or both are absent. Trees found in-
fected are confined to young plantings which were verified to be in-
troduced from Zone I as budded or unbudded seedlings. Healthy economic
groves exist in this zone. The provinces of Mindoro Oriental, Davao,
La Union, Cagayan, Mountain Province, Camarines Norte, and part of
Camarines Sur and Cebu are included in this zone.
The indexing program showed that the incidence of leaf mottling in
the Philippines has increased to include new areas previously rated as
disease-free. Evidently, the high rate of spread of the disease particular-
ly in the province of Batangas is due to the numerous infected trees serv-
ing as sources of inoculum and the high population of the psyllid vector
in the area. On the other hand, natural spread is not taking place in
many groves because either the vector or the source of inoculum, or both are
Diaphorina citri is widely distributed throughout the country but its
population density has been observed to vary considerably within one
Transmission of different citrus leaf mottling types by a psylla. -
R. E. Cortez and C. S. Celino. In the indexing of citrus trees from
different parts of the Philippines, various types of leaf mottling were en-
countered. Three leaf mottling isolates (LMT) were of particular signi-
ficance because of their origin and possible relationship with the citrus
virus diseases reported in other parts of the world. LMT-1 was obtained
from a Valencia Orange tree growing in San Juan, Batangas. LMT-2 was
obtained from an Eureka Lemon tree in Hacienda San Jose, Dadiangas, Cota-
bato, grown from budwood imported from California. LMT-3 was obtained
from a Gayonan tree grown in the collection field of the BPI Lipa Experi-
ment Station, Batangas, from an importation from China.
Non-viruliferous psyllids (Diaphorina citri Kuway) were first given an
acquisition feeding period of 1 week on experimentally-infected seedlings
of Sexton Tangelo and then transferred to healthy seedlings for an in-
fection feeding period of 1 week. Results showed that D. citri was able
to transmit all the 3 LMT.
The blighting of coconut and other plants caused by salt water.-
Lydia C. Crisostomo, S. B. Malimban, P. A. Santamaria, and A. C. Pizar-
ro.- The row of palms and ornamental plants planted along Roxas Boule-
vard facing Manila Bay has been a disturbing sight to passers-by. Exam-
ination of these plants revealed blighting symptoms. In coconut, Cocos
nucifera L. and in bunga de china, Adonidia merilli L., the blighting
symptoms were found to be similar. The latter plant appeared to be
more sensitive to the injury than the former. Bo-trees, Ficus religiosa L.
and calachuchi, Plumeria acuminata L. were also severely affected. Anii,
Erythrina fusca L. was less affected. Because salt water from the sea
was suspected to be the primary cause of this injury, an investigation
was conducted. Experimental results showed that salt water induced plant
injury. Evidence of leaf burning and defoliation of plants was obtained
under simulated conditions. The high chloride content and total alkalinity
of salt water appeared to be responsible for leaf burning; the toxicity
level of salt varied with the kind of plants.
Assessment of yield loss due to rice blast.-O. R. Exconde and A. D.
Raymundo. Yield losses due to rice blast in IR-4, IR-8, and IR-60 were
studied at 3 levels of disease intensity and 3 stages of inoculation. A sta-
tistically significant reduction in yield of IR-8 was obtained during the
wet and dry seasons. During the wet season, a reduction in yield of 7.0,
8.4, 15.2% at disease intensities 1, 2, and 3, respectively, was obtained.
The reduction in yield during the dry season was 8.0, 14.0, and 15.2%
for disease intensities 1, 2, and 3, respectively. On IR-4, a significant
reduction in yield was obtained in the wet season only. The reduction
in yield was 4.6 and 5.7% for disease intensities 1 and 3, respectively.
No significant reduction in yield on IR-60 was obtained in either season.
IN OS. 1 & c z
P-UT InTC T'rnn-UnT nr-c Io -T
Histopathology of mango fruits infected by Diplodia natalensis. -
P. M. Halos and G. G. Divinagracia. Both young and mature pycnidios-
pores of Diplodia natalensis Pole-Evans germinated in 2.5 hr; penetrated
after 3.5 hr in ripe mangoes and after 6.5 hr in mature green ones. Un-
like in previous reports, the fungus entered through the intact cuticle of
the pedicel as well as the cuticle and lenticel of the pericarp. The
pathogen also penetrated through the exposed pedicel surface, injured pedicel
sides, abscission between the peduncle and the pedicel, pedicel scar and
wounded exocarp. The fungous hyphae ramified inter- and intra-cellular-
ly, invading possibly all tissues in the pedicel, exocarp, and mesocarp.
Host cells were killed in advance of the hyphae. Longitudinal spread of
hyphae was most rapid through resin ducts of the pedicel and laticiferous
glands of the pericarp. Diplodia stem-end rot symptoms were observed
on fully ripened mangoes 48 hr after inoculation and on mature green
ones 48 hr later. Intact green fruits on twigs artificially inoculated with
pycnidiospores and sectioned after 96 hr, exhibited histogenic defense re-
action. Tannins were detected in both healthy and initially infected
tissues but not in completely macerated ones. Histochemical tests demons-
trated the action of both pectic and cellulolytic enzymes in host-tissue
disintegration. Their exact role in pathogenesis has not been ascertained
since walls of parenchymatous mesocarp cells break down naturally in
ripening mangoes. The decrease in acidity and tannin content with a
concomittant increase in sugars and starch granules in ripening mangoes
favored successful invasion by D. natalensis.
The interrelated effects of some meteorological factors on the onset
and development of citrus scab at Catarman, N. Samar. F. D. Fuentes. -
This study was conducted to 1) determine the interrelated effects of tem-
perature, dew point, relative humidity, wind velocity, rainfall, and sun-
shine on the onset and development of citrus scab, and 2) establish a
prediction equation that will serve as a basis for timing spray applications
in the control of this disease.
A grove consisting of bearing mandarin trees was divided into 3 lots.
Each lot represented a replication. Weekly observation was made on 3
trees randomly tagged in each lot. Using a disease rating scheme with
categories ranging from 1 to 6, weekly observations on the onset and de-
velopment of the disease were started in July 1969 and terminated in
January 1970. The weekly ratings were transformed into percentages for
statistical analysis. Daily observations on the meteorological factors for
the same period were recorded and transformed into weekly averages.
Simple correlations revealed that temperature was associated with dew
point and significantly correlated with bright sunshine; dew point was
associated with relative humidity and rainfall and significantly correlated
with bright sunshine: relative humidity was signifirantlv correlated with
Nos. 1 c 2]
rainfall; wind velocity was associated with rainfall; and rainfall was as-
sociated with bright sunshine. The onset of the disease was associated
with dew point, wind velocity and rainfall. It was significantly correlated
with relative humidity. The development of the disease was significant-
ly correlated with relative humidity and rainfall at 1% and 5% level,
By multiple correlation and regression, the variance in onset of the
disease (R2) was 0.576. This indicated that these meteorological factors
could account for about 57.6% of the disease onset. The multiple cor-
relation coefficient (R) was found to be statistically significant, indicat-
ing that the set of variables used was adequate in estimating the rate of
the disease onset. About 50.45% of the disease development could be
explained by the interrelated effects of the different meteorological factors.
Quantitatively, dew point contributed the most to the onset while rainfall
contributed the most to the disease development. These 2 meteorological
factors should be critically considered in planning an effective spray pro-
gram to control this disease by the use of effective chemicals.
Soil fumigation for controlling plant parasitic nematodes on okra and
its carry-over effects on cowpea. A. R. Josue and C. P. Madamba. D-D
(dichloropropane-dichloropropene) applied at 74 gal per hectare (2.6 ml/
injection at 12-inch centers) effectively controlled root-knot nematodes,
Meloidogyne incognita, and induced significant yield increase in okra.
Cowpea, planted as a second crop in lots previously treated with D-D
responded markedly to the residual effects of this nematocide. Solvirex
5G (0,0 diethyl-S-2 ethylthioethyl-phosphorodithioate) and Temik 10G (2-
methyl 2 (methylthio) methyl carbamyl oxime) applied at 120 and 80 kg
per hectare (4 and 2.7 g/m row), respectively, also induced increase in
okra yield. There were indications that these chemicals exhibited carry-
over effects on cowpea. Treatments with 1, 2 dibromo-3-chloropropane
and ethylene dibromide significantly reduced root-knot infection but these
were not reflected in the yield of either okra or cowpea.
Host range of the rice-grassy-stunt virus. K. C. Ling and V. M.
Aguiero. In the search for alternate hosts of the causal agent of rice
grassy stunt, different weeds, Aneilema malabaricum, Cyperus iria, C. ro-
tundus, Dactyloctenium aegyptium, Digitaria adcendens, Echinochloa colo-
num, E. crusgalli, Eleusine indica, Eragrostis pilosa, Fimbritylis mi-
liacea, Imperata cylindrica, Ischaemum rugosum, Leptochloa chinensis,
Monochoria vaginalis, Panicum maximum, Paspalum conjugatum, P. scro-
biculatum, and Rottboellia exaltata, were collected from rice fields and
inoculated. None of these inoculated weeds became infected although
some of them served as temporary hosts of the vector, Nilaparvata lugens
Stal. The inoculated rice species that became infected were Oryza sativa,
0. alta, 0. australiensis, 0. breviligulata, 0. glaberrima, 0. granulata, 0.
subsp. balunga, O. rufipogon, 0. sativa f. spontanea, and 0. spontanea
(Australia. One line of 0. nivara showed a consistently low percentage
of infection when tested by mass screening method. This line is the
most resistant so far tested. Infected rice varieties Shan-san-sa-san and
Laka showed distinct striping symptoms. These two varieties can be used
as differential hosts of the virus.
Plant parasitic nematodes associated with banana in South Cotabato.--
C. P. Madamba and J. J. Walawala. Identification of plant parasitic
nematodes associated with banana in General Santos, South Cotabato re-
vealed the presence of 12 genera. The most predominant genera isolated
from soil samples were Meloidogyne, Helicotylenchus, and Hemicycliophora.
They occurred in 100, 80, and 70% of the soil samples, respectively, col-
lected from 10 randomly selected plantations. Extraction from 50% or
more of the soil samples revealed the occurrence in low numbers of
Aphelenchus, Hoplolaimus, Tylenchorhynchus, Xiphinema, and Trichodorus.
Isolated from roots were Meloidogyne, Radopholus, and Pratylenchus. Two
species of the root-knot nematode, Meloidogyne incognita and M. javanica,
Nematodes and other organisms associated with shredded leaves of
downy mildewed Saccharum spontaneum. C. P. Madamba and J. J. Wala-
wala. Five nematode genera viz., Aphelenchoides, Aphelenchus, Rhabditis,
Helicotylenchus, and Dorylaimus were found associated with shredded leaves
of downy-mildewed talahib (Saccharum spontaneum L.) collected from 20
locations, respectively. Aphelenchoides was the dominant genus from
shredded leaves obtained from Mabalacat (Pampanga), Luisiana (Laguna),
Lucena City, and Pagbilao (Quezon) while Rhabditis was prevalent in
samples collected from Los Bafios and Mabitac (Laguna), and Lucban
(Quezon). In addition, Rhabditis was the only genus isolated from speci-
mens collected from Los Bafios (Laguna), Gumaca (Quezon), and the Bicol
Region. Aphelenchus and Helicotylenchus were extracted only in speci-
mens collected from Mabalacat, Pampanga. Shredded leaves obtained from
Pililla (Rizal), Calauag (Quezon), and Labo (Camarines Norte) contained
no nematode, suggesting the role of temperature in the desiccation of this
organism. Ciliated protozoans, rotifers, mites, earwigs, and Fusarium spp.
were also isolated from the shredded talahib leaves.
Responses of trifoliate orange, its hybrids and some citrus relatives
to the greening pathogen. A. L. Martinez. Seedlings of Severinia buxi-
folia, a citrus relative, inoculated with the greening (leaf-mottle-yellows)
pathogen, developed symptoms of slight leaf yellowing, faint mottling,
and slight stunting. These symptoms did not develop on inoculated seed-
A .TRn A cTr
sub-inoculations from these symptomless plants to seedlings of Ladu
Ponkan, Szinkom, Sziwuikom, and Madam Vinous sweet orange resulted
in symptoms of severe yellowing, mottling and stunting, demonstrating
that the symptomless plants are apparently tolerant and that they car
serve as carriers and/or hosts of the greening pathogen. When Ladu
Ponkan, Szinkom, Sziwuikom, and Madam Vinous scions were grown or
trifoliate orange, Troyer, and Carrizo rootstocks and these were inoculated
with the greening pathogen, all the scion varieties showed severe symptom!
as when inoculated as seedlings. It is evident that trifoliate orange, Troyer
and Carrizo, which are considered to be tolerant of the greening pathoger
when inoculated as seedlings, do not impart tolerance to the scion varieties
Variation in virulence among single-colony subcultures of Xanthomona,
oryzae. S. H. Ou, S. D. Merca, and F. L. Nuque. Variation in virulence
among daughter colonies of single strains was studied, using strain BlH
(virulent) and B23 (weak). One hundred single-colony subcultures fron
each strain were isolated, cultured and inoculated by needle prickng tc
two rice varieties, Zenith (resistant) and JC-70 (susceptible). The 10(
subcultures from each strain showed varying degrees of pathogenicity or
both varieties. From the few most virulent and very weak subcultures
one representative colony was selected and 100 single-colony subculture!
were again isolated and inoculated on the two varieties. The process o
selection towards more virulent and less virulent strains was continued
for 3 to 4 generations.
From B15, the subculture B15-27 was more virulent than the original
and a further selection, B15-37-109, was still more pathogenic to boti
indicator varieties. Among the less virulent colonies, B15-1 was less patho
genic than B15 but a further selection, B15-1-99, did not show much fur
their decrease in virulence. The changes in pathogenicity among single
colony subcultures of B23 were similar to that of B15 but changes toward:
the less virulent were not obvious. This study indicates that a single
colony culture does not represent the entire range of pathogenicity o
this bacterial plant pathogen.
Control of rice blast with systemic fungicides. S. H. Ou, F. L. Nuque
and T. T. Ebron, Jr. -Five systemic fungicides: Benlate, Kitazin, Mil
curb, Polyoxin, and Thiabendazole were tested for controlling leaf anc
panicle blast by soil and seed treatment as well as by foliar spray. The
experiments were conducted in blast nursery and in a farmer's field. B'
soil treatment, Benlate and Kitazin showed systemic activity against blast
the latter was less effective. Plants, grown in pots where Benlate wa
added at 1/2 to 1 g per 10-20 liters of soil, were almost completely free
of blast both at seedling and maturing stages. In pots without thi
chemical, the seedlings were severely damaged or sometimes complete!
killed; panicle blast reached 92 to 95%. Experiments in a farmer's field
Nos. I & 2
PD-- -ODJAI PUV-n-----n -- \
indicated that soil treatment requires a high dosage of Benlate, which is
impractical. Pot test indicated that this chemical has a residual effect in
soil which is effective against blast for a period of more than 160 days.
Benlate, applied as seed treatment at the rate of 8 g/kg of seed,
protected the seedlings for about 25 days in our blast nursery where
inoculum density was heavy. Other chemicals were found ineffective.
Foliar spray with Benlate at the early stage of flowering and repeated
I week later reduced panicle blast from 61 and 98% to 7 and 10%,
and increased yield from 1.4 and 3.8 tons in the controls to 4.8 and 5
tons/hectare in the sprayed plot. Benlate was absorbed quickly by rice
plants. When applied to the soil 12 hr before inoculation, Benlate pre-
vented the development of lesions on leaves. Preliminary tests indicated
that Benlate has also insecticidal effect on nymphs of leafhopper and
Pathogenicity of Pyricularia oryzae isolates from resistant varieties.--
S. H. Ou, F. L. Nuque, T. T. Ebron, Jr., and Sonia P. Ebron. The
international rice blast nurseries have found several rice varieties with a
broad spectrum of resistance to blast. Occasionally, a few leaf lesions
occur on these varieties, indicating that new pathogenic races have evolved
and that the varieties begin to break down their resistance. Isolates of
the fungus from such lesions when inoculated to the original varieties
from which they were isolated, produced only a few lesions while on
the control (susceptible) varieties inoculated at the same time, many lesions
were produced. When single conidial subcultures from these isolates were
inoculated to race differential varieties, they were separated into many
races. From isolate FR-13 (isolated from variety Carreon; infected both
Carreon and Tetep), for instance, the 182 single conidial subcultures were
separated into 43 races. Of these 182 subcultures, 80 cannot infect Car-
reon and 67 cannot infect Tetep. It seems that the extreme variability
of pathogenicity is a built-in weakness of the fungus against varieties
with broad spectrum of resistance. Since many of the new races cannot
infect the original suscept and since the new races cannot effectively
build up in these varieties, such varieties may, therefore, be expected to
be more or less stable in their resistance.
Nuclear behavior of Sclerospora philippinensis. A. M. Pedrosa, Jr. -
Leaves of downy-mildewed corn (Zea mays L.) and sugarcane (Saccharum
officinarum L.) were incubated under laboratory conditions to induce
sporulation of the causal fungus. Surface scrapings from these leaves
yielded multinucleate, knob-like conidiophore initials whose nuclei come
entirely from the multinucleate intercellular hyphae embedded in the
mesophyll or stomatal cavity of the infected corn leaves. All the nuclei
of the conidiophore initial, commonly 95-200, were passed on to the
r---~ ----~-~ -- r---- ------
in 5 to 7 days. Gloeosporium was more virulent than Fusarium. Symp-
toms on the crown and fingers were discernible 2 to 3 days after inocula-
tion. When previously inoculated banana fruits were dipped in a suspen-
sion of Thiabendazole (Tecto 90) at 1 g/gal water, rotting of the crown
and fingers was not favored. This treatment delayed ripening of banana
fruits by 1 to 3 days. The banana varieties Gloria, Latundan, and Lacatan
showed varying degrees of susceptibility while Bongolan showed resistance
to the two fungous pathogens. G. musarum infected snapbeans, tomato,
and pepper but not Carabao mango and Ponderosa chico; it grew better
on PDA than on banana decoction agar. Fusarium grew slightly better
on either medium.
The cause and control of garlic tangle top. -A. C. Pizarro, Nenita C.
Baniqued, C. A. Calica, and H. T. Bergonia.- Tangle top of garlic has
been very serious in Batangas, Nueva Ecija, and the Ilocos Region. Af-
fected plants are stunted; their leaves dry faster than normal ones.
Characteristic yellow streaks appear on young leaves. Soon affected leaves
become twisted and fail to expand assuming a round appearance. The
tip of succeeding leaves remains inserted between the folded blades of
the preceding infected leaf. This results in the entanglement of the in-
fected leaves and hence the name "tangle top." Microscopic examination
of cloves and leaves of infected plants showed the presence of the eriophyid
mite tentatively identified as Aceria tulipae Keifer. The yellow streaks
on infected leaves seemed to be due to a toxin injected by the mites
Nos. 1 g& 21
experimentt station during tne iYwb wet season planting showed tfat ter-
cospora leafspot can reduce the yield of mungbean by as much as 23%
when 75% of the foliage are infected. Benlate, a new systemic fungicide,
when sprayed at 4 oz/100 gal water at 10-day intervals can adequately
protect mungbean against severe infection.
Yield obtained from sprayed plots gave a difference of 177.04 kg of
clean seeds per hectare over the unsprayed plots. At P75/cavan, this will
mean a difference of about P225 in favor of the sprayed plot. The
calculated cost of controlling this disease amounts to P20/hectare. This
field test showed that timely spraying can delay the rate and development
of Cercospora leafspot and thus prevent appreciable yield loss.
Evidence that tristeza virus is both stylet-borne and circulative. -
Magin L. Retuerma and W. C. Price. Results presented in this paper
demonstrate that transmission of tristeza virus by the tropical citrus aphid,
Toxoptera citricidus Kirk., is of the stylet-borne type and possibly also
of the circulative type. The experimental work was done with 25 aphids
for each test plant, rather than individual aphids, because of the scarcity
of test plants. In an extensive series of experiments, aphids allowed an
acquisition feeding period of only a few seconds picked up tristeza virus
from an infected leaf of West Indian lime (Citrus aurantifolia [Christm.]
Swingle) and transmitted it to a healthy test plant. Aphids also trans-
mitted tristeza virus after an infection feeding period of only a few
seconds. In other experiments, aphids that were given an acquisition feed-
Nos. 1 8& 2]
Comparative studies on incubation period, virus retention, and disease-
transmitting days showed that the adapted and ordinary forms of N. im-
picticeps were equally efficient in transmitting tungro virus. The effi-
ciency of adapted N. lugens to transmit grassy stunt did not differ from
that of its field population.
Effect of carbon and nitrogen sources in culture media on sclerotial
morphology and pathogenicity of Corticium sasakii. L. G. Santos and S.
H. Ou. An isolate of Corticium sasakii, R-11, was grown separately in
synthetic media with 20 different nitrogen sources. The effect of N sources
on its growth characters was determined mainly on the formation and
morphology of sclerotia. In media with proline and glycine, the number
of sclerotia ranged from 34-37; and with valine, and leucine, it ranged
from 5-8; the sclerotia in these media were large (3.54 x 3.08 mm and
2.52 x 2.2 mm). With alanine, glutamine and NaNO3, the sclerotia were
small and numerous (244, 237, and 218, respectively) and aggregating
throughout the surface of the medium. With tryptophan, lysine, and his-
tidine, poor mycelial growth and very few or no sclerotia were formed.
Six other isolates (R-5, R-10, R-ll, R-12, R-54, and R-55) grown in media
with 6 selected nitrogen sources (arginine, ammonium sulfate. NaNO3,
glycine, lysine, methionine, and histidine) demonstrated similar behavior.
The specific effect of these different nitrogen sources on the formation
and morphology of sclerotia of each isolate was not the same, i.e., on
glycine, small number but large sclerotia; on NaNO., large number of
aggregating sclerotia throughout the surface of the medium; on lysine
and histidine, sparse mycelial growth with very few and small sclerotia
or no sclerotia at all. Also, the growth and sclerotial formation on dif-
ferent carbon sources were different but not as distinct as that on nitrogen
The pathogenicity of isolated fungous cultures in media containing
different C and N sources was tested by inoculating 1-month seedlings
with mycelial discs. By disease indexing, the degree of pathogenicity of
cultures in media with different sources of nitrogen is shown in the fol-
lowing descending order: arginine, urea, threonine, glycine, leucine, and
alanine (59.4-51.1%); ammonium sulfate. NaN'O:, valine, proline, methio-
nine, aspartic acid, and glutamine (47.5-40.7%): serine, isoleucine, phenyla-
lanine, tryptophan, tyrosine, lysine, and histidine (36.4-17.1%).
The degree of pathogenicity of cultures in media with different car-
bon sources also varied. The highest disease indices were 45.0% for sucrose,
38.7% for fructose, and 38.3% for glucose.
Evaluation of systemic fungicides for the control of Philippine downy
mildew of corn.-O. E. Schultz and B. B. Manimtim. The effectiveness
of several systemic fungicides in the control of Philippine downy mildew
of corn was evaluated employing seed treatment, foliar application, and
A IJ3r I RAU I N
comUIIiunarLons o me LWU. 1-ppillcaIUo Uo DelClma, I ImuenCuazole ( J DL),
and Polyoxin to seed by steeping or dusting of dry kernels failed to reduce
downy mildew incidence. Subsequent pretreatment of seed with methyl
cellulose to assist fungicide adherence did not improve the performance
of either Benlate or TBZ. Neither foliar application of dilute suspensions
of the last-mentioned compounds immediately after seedling emergence- nor
their combination with seed treatment was successful in controlling corn
downy mildew. Uniroyal F 427, one of the few systemic compounds with
limited activity against Phycomycetous fungi was also evaluated. Treat-
ment of methyl-cellulose-coated seed with this fungicide at several dif-
ferent concentrations did not reduce disease incidence. Combination of
seed treatment and application of concentrated spray suspensions of F 427
as well as Benlate and TBZ to newly-emerged seedlings produced negative
results. These experiments were conducted under epiphytotic conditions
using hypersusceptible corn. Although it is conceivable that the com-
pounds tested may be partially effective on corn of average resistance under
conditions of less "disease pressure," their failure to control downy mil-
dews on crops other than corn suggests inadequate activity against the
causal fungi of this category of disease.
Plant parasitic nematodes in the corn-growing areas of Mindanao.-
J. J. Walawala and C. P. Madamba. -A survey of the principal corn-
growing areas of Mindanao conducted in June 1969 revealed identification
of 12 genera of plant parasitic nematodes associated with the crop. Those
identified from soil samples collected from 9 randomly selected farms in 5
provinces were Aphelenchus, Criconemoides, Dorylaimus, Helicotylenchus,
Hemicycliophora, Meloidogyne, Pratylenchus, Rotylenchus, Scutellonema, Tri-
chodorus, Tylenchorhynchus and Xiphinema. The most predominant and
widespread genera were Heticotylenchus, Dorylaimus and Tylenchorhyn-
chus. These were frequently encountered in most of the places surveyed.
Helicotylenchus and Dorylaimus were prevalent in Cotabato. Tylenchorhyn-
chus was common in Bukidnon. Pratylenchus, Trichodorus and Scutello-
nema were less common; they were found only in 5 locations. Although
Meloidogyne has been recovered from soil samples, no galling on corn
roots was observed. More genera of nematodes existed in the corn areas
of Cotabato than in Bukidnon.
LIUS. I ac 4J
reveals the elect
ol DmHAI M-45
on blight spores
These photographs, taken with the commercially important vegetables, for
scanning electron microscope show the example: asparagus, melons, summer
contrast between healthy late blight squash, carrots, celery, onions, tomatoes
spores and those sprayed with Dithane and potatoes. On potatoes, Dithane M-45
M-45 at recommended use dosages. The also prevents decay and common scab
untreated spores appear normal, while in seedpieces.
those sprayed with Dithane M-45 show Disease prevention, healthy plants,
signs of collapse and disintegration, and higher yield are vital requirements
These unusual photos are meaningful in profitable vegetable operations.
evidence of how Dithane M-45 stops the Dithane M-45 will help you reach these
development of blight infections on goals this season; get it at your farm
potatoes and vegetables, supply dealer now.
Dithane M-45 attacks blight spores
as well as the fungi causing many other
diseases: rust, leaf spots, anthracnose, ROHM r
downy mildew, scab, neck rot, purple IHARS r
blotch, Septoria and leaf mold. This . ....
effective fungicide protects numerous H lp keep/ Ameria qree .
Late blight spores before Dithane M1-4. spray spores after Dithane M-45 spray
Photos from Rohm and Haas research laboratories 1700 X Enlargement
6762 AYALA AVE., MAKATI
SUBSIDIARY OF ROHM AND HAAS COMPANY I PHILADELPHIA
y o pro
HISTOPATHOLOGY OF MANGO FRUITS INFECTED
BY DIPLODIA NATALENSIS
P. M. HALOS AND G. G. DIVINAGRACIA
Instructor and Assistant Professor, Department of Plant Pathology, UPCA, C
Portion of an M. Sc. thesis by the senior author. Supported by UPCO Re
Fund under Project No. 285.
Young and mature pycnidiospores of D. natalensis germinated in 2.5 1
and penetrated after 6.5 hr in mature green mango fruits and after 35 1
in ripe fruits. The fungus entered directly through the intact cuticle of tl
pedicel and into the cuticle and lenticel of the pericarp. The pathogen
also entered through the exposed pedicel surface, injured pedicel side
abcission zone in the pedicel, pedicel scar and wounded exocarp. TI
hyphae ramified inter- and intra-cellularly, invading probably all tissues
the pedicel, exocarp and mesocarp. Host cells were killed in advance
the hyphae. Longitudinal spread of hyphae was most rapid through res:
ducts of the pedicel and laticiferous glands of the pericarp. Steam-end re
symptoms were observed 48 hr after inoculation in fully ripened mango
and 96 hr in green ones.
Green fruits intact in twigs that were artificially inoculated with pycnidio
pores and then sectioned after 96 hr exhibited histogenic defense reaction:
Tannins were detected in the healthy as well as in the initially infected bi
not in completely macerated tissues. Histochemical tests for pectins an
celluloses revealed the action of pectic and cellulolytic enzymes in host tissue
disintegration. The exact role of these materials in pathogenesis is nm
certain, however, because walls of parenchymatous mesocarp break down nm
turally in ripening mangoes.
The decrease in acidity and tannin content and the concomittant increase
in sugars and starch granules in ripening mangoes favor successful invasion
by D. natalensis into their tissues.
The stem-end rot of mango fruits, caused by Diplodia natalensis
Evans is a serious post-harvest disease. It has been reported in Bu
Ceylon, Mauritius, USA, India and the Philippines. In India, the e<
mic loss due to Diplodia infection in a New Delhi market is 4.3%
Langra variety (Pathak and Srivastava, 1967b); in the Philippines. it v
from 2 to 6% (Alicbusan and Schafer, 1958).
A sound post-harvest disease control program necessitates an acci
assessment of the mode of entry, time of infection and pathogen dev
DIVINAGRACIA: DIPLODIA RoT
nt in host tissues. Very little is known about the host-parasite relation-
p of the mango infected by the stem-end rot organism, hence, a detailed
dy on the histopathological development.
Alicbusan and Schafer (1958) were able to induce infection on mango
spraying a spore suspension of D. natalensis on a heap of ripe fruits.
vastava and Durgapal (1965) showed that the fungus could infect
dily when inoculated in cavities on the pedicel of the fruits. Chakra-
'ty and Srivastava (1964) successfully reproduced the disease by inoculat-
Sthe organism on fruits wounded near the pedicel base. Pathak and
vastava (1967b) infected mature unripe fruits of the Langra variety by
Cculating the exposed surface and injured pedicel sides, pedicel scar and
ured epicarp near the stem-end with a spore suspension of the fungus.
t no infection occurred on the uninjured epicarp near the pedicel base.
This investigation was conducted to determine the mode of penetra-
n by D. natalensis into the pedicel and pericarp of mango, the his-
)athological development incited by the pathogen on the host at various
ges of infection, the cytochemical changes that accompany infection, and
toxic effect of the sap as a possible cause of rotting.
MATERIALS AND METHODS
Preparation of inoculum and inoculation of mangoes. Green mature
I ripe carabao mangoes were surface sterilized with 1:1000 HgC12 and
!n rinsed with sterile distilled water. One loopful of pycnidiospore sus-
nsion of the most virulent isolate of D. natalensis containing approximate-
50,000 pycnidiospores/ml was inoculated on each of the following: 1) ex-
sed pedicel ends, 2) pin-pricked pedicel sides, 3) uninjured pedicel sides
:h wax-sealed ends, 4) pedicel scars, 5) uninjured shoulders and sides,
d 6) pin-pricked exocarp shoulders and uninjured side. Another set of
: same variety were treated with mango sap at the basal sinus and
)ulders. All the inoculated mangoes were placed in moist chambers lined
th moistened tissue papers.
Pathogenicity test. Pycnidia crushed aseptically in sterile distilled water
re used as inoculum. One loopful containing approximately 50,000
cnidiospores/ml was inoculated on the excised stem-end scar of green
Lture Pico mangoes. The inoculated fruits were kept in previously disin-
ted boxes for a period of 4 days.
Mode of Ingress and Internal Spread
Fresh sectioning with the freezing microtome. Cross and longitudinal
:sh sections of the pedicel and pericarp were made 3, 6, 9, 12, 24, 48,
,and 96 hr after inoculation. Microtome sections of 20 I. thick were
ined with phloxine and lactophenol cotton blue.
I uufjifl tcLtflll Luc.l-.- II IU IlxACUI 111 l>i~ri UCIIyUIdlcLu Ill il IID~. Sel
infiltrated and embedded in paraffin were sectioned 15 t thick in
rotary microtome, stained, and mounted in Canada balsam. Several st;
combinations were used. These were safranin-fast green, safranin-anil
blue, tannic acid-ferric chloride-safranin (Jensen, 1962) and Pianeze III
Histochemical tests for lignin and suberin in the pedicel.- Lignin I
nation in healthy and pathogen-invaded cells was tested using the pl
roglucinol test (Johansen, 1940; Siegel, 1959), chlorine-sulfite test (Siej
1959), and Schiff's reaction (Mclean and Cook, 1941). Suberin was tes
with Sudan dye (Jensen, 1962).
Histochemical tests for tannins. The presence of tannins in thi:
sectioned non-infected, newly infected, and diseased green and yellow exoc;
tissues was tested using the nitroso reaction (Reeve, 1951) and the fer
sulphate reaction (Reeve, 1959b).
Histochemical test for pectins and celluloses. -Pectins were tested usi
the hydroxylamine-ferric chloride reaction (Reeve, 1959a). Celluloses w
tested using the celluose-zinc-chlor-iodide reaction (Rawlins and Takahas
1952) and the IKI-H2SO4 method (Johansen, 1940).
Pathogenicity test. The typical stem-end rot symptoms on mange
induced by the different isolates are shown in Fig. 1. Iloilo 3 isolate '
the most virulent followed, in decreasing virulence, by Caloocan 2, S
Cruz 1, Antipolo 2, Los Bafios 1, Cebu 1, Balagtas 1, and Damortis
Mode of Ingress and Internal Spread
Fresh sectioning with the freezing microtome. This method did i
successfully differentiate the fungus from its host tissues. Starch granu
of varying sizes were observed especially at the shoulders and basal sinu
of the pericarp in non-stained sections. Accumulation of granules a
sugars increased correspondingly with the ripening process.
Paraffin technique. Safranin-fast green stain combination was b
suited for demonstrating penetration and internal spread of D. natalen
Young and mature pycnidiospores germinated 2.5 hr after being inoculai
to the moistened pedicel and pericarp surfaces. Young spores germinal
more frequently than the old ones. Penetration was observed 3.5 hr la
in ripe mangoes and 6.5 hr later in the mature green ones. The funj
entered its host tissues through the following: 1) the exposed pedi
THIiLi'I'IN THY I 1UYAItHULU.ix
: DIVINAGRACIA: DIPT.ODIA Re
Fig. 1. Typical symptoms of stem-end rot produced by 8' isolates of Diplodia
talensis on Pico mangoes 4 days after inoculation: (1) Ilolo (2) Caloocan 2,
1) Sta. Cruz 1, (4) Antipolo 2, (5) Los Baios 1, (6) Cebu 1, (7) Balagtas 1, and
I) Damortis 1. (9) Uninoculated fruits as checks. Note the whitish tufts of mycelium
the excised stem-end scar on most of the infected fruits.
id, 2) the injured pedicel side, 3) the uninjured pedicel side with wax-
aled end, 4) the pedicel scar, 5) the uninjured pericarp shoulder and
de, and 6) the injured exocarp shoulder and side (Fig. 2).
Entry through the exposed pedicel ends occurred mainly through the
:ssels and resin ducts in the phloem region. Aside from the injured
edicel sides, the pathogen hyphae also penetrated through an abcission
yer in the pedicel. Direct mechanical ingress was observed through the
>idermal cuticle of the pedicel. The point of entry of the germ tube
ained darker than the nearby area. The hyphae invaded the radially
ongated epidermal cells. They penetrated inter- and intra-cellularly
rough the thick-walled and sclerosed cells of the cortex. Also, they
mified progressively through the cells of the endodermis and pericycle,
-ch-shaped fiber cells of the bundle cap, phloem resin duct, phloem
irenchyma, sieve-tube member and companion cells, ray cell, annularly
id spirally thickened xylem tracheary elements, xylem parenchyma and
ally, into the parenchyma cells of the pith (Fig. 3). Infected cells in
Le phloem, xylem and pith regions contained granular substances sur-
'unded by swollen and darkened walls. In 48 hr, pedicel tissues, espe-
Fig. 2. Carabao mangoes 3 days after inoculation with isolate Iloilo 3 at variot
sites (arrows): 1) exposed pedicel end, 2) injured pedicel side, 3) uninjured pedio
side with wax-sealed end, 4) pedicel scar, 5) uninjured pericarp shoulder and sid
6) injured exocarp shoulder and side, and 7) sap-treated at the basal sinus an
cially the parenchyma in the phloem and pith, were completely invade
by the hyphae. Longitudinal spread of the pathogen to the fruit pericar
was primarily through the resin ducts.
The germ tube also penetrated directly through the injured exocarl
intact cuticle of the exocarp and lenticel. The hyphae spread inwar
through the small and irregularly-set epidermal cells and the oblong an
tangentially compressed hypodermal cells. Inter- and intra-cellular hypha
were observed in the exocarp and mesocarp cells. The fungus sprea
longitudinally chiefly through the laticiferous canals. Hyphal cells ram
fied laterally in the large, homogeneous and oblong mesocarp cells. The
also occurred in the small bundles embedded in the ground tissue (Fig.
Host cells were killed in advance of the hyphae. In infected cell
the nuclei disintegrated and the cytoplasm became granular. Cell wal
became swollen and then collapsed.
Fungal invasion was more rapid in ripe than in tissues of unril
fruits. Symptoms of stem-end rot infection were observed 48-96 hr afti
HALOS & DIVINAGRACIA: DIPLODIA ROT OF MANGO
. Fig. 3. Diagramatic drawing of a transverse section of a pedicel portion showing
the progress of the pathogen into the suscept tissues.
Nos. 1 & 2]
22, PHILIPPINE PHYTOPATHOLOGY -[VOL. 6
Iatic ife us
OS. 1 & 2] HALOS & DIVINAGRACIA: DIPLODIA Rar OF MANGO 23
r IQ J1 }...c 7
'24 PHILIPPINE PHYTOPATHOLOGY [VOL. 6
spore. Hyphae in the pin-pricked area of the mesocarp were delimited by
a gummy granular substance of the surrounding cells. Rapidly dividing
cork cells appeared around the wound that harbored D. natalensis hyphae.
Histochemical tests for lignin and suberin in the pedicel.- Neither
lignification nor suberization occurred in the pathogen-invaded pedicel cells
of newly harvested ripening mangoes. Lignins were detected only in the
cell walls of vessel elements in both healthy and inoculated pedicels.
Histochemical tests for tannins.- Tests were positive in non-infected and
newly infected exocarp, but negative in the diseased tissues. Blue precipitate
was noted in the ferric sulphate reaction and cherry-red color in the nitroso
reaction. The color intensities in both cases varied. Boundaries between
the healthy and infected areas were more intensely colored than the unin-
fected areas. Moreover, stain was darker in the unripe than in the ripe
tissues, suggesting that there are more tannins in unripe mangoes.
Histochemical tests for pectins and celluloses. The cuticle, epidermis,
cortex, endodermis, pericycle, phloem, xylem, exocarp and mesocarp have
undergone various stages of maceration. Parenchymatous cells were parti-
cularly most sensitive to maceration. Both the healthy and the partially
disintegrated cell walls appeared reddish when stained with hydroxylamine-
ferric chloride. The completely macerated walls appeared faint red and
highly disorganized. Similar tissues in different stages of disintegration were /
The cuticle did not provide a decisive mechanical barrier. This in-
:ated that resistance of mangoes to this disease is located in the cellular
sues. This agrees with the view of Martin (1964) on the role of cuticle
ainst plant disease.
Our observation of germinating D. natalensis pycnidiospores that en-
red directly through the intact cuticle of the pedicel and the cuticle
d lenticel of the exocarp appears to contradict the findings of earlier
vestigators (Frossard, 1964; Srivastava and Durgapal, 1964; Chakravartv
d Srivastava, 1964; Pathak and Srivastava, 1967b; Vir, Raychaudhuri
iirumalachar, 1967; Vir and Thirumalachar, 1968). Penetration through
! exposed pedicel surface, injured pedicel sides, and the abcission in the
dicel was also observed in studies by Pathak and Srivastava (1967b),
lakravarty and Srivastava (1964), Srivastava and Durgapal (1964), and
own and Wilson (1968). Penetration on both the pedicel scar and the
funded exocarp was reported by Pathak and Srivastava (1967b) and
icbusan and Schafer (1958).
The fungous hyphae ramified rapidly when the pH, starch granules,
d sugar increased in the mango tissues. These changes which accom-
nied the ripening process, occurred faster at the stem-end than at any
ler portion of a fruit, thus infection frequently occurred on such part.
Host cells were killed in advance of the hyphae. In 48 hr after
oculation most pedicel tissues were invaded by the fungus at the phloem
d pith regions. Longitudinal spread was most rapid through the resin
cts and subsequently through the laticiferous canals in the pericarp.
lis type of development suggests that the pathogen sought the paths of
s resistance and abundant food supply.
Information on the mode and time of infection and the ensuing
thogen development in suscept tissues are of vital importance in the
emulation of successful post-harvest treatments. These post-harvest treat-
ents include chemical treatments to prevent infection and suppress
thogen development on surfaces of the fruit; heat treatment to get rid
incipient infections; and gamma irradiation to inactivate or retard the
velopment of deep-seated infections (Eckert, 1967). The effectiveness of
given treatment is dependent on the number of pathogen cells and their
:ation in the host tissue. Most fungicides have limited capability to
netrate the host tissues. In a favorable environment, the rapidly spread-
g hyphae of D. natalensis may be beyond the reach of non-systemic
ngicides within 24 hr of inoculation. The eradicant should therefore
applied immediately after harvesting green mangoes to arrest latent in-
:tions. Contaminated fruits may be refrigerated at 10 C or less to in-
bit pycnidiospore germination. Irradiation therapy which can penetrate
to the host tissues may be useful in suppressing deep-seated infections.
The invaded unripe tissues of both pedicel and pericarp became histo-
ogically delimited by cicatrice or cork layer. The metabolic products
elaborated by D. natalensis may have stimulated the cicatricial demarcation
outsidee the artificially infected cells. This anti-toxic defense reaction may
:ut off the influence of toxic substances produced by the fungus which
diffused from the infected area (Gaumann, 1950). In advanced stages, the
:ork layers may inhibit further invasion by the fungus into the starchy
nesocarp. The accompanying gum deposition surrounding the artificially-
nduced lesion served as a protective demarcation. When the invading
fungus secretes hydrolytic enzymes to break down the cell walls, the
damaged cells release phenolic substances. In the presence of high oxidase
activity, they formed highly reactive quinones which may inhibit the ex-
:reted enzymes (Norkrans, 1963). Thus, the fungus have been prevented
mechanically and chemically from further spread.
Tannins are termed polyphenols (Robinson, 1955) which imply the pre-
sence of more than one hydroxyl group on a benzene ring. If the color
intensities in the nitroso and ferric chloride reactions were indicative of
quantity, then tannins were more concentrated in the unripe than in ripe
:issues. Moreover, cells near the initially infected tissues had more tannins
:han the distant healthy cells. Tannins were absent in completely macerated
.issues. Tomiyama (1963) alleged that upon death of the infected host cell.
phenolic compounds, phytoalexins or other abnormal metabolic products
nay be formed. The metabolic activity in healthy tissue adjacent to the
infected cells may provide the plant with inhibitory materials against fur-
:her development of the invading hyphae (Tomiyama et al., 1967 and
Kosuge, 1969). Common phenolic compounds play a role in this process.
Generally, tannins which arise from oxidative polymerization of phenols may
participate in defense mechanisms by denaturing proteins (Rohringer and
Samborski, 1967). During the ripening process, tannins decreased in con-
:entration (Barnell and Barnell, 1945). The polyphenols in healthy mango
:issues are therefore, important in disease resistance. Our histochemical tests
for pectins and celluloses in infected mango tissues suggested the action of
)ectin and cellulolytic enzymes. However, Bateman and Millar (1966) cau-
:ioned that staining techniques used to demonstrate the extent of degradation
Af pectic substances in infected tissues may have a limited value. A positive
-eaction may occur even in instances where there is extensive but incomplete
degradation of pectin or pectic substances. While these substances are in-
volved in pathogenesis, experimental proofs that the pathogenicity of an
)rganism is a function of its ability to produce pectic enzymes are lacking.
[n the case of mango, this is further complicated by the observations of
fuliano and Cuevas (1932) that when mangoes became ripe, the walls of
:he parenchymatous mesocarp cells break down, a sign of the action of
&C UIVINAGRACIA: UIPLODIA 1~
Pectic enzymes are among the complex factors involved in phytopatho-
ogical processes. The celluloses do not appear to contribute significantly
o the disintegration process (Bateman and Millar, 1966). -Our results sug-
,est that their role should not be discounted for they may aid in the
aaceration of the infected mangoes.
The infected tissues darkened with the advance of the D. natalensis
typhae. The healthy tissues near the infected parts of unripe and ripe
nangoes were positive to the nitroso reaction indicating the presence of
atechol tannins. This reaction, however, was negative in the completely
lacerated tissues. Thus, in the development of the stem-end rot symptoms,
he complex enzyme tyrosinase are probably synthesized or caused to be re-
eased by the pathogen.
LLICBUSAN, R. V. & L. A. SCHAFER. 1958. Diplodia rot of mango. The Phil. Agr.
1ARNELL, H. R. & E. BARNELL. 1945. Studies in tropical fruits. Ann. Bot. 9: 77-99.
BATEMAN, D. F. & R. L. MILLAR. 1966. Pectic enzymes in tissue degradation. Annu.
Rev. Phytopathol. 4: 119-146.
hROWN, G. E. 1968. Germination of immature and mature spores of Diplodia natalensis.
Phytopathology 58: 1044-(Abstr.)
I~OWN, G. E. & W. C. WILSON. 1968. Mode of entry of Diplodia natalensis and
Phomopsis citri into Florida oranges. Phytopathology 58: 736-739.
AHAKRAVARTY, D. R. & D. N. SRIVASTAVA. 1964. Stem-end rot of mango and orange
fruits incited by Diplodia natalensis Pole-Evans. Curr. Sci. 73: 285.
SCKERT, J. W. 1967. Application and use of post-harvest fungicides, p. 281 to 378.
In D. C. Torgeson [ed.] Fungicides, an advanced treatise. Academic Press, New
'RossA D, D. 1964. La pourriture pedonculaire des avocats en cote d'Ivoire. In-
fluence de la temperature sur le developpement de Diplodia natalensis. Fruits
d'outre mer. 19: 401-403.
3AUMANN, E. 1950. Principles of plant infection. Hafner Publ. Co., New York.
ENSEN, W. A. 1962. Botanical histochemistry. W. H. Freeman and Co., San Fran-
cisco. 408 p.
OHANSEN, D. A. 1940. Plant microtechnique. McGraw-Hill Book Co., New York.
ULIANO, J. B. & N. L. CUEVAS. 1932. Floral morphology of the mango (Mangifera
indica Linn.) with special reference to the Pico variety from the Philippines.
Philippine Agr. 21: 449-472.
CoSUcE, T. 1969. The role of phenolics in host response to infection. Annu. Rev.
Rev. Phytopathol. 7: 195-222.
IARTIN, J. T. 1964. Role of cuticle against plant disease. Annu. Rev. Phytopathol.
McLEAN, R. C. & W. R. I. COOK. 1941. Plant science formulae. Macmillan & Co.,
London. 203 p.
JORKRANS, B. 1963. Degradation of cellulose. Annu. Rev. Phytopathol. 1: 325-350.
PATHAK, V. N. & D. N. SRIVASTAVA. 1967a. Mango losses due to Diplodia stem-end
rot. Trop. Agr. 123: 75-77.
K, V. N. & D. N. SRIVASTAVA. 1967b. Mode of infection and preve
iplodia stem-end rot of mango fruits (Mangifera indica). Plant Dis. R
naria rot ot tomato truits during transit. Indian rnytopatnol. zu: uji- tju.
VIR, D. & M. J. THIRUMALACHAR. 1968. Studies on Diplodia rot of mango and A
ternaria rot of tomato fruits and their control. Hindustan Antibiot. Bull. 1(
TURAL, PHYSIOLOGICAL, AND BIOCHEMICAL PROPERTIES
ISOLATES OF PHILIPPINE PSEUDOMONAS SOLANACEARUM
ELDON I. ZEHR
ormer Graduate Assistant, Department of Plant Pathology, Cornell University,
L, New York 14850. Present address: Department of Plant Pathology and Phy-
y, Clemson University, Clemson, South Carolina 29681.
portionn of Ph. D. thesis.
'he interest and adivce of R. S. Dickey, K. G. Parker, and H. D. Thurston is
'his research was supported in part by Ford Foundation funds under the Uni-
r of the Philippines-Cornell Graduate Education Program.
The cultural, physiological, and biochemical properties of 85 isolates of
'seudomonas solanacearum from the Philippines and one isolate each from
Jorth Carolina and Colombia were comparatively investigated. Among iso-
ates from the Philippines, differences were observed in colony characteris-
ics, production of brown pigment in several media, hydrogen sulfide pro-
luction, reaction in litmus milk with cream, and utilization of maltose, lactose,
nd cellobiose. Philippine isolates differed from North Carolina isolate K-60
a colony characteristics and utilization of dulcitol, mannitol, arabinose, and
artrate; and from Colombia isolates S-208 in colony characteristics, pellicle
formation in nutrient broth, optimum temperature for growth, diffusible pigment
production in certain media, sensitivity to sodium chloride, growth characteristics
n nutrient broth, and utilization of sorbitol, dulcitol, mannitol, i-inositol,
!thanol, and arabinose. Among Philippine isolates, there was no apparent
!orrelation between cultural, physiological, or biochemical properties, and host
Many investigations on the cultural, physiological, and biochemical pro-
es of Pseudomonas solanacearum E. F. Smith have been made, but
spread disagreement concerning these properties remains unsettled (Kel-
1953). No doubt, some differences resulted from the use of different
liques and/or methods by various investigators, but several investiga-
that utilized uniform techniques and environmental conditions indicate
there are true differences among strains. Hayward (1964) observed
isolates differ in utilization of arabinose, lactose, maltose, cellobiose,
tol, dulcitor, mannitol, malonate, and i-inositol as carbon sources in
misynthetic medium. Harrison (1961) observed that isolates collected
.ustralia differ in certain properties, including utilization of lactose, mal-
and xylose; starch hydrolysis; and production of a diffusible brown
ient in agar media. In other comparative studies, Vasudeva (1955)
observed that isolates from India differ in utilization of xylose, raffino:
and salicin. In Japan (Okabe and Goto, 1952) some isolates produce g
from mannitol and dextrose, while others do not; and they differ qua
tatively in utilization of mannitol and lactose.
In the Philippines, isolates of P. solanacearum differ considerably
virulence to tobacco and ginger, and to a lesser extent to peanut, peppi
and tomato (Zehr, 1971). An investigation was undertaken to determi
whether these isolates also differ in cultural, physiological, and biochemic
properties, and to determine whether such differences might be related
differences in virulence.
MATERIALS AND METHODS
Thirty-five isolates of P. solanacearum from the Philippines, isol
K-60 from North Carolina, and isolates S-208 from Colombia, which we
used in host range studies (Zehr, 1971), were compared in cultural, phys
logical, and biochemical tests. Philippine isolates were identified as P. so
nacearum by pathogenicity tests on tomato (Lycopersicon esculentum Mil
or other known suscepts of this pathogen, gram reaction, and colony a
pearance on tetrazolium medium (TTC) (Kelman, 1954). Stock cultui
maintained in distilled water at room temperature (23-32 C) were test
periodically for virulence and for purity by streaking on TTC medium
Before each test, a loopful of bacterial suspension was transferred frc
stock cultures to nutrient agar slants and incubated at 32 C for 48 1
r color was observed (Society of American Bacteriologists, 1957).
Gelatin utilization.- Nine isolates were tested at 20 C for lique-
:tion of gelatin at a 12% aqueous concentration, however, incorporation
gelatin (0.4%) in nutrient agar proved to be a superior method for
is test. After growth appeared (48-72 hr), the medium was flooded with
mercuric chloride-HCl solution (Society of American Bacteriologists, 1957)
precipitate the gelatin in the medium. A clear zone surrounding the
cterial growth was interpreted as a positive test for gelatin utilization.
Steamed potato slants. Fresh potatoes were cut into slants 5-7 mm2
the base, placed in test tubes with 1 ml of 1.5% water agar to hold
e slants in position, and sterilized for 15 min at 121 C. Tests were
ide after incubation at room temperature (23-30 C) and at 32 C.
Motility. Motility was tested in the Difco motility medium (Anony-
)us, 1953), to which 0.005% 2, 3, 5-triphenyl tetrazolium chloride was
ded to aid detection of motility (Gershman, O'Meara, and Chute, 1959).
Sodium chloride tolerance. Growth was determined in Difco nutrient
oth containing 0.5, 1.0, and 2.0% sodium chloride.
Hydrogen sulfide production. Hydrogen sulfide (H2S) production from
' tryptone broth was tested by the lead acetate-impregnated filter paper
method. Sixteen isolate were tested for H2S production from cystine, using
e Ayers, Rupp, and Johnson basal salts medium with dextrose as a car-
In source (Rhodes, 1959). Nine isolates were tested further, using pep-
ne-iron agar as an indicator of H2S production.
Pectolytic activity.- Starr's method (Starr, 1947) for testing the pre-
ice of pectolytic enzymes was used. Sodium polypectate (Nutritional Bio-
emicals Corp.) was used as the substrate for this test.
Indole production.- After growing the bacteria in 1% tryptone broth
r 24 hr, Kovacs' reagent was added to test for the presence of indole.
Catalase production. Isolates were transferred from stock cultures to
itrient agar slants and grown at room temperature (23-30 C) for 48 hr.
wo ml 10-vol (3%) hydrogen peroxide was added to each tube and
served for evolution of gas, indicating catalase activity.
Kovacs' oxidase test. One loopful of bacteria from 4-day-old nutrient
ar cultures was transferred to filter paper moistened with a drop of
o aqueous N,N,N',N'-tetramethyl-p-phenylene-diamine dihydrochloride.
3pearance of a violet color was considered a positive oxidase reaction
Nitrate reduction. Reduction of nitrates was tested in nutrient br
plus 0.1% sodium nitrate. Sulfanilic acid and a-naphthylamine reage
were added after 12 hr, 24 hr, and one week to test for nitrites.
Starch hydrolysis. Nutrient agar containing 0.2% starch was pou
in plates. A single streak of bacterial suspension was made on the
dium, and incubated at 32 C for 3 days. Plates then were flooded v
Gram's iodine solution to test for starch hydrolysis.
Methyl Red-Voges Proskauer test. The medium and reagents for
test were prepared as suggested in the Manual of Microbiological Meth
(Society of American Bacteriologists, 1957). Reagents were added two <
after setting the test.
Optimum temperature. -This test was made in nutrient broth at t
peratures of 15, 21, 27, 33, and 39 C. Optimum temperature for gro,
was determined by observing turbidity after 16 hr, 24 hr, 2 days, an(
Utilization of amino acids as sources of carbon and nitrogen
Utilization of asparagine was tested according to the method of Starr
Weiss (1943) in a nitrogen and carbon-free basal medium. Three c
secutive transfers were made to tubes of fresh medium to ensure t
growth did not result from nutrient carry-over in the initial trans
Glassware used in the experiment was washed in acid, rinsed thorough
first in tap water and then in distilled water. Asparagine was sterili
by filtration and added aseptically to the autoclaved basal medium foi
final concentration of 0.5%.
Tyrosine was tested similarly as a substrate for carbon and nitrog
but repeated transfers from the tyrosine medium were not made. 1
concentration of tyrosine used was 0.025%.
Organic sources of carbon. -The synthetic basal salts medium of Ay
Rupp, and Johnson (Society of American Bacteriologists, 1957) was u
to test the utilization of 23 different materials as sources of carbon. Tl
were: dextrose, sucrose, mannose, galactose, raffinose, levulose, malti
rhamnose, lactose, cellobiose, salicin, sorbitol, dulcitol, mannitol, i-inosi
arabinose, ethanol, glycerol, tartrate, acetate, citrate, malonate, and form;
Brom thymol blue was added to the medium initially as a 1.6% alcoh4
solution but investigation showed that the alcohol could support bactel
growth. Therefore, the indicator was added as an aqueous suspension
most of the tests. Glassware was washed thoroughly in an acid clean
solution and rinsed in distilled water before use. The basal medium ,
dispensed in test tubes and autoclaved. while the carbon sources w
sterilized by Millipore filtration and added aseptically to the sterilized hb
medium. Organic acids were tested at 0.5% concentration; other mater
were tested at 1.0%. All tube cultures were incubated at 32 C. exc
r brief intervals during hot weather when the incubation temperature
se to 36 C. A maximum of 3 weeks was allowed for completion of
Gram stain.-All isolates were short, gram-negative rods.
Colony characteristics. Differences in colony size, pigmentation, vis-
sity, and production of a diffusible brown pigment were apparent among
any of the 37 isolates when they were grown on TT or PDPA media.
a TTC medium, most Philippine isolates grew rapidly and in 48 hr
oduced convex, irregularly round colonies (4-6 mm diam) that tended to
w readily. A few isolates, notably LB-6 and LC-I, always produced
taller colonies (2-3 mm diam) that remained discrete. All Philippine
dates produced colonies that were white with glistening, reddish-pink
enters. Colonies from isolates K-60 (North Carolina) and S-208 (Colom-
a) were similar, but had dull, red centers and were smaller than Phil-
pine isolates (except LB-6 and LC-1). Colonies of I-60 were more fluid
an were those of S-208. All isolates, except S-208, produced a diffusible
own pigment in the medium, but isolates DC-3. DC-4, V-l, and K-60
,re especially proficient in production of this pigment (Table 1).
On PDPA, greater variation among Philippine isolates was noted, espe-
illy in pigmentation of the colonies. Colonies after 48 hr were 3-5 mm
am and very fluid. Some were pinkish-buff, others were white and opaque,
.d still others grayish-white and translucent. Colonies derived from isolate
-1 were smaller (1-3 mm) on this medium, as was observed on TTC, but
e diameter of those from LB-6 did not differ from other isolates. Isolates
208 and K-60 were brownish-white, relatively small (1-3 mm diam for
208; 2-4 mm diam for K-60), and less fluid than Philippine isolates. As
is observed in TTC medium, isolates DC-3, DC-4 V-l, and K-60 were
table in production of a diffusible brown pigment in PDPA.
On nutrient agar, colonies were circular, convex with irregular margins,
lid, grayish-white or brownish-white, 2-3 mm diam, and there were few
parent differences among isolates. Little diffusible brown pigment was
oduced when Difco nutrient agar was used, but on BBL nutrient agar
gment production was similar to that noted with TTC and PDPA media.
Growth in nutrient broth.- All isolates grew well in nutrient broth
id produced abundant, diffused growth with white sediment. All isolates
cept S-208 produced a thin pellicle (S-208 produced none), and cultures
S-208 and K-60 were less turbid than those of other isolates. Isolates
C-3, DC-4. V-l, and K-60 produced brown pigment in the medium after
Reaction in litmus milk.- Smith (1914) described as "var. asiaticum"
train of P. solanarcarum that nrndnrlpd arid from litmnm milk with
TABLE 1 Summary of differences observed in cultural, physiological, and biochemical properties of 37 isolates of
2 Diffusible brown H2S produced Acid from carbon sources
-c _____ __ -,-_._
a 0 0)
-2 3 + + ++ + +
A- 3 < + + + + t + o + o + + + + + + + ++
SY ou 0 0 0 o
A-2 33 + + + + + + + ++ + + -H- ++ ++ -H + + ++
A-3 33 + + + + + + + ++ ++ ++ ++ -H- ++ + + -H++
Ca-1 33 + + + + + + + + -H++ ++ ++ + + 4+
Ce-1 33 + + + + + 4 ++ + ++ ++ ++ ++ + + ++
CO-7 33 + + + + + + + + + + + + + -+ + + 4+
Do- i 33 + + + + + + 4 44 + 4 + + +
Co-1 33 + + + + + + -H + -- -H ++ + + +-
Ce-1 33 + + + + + + + 4-+ + -++ ++ ++ -+- + + ++
DC-1 33 + + + + + + ++ + ++ ++ ++ ++ + + ++
DC-7 33 + + + + + + + + ++ + ++ ++ ++ ++ + + ++
TABLE 1 .-contd 2
Diffusible brown H2S produced Acid from carbon sources
2e pigment _
-- - -'"o -
*0 0 )
E | r: .E aI
- + +
S+ +-I 4 44 4-
-I 4- 44 44 4+
44 44 444 44t
4 + -I 4- -H-
4 4+ 44 4+- 4
4-t -+f -+-+ +-I -I+
4+ + +44 +4
44 + 44 44 44
f + ++ -H4 4
+ + +- .-- -+
-+F 4+ + -- "+
.-. Diffusible brown
Diffusible brown 2S produced Acid from carbon sources
U Pigment -
Li-1 33 + + + + + + -+ + ++ + +-
S-2 33 + + + + + + + + + + + + ++ + ++
S-3 33 + + + + + t +- ++ + ++ ++ ++ ++ + + -
Ab- 33 + + + + + + + -
Ab-2 33 + + + + + ++ ++ + ++ + + -+-
CO-4 33 + + + + + + ++ ++ + + ++
E 0 C c0 a
CO-8 33 + + + + + ++ -++ 4- ++ + + --
D-2 33 + + + + + ++ + + +
L g L ) 1 a z o
SL-1 33 + + + + ++ + + - ++ ++ ++ + + ++
D-3 33 + ++ + + + + + + -H++ + ++ + + ++
DC-4 33 + + + + + + + ++ ++ ++ -+ + + ++
Ab-2 33 + + + + + + --H 44 + + 44
CO-4 33 + + + + + 4- + 4- 4- + + 44
CO-8 33 + + + + + -- 4 -I4 + + +F
D-2 33 + + + + + -- +- 4I 44 + + 44
Ta-i 33 + + + + + + + F *+ -4 + + 44
SI-i 33 + + + + + +I- +4 4 + + + +4
DC-3 33 +444+ + + -4 + + + + 4+ +1 -+ +4 + + 44
DC-4 33 +4444+ + 44 + + + 444444 44 + 4- 44
Diffir;hi krnm MW C
U -o pigment
a- 0 a
I so 2s 2
V-1 33 + .-+ ++ + ++ + + + -
K-60 33 + + + + + -
a/ += positive test; +H= strongly positive test; = results differ among replicates;
b/ For carbon sources: -H-= acid produced in synthetic basal medium within one week
E 0 of 0eg, 1 07
= no acid produced in three weeks.
Isolates DC-3, DC-4, and V-1 produced acid from cellobiose in Hayward's medium
and Johnson medium (Society of American Bacteriologists, 1957.
among bacteriologists. This experiment was made to determine whether
Philippine isolates produce acid from cream-free litmus milk, litmus milk
with cream, or both.
In cream-free litmus milk, all isolates produced an alkaline reaction
in 3-4 days which gradually increased in intensity for 2-3 weeks without
clearing or litmus reduction. In litmus milk with cream, all isolates pro-
duced an alkaline reaction initially, but some changed to acid after one
week. The acid reaction began in the cream layer of the surface, gradually
spreading throughout the medium. Sometimes, however, the acid reaction
was weak and restricted to the surface layer. Isolates with an alkaline
reaction did not reduce litmus, but change to acid was accompanied by
litmus reduction and sometimes by acid curd formation. Twelve isolates
produced an acid reaction. However, the acid reaction of isolates GS-4 and
LB-5 reverted to alkalne within 3 weeks, and some others were variable
Gelatin utilization. All isolates utilized gelatin when it was incor-
porated in nutrient agar. All 9 isolates that were tested for gelatin lique-
faction produced crateriform liquefaction after 4 weeks.
Growth on steamed potato slants. -Production of a tar-black pigment
on steamed potato slants has been reported as a useful diagnostic tool for
P. solanacearum (Harrison, 1961; Hutchinson, 1913; and Smith, 1896). This
experiment examined the usefulness of this technique for Philippine isolates.
Glistening white bacterial growth appeared on the slants in 2 days,
and a golden-brown pigment was present after 1 week. Sometimes a tar-
black pigment also appeared within 1 week. Twenty-five of the 37 test
isolates produced the black pigment within 3 weeks, but production was
variable among replicates. Isolates K-60, S-208, and 10 Philippine isolates
produced no black pigments on steamed potato slants.
Motility. All isolates were motile in the semi-solid medium. How-
ever, growth of S-208 was poor in the medium, perhaps due to sodium
Sodium chloride tolerance. The rate of growth of all isolates was reduced
when NaCI added to the medium. Most isolates grew fairly well at concentra-
tions of 0.5% and 1.0% NaCI, but none grew at 2.0% NaC1. However, with
S-208, growth was much reduced at 0.5% NaCI, and no growth was visible at
the 1.0 or 2.0% concentrations. Sodium chloride was bacteriostatic at the
2.0% concentration. The bacteria settled to the bottom of the medium in -
a stringy mass, but when streaked on TTC medium after one week, they
produced normal virulent fluidall) colonies.
Hydrogen sulfide production. Only 4 of the 37 isolates tested produced
hydrogen sulfide (H2S) from tryptone, and these were only weakly
positive. The 4 positive isolates also produced considerable brown pig-
'1 -- 'n -_ - -
ment in the medium (none was produced by all other isolates). A strong
positive H2S reaction was obtained when cystine replaced tryptone as tl
substrate, and some isolates that had given a negative test for H2S wi
tryptone were positive in this test. However, other isolates were negati
for H2S production in both media. In peptone-iron agar all 9 isolat
tested were negative for H2S production.
Pectolytic activity. -Most isolates grew well in the pectin medium ai
produced acid with crateriform liquefaction within 1 week. Isolates GS
and S-208, however, grew very slowly in this medium and produced little
no liquefaction or change in pH.
Indole production. All isolates were negative for indole production
Catalase and oxidase reactions. -All isolates were positive for both tl
catalase and oxidase tests.
Production of green fluorescent pigment. All isolates grew well
Clara's medium, but none produced a green fluorescent pigment in tl
Nitrate reduction. All isolates reduced nitrates to nitrites.
Starch hydrolysis. None of the isolates hydrolyzed starch.
Methyl red-Voges Proskauer test. All isolates gave negative reaction
in the methyl red and Voges Proskauer test.
Optimum temperature for growth.-The most rapid growth occur
at 33 C for all isolates except S-208, which grew best at 27 C. However
the range of temperatures in which isolates grew differed considerable
Most isolates grew slowly at 15 C, and grew well from 21-33 C, but isolal
GS-2 and Li-1 produced no visible growth in eight days at 15 C and grn
slowly at 21 C. Isolates K-60, La 2, Ki-1, 1-3, 1-4, and Da-1 grew well
39 C, but the other isolates did not grow at this temperature.
Utilization of amino acids as sources carbon and nitrogen.- When
paragine or tyrosine was the sole source of carbon and nitrogen, all isolal
grew rapidly. No pigment formation was apparent from asparagine, but
the tyrosine medium isolates V-I, DC-3, and DC-4 produced a pink p
ment within 3 days. After 5 days, other isolates also produced a pink p
ment in the medium and when the cultures were 12 days old, all we
positive for pigment production. At 12 days, when the last observati,
was made, the pigment had changed to brownish-orange.
Organic sources of carbon.- Of the 23 materials investigated as possil
sources of carbon, all isolates utilized glycerol, acetate, citrate, dextro
sucrose, mannose, galactose, and levulose. None of the isolates utiliz
rhamnose, raffinose, malonate, format, or salicin. Isolates differed in ul
ization of the remaining 10 materials, however. None of the isolates pi
dulcpd one from thp mnatri;al tpstped
XT-> 1 2
Hayward's (1964) medium and Ditco OF Basal Medium were compared
with Ayers, Rupp, and Johnson's basal medium of determine whether the
basal medium might influence results of tests with lactose, maltose, and
cellobiose. Twelve isolates that had given negative results for acid pro-
duction with these materials previously were tested in the 3 media and
compared with isolate LC-1, which readily utilized lactose, maltose, and
cellobiose in previous experiments. Hayward's medium was slightly more
sensitive than the other two, giving the most rapid indication of acid
production. In Hayward's medium isolates V-l, DC-3 and DC-4 produced
slight acid from cellobiose which was not detected in the other 2 media.
When no additional carbon source was added to the OF medium, growth
and a strong alkaline reaction occurred; but when the medium was covered
with melted petrolatum, no growth or change in pH occurred, even when
an additional carbon source was added. This indicated that all 13 isolate,
tested had strictly oxidative metabolism.
Examination of similarities of isolates. Results of 56 tests were
analyzed by computer for similarities among the 37 isolates. Thin
analysis showed a high percentage similarity (over 90%) among most isolate'
from the Philippines (Fig. 1). Philippine isolates were distributed in two
relatively distinct, homogeneous groups that are similar to Biotypes 3 and -1
(Hayward, 1964), respectively. However, isolates DC-3, DC-4, and V-1 wert
not consistent with any of the 4 biotypes that have been proposed. Isolate'
K-60 and S-208 were distinct from all Philippine isolates and also were ol
relatively low similarity (76.8%) to one another.
The 37 isolates of P. solanacearum used in this study differed in man,
tests, including colony characteristics, production of brown diffusible pig
ment, optimum temperature and temperature range for growth in nutrien
broth, sodium chloride tolerance, pigment production on steamed potat(
slants, acid production in litmus milk with cream, hydrogen sulfide pro
duction, and carbon sources utilized. Production of black pigment or
steamed potato slants, acid production in litmus milk with cream, anc
hydrogen sulfide production were not stable characters that might be use
ful for defining strains of P. solanacearum. The steamed potato and lit
mus milk reactions differed among replicates, and the hydrogen sulfide re
action depended on the substrate and method of testing.
Latin American workers (Buddenhagen, 1960; French and Sequeira
1970) have found colony characteristics on TTC medium useful for iden
tifying certain strains of P. solanacearum. Among the Philippine isolate
tested, colony characteristics were useful for identifying isolates LB-6 ant
LC-1. Although certain differences among other isolates were apparent
they were distinct and tended to disappear as the colonies became older
l O O.-UUlt M M M.- CO M M .D .0 0 0 -
BC-1 90- 94.9
C -1 :
Li aI 80- 84.9
DC-7 i 75- 79.9
GS -470- 7.9
S -2 65- 69.9
Fig. 1. Percentage similarity of 56 cultural, physiological, and biochemical proper-
es of 37 isolates (A-2, A-8, etc.) of Pseudomonas solanacearum.
solates K-60 and S-208 were. easily distinguished from Philippine isolates
Ry colony form and pigmentation on TTC medium.
The designation "var. asiaticum" solely according to acid production in
itmus milk with cream (Smith, 1914) is of dubious value. All isolates
produced alkaline reaction in this medium within 1 week and only after 1
reek did some of the isolates show an acid reaction. Designation, there-
ore, depends on the time that the observations are made. Perhaps, a
nore serious objection is that the reaction sometimes differed among repli-
ates of the same isolate.
Reactions of Philippine isolates in other tests were fairly uniform, ex.
ept in utilization of lactose, maltose, and cellobiose. Apparently, Biotypes
and 4, which differ in utilization of these 3 compounds (Hayward, 1964),
re common in the Philippines, but Biotypes 1 and 2 were not found.
and 4, were more active in the production of a brown pigment in var
types of media than were other Philippine isolates.
Isolates K-60, from North Carolina, and S-208, from Colombia, 1
distinct from Philippine isolates in a number of characters, especially
the range of carbon sources that were utilized. These 2 isolates also %
distinct from one another in these tests. Isolate S-208 differed so mark,
from other isolates in its cultural and physiological properties that
might be tempted to define it as a distinct species. However, in pa
genicity tests (Zehr, 1971), S-208 produced typical symptoms of bacte
wilt in tomato (Lycopersicon esculentum Mill.), potato (Solanum ti
rosum L.), pepper (Capsicum annuum L.), and diploid banana (A
balbisiana Colla). Therefore, the writer is not inclined to consider
isolate as another species.
Little correlation was found between physiological properties and ]
range (Zehr, 1971). Okave and Goto (1961) reported that Japanese isol
that attacked tobacco oxidized lactose as a carbon source. All Philip]
isolates of high virulence to tobacco also utilized lactose, but North C
lina isolate K-60, which is virulent to tobacco, did not utilize lactose.
was observed by Okabe and Goto (1961), lactose utilization was not a g
index of virulence to tobacco because many isolates of low virulence
tobacco also utilized lactose. Colony form was not related to pathogenic
and also, was not correlated with difference in physiological or biochem
ANONYMOUS. 1953. Difco Manual (Ninth Edition). Difco Laboratories, Detroit, 35
BUDDENHACEN, I. W. 1960. Strains of Pseudomonas solanacearum in indigenous I
in banana plantations of Costa Rica, and their relationship to bacterial will
bananas. Phytopathology 50: 660-664.
CLARA, F. M. 1934. A comparative study of the green-fluorescent bacterial I
pathogens. New York (Cornell) Agr. Exp. Sta. Mem. 159. 34 p.
FRENCH, E. R. & L. SEQUEIRA. 1970. Strains of Pseudomonas solanacearum from I
tral and South America: a comparative study. Phytopathology 60: 506-512.
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for an easily discernible sulfide-motility reaction. J. Bacteriol. 78: 739-740.
HARRISSON, D. E. 1961. Bacterial wilt of potatoes. I. Field symptoms of the
ease and studies on the causal organism, Pseudomonas solanacearum variety ,
ticum. Australian J. Agr. Res. 12: 854-871.
HAYWARD, A. C. 1964. Characteristics of Pseudomonas solanacearum. J .Appl.
teriol. 27: 265-277.
HUTCHINSON, C. M. 1913. Rangpur tobacco wilt. Mem. Dept. Agr. India, Bact.
KELMAN, A. 1953. The bacterial wilt caused by Pseudomonas solanacearum. N
Carolina Agr. Exp. Sta. Tech. Bull. 99. 194 p.
KELMAN, A. 1954. The relationship of pathogenicity of Pseudomonas solanacearun
colony appearance in a tetrazolium medium. Phytopathology 44: 693-695.
)S. I K& J LEHR: YSEUDOMONAS SOLANACEARUM t3
VACS, N. 1956. Identification of Pseudomonas pyocyanae by the oxidase reaction.
Nature 178: 703.
SSEL, E. C. & J. G. HOLT. 1970. Presenting and interpreting the results. p. 50-58.
In W. R. Lockhart and J. Liston (ed.) Methods for Numerical Taxonomy. Amer-
ican Soc. for Microbiol., Bethesda, Md.
ABE, N. & M. GOTO. 1952. Studies on Bact. solanacearum with special reference
to the kinds of strains and their classification. Shizuoka Univ. Fac. Agr. Rept. 2:
ABE, N. & M. GOTO. 1961. Studies on Pseudomonas solanacearum. XI. Patho-
types in Japan. Shizuoka Univ. Fac. Agr. Rept. 11: 25-42.
ODES, MURIEL J. 1959. The characterization of Pseudomonas fluorescens. J. Gen.
Microbiol. 21: 211-263.
ITH, E. F. 1896. A bacterial disease of tomato, eggplant, and Irish potato, U.S.
Dept. Agr. Div. Veg. Physiol. and Pathol. Bull. 12. 28 p.
-n T- n -i -- -. .- 1- 1 -I------ - -* T__-A'.-t-_ X7__l-
STRAINS OF PSEUDOMONAS SOLANACEARUM IN THE
PHILIPPINES AS DETERMINED BY CROSS-INOCULATION
OF HOSTS AT DIFFERENT TEMPERATURES
ELDON I. ZEHR
Former Graduate Assistant, Department of Plant Pathology, Cornell Univer
Ithaca, New York 14850, Present address: Department of Plant Pathology
Physiology, Clemson University, Clemson, South Carolina 29681.
Portion of a Ph.D. thesis.
The writer is pleased to acknowledge the advice and interest of K. G. Par
H. D. Thurston, and R. S. Dickey.
This research was supported in part by Ford Foundation Funds under the I
versity of the Philippines-Cornell Graduate Education Program.
In comparative studies, isolates of Pseudomonas solanacearum which were
obtained from eight different host plants in the Philippines differed in virulence
to inoculated tobacco, ginger,, pepper, and peanut plants. All isolates, how-
ever, were highly virulent to potato and eggplant. Isolates obtained from
diseased ginger plants were more virulent to ginger but less virulent to to-
mato than were isolates obtained from other hosts. All Philippine isolates
were of low virulence to diploid banana; and they appeared to be race 1 despite
the relatively low virulence of many isolates to tobacco. Philippine isolates
were of greater virulence to eggplant than was North Carolina isolate K-60
(race 1) or Colombia isolate S-208 (race 8).
Some isolates induced wilt symptoms at 27-32 C but not at 23-24 C.
Potato plants that were inoculated at cooler temperatures (18-24 C) did not
wilt when moved to warmer temperatures (27-82 C). Apparently plant
resistance to the pathogen was not affected by this practice. Temperature
was an important factor for wilt development in inoculated tobacco plants.
Symptoms in Bottom Special tobacco plants were more effected by tempera-
ture changes than were symptoms in Reax tobacco plants. A change of the
inoculum concentration from 1 x 106 to 6 x 108 cells/ml had little effect on
disease severity at 27-32 C.
Pseudomonas solanacearum E. F. Smith, the causal agent of bacter
wilt of tomato (Lycopersicon esculentum Mill.) and other plants, is a patl
gen of great diversity, and that many strains are known to differ in he
range (Buddenhagen and Kelman, 1964; Buddenhagen, Sequeira, and K
man, 1962; Harrison, 1961; Kelman and Person, Okabe and Goto, 196
Quifion, Aragaki, and Ishii, 1964; Thurston, 1963). Strains are numerous
relatively limited geographical regions (Okabe and Goto, 1961), but sort
s their number differ qualitatively in different parts of the world.
ns that are virulent to banana (Musa sp.) in some parts of Central
South America for example, have not been reported in many other bana-
rowing countries (Buddenhagen, 1961; Buddenhagen and Kelman, 1964).
Hawaii, there are 3 known strains of P. solanacearum attacking tomato,
er (Zingiber officinale Roscoe), and bird-of-paradise (Strelitzia reginae
cs), respectively (Quifion, Aragaki, and Ishii, 1964).
A review of extensive investigation on the influence of temperature
wilt development has been published by Kelman in 1953. Most in-
gators have concluded that relatively high temperatures (above 21 C)
r wilt development. Thurston (1963), however, demonstrated that
tin strains from high mountain elevations in Colombia can induce wilt
relatively low temperatures. He observed an epiphytotic of bacterial wilt
potatoes (Solanum tuberosum L.) at elevations with mean temperature
13.1 C. Strains that cause wilt at low temperatures also have been
rted in Portugal (Moraes, 1947) and in Kenya (Robinson, 1967).
In the Philippines, P. solanacearum has been recognized as a pathogen
ing wilt of tomato, tobacco (Nicotiana tabacum L.), eggplant (Solanum
mngena L.), pepper (Capsicum annuum L.), and potato since 1919 (Reink-
1919). Bacterial wilt of ginger was recently reported (Zehr, 1970b), and
a (Musa textilis Nee) is affected by the wilt organism in some localities
he Philippines (Zehr, 1970c). Infection of bananas (Musa spp.) has oc-
ed under certain conditions in the Philippines, but strains that are highly
lent to bananas and abaca have not been encountered (Zehr, 1970c).
, little information is available concerning the occurrence of pathogenic
ns of P. solanacearum in the Philippines; therefore, isolates collected
1 various plants were compared for virulence to selected host species.
I, the influence by temperature on the development of wilt symptoms
potato and tobacco plants inoculated with Philippine strains of the
logen was investigated.
MATERIALS AND METHODS
Thirty-five isolates of P. solanacearum were obtained from tomato,
:to, eggplant, pepper, tobacco, ginger, abaca, and banana. Isolates K-60
i North Carolina and S-208 from Colombia were kindly supplied by
Arthur Kelman, University of Wisconsin and used for comparison.
ased specimens were collected and the presence of P. solanacearum was
rmined by aseptically transferring a small portion of diseased vascular
ie to 10 ml sterile distilled water. The resulting suspension was sub-
lently streaked on tetrazolium medium (TTC) (Kelman, 1954). After
ir of incubation at 32 C, a single, discrete and fluidal colony was trans-
,A -. 14-l^ 1) ^ __ .4 1--tr- n tr In l c*-;?. r;t
ing the above
pared by tran
medium, incubated, and a single
distilled water. After repeat
times, stock cultures were pre
Single, discrete bacterial colony
at room temperature (23-32 C)
y the development of symptoms
ant species, including tomato (cv
(cv. 'Pimiento'), eggplant (cv
banana (Musa balbisiana Colla
hypogaea L. 'UPCA 101') were
partially wilted; 3 = 1 or more leaves entirely wilted; 4 = all leaves
6-1.4 eggplant Isabela H H H M H L M L
7 LB-5 pepper Laguna H H H M H L M L
8 Li-1 eggplant Cebu H H H M H L M L
9 Ki-1 eggplant Leyte H H H M H L M M
0 V-1 pepper Neg. Oriental H H H M H L M M
.1- LC-1 tomato Batangas H H H H H L L L
2-LB-6 potato Laguna H H H H H L M L
3-1-2 tobacco Isabela H H H H M L L L
4 A-2 tobacco La Union H H H H M L L M
5- A-3 tomato La Union H H H L M L L L
6 S-1 ginger Batangas H M H L M L L H
7 Ta-1 ginger Batangas H M H L M L L H
8 K-60 tomato e H H M M M L L L
potato --.-- e H H M M M L L L
a Relative virulence based on disease index obtained from 12 plants each for pota-
o:, tomato, eggplant, tobacco, pepper, and peanut; 6 or 8 plants for banana (Musa
balbisiana Colla); and 5 or 6 plants for ginger.
b Representative of 12 isolates from tomato, pepper, eggplant, and banana that
were alike in host range.
SHigh virulence (H) = disease index of 70-100; moderate virulence (M) =
disease index of 41-69; low virulence (L) = disease index of 40 or less 3 weeks after
d Representative of 8 isolates from tomato, pepper, eggplant, and abaca that were
alike in host range.
LI~~ 1 n n,
tobacco and ginger, and also were apparent in inoculated tomato, pepper, and
peanut plants. Although all Philippine isolates were highly virulent to potato
and eggplant, none of them caused wilt of bananas. When diploid bana-
nas were used in the present study, representative isolates did not induce
wilt of various cultivars of triploid bananas (Zehr, 1970c). Only isolates
from diseased ginger plants were highly virulent to ginger; these isolates
were less virulent to tomatoes than the isolates from other hosts. Follow-
ing inoculation with ginger isolates, wilt of eggplant developed slower than
those inoculated with isolates from other hosts. Ginger isolates killed
almost all the inoculated eggplants during the three-week test period. Many
highly virulent isolates from other hosts killed inoculated eggplants
within 10 days.
Differences between highly virulent and less virulent isolates usually
were apparent within 1 week. Moderately virulent isolates induced partial
wilting, distortion, and stunted growth in 1 week (Fig. 1), and such
symptoms intensified thereafter without recovery or death of the diseased
plants. Most isolates were highly virulent to the hosts from which they
had been originally isolated; but those from abaca and banana were of rela-
tively low virulence to their hosts (Zehr, 1970c).
Fig. 1. Pimiento pepper plants 7 days after stem-inoculation with Pseudomonas
solanacearum. Plant inoculated with isolate LB-5 wilted completely (left), while in-
oculation with isolate 1-2 (center) resulted in only partial wilt, reduced growth, and
J-.----- -- ---1 -C -L A-l
r Yhr &
NOS. 1 & ZJ
EHR: YSEUDOMONAS SOLANACEARUM STRAINS
Relation of temperature to disease development in potatoes. In inocu-
lation experiments, 17 of the 34 isolates did not induce wilt symptoms on
Katahdin potatoes at greenhouse temperatures of 23-29 C. However, many
of these isolates had caused severe disease symptoms in various potato cul-
tivars in the Philippines at temperatures higher than those in the green-
house. Several experiments were made with controlled temperature condi-
tions to determine whether temperature might account for the differences
observed. Greenhouse-grown plants of certified Katahdin potato (15-30 cm
in height) were placed in Cornell growth chambers (21 C day, 16 C night;
or 32 C day, 27 C night; 2000 ft-c, 14 hr/day). After 24 hr, 6 plants for
each isolate GS-2. GS-4. LB-6. and S-208 (1 x 106 cells/ml) were inoculated.
were dying 1 week after inoculation.
To determine whether potatoes inoculated with bacterial wilt organism
might develop resistance at relatively cool temperatures, the following ex-
periment was made. Thirty-five young greenhouse-grown Katahdin potato
plants (5-10 cm tall) were placed in a growth chamber (24 C day, 18 C
night; 2000 ft-c, 14 hr/day). When the plants were 10-15 cm tall (8 days
after), 20 were inoculated with isolate LB-6 (1 x 106 cells/ml), employing the
procedure used in other experiments. Fifteen plants were provided as con-
trol. Twelve days after inoculation, none of the plants developed symptoms;
they were then moved to another growth chamber (32 C day, 27 C night;
2000 ft-c, 14 hr/day). Ten of the inoculated plants were reinoculated 4-6
cm above the previous inoculation site with isolate LB-6. Ten of the 15
non-inoculated check plants were also inoculated. All remaining plants
that received the first inoculation and control were injected with sterile,
One week after the second inoculation, all the inoculated plants wilted
(Table 3). The check plants and those that were not reinoculated showed
no symptoms. After 2 weeks, all the plants that were reinoculated died.
All unaffected plants that received first inoculation were removed and dis-
TABLE 3. Influence of temperature and reinoculation on symptom development of Katahdin
potatoes stem-inoculated with Pseudomonas solanacearum
Treatment 7b 14
Inoculatede 0 0
Inoculatedd 80 100
Reinoculatede 80 100
Noninoculatedt 0 0
aDisease index after Winstead and Kelman (1952). All plants dead = 100.
b Days after last inoculation.
cTen plants inoculated, kept at 24 C day, 18 C night for 12 days, moved to 32 C
day, 27 C night without reinoculation.
d Ten plants inoculated only after moving to 32 C day, 27 C night.
f Five noninoculated checks.
sected. Those that were kept at the cooler temperature showed a small,
darkened, hydrotic lesion at the site of inoculation, but no vascular brown-
ing was apparent. The pathogen was successfully re-isolated from the hy-
drotic tissue of 4 randomly selected plants. All colonies of the reisolated
bacteria on TTC medium were virulent fluidall). Young potato plants
rapidly developed typical wilt symptoms after inoculation from these bac-
oEHR: PSEUDOMONAS SOLANACEARUM STI
to be highly virulent to tobacco (Table 1). However, tobacco plants inoct
lated with North Carolina isolate K-60 which has been reported by other
investigators (Kelman and Person, 1961; Sequeira, 1964) to be highly virt
lent to tobacco did not wilt severely in the present study. Tobacco cu
tivar, temperature, and inoculum concentration were studied to determine
whether these factors contribute to these apparently conflicting results.
Tobacco cultivars 'Reax' and 'Bottom Special' were comparatively sti
died. Seedlings of each cultivar were grown in Vermiculite until the
were 2 cm tall and then transplanted in 4-inch clay pots with sterilize
soil. These 5-leaf seedling (5-7 cm height) were inoculated using procedure
described previously, except that the inoculum concentration was 1 x 1(
cells/ml. Four to 5 plants of each cultivar were inoculated with eac
In the greenhouse (23-29 C) no difference was observed between
the cultivars inoculated separately with 6 isolates. Isolates LB-6 and A-
killed the inoculated plants of both cultivars. Isolates K-60 and Li-1 ii
duced mild wilt symptoms but did not kill the plants. Isolates Sl-1 an
A-3 induced no wilt symptoms. In the growth chamber (32 C day, 27 4
night; 2000 ft-c, 11 hr/day), however, marked differences occurred between
cultivars inoculated with K-60. All the inoculated Bottom Special tobacco
plants were killed. Some of the inocuated Reax plants wilted, but re
Two inoculum concentrations (6 x 108 cells/ml and 1 x 106 cells/m
were compared in the growth chamber, using 6 plants of Bottom Specid
tobacco for each isolate. Results indicated that inoculum concentration
at these levels did not influence evaluation of susceptibility. Bottom Spi
cial plants inoculated with 3 Philippine isolates wilted less severely tha
those inoculated with isolate K-60, regardless of the inoculum level.
All isolates of P. solanacearum used in this study were highly viruler
to Katahdin potatoes, but certain isolates required a relatively high ten
perature before disease symptoms appeared. Optimum disease developer
occurred when maximum temperatures were near the optimum temperature
for the pathogen in vitro, which was approximately 33 C for most isolate
(Zehr, 1970d). For Katahdin potatoes, however, factors other than optimum
growth for the pathogen appeared to be involved in the apparent resistant
of the plants at greenhouse temperatures, because tobacco and tomat
plants wilted rapidly under the same conditions. Philippine potato cu
tivars also wilted rapidly when they were inoculated under tropical climate
conditions where the average maximum temperature was about 32 C. Th
reaction of Philippine cultivars to inoculation under cooler conditions w,
not determined. If Philippine cultivars are resistant at cooler temper;
Nos. 1 &
In the Philippines, P. solanacearum apparently consists of a number
if strains that differ considerably in relative virulence to different plants.
[he most common isolates used in these studies were of low virulence to
tobacco, peanuts, and M. balbisiana, low or moderate virulence to ginger,
Lnd high virulence to tomatoes, potatoes, eggplants, and peppers. Sur-
wrisingly, few isolates that were highly virulent to tobacco were collected.
ind even in areas of the country where tobacco is commonly grown,
trains of low virulence to tobacco were found. Indications are that dif-
erent strains might occur simultaneously in a single field. Isolates LB-4
Ind LB-5, from eggplant and pepper, respectively, were collected in the
ame field, but they differed in virulence to tobacco and peanut. Isolate
.B-6, collected from a potato field located 1 km away from the above
ield, was of much higher virulence to tobacco than either LB-4 or LB-5
yven though tobacco has not been grown in the area for many years. Al-
hough isolates I-2 and I-3 were collected within 300 m away from one
mother in adjoining fields, they differed greatly in relative virulence to
tobacco, peppers, and peanuts. In Japan, 2 or more strains of P. solana-
,earum nro not uncommon in a given field (Okabe and Goto. 1961L.
R: PSEUDOMONAS SOLANACEARUM STRAIN
Presumably, all Philippine isolates of P. solanacearum tested are race
1, because they are virulent to tomato, eggplants, pepper, and potato.
North Carolina isolate K-60, also race 1, was less virulent to eggplant than
the Philippine isolates, and was somewhat less virulent to pepper than
most of the other isolates. The Colombian potato isolate (S-208) was
typical of race 3, except for its high virulence to M. balbisiana. This
isolate, however, was not virulent to triploid bananas (Zehr, 1970c).
The isolation of virulent cells of P. solanacearum from artificially-
inoculated potatoes with no visible symptoms was surprisingly an interest-
ing development. Digat (1966) isolated virulent cells from inoculated to-
matoes that had no apparent symptoms of wilt; his studies indicated that
a form of induced resistance might occur in inoculated tomatoes under
certain conditions. The results herein presented, however, gave no evi-
dence of generalized induced resistance in potatoes under cool tempera-
ture conditions, but a form of localized, induced resistance might have
BREED, R. S. & J. D. BREW. 1916. Counting bacteria by means of the microscope.
New York Agr. Exp. Sta. Tech. Bull. 49.31 p.
BUDDENGAGEN, I. W. 1961. Bacterial wilt of bananas: history and known distribu-
tion. Trop. Agr. (Trinidad) 38: 107-121.
BUDDENHAGEN, I. & A. KELMAN. 1964. Biological and Physiological aspects of bac-
terial wilt caused by Pseudomonas solanacearum. Annu. Rev. Phytopathol. 2:
Vos. 1 8& 21
Dy Fseuaomonas solanacearum. rnytopatnology o4: iuo0-iuoz.
THURSTON, H. D. 1963. Bacterial wilt of potatoes in Colombia. American Potato J
WINSTEAD, N. N. & .A. KELMAN. 1952. Inoculation techniques for evaluation of re
distance to Pseudomonas solanacearum. Phytopathology 42: 628-634.
ZEHR, E. I. 1970a. Studies of the distribution and economic importance of Pseudo
monas solanacearum E. F. Smith in certain crops in the Philippines. Philippin(
Agr. 54: 218-223.
ZEHR, E. I. 1970b. Bacterial wilt of ginger in the Philippines. Philippine Agr. 54:
ZEHR, E. I. 1970c. Isolation of Pseudomonas solanacearum from abaca and banana.
in the Philippines. Plant Dis. Reptr. 54: 516-520.
ZEHR, E. I. 1970d. Cultural, physiological, and biochemical properties of Pseudomo
nas solanacearum in the Philippines. Philippine Phytopathol. 6: 29-43.
A BACTERIUM CAUSING VEIN NECROSIS OF RAMBUTAN
A. N. PORDESIMO AND F. C. BARRED
Assistant Professor and Graduate Research Assistant, Department of Plant Pathology,
UPCA, College, Laguna.
The bacterium causing vein necrosis of rambutan (Nephelium lappaceum
L.) is gram-negative, rod-shaped, with both ends round, motile by monotrichous
flagellum, has an average cell size of 2.14 x 0.94/p, no endospores, non-
capsulated and not acid fast. Agar colonies are light yellow. It produces
H2S but not indole; not capable of liquefying gelatin, hydrolyzing starch, re-
ducing nitrate to nitrite, and producing ammonia; produces acid without gas
in L(+)arabinose, D-xylose, D(-)glucose, and d-mannose. Neither acid nor
gas was formed in sucrose, d-dulcitol, glycogen, raffinose, starch, rhamnose,
inulin, adonitol, cellobiose, and salicin; it is inactivated at a temperature
between 52 and 53 C for 10 min and has a pH tolerance between 6 and 9.
The degree of infection on artificially-inoculated rambutan seedlings varied
with the methods of inoculation; the prick method was the most effective.
Based on its morphological, cultural, and physiological characters and in ac-
cordance with the "new host-species concept," the binomial Xanthomonas
nepheliae sp. n. is proposed to designate this bacterium pathogenic on rambutan.
Rambutan (Nephelium lappaceum L.) is fast becoming an important
commercial fruit tree. Its area of cultivation has expanded in some parts
of the Philippines in recent years. But expansion of its cultivation ha!
brought about some plant disease problems. One of these was a foliag(
disease found in Pakyas, Victoria, Oriental Mindoro in September 1965
(Pordesimo, 1968). This disease was noted on seedlings and bearing trees
Critical examination of infected leaves revealed characteristic symptom anc
signs of bacterial infection. Infected leaves were characterized by vein necro
sis and angular spots and affected tissues remained intact. Lesions were
light to dark brown and not limited to veins that appeared yellowish t<
brownish with transmitted light.
Evidently, this has been regarded as a new disease of rambutan since
no earlier report on its occurrence has been published. This study wa
aimed to determine the primary causal agent of this disease and its identity'
based on morphological, cultural, and physiological characters.
MATERIALS AND METHODS
The causal bacterium was isolated from infected rambutan leaf by th,
streak plating method (Riker and Riker, 1936). Eight single colony isolate
rHILIFINE r-1Y ILY OAAlULULbY
were sepairatly LtanIsiCereu LU iuLeICIiL-agad slaILs aIlu1 CadL was IniallalneuI
in tubed sterile distilled water for use in subsequent studies.
Two-month old seedlings of rambutan var. Maharlika, supplied by the
Nursery Division, Department of Agronomy were used for inoculation ex-
periments. They were inoculated by 1) first wounding the leaves either by
pricking or abrasing then swabbed with cotton wet with the bacterial sus-
pension, and 2) directly swabbing the inoculum over the upper and lower
surfaces of unwounded leaves. Uninoculated seedlings served as control.
All test plants were sprayed with sterile rain water and incubated in
polyethylene bags for 48 hr. After incubation, they were uncovered and
placed on outdoor benches for observation of symptoms.
Reisolation from heavily infected seedlings was made by streak plating
15 days after inoculation. Six single colony isolates were thus obtained
and maintained separately in tubed sterile distilled water. These were used
in parallel tests with those originally isolated. Periodic transfers to nutrient
agar slants were made to study the morphological, cultural and physiolo-
gical characteristics of the bacterium.
The formulae and composition of the various culture media tested and
the routine techniques employed for studying bacterial plant pathogens
were essentially those of the Society of American Bacteriologists (1957) and
Difco Manual (1953).
All the isolates incited symptoms similar to those occurring in naturally-
infected leaves. The degree of infection on the artificially-inoculated seed-
lings varied with the method of inoculation. Severity was noticeable on
seedlings that were inoculated after their leaves have been wounded by
pricking (Fig. 1). Distinct vein necrosis in an oak-leaf pattern developed
in the succeeding young leaves one month after inoculation (Fig. 2).
The bacterial cells were rod-shaped with rounded ends, occurring singly,
occasionally in pairs and in aggregates, with average size of 2.14 x 0.94 p,
motile by a single polar flagellum (Fig. 3), gram-negative, no endospores,
non-capsulated, and not acid fast.
Colonies on plated nutrient agar were circular, raised, smooth, shiny,
and yellowish. In nutrient agar strokes, the bacterial growth was filiform
and yellowish. Ring-type of growth developed in nutrient broth in 72 hr.
Colonies in PDA plates were light yellow, circular, raised and smooth. In
Endo agar, colonies were circular, flat and translucent with a pinkish halo
around the margin. Surface growth in Fermi's solution was a pellicle type
after 48 hr. Acid reaction was observed in litmus milk. No changes were
observed in the triple sugar agar medium.
I'lnrQT'In Z- RAInRrn RTTT MRIlTAN VVII
Fig. 1. Artificially-infected rambutan seedlings (Top) and the degree of infection
(bottom) 15 days after inoculation with 3 methods. A) Control, B) without wounds,
Nos. 1 e 21
,, -i ;1-i:
Fig. 2. Diagnostic symptoms of the disease by transmitted light. Note the oak-
leaf pattern of necrosis with transparent borders.
. 1 &8 2] PORDESIMO & BARRED: RUMBUTAN VEIN NECROSIS 59
. .. ...
nas employing the usual determinative tests has been regarded as inadeq
(Dye, 1962). In his comparative study of 209 phytopathogeoic Xanthi
nas cultures, 57 of which have been identified, Dye (1958) concluded
the standard method used for identification in the laboratory revealed :
lar characteristics making it difficult to identify species.
Stolp et al. (1965) pointed out that naming an unidentified bact
plant pathogen raises the question as to whether that bacterium is iden
with another that has previously been isolated, described and named
whether it represents a "new" bacterium possessing distinct or difft
characters from those already known.
Hayward (1966) suggested that the identification of an unknown is,
should be made by comparing the fundamental characteristics of iso
fresh from the suscepts with those of authentic cultures isolated from
same plant. He, however, admitted that the distinction of species is
plausible using the above tests. He suggested that species must be ado
based on host specificity, since it is difficult to identify a species by
time it is separated from its host.
Inasmuch as the disease under consideration has recently been rep(
(Pordesimo, 1968) and that no bacterial pathogen isolated from ramb
has been reported or described elsewhere, comparative tests as suggested
Hayward (1966) were not made possible, except with authentic culture
Erwinia carotovora and Xanthomonas citri which were used only as
tive or negative indicators for certain tests on cultural and physiolo:
characters. Furthermore, the rambutan bacterium can be distinguished J
the other species of Xanthomonas that have been described in the li
ture (Breed et al., 1957; Hayward, 1966), hence, it can be regarded
On the bases of pathogenicity tests, morphological, cultural, and
biological characters, and in accordance with the "new host-species conc
of Stolp et al. (1965), we propose the name Xanthomonas nepheliae sl
for this bacterium pathogenic on rambutan.
BBEED, R. S., E. G. D. MURRAY, & N. R. SMITH. 1957. Bergey's manual of (
minative bacteriology. The Williams and Wilkins Co., Baltimore, Md. 1094 p
BURKHOLDER, W. H. & M. P. STARR. 1948. The generic and specific character
phytopathogenic species of Pseudomonas and Xanthomonas. Phytopathology
DIFCO LABORATORIES. 1953. Difco manual (9th ed.). 350 p.
wARD, A. C. 1966. Method of identification in the genus Xanthomonas. p. 9-14.
In Gibbs, B. M. and F. A. Skinner [ed.] Identification methods for microbiologists.
Part A. Academic Press, New York.
DESIMO, A. N. 1968. Chlorotic ring spot and vein necrosis of rambutan (Nephe-
lium lappaceum L.). Philippine Phytopathol. 3: 60-62.
ES, A. J. & REGINA S. RIKEn. 1936. Introduction to research on plant diseases.
John S. Swift Co., New York. 117 p.
ETY OF AMERICAN BACTERIOLOGISTS. 1957. Manual of methods for pure culture
study of bacteria. McGraw-Hill Book, Co., New York. 315 p.
.P, H., M. P. STARR, & NANCY L. BAIGENT. 1965. Problems in speciation of
phytopathogenic Pseudomonas and Xanthomonas. Annu. Rev. Phytopathol 3:
results prove that tissue incompatibility rather than a virus is the main cause
of this bud-union crease.
Various studies (Olson, 1954; Weathers et al., 1955; Weathers and C
van, 1959; Olson and Frolich, 1968) on bud-union crease of calamor
(Citrus madurensis Loureira) grafted with certain citrus varieties have
to the general conclusion that this disorder probably resulted from vari
incompatibility rather than from virus infection. This paper presents a
tional evidence that tissue incompatibility is the cause of this bud-ur
MATERIALS AND METHODS
All experiments were conducted in the greenhouse of the Bureau
Plant Industry at Lipa City. Tests included budlings of calamondin
calamondin, calamandarin, Batangas, Ladu, Cleopatra, Ransas, Ponkon, I
kom, Sziwuikom, Sunki, Florida rough lemon, Eureka lemon, Key I
Palestine sweet lime, Rangpur lime, Citrus macrophylla, Pera, Ma4
Vinous, Shamouti, sweet orange of an unknown variety, grapefruit, ,
native pummelo, Troyer and Carrizo citranges, and trifoliate orange
cks. The calamondin scion buds were obtained from greenhouse-
seedlings. Some of the seeds of calamondin, Eureka lemon and an
vn variety of sweet orange were hot-water-treated at 60 C for 30
before germination and others were untreated. Calamondin was used
as a rootstock or as a scion with Eureka lemon and an unknown
of sweet orange. Calamandarin, Ladu mandarin and Florida rough
were used as interstocks between calamondin and Eureka lemon and
n calamondin and the unknown variety of sweet orange. Repeated
ttions were made from budlings severely affected with bud-union
lorida rough lemon rootstocks. T
Ltion, the experimental, budlings
hree years after propagation and/or
were examined for bud-union crease.
in combination with Eureka lemon and an unknown variety of sweet
Produced bud-union crease. Necrotic depressions or constrictions in
ood at the bud-union with corresponding necrotic projections on the
Il face of the bark (Fig. 1) characterized this disorder. These necro-
sues in the wood and bark were impregnated with a gummy sub-
The other varieties that produced bud-union crease when used as
>cks for calamondin were grapefruit, native pummelo, Citrus macro-
,Madam Vinous, Pera, Shamouti, Palestine sweet lime, sour orange,
r and Carrizo citranges, and trifoliate orange. No bud-union crease
ped on the 3-year old budlings of calamondin on calamandarin, Ba-
, Cleopatra, Ransas, Ponkan, Sunki, Szinkom, Sziwuikom, Key lime
.angpur lime. Despite repeated inoculations from the severely af-
budlings of calamondin on Eureka lemon rootstock, no bud-union
was produced within 3 years on budlings of calamondin on calamon-
alamandarin, Ladu and Florida rough lemon rootstocks, combinations
formally develop no bud-union disorder. Also, no bud-union crease
ped when calamandarin, Ladu and Florida rough lemon were used
erstocks between calamondin and Eureka lemon and between calamon-
nd the unknown variety of sweet orange.
DISCUSSION AND CONCLUSION
he bud-union crease of the calamondin combinations tested in these
s is apparently the same as that described by Weathers and Calavan
, and Olson and Frolich (1968). Similar bud-union effects on other
varietal combinations have been studied by Olson (1954, 1958),
n. Grant and Childs (1955), Weathers et al' (1955), Cooper and Olson
Pmnn ~P~~l~~- ~~nrr
Fig. 1. Bud-union crease of calamondin scion on Eureka lemon rootstock.
bark intact; Right: bark removed to show necrotic constriction in the wood a
(1956), Grant, Moreira and Costa (1957), and Salibe (1961, 1965). 1
none of these investigations completely eliminated viruses as causes,
general conclusions suggest that citrus bud-union disorders of this
were probably a result. of varietal incompatibility. Results of my st
provide additional evidence that the bud-union crease of trees of cc
combinations of calamondin and other kinds of citrus is not virus-ind
Certain combinations of calamondin and other kinds of citrus always
veloped crease; others did not. Repeated inoculations of the normal bu
trees had no discernible effects on them. Also, interstocks of calamo
compatible varieties such as calamandarin, Ladu and Florida rough le
between calamondin and Eureka lemon and between calamondin and
orange prevented the development of bud-union crease. The writer
cludes that tissue incompatibility is the cause of bud-union crease of
mondin when grown as a scion or rootstock with certain citrus vari
I II1YI1III~Y~IIIV1 ~II1VIVU
z: CITRUS BUD-UNION CREASE
Sci. 63: 131-136
with some kum.
). A bud-unii
Jniv. Calif. Div. Agr. Sci., Berkeley.
THEBS, L. G., E. C. CALAVAN, J. M. WALLACE, & D. F. CHRISTIANSEN. 1955. A
bud-union and rootstock disorder of Troyer citrange with Eureka lemon tops.
Plant Dis. Reptr. 39: 665-669.
I and 15.2% at disi
a yield of same var
disease intensities 1,
in yield of IR-8
gained in the wet sea
n be made b)
.on, stages of I
e amount of
E cron develooi
LAVlfaII 1 11 uL. u s l i All -.-alnll-JljALy Lt Lv la.t- IlAu LIJAJ L At tl O, o-s, JlIL aJL .
of yields of susceptible and resistant varieties of the same yield poten
1 & 2] EXCONDE & RAYMUNDO: RICE BLAST 67
,arison of yield of plots infected and plots protected by fungicides,
weighing healthy and diseased tillers. Systems of grading have been
to distinguish degrees of infection in estimating yield losses by some
tgators (McKinney, 1923; Jennings, 1963; Chenulu et al., 1966).
The blast disease of rice has always been a threat to the profitable
auction of rice in many rice-producing countries. Goto (1965) reported
this disease reduced the yield of rice by 24.8% in Japan in 1960. In
'1 N-methyl carbamate) was sprayed ;
IL UitL u&u(6UT. X 1 lCtidlI1y LCtL plllLS WCIt IIIULUliLCt U ay sulIIUil
them with blast-infected rice seedlings. Diseased leaves were also insei
between th foliage of the experimental plants. In addition, calibre
spore suspension from 2-week-old cultures of Pyricularia oryzae in w;
hyacinth (Monochoria vaginalis Presl.) was sprayed to the plants. The
sired disease intensities 1, 2, and 3 were attained by inoculating pl;
equivalent to 25, 50 and 75% of the total number of hills per subp
respectively. The control was sprayed with Blasticidin SM (2% blasticil
benzil amino sulfonate) at the rate of 15 g/gal of water.
Yield loss data.- When 85 to 95% of the grains were ripe, the boi
plants in each sub-plot were cut. The remaining experimental plants v
then harvested, threshed and sun-dried for 2 days. Determination '
a Steinlite Moisture Tester of 100-g samples from each treatment gave
moisture content of 13%. Yield losses of experimental plants with var)
degrees of infection were determined by weighing the grains and compal
them with the yield of the control. Yields of the plants at different st;
of inoculation were also compared for losses at different stages of groin
RESULTS AND DISCUSSION
The effect of intensity of rice blast infection on the yields of I]
IR-4 and IR-60 during the wet and dry seasons is shown in Fig. 1. Si
there was no significant interaction in yield between the 3 stages of in(
lation and disease intensities, the yields obtained from the 3 stages
inoculation were pooled.
IR-8 (susceptible). Highly significant reduction in yield was obtai
in all disease intensities during wet the season (Table 1). For instai
the mean yield of the control was 784 g/1.35 sq m compared to the m
yields with intensities 1, 2 and 3 which were 729.3, 718.2 and 686.7
respectively. These represent yield losses of 54.7, 65.8 and 97.3 g or
8.4 and 12.4% at disease intensities 1, 2 and 3, respectively. Signific
TABLE 1. Yield loss of IR-8 as affected by Pyricularia oryzae at 3 disease intense
Wet Season 1968 Dry Season 1969
Yield % Loss Yield % Loss
0 (control) 784.0 747.4
1 729.8 7.0* 687.8 8.C
2 718.2 8.4** 642.9 14.C
3 686.7 12.4** 633.7 15.I
auction in the weight of grains of brown rice and the weight of brown
:e per hill. Fazli and Schroeder (1966) recently found that the average
During the dry season, no significant reduction of yield was attribi
to disease intensities (Table 2). Observations from stages of inoculat
gave no significant comparison of yield losses.
IR-60 (resistant). During the wet and dry seasons (Table 3)
slight and statistically insignificant reductions in yield were obtained
the 3 levels of disease intensities compared to the control; these corrobo
the earlier findings of Exconde et al. (1968).
Our results show that the highest yield loss when compared to
control occurred in the susceptible variety (IR-8). Although there
TABLE 3. Yield loss of IR-60 as affected by Pyricularia oryzae at 3 disease intensi
Wet Season 1968 Dry Season 1969
Yield % Loss Yield % ]
0 (control) 840.2 -800.2
1 798.8 4.9ns 794.1 0.,
2 806.0 4.1ns 784.6 1.5
3 804.9 4.2ns 781.2 2."
a Each figures is an average of S replicates and 3 stages of inoculation; it is
pressed in g/1.35 sq m.
r~.wrw - - ---- --- N
---- IR -8
*---* IR -4
k------A IR 60
iITY R&TIM drI SWeaH)
775.01 30.31 X
y = 735.88 38.622 X
1 2 3 4
DISEASE INTENSITY (& season)
Fig. 2. Linear relationship between disease intensity and yield of IR-8.
1 2 53 3
INTENSITY (wet season)
a significant reduction in yield in the moderately susceptible variety (IR-4)
during the wet season, it was not as marked as in IR-8. No significant
loss in yield was obtained in IR-4 during the dry season and in IR-60 (re-
sistant variety) during the wet and dry seasons. Obviously, such differences
blast than either IR-4 or IR-60.
The absence of substantial yield loss in IR-4 and IR-60 could be due
in yield losses were due to the more susceptible reaction of IR-6 to rice
to the minimal infection that occurred in the plots. The lesions that
,rlrAlnnrld nairl from heino fewer in number. were minute sDecks. esDe-
LECLERG, E. L. 1964. Crop losses due to plant diseases in te united states. rnyto-
pathology 54: 1039-1313.
MATHUR, R. S., G. K. BAJPAI, L. S. CHACHAN, & S. C. VERMA. 1964. Assessment
of loss caused by paddy blast. Plant Dis. Reptr. 48:. 711-713.
MoKINNEY, H. H. 1928. Influence of soil temperature and moisture on infection of
wheat seedlings by Helminthosporium sativum. J. Agr. Res. 26: 195-217.
McNEAL, F. H. & E. L. SHARP. 1963. Effect of striped rust on yield and test weight
of white spring white varieties at Bozeman, Montana in 1962. Plant Dis. Reptr.
PADMANABHAN, S. Y. 1965. Estimating losses from rice blast in India. p. 205-221.
its sporadic o<
Vlr'M' i ur V Z-Au & kIuMr ri.nirJS.U ILN I1iuiI. ,1. *IVJl ,I uJ.v'
CERCOSPORA FULIGENA SPORE GERMINATION,
PENETRATION, INFECTION AND
SPORULATION ON TOMATO
T. R. MAGDA AND F. C. QUEBRAL
Former undergraduate thesis student and Assistant Professor, respectively, Depart-
ient of Plant Pathology, UPCA, College, Laguna.
This work was financed by the UPCO Vegetable Project 234.
Spores of Cercospora fuligena Roldan germinated in 5 hr on tomato ex-
posed in the moist chamber with 100% R.H. at ordinary room temperature.
Germination was faster within 5 to 10 hr of exposure; average germination
of 92.8% was obtained after 48 hr exposure. Penetration was observed
in 70 hr of exposure; an average of 7.94% was noted. The rate of penetra-
tion was much faster within 70 to 77 hr of exposure. The pathogen entered
the suscept by the penetration of conidial germ tube through stomatal open-
ings. Evidence of infection was the production of spots that were discernible
as early as the fourth day of exposure. These spots became more visible
after 5 days of exposure in the moist chamber. Sporulation was not observed
in infected plants allowed to remain inside the moist chamber; hence sporula-
tion was studied on tomato plants placed outside the moist chamber after
Leaf mold, caused by Cercospora fuligena Roldan, can be a serious
disease of tomato in the field. In the Philippines, no study has yet been
conducted on the pathogenesis of this leafmold pathogen. This paper
describes the effect of exposure period in the moist chamber on spore
ermination, penetration, infection, and sporulation of this fungous pathogen
Related studies by Bean and Wilcoxson (1961) on the development of
pring black stem on alfalfa and red clover have demonstrated that trace,
ight, and moderate infection resulted when experimental plants were ex-
losed for 24, 48, and 72 hr, respectively, at 100% relative humidity in the
noist chamber. These workers concluded that disease severity vary with
he amount of inoculum applied an exposure period in the moist chamber.
leaves to separate tne epicermal tissue trom cne orner ieai tissues. i in
adhering epidermal tissue on the tape was stained with methylene blui
and acid fuchsin. This was mounted on a microslide and examined unde
the microscope periodically.
Sections of infected tomato leaf were decolorized in chloral hydrate so
lution, washed in 95% ethyl alcohol and stained with methylene blue anc
basic fuchsin. These sections were examined under the microscope to de
termine penetration of germinated spores.
Visible lesions on infected leaves were counted to determine the num
ber in relation to exposure period in the moist chamber (Fig. 1).
The germination of spores of C. fuligena exposed at varying period!
in the moist chamber with 100% R.H. is presented in Table 1. The
highest germination percentage was obtained after 48 hr. The average rate
of germination/hr was 3.44% between 0 and 5 hr, 6.82% between 5 and
10 hr, 0.9% between 10 and 20 hr, 2.08% between 20 and 30 hr, and
0.63% between 30 and 48 hr. The rate of germination appeared to be
faster between the 5- and 10-hr exposure period.
. .-A .I1-,
2 QUEBRAL: CERCOSPORA I
ne average rate ot penetration/hr was U.2% trom 0 to 70 hr, 1% from
0 to 77 hr, 0.21% from 77 to 95 hr, and 0.19% from 95 to 140 hr of
Nos. I & 21
pient spots bet
C. fuligena on
Number of S
:n 6 a
narm;~n,;,rr :Ilrra~.a~ rrr;tl, ~kn rlr~t;nn nF
oC OUEBRAL: CERCOSPORA Ft
nearby stomates thus giving a high rate of infection per unit area of the
At an early stage, infection by C. fuligena was characterized by the
appearance of incipient spotting on the under surface of the leaf. Only
few visible spots appeared. These incipient spots were actually made up
of branched germ tubes ramified in the epidermal tissue. With increased
period of exposure, the number of visible spots also gradually increased
and then general vellowing of the leaves resulted.
Nos. 1 & 2]
Fig. 3. Excised tomato leaves inside Petri dishes used as moist chambers.
A 100% R.H. favored spore germination, penetration, and infection bu
not sporulation. At 100% R.H., water vapor condensed in the mois
chamber which made the leaves turn yellow before sporulation could tool
place. Sporulation took place on inoculated plants placed outside th
moist chamber after incubation in 14 to 15 days. Sunlight was found t(
affect the color of spores. Spores on leaf exposed to sunlight were darl
while spores under the shade appeared grayish. Fungous colony on leave
issues, after 7-day exposure in moist chamber. (970 x)
Fig. 1. Helminthosporium leaf spot showing typical symptom in the field; A) a
close-up of leaf symptoms, B) another type of symptom, C) target-spot pattern, and
D) spores of the causal fungus (x 160).
Fig. 2. A) Diagnostic symptom of Rhioctonia disease on the leafsheath, and
B) the sclerotial bodies of Rhizoctonia solani.
a faster rate resulting to the drying and death of the entire foliage. In
severe cases, the stalk is infected, thus, causing stalk rot. This disease is
caused by Rhizoctonia solani Kuhn which forms sclerotial bodies on infected
tissues (Fig. 2B). This fungous structure enables the pathogen to survive
from season to season and serves as inoculum for the next crop.
Leaf rust. This disease has been observed plants were about 2-'
months old. It is more severe during the dry season. This is readily re-
cognized by the brown to reddish-brown elliptical spots with blisters or
pustules (Fig. 3A). Upon maturity, the pustules break open exposing the
brown colored spores (Fig. 3B) which serve as inoculum for secondary in-
fection. The causal fungus is Puccinia purpurea Cke.
Anthracnose.- This is characterized by the presence of reddish specks.
Some of these specks may progress into spindle-shaped reddish lesions. As
the disease progresses, the lesions become straw-colored with depressed cen-
ter and reddish brown border .(Fig. 4A). Observations on inoculated plants
in the greenhouse revealed that this disease does not spread from leaf to
leaf. It is caused by Coll totrichum graminicolum (Ces.) Wilson (Fig. 4B).
Fig. 3. A) Leaf symptoms of rust, and B) spores of Puccinia purpurea (x 400).
w '-. ^
Fig. 4. A) Leaf symptom of
inicolum (x 400).
anthracnose, and B) spores of Colletotrichum gram-
is Gleocercospora sorghi Bain and Edg. which produces needle-like spores
Fig. 5. A) Leaf symptom of zonate spot, and B) spores of Gleocercospora sorghi
Bacterial stripe. This disease has been known to be caused by
Pseudomonas andropogoni (EFS) Stapp. It was observed to affect sorghum
plants in all stages of plant growth. It is characterized by long narrow
reddish irregular lesions starting from the base and spreading upwards,
thus affecting the entire leaf (Fig. 6).
r YHILIPPINE "HYTOPATHOLOGY LV(H1 0
Fig. 6. Diagnostic symptom of bacterial stripe of sorghum.
DISEASES OF CORN UNREPORTED IN THE PHILIPPINES
S. C. DALMACIO AND O. R. EXCONDE
Instructor, and Associate Professor and Chairman, Department of Plant Pathology,
UPCA, College, Laguna.
This study was supported in part by Corn Project 3.3.
During the 1969-70 wet and dry seasons, 5 diseases of corn believed to
be new in the country were noted. Three of these diseases have been
observed every season. Their prevalence has attracted the attention of corn
Border Leaf Blight.- This disease is now very common; it occurs in
any corn field at the Central Experiment Station. Only mature plants
appear to be affected. Infection always starts from the lower leaves. At
first it is manifested by small, watersoaked lesions at the margin of lower
leaves. These lesions -gradually enlarge to a size measuring more than 10
cm long and 2-3 cm wide. Old lesions are greyish with distinct greyish-
green border (Fig. 1A and B). During continuous rain for several days,
fan-like, sessile basidiocarps, about 0.5 cm in diam can readily be seen at
the center of old lesions.
The disease is caused by a basidiomycetous fungus. No fructification
has so far been produced on potato dextrose agar (PDA). The mycelium
is white and cottony and clamp connections can readily be observed under
the microscope (Fig. 2A and B). Inoculation of mature plants revealed
that the fungus is capable of infecting the ears.
Curvularia Leaf Spot. This disease is more commonly observed on
mature plants than on young ones. The disease is manifested at first by
small, numerous, circular spots with chlorotic borders. Spots resemble the
early symptom of Helminthosporium leaf spot and leaf blight of corn. Such
spots, however, do not increase further in diameter. Old spots seldom ex-
ceed 2 mm in diam with greyish center and brown border (Fig. 3).
This disease is caused by Curvularia sp.
Bacterial Stalk Rot.- About 10% of the corn population in a 400-sq m
area was affected by the disease during the wet season of 1970. The leaf
sheaths are cemented to the stem by slimy bacterial exudates. When the
stem is cut and submerged in water, milky mass of bacteria can be seen
oozing out of the vascular bundles. Corn stubbles left after cutting the
i _. __ i .-
A : B
Fig. 1. Corn leaves showing the early (A) and advanced stage (B) of border leaf
Affected plants are easily toppled down by slight wind although some
may remain standing. Plants that do not fall over appear wilted and die
before reaching maturity (Fig. 4). Stalks of affected plants are rotted at
the base near the soil line although rotting may also occur at the base
of the ear shoot. Affected plants emit foul or fermented odor.
The disease is caused by a still unidentified bacterium. When stems
of healthy plants are punctured with needles previously pricked into the
stem of infected plants, rotting proceeds rapidly. Inoculated plants fall
over within 5 days after inoculation.
Ear Rot. This disease is still of minor importance. It is manifested
by rotting of corn ears which are about to mature. Symptoms are first
observed on the husks as blotches which are irregular in outline. As
the disease advances the entire ear becomes dry. Numerous, pinkish to
black, hemispherical, sclerotial bodies about 1 mm in diam are formed on
Fig. 2. The fungus causing border leaf blight: A) growth on PDA, and B) clamp
connection (Indicated by an arrow; x 1000).
TO~r 1 0. 0-
ny mycelium which are also found in between and over the kernels
The disease is caused by Rhizoctonia zeae Voorhees. The fungous
*lium on PDA is similar to that produced on the infected ear. Nu-
)us brown sclerotial bodies lump together to form bigger sclerotial
es. Sclerotia produced by R. zeae are soft and are easily crushed
s. 6A and B) unlike sclerotia produced by R. solani.
Fig. 4. A) Corn plants affected by bacterial stalk rot. B) Corn stalk showing
r-soaked lesion (indicated by an arrow) of bacterial stalk rot.
Charcoal Rot.- This disease is also of minor importance. Affected
its, especially those about to mature, topple down and die. When the
is of such plants are longtiudinally cut the vascular tissues are shredded
greyish black portions of the stem can be observed (Fig. 7). Minute
k specks, or sclerotia of the fungus are scattered over the surface of
Ik- _- I l- _.ai -
Fig. 6. Rhsoctonia zeae: A) growth on PDA (left) and sclerotial bodies (right).
manral e~. amntial hinrlv tY 1in000
Fig. 7. Splitted corn stalks showing symptoms of stalk, rot caused by Macrophomi