I: YtLI I "iII1 Pe
distracts of Papers Accepted for Presentation at the Ninth Annual
Meeting of the Philippine Phytopathological Society ......... 1
latoxin Contamination on Raw Agricultural Crops and Their By-
Products in the Philippines
P.A. Santamaria, A.C. Pizarro and C.R. Jackson ........ ... 12:
)gress Report on Whitefly Transmission of Tobacco Leaf Curl Virus
to Vegetables and Weeds
F.M. Olivares, Jr., Aurelia A. dela Cruz and Lagrimas B. Salisi 21
lyldhe Synthesis by Penicillium expansum Link in Relation to
Lina L. Ilag .......... .......... ........ .................. 30
hogenicity Tests of Species of Praytylenchus, Tylenchorhynchus
and Helicotylenchus on Corn
J.J. Walavyala and C.P. Madamba .................. ........ 35
Tie Biochemical Changes During Pythium Infection on Cotton Seed-
Tricita H. Q uim io ......................... ....... 41
iergistic Reactions Between the Pathogens of Citrus. reenhqg and
A.L. Martinez ............................... .. -.. 49
racellular Enzymes Produced by Pyricularia oryzae Caiw-
R. Sridhar and S. H. Ou .......................... .... 2
le of Phenolic Compounds and Polyphenol Oxidase in Rice Blast
R. Sridhar and S. H. Ou ................................. 57
* Role of Red Spider Mites on Infection of Soybean with Pseudo-
monas glycinea Coerper
D B. Lapis .......................... ............ ..... 66
ore Density of Sclerospora philippinensis in Relation to Field Tem-
perature, Relative Humidity and Downy Mildew Incidence
S.C. Dalmacio and A.D. Raymundo ......................... 72
YTOPATHOLOGICAL NOTE: Influence of Root--Knot Nematodes
on the Severity of Bacterial Wilt and Fusarium Wilt of Tomato
R. G. Davide ......................................... 78
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ABSTRACTS OF PAPERS ACCEPTED FOR PRESENTATION
AT THE NINTH ANNUAL MEETING OF THE PHILIPPINE
BAGUIO CITY, 17-19 APRIL 1972
(Arranged alphabetically according to first author's surname)
Xanthomonas oryzae resistant to Celdion S.-K. C. Agrawal and S. H.
u.-Celdion S (TF 130) has been found effective in controlling bacterial
if blight, either by soil application or by foliage spraying.
The bacterium (Xanthomonas oryzae) is capable of building up resis-
nce to the chemical. When plated on agar media containing varying
counts of Celdion S, resistant colonies gradually appeared in successive
ys-sooner in low and later in high concentrations of the chemical. Colo-
es resistant to higher concentrations could be obtained by further plating
higher concentration of the chemical. Strains resistant to 1000 ppm of
e chemical were developed.
Experiments also demonstrated that a much higher concentration of
emicals was required to control the disease when resistant strains were
aculated to the plants. Chemical-resistant strains were more virulent than
e original strain from which they developed, as determined by the rate
lesion development. No resistant strain was developed on media contain-
g autoclaved Celdion S above 200 ppm.
Dual infection of tungro and grassy stunt.-V. M. Aguiero, C. T.
vera and D. F. Dimasuay. In 1971 we found that some diseased plants
the IRRI fields had dual infection of tungro and grassy stunt. A total
54 individual infected plants, consisting of samples from IRRI and Sta.
)sa, were caged with Nephotettix virescens and Nilaparvata lugens. About
% of the diseased plants had mixed infection of tungro and grassy stunt
iile the rest had either tungro or grassy stunt alone. Similar results'
!re obtained with artificially-inoculated plants. However, a higher per-
ntage of tungro transmission was obtained from plants infected first
th tungro and later with grassy stunt than on plants infected first with
assy stunt and later with tungro. In the same manner, a higher per-
ntage of grassy stunt recovery was obtained from plants infected earlier
th grassy stunt and later with tungro. Virus recovery was done 20 days
:er inoculation of the second virus.
Symptoms of dual infection, which usually became severe one month
:er the introduction of the second virus, were distinguishable at the
early stage of development. Infected plants normally died as a result
Varietal resistance to tungro and grassy stunt. V. M. Aguiero, C.
Rivera and K. C. Ling--Screening of rice varieties and selections for th
reaction to tungro and grassy stunt by mass screening was continued
A total of 4,042 entries, consisting of 275 varieties, 2,828 selecti4
of IRRI and UPCA, 101 Indonesian and Thailand selections, 689 ge
tical materials and 149 duplicates, were tested against tungro. Most val
ties were susceptible. A variety, Habigonj 8(3) was highly resistant, simi
to Pankhari 203. IRRI crosses with Pankhari 203 had the highest num
of selections in the resistant group.
A total of 8,042 entries, consisting of 2,274 varieties, 2,423 entries
IRRI selections, 351 Thailand and UPCA selections, 76 Oryza nivara
tries from India, 2,172 entries of genetical materials, and 746 entries
duplicates were tested against grassy stunt. A line of 0. nivara, a -
Oryza species, was highly resistant to grassy stunt. IRRI materials cros
with this wild rice have a high percentage of selections in the resist
Virus recovery from green and yellow leaves of tungro-infected plan
M. P. Carbonell and K. C. Ling.--A study was conducted to determ
the difference in the recovery of tungro virus by insects fed on yol
green and on old yellow leaves of an infected plant. Virus-free adi
of Nephotettix virescens were given 24-hr acquisition feeding and 3-1
serial inoculation feeding. The percentage of infection and the virus rel
tion period were both higher and longer (62.6% and 3 days, respective
with the insects feeding on green leaves than those on yellow leaves (19.0
The control of mungo mosaic by insecticide spray against the aphic
R. V. Cortado.- Mosaic, a serious disease of mungo in Cagayan Val
can be greatly minimized by spraying insecticides to control the ap
All of the insecticides tested, viz., Thiodan, Endrin, Metacide 4
Ultracide, Basudin 60 and Dimecron significantly reduced the percent
of infected plants compared with the control. There was no statistic
differences among the insecticide treatments. Disease control ranged fr
77.7 to 86.88%. The sprayed plants gave higher yields than the unspra
ones. Yields obtained from the sprayed plants did not differ significant
The increases in yield over the control ranged from 44.90 to 67.35%.
Evaluation of three nematocides as pre-planting treatment for the cont
of root-knot nematodes on banana.- R. G. Davide.- Three nematoci
- -I- -- -- - -------I-- ------- _---.. __
treaunent or oanana, var. Lacatan, ior me control ot root-Knot nema-
e, Meloidogyne incognita Chitwood.
In the first trial the nematocides were used as water-dip and soil in-
poration at the rate of 1 g/gal and 1 g/8-inch day pot, respectively.
e small banana suckers were dipped for 15 min in the nematocide sus-
ision and later planted in 8-inch clay pots filled with soil infested with
incognita by placing 3 g of galled tomato roots per pot. Another 5
kers were planted directly to the 8-inch clay pots with soil infested
lilarly with the nematodes. All the treated and control plants were
)t in the greenhouse.
The results showed that the water-dip treatment gave better nematode
itrol than soil incorporation based on the mean gall index taken 6
nths later. In all the nematocide treatments the mean gall index was
,hin the slight to moderate galling range while those of the soil in-
poration was within moderate to severe galling. Likewise, the data on
System growth rate of the plants showed increased growth in the water-
treatment by 66.14% in Mocap 10G, 60.5% in Nemagon 20G and
23% in Temik 10G. However, no growth increase was observed in the
I incorporation treatment, indicating that the rate for soil treatment
y be too low.
In a second trial the rate was increased from 1 g to 5 g/gal and 5 g/8-
h clay pot for the water-dip and soil incorporation treatments, respective-
Each pot of soil in all treatments was infested with 40 egg masses of
incognita. The results revealed that the soil treatment gave better
natode control than the water-dip treatment. The data on the stem
)wth rate of the plants taken 4 months after treatment also indicated
it the plants in the soil incorporation treatments had higher percentage
growth rate than those in the water-dip treatment.
Another experiment was conducted to determine the effect of time of
)ping banana suckers, var. Lacatan, on the control of M. incognita. The
e used for the three nematocides was 5 g/gal. The banana suckers
re taken from heavily root-knot-nematode infested area. These suckers
re dipped in the nematocide suspension at 5, 10, and 15 min and then
re planted in 8-inch clay pots filled with decontaminated soil. Five
)licate pots were assigned for each time of dipping. The same number
pots were also provided for the controls. The plants were kept and
served under greenhouse conditions. Five months after treatment the
ta on the stem growth of the plants were taken and the roots indexed
Sroot-knot-nematode infection. Trace to slight galling were obtained in
ints dipped for 5 min in the nematocide suspension; similar results were
trained in 10 and 15 min dipping. However, based on the stem growth
te of the plants there were indications that dipping 10 or 15 min in the
matocide suspension inhibited the early growth of the plants. Thus,
in some treatments the plants dipped in 10 or 15 min were relatively shor
than those dipped in 5 min but this difference was not statistically sig
Bacterial stalk rot of corn and sorghum.- Asuncion D. Karganilla a
O. R. Exconde.- Bacterial stalk rot has been reported to occur at I
Central Experiment Station, U.P. College of Agriculture, College, Lagu
No study, however, was made on symptomatology. During the last i
season, the disease was observed in most corn varieties at CES, UPCA a
in Batangas. On sorghum, it was first observed on var. Darso during i
last wet season. Actual count of infected corn plants in the field ran(
from 2 to 5% while of infected sorghum plants it was less than 6.1%
Field observation indicated that rotting usually starts from the b
progressing upward and eventually causing the leaves to wither start
usually from the lowermost leaves. Infected old corn plants do not ea,
topple over like infected young plants. The infected inner tissues of ol0
plants deteriorate, become soft but later dry up into easily disjointed fib4
They either topple over or remain standing until maturity.
The same symptom pattern was also observed on sorghum. Isolati
and tests of pathogenicity showed that the causal organism is a bacteria
the identity of which is presently being studied.
A rapid method for bacterial leaf blight inoculation. H. E. Kauffm
A. P. K. Reddy and S. D. Merca. A rapid method of inoculating r
plants with Xanthomonas oryzae, the bacterial leaf blight pathogen, has be
standardized. Scissors are dipped into a bacterial suspension and the 2
of the tips of rice leaves are clipped off; 30-40 leaves are clipped; sim
taneously in each plant at maximum tillering stage. One person can ea:
inoculate up to 1000 plants/hour in the field. Infection results in 10(
of the inoculated leaves if adequate bacterial populations are used. Dise
reaction is evaluated 14 days after inoculation on the basis of lesion mo
ment downward from the inoculated cut at the tip of the leaf.
The clipping method has advantages over earlier methods in that i
inoculation and scoring is more rapid and much simpler. In addition I
method gives equally good results when the inoculum suspension consi
of diseased-leaf extract or artificially-cultured bacterium. With this methe
varietal screening trials can be conducted effectively in any location wh,
natural inoculum is available. By using naturally-infected leaves as I
inoculum source prevalent strains of the bacterium in each area can be us
to test varietal resistance.
The role of red spider mites on infection of soybean by Pseudomoi
glycinea. D. B. Lapis. Soybean plants infested with red spider mi
(Tetranychus telarius L.) showed profound resistance or immunity to
fection by Pseudomonas glycinea. This response of the plant, howev
is dependent on the number of mites and length of time the plants had
en exposed to mite infestation prior to inoculation with the bacterium.
resistance or immunity increased with the increase in number of mites and
riod of exposure of plants to them.
Media for single cell growth and recognition of Xanthomonas oryzae.-
arina P. Natural, I. W. Buddenhagen and Asuncion Karganilla.- Among
e different media tested for culture and single colony growth of Xantho.
onas oryzae (Uyeda et Ishiyama) Dowson, the improved Wakimoto me-
um, i.e., 0.05% FeSO, added and potato eliminated (WF-P) and Silva-
iddenhagen (SB) medium gave the highest colony recovery. SB medium
is slightly inferior to WF-P in colony recovery but since it is a clear me-
um, the colonies of X. oryzae were of consistent size that could be dif-
rentiated from X. translucens. Attempts to incorporate FeSO4 and the
It components of Wakimoto medium failed to substantially increase the
lony recovery of SB medium. Results showed that FeSO4 is necessary to
ercome the inhibitory effect of the other essential salt components of
akimoto medium. FeSO, is not colony-recovery enhancing with SB me-
am. SB medium was improved by replacing 5 g glucose with 5 g sucrose
d 1 g glutamic acid with 1 g sodium glutamate. Sucrose is more stable
ider excessive autoclaving and sodium glutamate is cheaper and less acidic.
Effect of three systemic fungicides on the life span and reproduction of
ephotettix virescens and Nilaparvata lugens.- F. L. Nuque, V. M. Aguiero
.d S. H. Ou. In our preliminary trials to control tungro with systemic
ngicides, we observed that plants grown in treated soil with Benlate,
opsin M and NF-48 had many dead nymphs attached to the plant's sheaths.
his observation led us to study the effect of the three chemicals on the life
an and reproduction of both green and brown hoppers. Two chemical-
il ratios of 1/3500 and 2/3500 were used. The results showed that there
is no significant difference in the life span of both insects in the treated
ants and the control. However, in the different chemical treatments the
amber of progenies that became adults were significantly lower on plants
own in the treated soil than in the control. These chemicals seem to
rye as insecticides for the nymphs of the two insects.
Field evaluation of Celdion S (TF 130) to control bacterial blight of
-e. F. L. Nuque, S. D. Merca and S. H. Ou. Bacterial blight of rice
ie to Xanthomonas oryzae (Uyeda et Ishiyama) causes varying degree of
sses in yield in the tropics depending upon the variety and the season.
any chemicals in Japan have been reported to be effective in the control
the disease but failed in the tropics probably due to differences in bac-
rial strains and environmental conditions.
In our earlier pot experiments under greenhouse conditions it was
und that Celdion S (TF 130) appeared to be promising in the control
h n~mn r ~~c~
Field results of Celdion S showed that soil treatments of 30 to 6(
kg/ha of the compound provided good bacterial blight control as indicated
by a 22-31% increase in the yield of two treatments over the untreate(
plots. Foliar sprayings which were done twice at booting and early flower
ing stages at the rate of 2-4 kg of the materials showed that the disease
incidence was considerably lower in the treated plots than the control. Th
yield of treated plots was 27-30% better than that of the control.
The effect of Kasumin and Hinosan spray on rice blast (Pyriculari,
crea ior cuiurmng Elis fungus in artuiciai meala ana aeveloping a
thod of artificial inoculation so that varietal resistance could be evaluated.
Several studies on germination of the chlamydospores have been made
earlier workers but culturing the fungus has not been reported. From
'se studies, it was found that light and oxygen are essential for germina-
In our trials chlamydospores germinated readily in soft plain agar
)osed to direct sunlight for 4 hr. The germinated spores grew well on
A. Numerous secondary and tertiary sporidia were produced on PDA
Sr 3 weeks.
Successful inoculation was obtained by either (1) dipping the flower-
panicles in the sporidial suspension, (2) spraying the suspension on
wearing panicles, or (3) injecting the suspension into the boots before
iicle emergence. The injection method produced more infected kernels
.n either the dipping or spraying method.
Estimates of rice yield losses due to bacterial leaf blight in the field.-
H. Ou, F. L. Nuque and S. D. Merca. No method is known to estimate
Id losses due to bacterial leaf blight in fields where its degree of severity
We tried to correlate the diseased areas of the top three leaves to
Losses in grain yield. From a naturally-affected field, 200 to 300 hills
h varying degrees of infection were selected. From each hill, the three
lest tillers were picked. The diseased areas (by percent of total leaf
a) of the three top leaves of each tiller were estimated. The weight
filled grains, number of empty spikelets, and a 1000-grain weight were
:ermined. The percentage of losses of grain weight (compared with un-
ected plants) was plotted against the percentage leaf-area damage to form
Two such estimates showed that when the leaf-area damage reaches
)% the yield loss is about 50% and a 50%, leaf damage caused 25%
s on grain yield.
By making a number of such estimates, it is hoped that a more or
i standard curve may be developed. Then, by sampling leaf damage in
y field, the yield losses may be roughly estimated.
Further studies on stable resistance to rice blast. S. H. Ou, F. L.
ique, J. M. Bandong and T. T. Ebron, Jr.- Several highly-resistant varie-
s selected from the international blast nurseries were found to be infected
certain races of Pyricularia oryzae in our blast nursery as few lesions
The fungus was isolated from the lesions, and a number of single conidia
subcultures were obtained from each of the isdlates. The subcultures wer
inoculated on their respective hosts, resistant varieties listed above, and Car
reon and Tetep, as well as on the international and Philippine differentia
varieties. The results showed that the subcultures of each isolate consisted
of several races. Only very small number of lesions appeared on thesis
varieties as compared with variety Khao-teh-haeng 17 which was a susceptible
This confirms our earlier study on variety Tetep, indicating that varied
ties having broad spectrum of resistance to blast are stable. Only few lesion
may be produced on their leaves even when pathogenic races are inoculated
.Evaluation of four fungicides for the control of foliage diseases o
grapes. -A. N. Pordesimo and R. V. Valmayor.- Four fungicides, viz., Thia
bendazole (Tecto 90) at 1 Ib + 2 oz/100 gal, Copper oxychloride (Vitigrai
Blue) at 4 llb/100 gal, Benomyl at 1/2 lb a.i./100 gal, and Mancozel
(Dithane M-45) at 2 lb/100 gal were tested against foliage diseases of twi
grape varieties, viz., Cardinal and IAC (Instituto Agronomico Campenas
971-1 at UPCA. Sevin (insecticide) at 1 1/2 lb/100 gal and Dupont Spreade
Sticker at 4 oz/100 gal were added to each fungicide suspension and at thi
same time used as control.
Nine weekly sprays starting 15 October thru 14 December 1971 were
applied using a Knapsack sprayer (Birchmeier Senior).
Downy mildew and leaf rust were common on the two varieties whili
anthracnose occurred only on IAC 971-1. Mancozeb and Copper oxychloride
(Vitigran Blue) gave better control of downy mildew and rust on both varie
ties. All test fungicides gave good control of rust. The effectivity of the
fungicides to control foliage diseases was also reflected in number of terminal
and green leaves. Prolonged wetting periods decidedly favored disease deve
lopment and tended to break down effectivity of the fungicides.
Pre-harvest spray and post-harvest dip of mango fruits against anthrax
nose and fruit rots. A. N. Pordesimo, E. N. Imperio and P. M. Halos.-
Five fungicides were tested as pre-harvest spray and post-harvest dip for the
control of anthracnose and rots of mango fruits. The pre-harvest spray wa
applied 3 weeks before harvest at Yuson's Farm in Jaen, Nueva Ecija
Benomyl (Benlate) was used at 1/2 lb, Thiabendazole (Tecto 90) at
lb + 2 oz, Copper oxychloride (Vitigran Blue) at 4 Ib, and Mancozel
(Dithane M-45) at 2 lb per 100 gal. A surfactant was used at 4 oz/101
gal as control and as wetting agent for all the test fungicides. Four single
tree replicates were used per treatment. At harvest, 30 fruits from eacl
tree were carefully picked at random, properly identified, and brought t<
the laboratory for the post-harvest treatment with the same fungicides am
dosage, and swabbing with 5% NaCI.
ula.tu ..a lLa UniI.ILcua 1un uIuluc I 0 UL icatUlIS pJ III11CLCU IIFUL, anu num-
er of clean fruits at 7 and 14 days after harvest were obtained among
ingicide-treated fruits. Control fruits had the most number of lesions.
itigran Blue and Mancozeb-treated fruits either before or after harvest or
)th had the least number of lesions per fruit.
Swabbing pre-harvest sprayed fruits with 5% NaCI helped to remove
.e unsightly dried latex stains on the fruit skin.
Survey of pesticide needs and application in some selected vegetable
rms in Atok and Bugias, Benguet Province. F. C. Quebral and M. I.
iramancion. The use of pesticides is one of the indispensable factors in
e successful operation of any vegetable enterprise in Benguet province.
withoutt pesticides, the vegetable industry would suffer tremendous loss from,
sect pests and diseases and implicitly discourage vegetable farmers.
Limited funds and high cost of farm chemicals are the common prob-
ms that apparently hinder the intensive and proper use of pesticides.
sound financing scheme can help vegetable farmers ensure the appropriate
id judicious use of pesticides and at the same time save these crops from
aladies and pests.
An anatomical study of leaves from coconut affected by cadang-cadang.-
linda P. Rillo and W. C. Price. An anatomical study was made of
lives from coconut palms in various stages of cadang-cadang in compari-
r with leaves from a healthy tree. Differences between leaves in an
rly stage of the disease and those of a healthy tree were confined to the
[low spots, no differences being found in the green portions of the leaves.
more advanced stages, the differences extended even to the green areas.
aaves from trees in medium and advanced stages of disease were signi-
antly thinner than those from a healthy palm. The difference in thick-
ss is due to the small size of all types of cells, there being little or no
Reference in their numbers. The palisade cells are not only small but are
regularly arranged rather than in tiers. Mesophyll cells are rounded rather
an angular. The differences observed seem to be due to incomplete or
rested development of all types of cells in the leaf and to necrosis of
ne of the cells in the characteristic yellow spots.
Strains of rice tungro virus during its outbreak in 1971.-C. T. Rivera
d K. C. Ling. Three strains of the virus causing tungro disease of rice
re identified in the Philippines in 1966 and 1970. In 1971, tungro broke
t in several provinces in Luzon and in many places in Cotabato and
ikidnon. To determine whether the virus that caused tungro was the
ne in all affected areas, diseased rice plants and suspected weeds were
elected and tested.
The virus was recovered from diseased plants and inoculated to dif-
rential rice varieties by feeding Nephotettix virescens (= N. impic-
10 PHILIPPINE PHYTOPATHOLOGY [VOL. 8
ticeps). Our results showed that the isolates, consisting of 93 diseased
plants from main crop and 16 from ratoon fields, produced symptoms
similar to the virulent strain of tungro on FK-135, C4-63G, IR20, Pankhari
203, and Taichung Native 1. Chlorotic stripes were incited on the leaves
of FK-135 which are diagnostic symptoms of the S strain of tungro.
A comparison of daily serial transmission of the various isolates of
tungro using laboratory-reared and naturally occurring N. virescens indicated
that incubation period and disease-transmitting days were similar to that
New vector of rice tungro and yellow dwarf. C. T. Rivera, V. M.
Aguiero, D. F. Dimasuay and K. C. Ling.--A new leafhopper species,
Nephotettix parvus Ishihara and Kawasa, was found to transmit tungro
and yellow dwarf diseases of rice. The active transmission by the insect
was 8% for tungro and 62% for yellow dwarf. This indicated that the
leafhopper was an efficient vector of yellow dwarf, but a poor vector of
tungro. Results of virus-vector studies were similar to those of N. virescens
(= N. impicticeps) and N. nigropictus (N. apicalis) for the same diseases.
Some factors influencing blast resistance in rice plants. R. Sridhar
and S. H. Ou. A comparison of metabolic changes in both resistant and
susceptible varieties of host plants due to infection by a particular pathogen
has yielded some information on the mechanism of resistance. Such studies
conducted on rice blast caused by Pyricularia oryzae Cav. does not explain
the resistance or susceptibility of rice varieties to blast. An attempt was
made to explain the resistance of rice plants to blast, using a single variety,
Nos. 1 & 2] ABSTACTS 11
lated initially in the susceptible host. A wider ratio between total phenol
and free amino acid content for a longer time was associated with sus-
The post-infection changes in the metabolism of the host plant was
influenced by the type of race with which the variety is infected. The
resistance was expressed by more rapid accumulation of total phenolic com-
pounds, increased peroxidase activity and enhanced depletion of ascorbic
AWT ArMIMT T 'n3TTrAMUTWArTnT#Z nWhT 1Aw ArfDTV'FlTr1T0AT iDl~nDQ
AND THEIR BY-PRODUCTS IN THE PHILIPPINES
P. A. SANTAMARIA, A. C. PIZARRO AND C. R. JACKSON
Plant Pathologists, Bureau of Plant Industry, Manila and U.S. AID Consultant,
concurrently Assistant Director, Georgia Agricultural Experiment Station and Resident
Director, College of Agriculture, University of Georgia.
Raw agricultural crops and their by-products collected from different areas
of the Philippines were found to contain varying amounts of aflatoxin and the
fungus (Aspergillus flaus) that produced it. Some of the test samples showed
no Wsible signs of A. flavus, but contained high levels of aflatoxin. Converse-
ly, other samples yielded the fungus in agar plates but contained little or no
Corn grains on the cob were high in aflatoxin, the latter apparently con-
centrated in the germ. Corn grown in the Visayas and Mindanao had more
aflatoxin than those grown in Northern Luzon.
Dried peanuts from the Visayas and Mindanao generally contained less
amount of aflatoxin. Sorted out peanuts with wrinkled kernels unfit for com-
mercial processing, however, had as much as 144 ppb of the toxin.
The processes in copra-making like drying and storage predispose the
coconut meat to infestation by A. flavus and other microorganisms. However,
a finished product, copra pellet, contained no aflatoxin when treated with
Sorghum, palay, soybean and millet are poor media for aflatoxin production.
r'-.tt- ..-- D l,L..->- t-~~ it...... -- i.:__J Li-L. l-.-rl -t -tl-t-*_
OS. I & 'Z SANTAMARIA ET AL.: AFLATOXIN ON AGRICULTURAL LROPS I3
tIIULUAXIII IS UlIILCUIL LU uesCroy UCcause oU ILs nIlgn neat SiauIILly.
)ur hr of continuous heating at 125 C and 25 psi is usually required to
ing about a significant destruction. Aflatoxin has been designated into
veral identifiable structures whose toxicity varies on the basis of duckling
The effect of aflatoxin on animals depends upon the kind of animals,
iount of toxin, time of toxin exposure, and age of animals (Engel, 1970).
nly 1 to 2 ppb is required to produce hepatoma in rainbow trout finger-
igs, 30 to 50 ppb in day-old duckling and 500 to 700 ppb in the re-
itant laboratory rat (Engel, 1970). Sheep are quite resistant. In humans,
) direct evidence has been obtained to link aflatoxin and liver cancer.
nce aflatoxin has been shown to produce carceroma in fish, poultry and
her animals, there is an apprehension that man may be affected.
MATERIALS AND METHODS
A survey of A. flavus infection and the extent of aflatoxin contamina-
on in some local raw agricultural crops and their by-products was con-
icted. The 70 different samples used were of corn and its by-products,
!anuts, coconut products, sorghum, rice seeds, soybean and other assorted
Table 1 shows that aflatoxin in whole corn seeds was concentrated in
he germ and that corn "tiki-tiki" (dust and fine particles) had more toxin
han milled corn. The corn samples collected from the South contained
higher amounts of aflatoxin than those from Luzon. This may be due
o the different weather conditions in these areas during the drying process.
Fresh, mature corn in the field contained no toxin. However, over-
natured, insect-infested ones contained a high level of aflatoxin. A corn
:ake, after the oil was extracted from the germ, contained the toxin but
he filter residues from the crude oil had more. Corn grits intended
primarilyy for human consumption also contained the toxic compound.
Poor quality corn and corn products, and even corn refuse intended for
ise as ingredients in animal feed contained aflatoxin.
Table 2 shows that fresh peanut did not contain high levels of afla-
oxin even if A. flavus was isolated from them. Some of the dried peanuts
rom Iloilo, Bacolod and General Santos contained little amounts of afla-
oxin; the others did not. The low toxin level obtained from the samples
vas perhaps due to proper handling, harvesting and drying of the crop.
The results of the study on copra and sorghum are shown in Table 3.
,opra appeared to be a suitable substrate for growth and development of
1. flavus. Without any visible signs of fungal growth, it showed no afla-
oxin. Apparently, the fungus does not attack new and fresh coconut
when the moisture content is still high. Copra which was used for oil
extraction contained traces of aflatoxin. The expeller cake extracted with
lexane and made into copra pellets did not contain aflatoxin although
iO% of the sample were infected with A. flavus. A large amount of afla-
oxin was detected in the filter residue from the copra oil extraction
Properly handled and dried sorghum grains were totally free of mold
and aflatoxin contamination. Sorghum seemed to be a poor substrate for
aflatoxin production. However, when the fungus was allowed to grow on
he seeds, aflatoxin was produced rapidly.
Table 4 shows that palay was another poor medium for aflatoxin
reductionn. Evidence was obtained that very moldy samples contained no
Slightly mechanically injured soybean had low toxin contents. Millet
and "gabi" roots were free of aflatoxin. Coffee, variety Robusta, contained
a high amount of aflatoxin. The coffee samples were taken from the ship-
nent from Davao.
D-I wnole corn on coos, aryig yard, rural area
B-2 Whole corn, shelled, drying yard, related to B-1
B-5 Corn grits for human consumption, mill in rural areas
C-1 Whole corn, shelled, used in milling procedure
C-2 Corn starch derived from C-1
C-3 Corn gluten meal derived from C-1
C-4 Corn gluten feed derived from C-1
C-5 Corn gluten press cake
C-11 Whole corn on cob standing in field, improved varieties,
overmature, some ears on ground
C-12 Whole corn on cob, drying on cement, private drying yard,
rural area, mixed varieties
C-13 Whole corn on cob showing evidence of advanced decay by
fungi other than A. flavus. Related collection to C-12
C-14 Whole corn, shelled, representative of 'animal feed' grade
available on Cebu market
C-15 Whole corn, shelled, 'animal feed' grade A available on
C-16 Trash removed from corn prior to milling process, consisting
of seed coats, cob fragments, light kernels and dust. Use in
C-20 Wet milling corn germ
D-1 Whole corn, shelled, dry, ready for milling into human food.
Related to D-2, D-3, D-4
D-2 Corn grits taken from mill at bagging spout
D-3 Corn germ, used for oil milling or feed
D-4 Corn "tiki-tiki" (dust and fine debris) animal feed
D-5 Whole corn, shelled, dried very slowly in tightly packed bags,
locally called 'Atabrine' and consigned to animal feed
D-6 Whole corn, shelled, wet sample dried rapidly by collectors 20 35 trace
D-8 Whole corn, shelled, from sacks destined for grits 21 33
D-9 Corn debris, germ & "tiki-tiki" 30 90
D-11 Whole corn on cob, wet, stored in sacks in barrio "bodega"
awaiting shelling 20 30 314
D-13 Whole corn on cob, wet, stored in sacks in barrio "bodega"
(different from D-11) 20 35 948
GS-1 Corn grits, freshly milled 20 25 86
GS-2 Corn "tiki-tiki" related to GS-1 20 80 126
GS-3 Corn germ ready for oil extraction 40 0 67
GS-4 Corn germ oil cake resulting from expeller mill 20 80 83
GS-5 Corn oil filter residue from oil refining 600
GS-6 Whole corn, shelled, drying on slab of barrio bodega, Tupi
Poblacion 20 0 trace
GS-7 Corn standing in field, native variety in rural area, ready
to harvest 20 5
I-4 Whole corn, shelled, dry, UPCA Var 2, pickouts from corn
seed processing 20 75 314
GS-9 Whole corn, shelled, dry, in storage, variety BPI No. 2,
Tupi 20 30 -
I-5 Cracked corn, yellow, dry, for animal feed obtained at mill 15 100 393
1-6 Corn germ and bran from milling procedure 18 100 367
I-7 Corn grits, human food, made from local corn, obtained at
mill 15 60 43
I-8 Whole corn on cob, dry, obtained from farmer's own stock,
presumably for feed but probably also for food 20 40 378
I-9 Whole corn, shelled, dry, from farmer (see I-8) who will
sell for human food. Farmer admitted drying problem with
this corn 41 12 346
IS-1 Whole corn on cobs, wet, stored in shucks, UPCA-1, rotten
cob symptoms, animal feed 20 10 10
IS-2 Whole corn on cobs, wet, stored in shucks, UPCA-1, firm
cobs, animal feed 20 5 7
a, bSee footnotes in Table 4. o0
4C-3 Production line pickouts removed from stocks of CMC-1
.3 Freshly dug peanuts, native selection representative of Isabela
province production 20
4 Farmer stock peanuts of commerce, in bulk storage, dry,
1970 crop 20
.5 Pickouts from farmer stock (IS-4) sorted at sheller, shelled 20
-6 Whole peanuts in shell, nuts too small to be shelled, derived
from IS-4 20
0 Peanuts in shell, sample obtained for us by "bodega" owner,
1970 crop from Iloilo area 20
Peanuts in shell, purchased in Cebu market, wet, presumed
origin in Cebu 35
I Peanuts in shell, dry, storage, 1970 crop, Variety CES-101 20
"-8 Peanuts in shell, dry, from storage 1970 crops, Variety
IC-1 Large segregated peanuts separated from smaller discards,
used for peanut products 20
IC-2 Small segregated peanuts sold on market for food use, residue
from sorting (see CMC-1)
a. b See footnotes in Table 4.
TABLE 3. Aspergillus flavus infection and aflatoxin content of sorghum and copra and its by-products
No. of % of units Aflatoxin
Codea Sample Description units yielding pph of
platedb A. flavus B,
C-17 Copra 'pellet' after solvent extraction, animal feed ingredient 26 50 0
C-18 Filter residue from copra oil extraction process and oil
clarification -- 357
C-19 Crude ground copra, oil meal used as basic raw material
for oil extraction traces ,
C-21 Expeller cake from expeller mill, copra oil process 17 64 traces
D-10 Crude copra infested with fungi, in pile 20 100 314
D-14 Crude copra without visible A. flavus 20 45 0 4
B-6 Sorghum, mixed varieties in large molding pile, very moldy
sample obtained 44 25 208
C-10 Sorghum, dry in storage, Variety Darso, 1970 crop 30 0 0
GS-10 Sorghum, dry in storage, Variety Darso 40 3 0
a, b See footnotes in Table 4.
- --- A
Codea Sample Description
D-7 Moldy unmilled rice from milling company bodega
I-1 Unmilled rice of poor germinability, 9 months old, Variety
I-2 Rice, exposed in field experiment, improperly dried, Variety
I-3 Rice, 2 years old, stored in bodega, Variety IR-8-68
C-7 Cebu market, 'Dawa' millet
C-8 Cebu market, 'Kabog' millet
B-3 Soybean, drying on mat, Variety CES-101
D-12 Gabi, moldy root crop obtained in public market, Toril, Davao
NP-1 Coffee beans on sacks, Davao
a Code letters refer to geographic areas where samples were collected as follows
eral Santos: I, Iloilo; IS, Cauayan, Isabela; and NP, CMC, Manila market. All crop!
b Refers to number of seeds or grains plated. For pulverized materials, clumps
.~~,...., :~~an~:rrr nrrl n~ln~nv:~ ~n~~a~~ ~F -n~
Regulate the temperature to control the production ot atlatoxin
A. flavus. The toxic compound is not produced at 2, 7, 40 and 52
(Schindler, Palmer and Eisenberg, 1967).
Studies on relative humidity show that below 65% RH no molds w
grow while from 75% RH and above molds grow rapidly (Schroeder a
Hein, 1967). Corn, rice or sorghum with 14% moisture content will ha
75% RH in the air immediately adjacent to the kernel. Under this c<
edition, a low moisture level or a cold warm temperature may influer
and control the activity of mold growth. Corn, soybean and other crc
that mature in the air are less susceptible to infection compared to pear
which grows under the soil and in very moist conditions.
SCHROEDER, H. W. and H. HEIN, JR. Aflatoxin: production of the toxins in vitro
relation to temperature. J. Appl. Microbiol. 15: 441-445.
&fgsntu aillUu Upmu s UHlrru.w, .~U
igna sinensis var. sesquipedalis, I
andeliana, Acanthaceae. All exce
rus. Percentage infection was 2
sease was 7-41 days.
rkIJIk'k'INh rI-X' InrA1HU~kIVLL(
L., then reared on hyacinth bean, Dolichos lablab L. inside an ii
were transferred for es
ferous insects were allo
of 6 plants for each
IVARES, ET AL.: IOBACCO LEA)
* oleracea var. capitata
. oleracea var. botrytis
ze and cupped downward due to twisted veins (Fig. 6).
Fig. 1. Small cages made of celluloid, nylon tulle and nylon stocking used in
Fig. 2. Infected Brassica chinensis L. (left) showing pronounced downward curling
-C &Lh 1-af l-r4ne -nmnnrarl tn hllAhhr niant lritlf
N &S. rT AL.: TOBACCO LTEAF Cu
Fig. 3. Infected Brassica iuncea Goss (left) showing intense downward cupping
of leaves compared to healthy plant (right).
Fig. 4. Infected Raphanus sativus L. (right) showing mild downward cupping ol
n...... anmnoneal tn hanlthv nlnt /lIfts
Nos. 1 & 2'
Fig. 5. Infected Lycoperslcum
esculentum Mill. (left) showing
marked stunting, cupping and
puckering of leaves as compared
to healthy plant (right).
Fig. 6. Infected Solanum melongena L. (left) showing pronounced stunting an(
NS. ET AL.: TOBACCrn T.'A CfIT
Fig. 7. Infected Capsicum annum L. (left) showing young leaves greatly curled,
distorted and reduced in size as compared to healthy plant (right).
Fig. 8. Infected Cucurbita
maxima Duch. (left) showing
stunting, curling and cupping of
youngest leaf compared to healthy
Nos. 1 & 21
28 PHILIPPINE PHYTOPATHOLOGY [VOL. 8
Fig. 9. Infected Vigna sinesis
var. sesquipedalis Frunv. (left)
showing marked stunting, down-
ward curling and cupping of -O
leaves as compared to healthy
ceased to grow and only tiny leaves appeared at the apex. The older
leaves were curled and small-sized (Fig. 7).
C. maxima Plants were generally stunted. Leaves were slightly curled
and cupped with scattered yellowish areas. Outgrowths or nations deve-
loped on affected leaves as the plants grew older (Fig. 8).
V. sinensis var. sesquipedalis Plants were markedly stunted. Leaves
were greatly reduced in size, curled downward and cupped (Fig. 9).
A. coromandeliana Plants were generally stunted. Youngest leaves were
much reduced in size and cupped downward (Fig. 10).
The percentage infection in all affected test plants ranged from 22 to
72%. The incubation period of the disease ranged from 7 to 41 days.
Symptoms of virus infection were not observed on Brassica oleracea var.
capitata, B. oleracea var. botrytis (Cruciferae), S. tuberosum, (Solanaceae),
M. charantia, (Cucurbitaceae) and A. esculentus, (Malvaceae).
Pechay, mustard, radish, eggplant, sweet pepper, squash, "sitao" and a
weed, A. coromandeliana, are reported here as new suscepts of the tobacco
leaf curl virus. Tomato had earlier been reported susceptible to this par-
MCCLEAN, A. P. D. 1940. Some leaf curl diseases in South Africa. I. Leaf curl
disease of tobacco. I. A new "Petunia" strain of leaf curl and a note on the
occurrence of a leaf curl disease of Hollyhock. Sci. Bull. Dept. Agr. S. Afr. 225:
72. In Rev. Appl. Mycol. 21: 167-168.
NUQUE, F. L. 1959. Notes on the occurrence of leaf disease of tobacco Golder
Simox variety. Araneta J. Agr. 6: 206-209.
OLIVARES, F. M., JR. and M. O. SAN JUAN. 1966. The tobacco leaf curl virus: Its
transmission, virus-vector relationship and host range. The Eleventh Pacific Science
Congress, Tokyo, Japan 1966: 283-297.
PaRUTH, H. S. and C. K. SAMUEL. 1942. Entomological investigations on the leal
curl disease of tobacco in Northern India. V. Biology and population of the
whitefly vector, Bemisia tabaci (Genn.) in relation to the incidence of the disease
Indian Jour. Agric. Sci. 12: 35-57. In Rev. Appl. Mycol. 24: 122.
SAN JUAN, M. O. 1959. Occurrence of the leaf curl disease of tobacco in the Phil
ippines. Philippine Agr. 41: 527-529.
ETHYLENE SYNTHESIS BY PENICILLIUM EXPANSUM LINK
IN RELATION TO DISEASE PRODUCTION
LINA L. ILAG
Assistant Professor, Department of Plant Pathology, UPCA, College, Laguna
Portion of a thesis submitted in partial fulfillment of the requirements for graduation
with the degree of Doctor of Philosophy from Purdue University, Lafayette, Indiana
The relationship between the in vitro production of ethylene by 5 isolates
of Penicillium expansum Link and their virulence was investigated. The isolates
caused varied degrees of rotting in apples; the extent of rotting was not related
to the amount of ethylene each isolate produced in vitro.
The addition of exogenous ethylene had no effect on the size and rate
of increase of lesions.
Ethylene production by apple plugs inoculated with the isolates were not
enhanced over that of healthy tissues.
It has been suggested that ethylene increases both resistance (Stahmann
Clare and Woodbury, 1966) and susceptibility (Pratt and Goeschl, 1969,
to plant pathogens. Ethylene induces senescence (Burg, 1968), and thus ii
was surmised by Pratt and Goeschl (1969) that ethylene may make a
plant more prone to pathogen invasion. On the other hand, ethylene was founc
to stimulate the production of isocoumarin by carrot invaded by Cerato
cystis fimbriata (Chalutz, DeVay and Maxie, 1969) as well as the synthesis!
of the enzymes, polyphenoloxidase and peroxidase, that inhibit the path
gen's invasion (Stahman, et al., 1966) so that the gas may also play a role
in increasing resistance.
Ethylene has been associated with various plant disorders (Burg, 1962;
Dimond and Waggoner, 1953). It has not been conclusively determined
which of the host-pathogen complex produces the ethylene in diseased tissues
which evolve the gas. If both are known to produce ethylene, the amount
contributed by each in vivo has not been ascertained.
This study was conducted to determine if the amount of ethylene
produced by each of 5 isolates of Penicillium expansum is related to the
relative ability of the fungus to cause rotting in apple.
MATERIALS AND METHODS
In vitro production of ethylene by Penicillium expansum.- Five isolates
of P. expansum pathogenic on the apple were grown for 7 days in corn.
ILAG ETHYLENE SYNTHESIS
steep cerelose medium (corn steep liquor, 40 g; cerelose or technical glucose,
40 g; CaCO3, 3.5 g; NaNIO, 3.0 g; K2HPO4, 0.5 g; MgSO4, 0.25 g; deionized
water, 1 liter). The cultures were filtered and half-gram (wet weight)
were surtace-sterulzea wlmn
in sterile deionized water a
by pricking each fruit 10 t
old PDA cultures were insel
.UIt Wa CvaILUar.LL uL W a LA
c ethylene (1, 10, and 100
was added. Air was then
, ;-r nr-toclrP 'Pnr rnntrnl.
Nos. 1 & 2]
1.11 nl/hr). No marked difi
the amount of ethylene tha
- -- ..-- --. - .-..J
e with chemicals that probate
contained diseased as well
ene than the completely rott
Ale frp occantial fFr othlo.
iene it produces an vitro. 1nar rise abiHLY 01 CRCH
Ethylene Concentration (ppm)
1 10 100 0
Nos. 1 & 2]
ILAG ETHYLENE SYNTHESIS
2 that the total ethylene produced by both pathogen and host in the diseased
tissues is not related to the amount produced by the fungal isolates in vitro.
Effect of added ethylene on disease expression.--Since the amount of
ethylene produced by the fungus in vitro may be considerably different
from that produced by the host-parasite complex the effect of exogenously
added ethylene on disease expression was examined. It was observed that
ethylene had little or no effect on the ultimate size of the lesions produced
by P. expansum (Table 3).
TABLE 2. Ethylene production by apple tissues infected by 5 isolates of P. expansuma
Ethylene produced by tissues
State of apple tissues with intected by isolates:
respect to infection
1 2 3 4 5
Completely rotten .06 .04 .09 .13 .36
Rotten plus apparently healthy 2.95 3.22 1.77 3.55 2.32
Apparently healthy 4.77 5.12 4
t.37 5.30 ,
Ice of P.
nsum is not relate
to the amount of ethy
isolate to produce the
as a pathogen was not
ethylene did not affect
the isolates on apple.
not function in this
tibility. The gas appi
pathogenicity of P. ex
also found no correlati
lene by C. fimbriata
onnr mprl;i andr when
ported by t]
Le size and
r these reas,
'stem in ii
to be of
sum on apF
:n several i:
s ethylene o0
s in i
1 10.2 9.8
o 11 4 11 1i
3 8.0 8.6 7.8 8.5
4 11.3 11.7 11.6 12.1
5 9.1 8.9 10.2 9.2
aAverage of 10 lesions. Values are diameter of lesions (mm) 4 days after inocu-
34 PHILIPPINE PHYTOPATHOLOGY [VOL. 8
BURG, S. P. 1962. The physiology of ethylene formation. Ann. Rev. Plant Physiol.
S1968. Ethylene, plant senescence and abscission. Plant Physiol. 43:
CHALUTZ, E. and J. E. DEVAY. 1969. Production of ethylene in vitro and in vivo by
Ceratocystis fimbriata in relation to disease development. Phytopathology 59:
CHALUTZ, E., J. E. DEVAY and E. MAXIE. 1969. Ethylene-induced isocoumarin for-
mation in carrot root tissue. Plant Physiol. 44: 231-241.
DIMOND, A. E. and P. E. WAGGONER. 1953. The cause of epinastic symptoms in
Fusarium wilt of tomatoes. Phytopathology 43: 663-669.
IMASEKI, H., I. URITANI and M. A. STAHMANN. 1968. Production of ethylene by in-
jured sweet potato root tissue. Plant and Cell Physiol. 9: 757-768.
PRATr, H. K. and J. D. GOESCHL. 1969. Physiological roles of ethylene in plants.
Ann. Rev. Plant Physiol. 20: 541-584.
STAHMANN, M. A., B. G. CLARE and W. WOODBURY. 1966. Increased disease resistance
and enzyme activity induced by ethylene production by black rot infected sweet
potato tissues. Plant Physiol. 41: 1505-1512.
PATHOGENICITY TESTS OF SPECIES OF .PRATYLENCHUS,
TYLENCHORHYNCHUS AND HELICOTYLENCHUS
J. J. WALAWALA AND C. P. MADAMBA
Formerly Upland Crops Fellow and Associate Professor, Department of Entomology:
Paper presented at the Second National Pest Control Conference, Zamboanga City,
May 3-5, 1971.
Greenhouse pathogenicity tests were conducted with three nematode species.
Pratylenchus coffee and Tylenchorhynchus martini reproduced in and caused
appreciable damage to corn varieties UPCA var 1, 2 and Philippine Hybrid
801. The third species, Helicotylenchus erythrinae, did not reproduce in nor
cause any apparent damage to the plants.
Corn plants inoculated with P. coffee exhibited extensive root lesions
and necrosis. UPCA var 1 was apparently most suitable, supporting a 237%
increase in nematode population 60 days after inoculation. Philippine Hybrid
801 was the least suitable host among the varieties tested.
No root lesions and necrosis were observed among varieties inoculated
with T. martini. All inoculated plants, however, had sparsely developed and
shrivelled roots. UPCA var 2 was most suitable, supporting a population
increase of 97% 60 days after inoculation with the nematodes.
Corn, Zea mays L., is second to rice as a staple crop in the Philippines.
It is used as food by more than 8 million Filipinos, and as feed for poultry
and livestock. In 1952, the total area planted to corn was 969,000 ha with
an average yield of 720 kg/ha. In 1966, the area was increased to 2,167,000
ha with an average yield of 660 kg/ha (FAO, 1967). In spite of the use
of improved varieties, the unit yield in 1966 compared to 1952 was ap-
proximately 10% lower.
The loss incurred may have been due to pests and diseases. Least
known are the plant parasitic nematodes which cause considerable damage
in most agricultural crops. Whitehead (1956) noted that the pathologic
activities of these pests reduce crop yields by as much as 50%. In terms
of money, such activities cost the U.S. farmers half a billion dollars an-
nually (McGlohon, 1965). Tropical countries apparently suffer more from
nematode infestations than temperate ones.
According to Nelson (1955) and Perry (1956), Pratylenchus spp. are
most destructive among plant parasitic nematodes attacking corn. Praty-
lenchus penetrans prefers corn over potato as host (Dikerson, Darling and
Griffin, 1964). High populations of these nematodes are known to stunt
corn in Texas and Wisconsin (Young, 1951, 1953; Anon., 1951). In Egypt,
Oteifa and Taha (1964) observed P. zeae to be the major cause of deterio-
ration of maize. According to Miller, Boothroyd and Mai (1962) popula-
tion of certain lesion nematode species increased 3- to 7-folds on corn
grown in New York.
Other nematode groups reported to be potentially destructive are Heli-
cotylenchus spp., Tylenchorhynchus spp., Xiphinema spp. and Heterodera
In the Philippines, no study has been conducted on the pathogenicity
of nematode pests on corn. Survey and identification studies indicate the
presence of potentially destructive species in several areas of the Philippines
(Timm, 1964; Walawala and Madamba, 1970, 1971).
The present study was conducted to determine the pathogenicity of our
nematode species on corn.
MATERIALS AND METHODS
Three nematode species, viz., Pratylenchus coffeae, Tylenchorhynchus
martini and Helicotylenchus erythrinae were isolated from soil samples col-
lected from the Central Experiment Station, University of the Philippines,
College of Agriculture (UPCA). The nematode contents were extracted
using a combination of sieving and Baermann funnel methods as reported
by Christie and Perry (1951). The nematode inocula were individually
picked and each species kept in separate containers until ready for use.
WALAWALA & MADAMBA NEMATODES ON CORN
plants inoculated with T. martini. The roots, however, were shriveled and
sparsely developed. The feeder roots were much reduced. No apparent
symptom was observed in plants inoculated with H. erythrinae.
Fig. la. Root systems of corn
varieties free of nematode infec-
tion. Form left, UPCA var. 1,
var. 2 and Philippine Hybrid
Fig. lb. Root systems of corn
varieties infected with Pralylen-
chus coffeae. From left, UPCA
var. 1, var. 2 and PH 801.
Nos. 1 & 2]
Fig. 2. Root systems of corn
varieties infected with Tylen-
chorhynchus martini. From left, ;
UPCA var. 1, 2 and PH 801. /
Fig. 8. Root systems of corn
varieties infected with Helicoly-
lenchus erythrinae. From left,
UPCA var. 1, var. 2 and PH
WALAWALA & MADAMBA NEMATODES ON CORN
Population counts. The data on the population of nematode in the
soils of experimental pots 60 days after the plants were inoculated are sum-
marized in Table 1. The results show that the lesion nematode, P. coffeae,
had increased its population on all corn varieties. Both UPCA varieties
appeared very suitable hosts for the nematodes. An increase of 237 and
153.4% from the initial population of 1,500 individuals was observed on
UPCA var 1 and UPCA var 2, respectively. On the other hand, only an
increase of 54.8% from the initial population was noted on Philippine
T. martini had also reproduced and increase its initial population on
both UPCA varieties. However, its population did not increase on Philip-
pine Hybrid 801 at 60 days after inoculation. The final population on
both UPCA varieties was 79 and 97% more than the initial populations.
No increase in the initial population of the spiral nematode, H. ery-
thrinae, was observed on all corn varieties 60 days after inoculation. This
suggests that the varieties were unsuitable for the
TABLE 1. Mean counts of
nematodes recovered from soil planted to
corn 60 days
UPCA var 1
UPCA var 2
UPCA var 1
UPCA var 2
UPCA var 1
UPCA var 2
Final Per Cent
On the basis of the results, P. coffeae and T. martini appeared patho-
genic on the corn varieties tested. It is
tode species are potential pests that may
highly probable that both nema-
cause appreciable damage to our
recommended corn varieties. It would be interesting to determine under
field conditions the influence of natural populations of these nematode
species on corn yields. Similarly, it may be worthwhile working on the
pathogenic effects of other Helicotylenchus species since, according to sur-
veys, more than three species are found associated with corn.
Nos. 1 & 2]
kNON. 1951. Corn plants stunted by nematodes. Crops and Soils 3: 29.
HIRISTIE, J. R. and V. G. PERRY. 1951. Removing nematodes from soil. Proc. Hel-
minthol. Soc. Wash. 18: 106-108.
DICKERSON, O. J., H. M. DARLING and G. D. GRIFFIN. 1964. Pathogenicity and popula-
tion trends of Pratylenchus penetrans on potato and corn. Phytopathology 54:
FAO. 1967. Production Yearbook. Vol. 21.
McGLOHON, N. 1965. Room and board for nematodes costs farmers a half a billion
dollars annually. Crops and Soils 10:12.
fILLER, R. E., C. W. BOOTHROYD and W. F. MAI. 1962. Plant parasitic nematodes
associated with corn roots in New York. Phytopathology 52: 22 (Abstract).
NELSON, R. R. 1955. Nematode parasites of corn in the coastal plain of North Caro-
lina. Plant Dis. Reptr. 39: 818-819.
)TEIFA, B. A. and A. TAHA. 1964. Significance of plant parasitic nematodes in maize
deterioration problem. I. Nematodes species involved in the syndrome of diseased
plants. Bahtm. Expt. Sta. Egypt. Agric. Org. Tech. 73, 16 p.
PERRY, V. G. 1956. Nematode affecting corn in Florida, Alabama, Maryland and
Wisconsin. Phytopathology 46: 23 (Abstract).
[IMM, R. W. 1964. A preliminary survey of plant parasitic nematodes of Thailand
and the Philippines. Sambhand Printing Press, Bangkok. 71 p.
VALAWALA, J. J. and C. P. MADAMBA. 1970. Plant parasitic nematodes in corn-grow-
ing areas of Mindanao. Proc. First Nat. Pest Control Conference 1970: 147-153.
and 1971. Plant parasitic nematodes in corn-growing
areas. II. Luzon and Visayas. Philippine Phytopathol. 7: 40-43.
VHITEHEAD, A. G. 1956. Plant parasitic nematodes important pathogens in tropical
agriculture. East African Agric. J. 22: 92-96.
'oUNG, P. A. 1951. Corn plants stunted by meadow nematodes. Crops and Soils 3:
'OUNG, P. A. 1953. Damages caused by meadow nematodes to corn in West Texas.
Plant Dis. Reptr. 37: 599-600.
SOME BIOCHEMICAL CHANGES DURING PYTHIUM INFECTION
ON COTTON SEEDLING RADICLES
TRICITA H. QUIMIO
Former Graduate Research Assistant, Botany Department, North Carolina Stal
University, Raleigh, North Carolina; now Assistant Professor, Department of Plai
Portion of a Ph.D. thesis. The author is indebted to Drs. G. R. Noggle ar
R. C. Fites, Botany Department, NCSU, and Drs. N. T. Powell and L. F. Granm
Department of Plant Pathology, NCSU, for their valuable suggestions and advice.
The effect of Pythium infection on chilled (nub) and unchilled (normal)
radicles was studied. Total protein and sugars decreased while amino acids
increased in infected radicles of both types. Increased proteolytic activity in
inoculated radicles indicated hydrolysis of proteins to amino acids during
Gel electrophoresis of some enzymes showed varied patterns and degree
of staining due to infection. Wider bands and more intense staining in in-
fected radicles suggested increased enzyme activity.
Relative susceptibility test suggests that the organism can penetrate nor-
mal radicles faster than nub ones. Once inside the host tissues, however,
infection rate was faster in the nubs. This suggests that chilling predis-
poses the tissues to more rapid infection and tissue disintegration.
Seedling disease is caused by a complex of seed-borne and soil-ir
habiting organisms. This may occur as seed rot, root damage, or seedlin
death before or after emergence. Death in the early stages of growth
is often referred to as "damping-off".
Seedling diseases of cotton are usually more severe in fields with mois
cool conditions. The seeds are prone to decay and seedling diseases a
low temperatures. Among the common organisms involved are Glomereli
gossypii, Rhizoctonia solani, Thielaviopsis basicola, and species of Fusariun
Pythium and nematodes. Pythium debaryanum Hesse and P. ultimui
Throw are commonly associated with the seedling disease of cotton i
the United States when specific identifications are indicated. Few definite
reports, however, are available concerning the pathogenesis of Pythiui
species on cotton plants, especially in moist cool conditions. It is then
fore the purpose of this study to contribute to our knowledge of th
biochemical changes during infection by Pythium debaryanum and ho'
these changes are affected by pre-chilling exposure.
12 PHILIPPINE PHYTOPATHOLOGY [VOL. 8
MATERIALS AND METHODS
The seeds of Gossypium hirsutum L. (var, Carolina Queen Coker)
.sed in this study were grown in Georgia in 1967 and coated with Ara-
san-Demosan mixture. The culture of P. debaryanum, reported to cause
lamping-off of cotton seedlings in North Carolina, was obtained from the
late Dr. W. E. Cooper, Department of Plant Pathology, North Carolina
State University. The fungus was grown at 25 C in 100 ml potato-dex-
trose broth in 250 ml flasks. The mycelial mats formed after 5 days were
washed in sterile distilled water and homogenized for 10 sec in 100 ml
water at medium speed of a Virtis 45" homogenizer.
3f protein as compared to the uninoculated control.
Total amino acids rose in both inoculated and uninoculated normal
radicles (Fig. 2); however, more amino acids were detected in the former.
Similar general increases in the concentration of amino have been reported
in plants infected by a virus (Diener, 1960), a bacterial pathogen (Patel
and Walker, 1963), a fungus (Shaw and Colotelo, 1961) and plant parasitic
nematode (Hanks and Feldman, 1966). Such general increases in organic
nitrogen thus appear to be a fairly common result of pathogenesis. That
:he increase in amino acid during the first 12 hr resulted from protein
breakdown is precluded because there was no significant difference in total
proteins between inoculated and uninoculated normal radicles during this
period (Fig. 1). From the 12th to the 24th hr, however, the increase in
imino acids was evidently due to protein breakdown because during this
period, total protein decreased in the inoculated radicles. Another evidence
lor protein breakdown was the greater proteolytic activity of extracts from
:issues (Table I).
TABLE 1. Proteolytic activity of normal and nub radicles of cotton 24 hr after
inoculation with Pythium debaryanum
OD (280)/radicle 90 min
Results on the total protein and amino acids of inoculated and un-
inoculated nub radicle parallel those of normal radicles.
Effect on sugar content. Figure 3 shows the correlative trends of su-
gar content in inoculated and uninoculated radicles (nub and normal).
Within 24 hr after inoculation, there was a remarkable decrease in sugar
content in inoculated chilled and normal radicles in contrast to the in-
crease in the uninoculated radicles of both types. Apparently the sugars
were rapidly utilized by the invading fungus in both normal and nub
Relative susceptibility to Pythium infection. -To determine what ef-
fect chilling has on the penetrability and susceptibility of cotton radicles,
a block of water agar containing a single hyphal tip of P. debaryanum
was placed on the top of each nub or normal radicle at the radicle-hypo-
n00 td Inoculated un2 d ed
S nanocu lated unchlld
0oo 800 -/ -
0-w- "''o 00 / --"InoctieId chl. .d
i .600 0-0-0 .Zt - - -
S ninoculateO chilled
-i!2 i1 6 12 18 24 "
Hor After Inculaton Hours After Inoculation
Hour After Inoculatlon
Fig. 1. Changes in total protein of Fig. 2. Changes in total amino ac
chilled (nub) and unchilled (normal) by chilled and unchilled cotton radic
cotton radicles following infection by following infection by Pythium debari
Pythium debaryanum. num.
S80 TJninoculated nub
Hour- Afer Inoculation
Fig. 3. Changes in total sugars of chilled and unchilled cotton radicle follow
infection by Pythium.
OUIMIO Phuthium oN COrTTm RAmnTCr.V
Observations were made at 4, 6, 8, 16 and 24 hr after inoculation.
'he degree of yellowing was recorded (Table 2). Results suggest that the
rganism can penetrate normal radicles faster than nub ones. But once the
rganism is within, infection appears to progress at a relatively faster rate
i the nub than in the normal radicle. Presumably, the altered metabol-
;m of nub radicles predisposes the tissues to more rapid infection and
TABLE 2. Relative susceptibility of normal and nub radicles to Pythium infection
No. yellowing radicles/100 Extent of yellowing
Hr after (mm)
Nub Normal Nub Normal
4 8 14 -1-3
6 16 28 5-10
8 38 52 1-3 5-10
16 77 75 5-10 10-15
20 86 82 10-15 10-15
24 100 94 15-20 10-15
Alterations on proteins and isoenzyme pattern.-Figure 4 shows the
amparative alterations in total proteins and isoenzymes of normal and nub
adicles. Infection in some cases resulted in increased enzyme activity. The
activity of cytochrome oxidases of both nub and normal radicles is apparent-
Senhanced following infection. This may explain partly the general in-
rease in respiration rate during pathogenesis (Daly, Bell and Krupka, 1961;
,ristev, 1961; Millerd and Scott, 1956). Peroxidase activity also seems to
,e activated in the inoculated radicles. Similar findings were observed by
'u and Hampton (1964) on the peroxidases of tobacco tissues infected with
Jos. 1 & 2]
IVAUt L UK LAAI Al '-' ^- --- --- - -
fection were from studies on rust (Staples and Stahmann, 1964; Wrigley
and Webster, 1966). Quantitative changes in proteins and isoenzyme pat-
Esterase Lactic Dehydrogenase
Rf a b c d e Rf a b c d e
Total Proteins Malic Dehydrogenase
Rf a b c d e Rf a b c d
Peroxidases Cytochrome Oxidases
Rf a b c d e RI a b c d e
Glutamlc Dehydrogenase Alcohol Dehydrogenase
I a b c d a Rf a b c d e
-- r -A
studies on enzyme activity were done by colorimetric methods. In most
of these studies, a change in enzyme activity is a common feature of the
parasitically altered metabolism. Based on modern genetic principles, the
idea of enzyme induction or repression, or both is offered to explain at
least part of the parasitically induced alteration in the host proteins (Far-
kas et al., 1964). While decreases in enzyme activity might be due either
to repression of enzyme synthesis or accumulation of some enzyme inhibitors
(Shaw, 1963), increased enzyme activities as shown ii 'some infected tissues,
may be due to induced enzyme synthesis. Farkas et al (1964) stressed the
importance of proteolytic and pectolytic activation of latent enzymes as a
probable mechanism contributing to increased enzyme activities in infected
tissues. There is also a possibility that enzymes secreted by the parasite
contribute to the protein or enzyme changes in the host-parasite complex.
DALY. J. M., A. A. BELL and L. R. KRUPKA. 1961. Respiratory changes during dev-
elopment of rust diseases. Phytopathology 51: 461-471.
DIENER, T. O. 1960. Free amino acid and amides in healthy and virus-infected cherry
and peach leaves. Phytopathology 50: 141-146.
DUBOIS, M., K. A. GILES, J. K. HAMILTON, P. A. REBELS and F. SMITH. 1956. Co-
lorimetric method for determination of sugars and related substances. Anal. Chem.
FARIAS, G. L., L. DEZSI, M. HORVATH, K. KISBAN and J. UDVARDY. 1964. Common
pattern of enzymatic changes in detached leaves and tissues attacked by parasites.
Phytopath. Z. 49: 343-354.
HANKS, R. W. and A. W. FELDMAN. 1966. Quantitative changes in free and protein
amino acids in leaves of healthy, Radopholus similis-infected and "recovered" grape-
fruit seedlings. Phytopathology 56: 261-264.
INGRAM, V. M. 1957. Gene mutations in human haemoglobin: the chemical difference
between normal and sickle cell haemoglobin. Nature 180: 326-328.
JOHNSON, L. B., B. L. BRANNAMAN and F. P. ZCHEILLE, JR. 1968. Protein and en-
zyme changes in wheat leaves following infection with Puccinia recondita. Phyto-
pathology 58: 578-583.
KRISTEV, K. 1961. On the role of pyrophosphate metabolism in the respiratory in-
crease of rust-infected wheat leaves. Phytopath. Z. 42: 279-295.
LowRY, O. H., N. ROSEBROUGH, A. L. FARR and R. J. RANDALL. 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.
MILLERD, A. and K. J. SCOTT. 1956. Host-pathogen relations in powdery mildew of
barley. II. Changes in respiratory pattern. Aust. J. Biol. Sci. 9: 37-44.
PATEL, P. N. and J. C. WALKER. 1963. Free amino acid and amide content of to-
bacco and oats infected by wildfire and halo blight bacteria. Phytopathology 53:
QuIMIo, T. H. 1971. Germination and chilling response of cotton seeds with special
emphasis on proteins and isoenzymes. Ph.D. dissertation, North Carolina State
University. 121 pp.
SHAw, M. 1963. The physiology and host-parasite relations of the rusts. Ann. Rev.
Phytopath. 1: 256-294.
The effect of stem rust on the nitrogen and amino acid of wheat leaves. Can.
Bot. 39: 1351-1372.
STAPLES, R. C. and M. A. STAHMANN. 1964. Changes in proteins and several e
zymes in susceptible bean leaves after infection by bean rust fungus. Phyt
pathology 54: 760-764.
WmGLEY, C. W. and H. L. WEBSTER. 1966. The effect of stem rust infection
the soluble proteins of wheat leaves. Aust. J. Biol. Sci .19: 895-911.
YEMM, E. W. and E. C. CocKING. 1955. The determination of amino acids wi
ninhydrin. Analyst 80: 209-213.
YoUNc, J. L. and J. E. VARNEn. 1959. Enzyme synthesis in the cotyledons of germinm
ing seeds. Arch. Biochem. Biophys. 84: 71-78.
Yu, L. M. and R. E. HAMPTON. 1964. Biochemical changes in tobacco infected wi
Colletotrichum destructivum. II. Peroxidases. Phytochemistry 3: 499-501.
~~y ~~ ~1~ IV1 ~IIIVYYI
L T -.
SYNERGISTIC REACTIONS BETWEEN THE PATHOGENS
OF CITRUS GREENING AND SEEDLING-YELLOWS
A. L. MARTINEZ
Senior Plant Pathologist, Bureau of Plant Industry, Lipa City. The author
wishes to express his gratitude to Dr. J. M. Wallace, University of California, Riverside,
for helpful criticisms of the manuscript.
Evidence has been obtained indicating synergistic reactions between the
pathogens of citrus greening and seedling-yellows. Irrespective of sequence
of inoculation to budlings of Ladu, Ponkan and Szinkom mandarin on Cala-
mandarin and Florida rough lemon rootstocks, combined infection of the
pathogens produced more severe leaf symptoms and more stunting; a greater
number of plants collapsed earlier than those infected with the greening
pathogen alone. The results demonstrate that the presence of the seedling-
yellows virus in greening-infected orchared trees in the Philippines might have
contributed to the decline and death of numerous citrus trees in the last 14
Simultaneous infections of two or more unrelated viruses are known
to induce a more severe effect or injury on the host plant than infections
of a single virus (Rochow and Ross, 1955; Ross, 1957, 1959). Weathers
(1959, 1960, 1961) demonstrated that citrus plants dually infected with
yellow-vein and vein-enation viruses exhibited more severe yellow-vein
symptoms than plants infected only with yellow-vein virus. Similarly, plants
doubly infected with the viruses of psorosis and yellow-vein developed more
severe leaf symptoms of psorosis and more stunting than those infected
with psorosis virus alone (Weathers, 1961).
This paper describes the study on the synergistic reactions between
the pathogens of citrus greening and seedling-yellows. The preliminary re-
sults have already been published elsewhere (Martinez and Wallace, 1969).
MATERIALS AND METHODS
Young budlings of Ladu, Ponkan and Szinkom mandarin on Calaman-
darin and Florida rough lemon rootstocks were inoculated separately and
simultaneously by tissue grafting with the seedling-yellows and greening
pathogens. Non-inoculated budlings of each stionic combination were main-
tained as control plants. In another test, budlings of the same stionic
combinations used in the preceding experiment were treated similarly ex-
cept that in the doubly-infected groups, seedling-yellows virus was intro-
duced 3 to 4 months before the greening pathogens, or vice versa.
The budlings inoculated with the seedling-yellows virus alone developed
no symptoms other than slight stunting and reduction in size of leaves
Those with the greening pathogen alone exhibited severe stunting, yellow
ing and mottling. Dual infection of both pathogens resulted in symptom
similar to but more severe than those of plants with the greening pathogen
alone. In addition, a greater number of doubly-infected plants collapse
The symptom expressions did not vary regardless of whether the citru
greening and seedling-yellows organisms were inoculated simultaneously o:
separately one alter the other alter 3-4 months. The control plants re
mained healthy in all cases.
The increase in severity of leaf symptoms, growth retardation anc
earlier collapse of dually infected plants is apparently due to an increase
activity of the greening pathogen in the presence of seedling-yellows virus
or to the combined ettect ot both pathogens. Various workers (Rochov
and toss, 1905; 1957, 1959; Weathers, 18bi) have reported interactions bet.
ween viruses in dual infections wherein there is an increase in the ac.
tivity of one virus in the presence of another. The synergistic reaction:
MARTINEZ, A. L., D. M. NORA and N. M. SEBASTIAN. 1965. The prevalence of
seedling-yellows virus disease of citrus in the Philippines as detected by indexing
procedures. Philippine Phytopathol. 1: 35-36.
MARTINEZ, A. L. and J. M. WALLACE. 1967. Citrus leaf-mottle-yellows disease in the
Philippines and transmission of the causal virus by a psyllid, Diaphorina citri.
Plant Dis. Reptr. 51: 692-695.
MARTINEZ, A. L. and J. M. WALLACE. 1968. Studies on leaf-mottle-yellows disease
of citrus in the Philippines. In J. F. L. Childs (ed.), Proc. 4th Conf. Intern.
Organization Citrus Virol. Univ. Florida Press, Gainesville. p. 167-176.
MARTINEZ, A. L. and J. M. WALLACE. 1969. Citrus greening disease in the Philip-
pines. In H. D. Chapman (ed.), Proc. 1st International Citrus Symposium, vol. 3,
Univ. Calif. Press, Riverside. p. 1427-1431.
RocHow, W. F. and A. F. Ross. 1955. Virus multiplication in plants doubly-infected
by potato viruses X and Y. Virology 1: 10-27.
Ross, A. F. 1957. Responses of plants to concurrent infection by two or more viruses.
Trans. N.Y. Acad. Sci. 19: 236-243.
Ross, A. F. 1959. The interaction of viruses in the host. In C. S. Holton, C. W.
Fischer, R. W. Fulton, H. Hart & S.E.A. McCallan (ed.) Plant Pathology-
Problems and Progress 1908-1958. Univ. Wisconsin Press, Madison. p. 511-520.
WEATHERS, L. G. 1959. Interference and synergistic reactions with reference to yellow-
vein and other viruses of citrus. Phytopathology 49: 554.
WEATHERs, L. G. 1960. Yellow-vein disease of citrus and studies of interaction be-
tween yellow-vein and other viruses of citrus. Virology 11: 752-764.
WEATHERS, L. G. 1961. Responses of citrus to concurrent infection with two or more
unrelated viruses. In W. C. Price (ed.) Proc. 2nd Intern. Organization Citrus
Virol. Univ. Florida Press, Gainesville. p. 187-196.
WEATHERS, L. G. and F. C. GREER, JR. 1968. Identification of variants of vein.
enation (woody-gall) virus through synergism. In J. F. L. Childs (ed.), Proc. 4th
Conf. Intern. Organization Citrus Virol. Univ. Florida Press, Gainesville. p. 232.
EXTRACELLULAR ENZYMES PRODUCED
BY PYRICULARIA ORYZAE CAV.
R. SRIDHAR AND S. H. Ou
Respectively Research Fellow and Plant Pathologist, The International Rice Re-
search Institute, Los Bafios, Laguna, Philippines.
Different races of the blast pathogen (Pyricularia oryzae Cav.) varied in
their ability to produce extracellular invertase, amylase and cellulase in cul-
ture. The most virulent race produced more of these enzymes than the
less virulent races.
The ability of plant pathogens to penetrate a host is commonly as-
cribed to the dissolution of the cell walls by enzymes secreted by the fungus
hyphae or the spores (Rosenberg and Wilkes, 1952; Davson and Danielli,
1943). The ability to parasitize a tissue depends on nutrients available in
the plant tissue (Wood, 1960). The hydrolytic enzymes of a pathogen
may play an important role in making nutrients available to the pathogen.
Under laboratory conditions Pyricularia oryzae Cav. grows and sporulates
well on autoclaved rice leaf bits or on the culm nodes of the rice plant.
Moreover, disintegration of tissue is a common feature of blast diseased
areas on rice leaves. These characters suggest that the fungus secretes en-
zymes which help it derive its nutrients from the plant parts.
The blast pathogen uses sucrose and starch effectively (Tochinai and
Nakano, 1940) but carboxymethyl cellulose supports the growth of the
fungus poorly (Manibhushanrao, 1971a). These carbohydrates cannot be
used directly by P. oryzae; they must first be converted to simpler monosac-
charides (Tanaka, 1965). Results at IRRI have indicated that the use of
sucrose and starch by different isolates of P. oryzae varies widely (Chinte,
1965). This suggests that races of the pathogen differ greatly in production
of hydrolytic enzymes to cleave the carbohydrates into simpler monosac-
Races of P. oryzae, the causal organism of the rice blast disease, have
a wide range of virulence (Ou, 1972). This paper reports on the in vitro
production of invertase, amylase, and cellulase by four different races of
the blast pathogen which differ in virulence.
MATERIALS AND METHODS
Two virulent Philippine races (P150 and P38) and two less virulent
ones P149 and P26) of P. oryzae were selected for this study on the basis
R 1O UU ,ZYMES DY r. urT
of their pathogenicity on variety reta. Cultures were maintained on potato
dextrose agar medium. One hundred ml of Tanaka's medium B (Tanaka,
1965) containing 20 g sucrose (Allied Chemical), 10 g potato starch (E.
Merck) or 10 g carboxymethylcellulose (CMC) (Matheson Coleman and
Bell) per liter as carbon source were placed in 250-ml Erlenmeyer flasks,
sterilized at 15 psi for 20 min. The required amounts of thiamine and
biotin were then added to each flask under aseptic conditions, inoculated
with a 4-mm culture disc of one of the four races of P. oryzae, and in-
cubated at 28 C for 15 days. The cultures were maintained in triplicate
in all the experiments. After incubation the mycelium was filtered off
through a previously dried and weighed No. 41 filter paper. The mycelial
mat and the filter paper were dried for an hour in an oven at 100 C and
weighed again to find the dry weight of the mycelium. The culture fil-
trates were centrifuged at 3,000 g for 30 min in cold, and dialyzed against
distilled, deionized water for 16 to 20 hr at 8 C. After dialysis, the sam-
ples were used for enzyme analysis. Culture filtrates without dialysis were
used for the cellulase assay to minimize the reduction in activity (Bateman,
Enzyme assays.- For invertase, the reaction mixture, consisting of 5 ml
of 6.5% sucrose in 0.1 M acetate buffer at pH 4.5 and 1 ml of culture fil-
trate, was placed in a boiling tube and incubated in a water bath at 30 C.
At the end of 0, 15, 30, and 45 min of incubation, 5 ml of 0.1 N NaOH
was added to the mixture to stop the enzyme activity (Summer and Somers,
1953). The amount of reducing sugars released was estimated (Nelson,
1944) from 1-ml portions of the reaction mixture. The invertase activity
in the culture filtrate is expressed as milligrams of reducing sugars released/
ml of reaction mixture. The enzyme source, heated at 121 C for 30 min,
was used in the control.
The method of Street (1965) was used to determine the amylase activity
of the culture filtrates. Five ml of 0.02 M phosphate buffer at pH 7.0,
2 ml of 0.1% potato-starch solution in 0.01 N NaOH, and 2 ml of 0.01 N
HCI were placed in volumetric flasks and kept in a water bath at 37 C
for 3 min. One ml of 1:10 diluted enzyme source was added to the con-
tents of the flasks which were allowed to stand for exactly 15 min in the
water bath at 37 C. One milliliter of the diluted filtrate heated to 121
C for 30 min was added to the control flasks. At the end of the incuba-
tion period 4 ml of iodine-potassium iodide solution (0.01 N iodine) was
added. The contents of the flasks were mixed thoroughly. The optical
density of the solutions was measured at 620 tt against distilled water blanks.
The amylase concentration in the culture filtrate was calculated and ex-
pressed in terms of Street-Close units per 100 ml of the filtrate.
The cellulase activity was determined by measuring the reduction in
viscosity of CMC solution (Hancock, Millar and Lorbeer, 1964). To 4 ml
Nos. 1 & 2]
54 PHILIPPINE PHYTOPATHOLOGY [VOL. (
of 1-2% CMC buffered at pH 5 with citric acid-NaOH (0.05 M), 2 m
of enzyme preparation was added in size 300 Fenske-Ostwald viscometers
Viscosity was measured at 30 C (Bell, Etchells and Jones, 1955) after re
action periods of 0, 10, 15, 30 and 45 min. The enzyme activity is ex
pressed in percentage of loss in viscosity. Filtrates heated at 121 C for 3<
min were used in the control.
RESULTS AND DISCUSSION
Comparative studies of four different races of P. oryzae revealed thi
highly variable ability of these races to produce invertase, amylase am
cellulase. The most virulent race, P150, produced higher quantities of al
the three enzymes than the less virulent races (Tables 1-3). Though thi
less-virulent race P26 produced the least amounts of the three enzymes it
culture, it grew as well as the virulent races. Hence, growth and thi
amount of enzyme produced by the pathogen is not correlated. The dif
ference in the capacity of different races of the pathogen to produce enzyme
should be viewed however in terms of enzyme inhibition by phenolic cor
pounds during pathogenesis (Lyr, 1965).
Extracellular fungal enzymes have frequently been associated with patho
genesis (Bateman, 1968; Hancock, 1967; Hancock and Millar, 1965; Han
cock et al., 1964). Virulent isolates of Colletotrichum falcatum Went an
Cytospora sp. produced higher amounts of sucrase (Singh, 1965) and cellu
lase (Gairola and Powell, 1971), respectively, than the less virulent isolates
But different isolates of P. oryzae did not differ in amylase production
(Otsuka, Tamari and Ogasawara, 1965). In a comparison between viru
lent and avirulent isolates of P. oryzae, cellulase production was not cor
related with the virulence (Manibushanrao, 1971a). The avirulent UX
mutants used in Manibushanrao's studies (1971b) did not penetrate int(
the host tissue upon inoculation while the less virulent races used in oui
study penetrated the host tissue and caused typical resistant-type spots upor
inoculation. The ability of the virulent race to produce higher amount;
of enzymes may help the pathogen establish itself on the host more easily
TABLE 1. Invertase activity in culture filtrates of different races of P. oryzae
(Mg/ml reaction mixture)
Dry wt of
Race Assay time (min) mycelium
15 30 45
P26 126 150 244 347
P149 156 210 302 227
P38 256 330 354 243
P150 256 350 410 351
TABLE 2. Amylase activity in culture filtrates of different races of P. oryzae
Street-Close units/ Dry wt of
Race 100 ml culture filtratea mycelium (mg)
P26 3.79 20
P149 20.50 13
P38 33.95 16
P150 48.20 20
a Hydrolysis of 2 mg of amylose in 15 min at pH 7.0 and 37 C by 0.1 ml
nl of 1:10 diluted solution) of culture filtrate = 100 Street-Close units/100 ml.
"ABLE 3. Cellulase activity in culture filtrates of different races of P. oryzae
Loss in viscosity (%) Dry wt of
Dry wt of
Race Assay time (min) mycelium
5 10 15 30 45
P26 3.3 6.7 10.0 20.0 26.7 14
P149 2.9 8.8 13.2 22.1 27.9 14
P38 5.9 14.7 17.7 26.5 35.3 15
P150 12.9 19.4 22.6 32.2 41.9 13
MAN. ). F 1964. Cellulase and the Rhizoctonia disease of bean. Phytopathology
EMAN, D. F. 19R8. The enzymatic maceration of plant tissues. Neth. J. Plant
Pathol. 74: 67-80.
L, T. A., J. L. ETCHELLS and I. D. JONES. 1955. A method for testing cucumber
salt-stock brine for softening activity. U.S. Dep. Agr. A.R.S. No. 72-5. 15 p.
tTE, P. T. 1965. The nutritional requirements and physiological response of
Piricularia oryzae Cav. in vitro. M.S. thesis, Coll. Agr. Univ. Philippines, Philip-
pines. 76 p.
'soN. H. and J. F. DANIELLI. 1943. The permeability of natural membranes. The
MacMillan Co., New York. 361 p.
.OLA, C. and D. POWELL. 1971. Extracellular enzymes and pathogenesis by peach
Cytosporas. Phytopath. Z. 72: 305-314.
rcocK, J. G. 1967. Hemicellulose degradation in sunflower hypocotyles infected
with Sclerotinia sclerotiorum. Phytopathology 57: 203-206.
rcocK, J. G. and R. L. MILLAR. 1965. Association of cellulolytic, proteolytic,
and zylolytic enzymes with southern anthracnose, spring black stem, and Stemphy-
lium leaf spot of alfalfa. Phytopathology 55: 346-355.
rcocx, J. G., R. L. MILLAR and J. W. LORBEER. 1964. Pectolytic and cellulolytic
enzymes produced by Bnfrutts allii, B. cinerea, B. squamosa in vitro and in vivo.
Phytopathology 54: 932-935.
SH. 1965. On the toxicity of oxidized polyphenols. Phytopath. Z. 52: 229-240.
quUSHANRAO, K. 1971a. Studies on the cultures of Pyricularia oryzae Cav. III.
In vitro production of pectolytic and cellulolytic enzymes. Phytopath. Z. 72:
IBUSHANRAO, K. 1971b. Studies on the cultures of Pyricularia oryzae Cav. I.
Mnrnhnlnav sanA nathnloaniitv TT Rien 0 9.-9.L07
.TTT T1DDTW PUiVTrDIATTruT .AV
NELSON, N. 1944. A photometric adaptation of the Somogyi method for the d
mination of glucose. J. Biol. Chem. 153: 375-380.
OTSUKA, H., K. TAAuI and N. OGASAWARA. 1965. Variability of Piricularia ot
in culture, pp. 69-109. In Proceedings of a symposium on rice blast disease,
1963, Los Bafios, Philippines. Johns Hopkins Press, Baltimore.
Ou, S. H. 1972. Rice diseases. Comm. Mycol. Inst., Kew, Surrey, England, 36
ROSENBERG, T. H. and B. G. WILKES. 1952. Enzymatic process in cell memi
penetration. Int. Rev. Cytol. 1: 65-92.
SINGH, P. 1965. Studies on the enzyme sucrase as secreted by the conidia of
letotrichum falcatum Went. Phytopath. Z. 54: 79-86.
STREET, H. V. 1965. Amylase, In H. U. Bergmeyer (ed.) Methods of enzyr
analysis, p. 854-858. Acad. Press, New York.
SUMNER, J. B. and G. F. SOMER. 1953. Chemistry and Methods of Enzymes
ed.). Acad. Press, New York. 462 p.
TANAKA, S. 1965. Nutrition of Pyricularia oryzae in vitro, pp. 23-33. In Proceec
of a symposium on the rice blast diseases, July, 1963, Los Bafios, Philipi
John Hopkins Press, Baltimore.
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XE OF PHENOLIC COMPOUNDS AND POLYPHENOL OXIDASE
IN RICE BLAST DISEASE
R. SRIDHAR AND S. H. Ou
Research Fellow and Plant Pathologist, Department of Plant Pathology, The
national Rice Research Institute, Los Bafios, Laguna, Philippines.
Several phenolic compounds tested were toxic to both spore germination
and growth of Pyricularia oryzae Cav. in culture. Races of the pathogen
differed in their toxicity to p-quinone. Addition of either ascorbic acid or
glutathione decreased the phenol toxicity. Germinating conidia produced poly-
phenol oxidase. The results are discussed in terms of symptom production.
The infection of rice leaf tissues with the blast pathogen, Pyricularia
ze Cay., is often associated with brown discoloration of the infected
s, suggesting an accumulation of colored polyphenol oxidation products.
(1960) found that the role of polyphenol oxidase produced by the
ogen is correlated with the production of symptoms. Reducing agents
as ascorbic acid and glutathione decrease the resistance of the tissue
ost plants to the invading pathogens by reducing quinones which are
ly toxic to pathogens to less toxic phenols (Scent-Gyorgyi and Vietorisz,
; Uritani and lechika, 1953). The effect of such reducing agents in
blast disease and the polyphenol oxidase produced by the pathogen is
MATERIALS AND METHODS
For spore germination tests the substances tested were dissolved in wa-
ind suitable dilutions were made. One-half ml portions of these solutions
placed in a series of test tubes together with 2 ml 0.05 M phos-
e buffer at pH 6.8, 1 ml of P. oryzae spore suspension (about 5 x 104
es/ml), and 1.5 ml of distilled water. The final concentrations of
compounds were 2 x 10-4, 5 x 10-4, and 10-3M. Each mixture was
en vigorously, then 100 Iz ml were placed in the wells of glass slides.
slides were placed in a moist chamber and held at 28 C for 12 hr.
;rol slides contained spores in distilled water. Counts were made on
-al hundred spores in each slide to determine the percentage germina-
For growth studies the test compounds were dissolved in 0.05 M phos-
e buffer at pH 6.8, filtered through a sterilized bacteriological filter
aseptically transferred to flasks containing Tanaka's B medium at pH
to give a final concentration of 5 x 10-4 and 1 x 10-M individually.
nty ml of agar medium was placed in each petri plate and allowed to
58 PHILIPPINE PHYTOPATHOLOGY [Vol
solidify. A 5 mm section of the culture disc, obtained from a week
culture, was placed in the center of the each plate. Control plates I
trained only the basal medium without the test compounds. The pl
were incubated for 10 days at 28 C. The diameter of the colony and
browning of the medium were recorded.
In another study 12 Philippine races of P. oryzae were inoculated
dividually on medium containing various phenolic compounds at 1 x
M concentration and growth was measured as described above.
To determine the production of polyphenol oxidase by germina
conidia 50-ml Erlenmeyer flasks, containing 10 ml chlorogenic acid
2 x 104M and 1 ml of P. oryzae spore suspension, giving 5 x 104 spores
of the mixture, were agitated under aseptic conditions in a horizon
shaker at 60 strokes/min. to allow the spores to germinate. Every 2
ABLE 1. Toxicity of different phenolic compounds to P. oryzae spore germination
mnolic compound Cone. of phenols (M)
2 x 10-4 5 x 10-4 10-8
techol 87.23 95.00 100.00
droquinone 81.65 95.49 96.62
luinone 64.80 78.85 100.00
loroglucinol 52.81 67.21 73.59
rogallol 64.46 73.59 80.48
aillin 66.71 76.47 86.61
icylic acid 66.46 75.72 91.49
itocatechuic acid 62.83 66.21 68.08
;oumaric acid 43.05 55.82 69.59
Efeic acid 74.59 80.85 90.36
-ulic acid 59.82 62.83 66.96
lorogenic acid 50.56 64.58 71.09
a Inhibition of spore germination compared with the control.
TABLE 2. Effect of different phenolic compounds on the growth of P. oryzae
Inhibitiona (%) Browning of the medium
nolic compound Cone. of phenols (M)
5 x 10-4 10- 5 x 10-4 10-s
*nol 7.67 16.22 -
techol 40.41 58.26 ++ ++++
orcinol 26.25 28.91 + +
Iroquinone 24.78 53.54 + ++
Iuinone 28.61 43.66 +
oroglucinol 24.78 44.25 +
icylic acid 30.68 32.15 -
tocatechuic acid 23.89 32.74 +
lic acid 5.60 6.19 + +
inic acid 21.83 22.12 -
lillic acid 11.50 22.12 -
oumaric acid 23.01 28.61 -
feic acid 27.43 33.31 + ++
ulic acid 20.06 33.33 +
orogenic acid 23.75 35.40 + +++
Optical density at 326 mI
2 X 10-4 M Chlorogenic acid
2 X 10-4 M Chlorogenic acid +
0.5 I I I I
0 2 4 6 8 10
Fig. 1. Changes in optical density of chlorogenic acid solution at 326 mu due
its oxidation by the germinating conidia of P. oryzae.
Polyphenols have been frequently described as factors of the chemic:
resistance of plant tissues to many pathogens because of their general a
cumulation near wounded and infected tissues and because phenols an
*U-tA- Avfflnf-^m *^*>Artc -**- 1^n:T1t fi-f:fnv-- /A~lltp- r C-UM,-m-nA 1QIK
(Suzuki, Doi and Toyoda, 1955), p-hydroxybenzoic, vanillic, p-couma-
ind feruluc acids (Kuwatsuka and Oshma, 1961), salicylic acid (Ishii et
962), and protocatechuic, cinnamic, o-ocumaric, and catfeic acids (Var-
970). Most of these compounds had a toxic effect on P. oryzae spore
nation and growth on agar medium (Wakimoto, Ikari and Yoshii,
S3. Toxicity of various phenolic compounds to growth of different races of
P. oryzae on agar medium
oryzae Phenolic compounds
Catechol p-Quinone Ferulic Chlorogenic acid
Catechol p- one acid acid Catteic
?26 62 52 33 29 18
'38 65 38 32 23 19
'189 62 36 24 21 19
'16 66 45 32 31 32
59 38 35
ITT T.DYTI PUVTV'DA'Ti -nT ni-V
tissues and their role in defense mecnamsms nave Deen snown oy j
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wnen mne pamogen overo-uiie Wie Ltoxi cticLt ui LIcac .i-uipoLAiu",
by degrading or tolerating them, it becomes pathogenic to the pa
host plant (Mahadevan, 1969). Such a situation may govern the
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fertilizers has been shown to decrease the amount of phenolic com
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1972) and has been correlated with increased susceptibility to c
The variation among different races of the pathogen and their toler;
of a variety in response to infection with different races of the pa
might govern the specificity of the races.
The presence of reducing agents such as ascorbic acid and gluta
decreases the resistance of the plants by reducing quinones which are
to pathogens to less toxic phenols (Szent-Gyorgi and Vietorisz, 1931; .
SRIDInAR & OU RTCE BL
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WITH 'aJUDIUMUNA6 GLYUIJNEA CU;UYrK
DELFIN B. LAPIS
Assistant Professor, Department of Plant Pathology, College of Agriculture, U.P.
at Los Bafios, College, Laguna.
Soybean plants infested with red spider mites (Tetranychus telarius L.)
show profound resistance or immunity to infection with Pseudomonas glycinea
Coerper. This reaction of the plants increases with increases in number of
mites and longer exposure of plants to them prior to infection by the bac-
Researchers working on the pathogenicity of Pseudomonas glycinea
Coerper on soybean have observed that plants infested with red spider
mites tend to show some degree of resistance or immunity to bacterial in-
fection. Red spider mites can adapt to different climatic conditions and
they are a common greenhouse pest occurring on a wide range of host
plants. This study was conducted to determine the effects of 1) proximity
of mites to the area of inoculation, 2) length of period of mite infestation
prior to inoculation, and 3) number of mites.
MATERIALS AND METHODS
General procedures. Seeds of soybean var. Acme were sown in 4" clay
pots containing sterile soil at the bottom and sand at the top. Through-
out the experiment, the plants were maintained on a sand-covered bench
in a greenhouse (24-27 C, 40-80%o RH).
Mites were collected from bean plants and reared on soybean. Periodic
transfers were made to maintain a large population of mites. This was
done by allowing the mites to crawl from mature to young soybean plants.
Mites were transferred to the experimental plants with a camel's brush
moistened with water. Only those moving were transferred so as not to
break the two chalecereae (prehensile claws) used to puncture the plant.
Bacterial inoculum was prepared by suspending in sterile distilled
water a 48-hr old culture of P. glycinea on yeast-dextrose agar. This was
adjusted to contain 10 x 106 bacterial cell/ml following MacFarland's
scale (Barret, 1964). With a Paasch H "3 in 1" air brush at 15 psi, plants
LAPIS RED SPIDER MITES
were inoculated by spraying the under surface of fully unfolded trifoliate.
With this device, the bacterial suspension is forced through the epidermis,
thus water-soaked spots appear on the leaflets.
Disease severity rating was taken 7-10 days after the separate or simul-
taneous introduction of mites and bacteria. A scale of 1-5 was adopted
for disease rating, where 1 represents no color change or water soaking,
and 5 severe water soaking and intense brown discoloration.
Each treatment was replicated 6 times.
Effect of the proximity of mites to the area of inoculation.-
Treatment I. The mites were introduced on each of the two
outer leaflets of the trifoliate, and the bacteria on the center leaflet.
Treatment II. The mites were transferred on the center leaflet
and the 2 outer leaflets inoculated with the bacteria.
Treatment III. Both the mites and bacteria were introduced on
the center leaflet of the trifoliate.
In the above treatments 15 mites were introduced per leaflet.
Both mites and bacteria were introduced at the same time. For the
check, trifoliates were maintained with either bacteria or mites only.
Effects of length of exposure of plants to mite infestation prior to
bacterial inoculation. Ten mites were allowed to feed on each leaflet of
the trifoliates for 0, 1, 2, 4, 14 and 21 days before inoculation.
Effects of number of infesting mites prior to inoculation. Zero, 12, 25
and 50 mites were allowed to feed on the trifoliates 48 hr prior to inocu-
lation with the bacterium.
Minute, white chlorotic areas were noticeable on leaflets where mites
had been feeding for 2 days. On leaflets inoculated with the bacterium
3-4 days earlier, infection appeared as very faint water-soaked areas which
became necrotic as infection advanced.
There was no striking difference in severity of the disease when mites
and bacteria were introduced on the same, or different leaflets of the tri-
foliates. Plants with both mites and bacteria on the center leaflet gave
an average rating of 4.6, while checks (no mites) had 5.0 (Table 1).
Exposure of plants to mite infestation for 0, 1, 2, 4, 14 and 21 days
prior to inoculation with the bacterium gave a disease severity rating of
4.6, 4.3, 4.0, 2.5, 1.8, and 1.5, respectively. This is clear indication that as
plants were exposed longer to mite infestation they acquired a certain re-
sistance or immunity to bacterial infection (Figs. 1-2).
Varying the number of mites but keeping the exposure time (48 hr)
constant indicated an inverse relationship. Experiments where 0, 12, 25
Nos. 1 & 2J
TABLE 1. Effects .of mites on infection of soybeans with I
Mites on 2 outer leaflets, center leaflet
inoculated with bacteria 5 4 4 5
Mites on center leaflet, 2 outer leaflets
inoculated with bacteria 4 5 5 5
Mites and bacterial inoculum on center
Check 5 5 5 5
See text for explanation.
b Bacterial inoculation and transfer of mites done at the same time.
LAPIS RED SPIDER MTES
Fig. 1. Soybean leaves showing the effects of varying exposure periods (in days)
to mite infestation prior to inoculation with P. glyclnea.
0 2 3 4 8a 6 7 e so It t1 ), as 17 I s 1 o 2
Fig. 2. Relation of length of exposure of soybean plants to mite infestation prior
to bacterial inoculation.
Nos. 1 & 2]
0 12 25 50
Fig. 3. Soybean leaves showing the effects of number of mites to infection:
5 10 15 20 25 30 35 40 45
NUMBER OF MITES
Fig. 4. Effect of the number of mites on bacterial infection with a 48
exposure period prior to bacterial inoculation
actively (Figs. 3-4).
Injury of the cells at the site of inoculation was experienced on all
.e inoculated leaves, and too high inoculum concentration may be the
n for the severe symptoms on all the inoculated plants.
Results of this study indicate that mite infestation gave soybean plants
rtain resistance or immunity to infection with P. glycinea. This
!pendent on the length of time the plant had been exposed to mite
:ation and on the number of mites on the plant prior to inoculation.
ability of P. glycinea to infect the leaves is not reduced if transfer of
and bacterial inoculation were done at the same time on the same
different leaflets of the trifoliate.
Experiments on the effects of length of exposure to and number of
corroborate with each other, clearly showing that more mites and
r exposure period have a striking effect on the ability of P. glycinea
The chlorotic areas on the leaves are indications that toxic substances
excreted by the mites. But this is doubted due to the very limited
it and localized site of feeding (Anon., 1968). It was postulated that
:hlorotic areas on the leaves are not due to toxic substances excreted
ie mites, but are formed when the chalecerae pierced the plant. After
had withdrawn their mouth, air then enters the empty cells resulting
te formation of light colored areas or collapse of cells.
)ne theory that may be worked out to correlate mite infestation with
ance, immunity, or both is to determine the role of guanine and uric
which were purified from fecal elements of mites (McEnroe, 1961).
line and uric acid mght have a role in preventing multiplication and
tion of the bacterium in the cell, due to changes in its physiologic
metabolic activities or changes in the pH of the sap.
Further studies are needed to pinpoint the exact role of mites in pro-
ig resistance or immunity to plant against infection by P. glycinea.
differentt levels of inoculum concentration should be tested further
it is suspected that the concentration used in this study was too high
cause injury to the leaves.
nMous. 1968. Spider mites- a world-wide plague. Chemagro Courier. February,
.T, J. 1964. Preparation of a bacterial vaccines. In Laboratory Manual in
immunology and Serology. Department of Microbiology, School of Medicine, Uni-
ersity of Missouri.
WOE, R. 1961. Guanine and uric acid excretion in mites. Enton. Soc. of Amer.
SPORE DENSITY OF SCLEROSPORA PHILIPPINENSIS
IN RELATION TO FIELD TEMPERATURE,
RELATIVE HUMIDITY AND DOWNY
S. C. DALMACIO AND A. D. RAYMUNDO
Respectively Instructor and Assistant Instructor, Department of Plant Patho
U.P.C.A., College, Laguna.
lined therefrom are useful in the formulation of control measures against
MATERIALS AND METHODS
Spore density in relation to temperature and relative humidity. Glass
!s coated with petrolatum and mounted on improvised spore traps
e exposed late in the afternoon and collected the following morning at
I. Eight slides, 2/spore trap, were exposed daily. Two cover slips were
ised against every exposed slide and counts made on the number of
bhilippinensis spores in the covered area(4.8 cm2/coverslip). Daily re-
Is of RH and temperature prevailing from 9:00 PM to 4:00 AM were
wished by the weather station adjacent to the experimental fields.
Another method involving inspection of individual diseased plants was
1. Ten one-month old infected plants were tagged. Every afternoon,
nants of downy growth produced the previous night were removed by
bbing the chlorotic leaves with moist cotton. These swabbed leaves
then individually examined the following morning for the presence
ndance of downy growth. Sporulation was recorded as follows: none, no
ny growth; slight, downy growth scanty; moderate, 1/2 of chlorotic area
,red with downy growth; heavy, profuse downy growth all over chlorotic
Spore density in relation to downy mildew incidence. Sweet corn seeds
[ 801) were planted in seedboxes daily for 5 days for every trial. One
k later, the seedlings were exposed overnight in the field in between
s of infected plants. They were then brought to the laboratory and
!rved for 15 days. Slides coated with petrolatum were also exposed to
rmine the relative density of spores during each exposure night. Plants
showed systemic symptoms of the disease were cut immediately. Plants
Also also sprayed with Dithane M-45 every other day to avoid further
action of neighboring plants. Percentage infection and spore count were
rded and compared.
RESULTS AND DISCUSSION
The effect of temperature and RH on sporulation of Sclerospora philip-
nsis Weston during 2 one-month periods in 1970 and 1971 is presented
tables 1-2. Results showed that spore production is dependent mainly
i relative humidity. Furthermore, the number of hours from 8:00 PM
:00' AM when RH was 90% or above appeared important. At least
)urs of this moist condition during the 8-hour period was required for
e production. On the other hand, the amount of spores produced was
directly related to RH. Spore count on January 12-13, 1970 was two
s more than that for the following nivht ITanuarv 1.-14M when RH
PUTT TDDIr m PUTVTrnmDA-TnT r1rV
TABLE 1. Effect of temperature and relative humidity on spore production of Scleros]
philippinensis, January-February, 1970
Temperature RH No. of Hr. Spore
Date (F) (%) RH 90% count'
Jan. 12-13 75.6 92.6 8 975.2
13-14 74.4 94.6 8 458.1
15-16 75.5 88.6 3 4.7
17-18 71.9 90.5 7 109.6
19-20 72.8 83.1 0 0
21-22 70.9 91.2 6 107.5
22-23 72.6 92.2 6 136.8
23-24 74.5 92.8 7 320.9
27-28 72.0 90.0 6 633.6
30-31 73.8 79.5 0 0
Feb. 2-3 71.6 87.8 2 0
3-4 72.8 90.6 7 160.0
4-5 74.8 90.4 5 625.0
5-6 76.3 85.8 0 0
10-11 73.4 86.5 0 0
11-12 72.4 87.8 3 30.3
a Average of 16 replicates; 4.8 cm2/cover slip.
TABLE 2. Effect of temperature and relative humidity on sporulation of Scleros
philippinensis, January-February, 1971
Temperature RH No. of Hr.
Date (F) (%) RH 90% Sporulation,
Jan. 28-29 74.00 94.1 8 slight
29-30 73.3 92.1 8 heavy
30-31 74.00 86.4 1 none
31-Feb. 1 75.4 89.8 4 slight
Feb. 1-2 74.00 87.3 0 none
2-3 73.00 88.4 1 none
3-4 73.9 84.1 0 none
4-5 72.8 85.8 1 none
5-6 72.8 78.8 0 none
6-7 71.9 78.8 0 none
7-8 70.8 92.4 7 slight
8-9 73.8 80.00 0 none
9-10 73.5 81.3 1 none
10-11 72.8 96.5 8 heavy
11-12 74.0 88.4 3 none
12-13 75.8 89.3 3 none
13-14 75.5 90.3 5 moderate
15-16 75.3 94.9 8 moderate
16-17 75.6 91.6 5 heavy
17-18 76.3 86.9 0 none
18-19 76.00 94.4 8 moderate
19-20 75.00 92.50 8 moderate
20-21 76.9 89.4 5 moderate
a See text for exDlanation.
The data in Tables 1-2 also indicate that at RH of 90% or above,
elation takes place regardless of temperature. Temperature becomes
rtant, however, when RH is below 90%. It can be noted in Table 3
Is 2 and 3), for example, that the nightly temperature always exceeded
; yet, heavy sporulation occurred from 30 April to 4 May 1970 despite
RH. Temperature during this period was relatively high (78-79 F).
Results of 3 trials conducted at monthly intervals are presented in
e 3. Spore production occurred even at relatively low RH although
pore was observed when the average night RH was below 80%. It
Id be emphasized here, however, that the average temperature during
period was relatively higher compared with that in Table 1.
2-3 75.6 86.7 0 467.3 100.0
3-4 76.3 84.7 0 230.3 100.0
29-30 78.3 85.7 0 5521.60 100.0
30-May 1 78.4 83.9 0 3888.0 100.0
1-2 78.0 82.9 1 3444.0 100.0
2-3 79.3 83.00 1 3645.3 100.0'
3-4 2172.0 100.0
L A--r-an 1 U rnhlratoe Thf Qlnrro ,F Nnr IltIAnn -ht- sfhA Gr- th4 thro fr;.le
Downy mildew incidence is not entirely dependent upon spore del
In trial 2, Table 3, disease incidence was 100% with spore counts of:
and 467.3; however, it was down to 97.5% in trial 1 when spore count
much higher (1161.9). Perhaps these differences could be attributed
other factors such as wind velocity, solar radiation, time of spore dispi
etc. It should be emphasized that conidia of S. philippinensis are very
sitive to dessication, thus penetration and infection by conidia prod
late in the morning are adversely affected by the early morning sun.
above results indicate that temperature and relative humidity are not
only main factors influencing sporulation of S. philippinensis in
field. Dew, the formation of which is influenced by temperature and
lative humidity, seems to be the most important factor. Spore produce
was thus always observed when dew or a thin film of moisture covered
surfaces of infected leaves. This could explain the observation of
sporulation occurring on infected plants in growth chambers even v
RH is over 90%. Such chambers are equipped with fans that continue
rotate to maintain the inside RH and temperature, thus preventing in
process the formation of a thin film of moisture on the leaf surface. 1
larly, under conditions when relative humidity is high and temperature
suitable, strong wind is a limiting factor in spore production.
Results obtained from this study may also help explain the obs(
tions on the prevalence of downy mildew in Mindanao and Ilagan, Isa
in Luzon (Francis, 1967). The even distribution of rainfall in t
localities favors the growth of "talahib" (Saccharum spontaneum) the
round. Infected "talahib" could then serve as a continuous source
inoculum from year to year or from one crop season to another. Furt
more, the moist condition in such areas not only favors sporulation
also infection by prolonging the viability of the spores.
Nos. 1 & 2] DALMACIO & RAYMUNDO DOWNY MILDEW 77
EXCONDE, O. R., EDNA A. EDRALIN and B. A. ADVINCULA. 1967. Some factors affecting
formation and germination of spores of Sclerospora philippinensis. Proc. Fourth
Inter-Asian Corn Improvement Workshop, Lyallpur, West Pakistan, Oct. 9-14, 1967.
EXCONDE, O. R., J. Q. ADVERSARIO and B. A. ADVINCULA. 1968. Incidence of corn
downy mildew in relation to planting date and meteorological factors. Philippine
Agr. 52: 189-199.
FRANCIs, A. C. 1967. Downy mildew disease of maize. M.S. thesis. Cornell Uni-
Sclerospora of maize. J. Agr. Res. 23: 239-278.
WESTON, W. H., Ja. 1923. Production and dispersal of conidia in the Philippine
versity, Ithaca, New York.
INFLUENCE OF ROOT-KNOT NEMATODES ON THE SEVERITY
OF BACTERIAL WILT AND FUSARIUM WILT OF TOMATO
R. G. DAVIDE
Assistant Professor of Plant Pathology, U.P.C.A.
Partly supported by UPCO research grant, project No. 234, and Vegetable research
During one of my disease surveys of tomato fields in Negros Oriental in
1968, I observed that in some areas bacterial wilt, Fusarium wilt and root-
knot nematodes were present together. Under this situation it was difficult
to appraise disease losses due to the wilt pathogens and neither was it pos-
sible to get estimate on losses due to nematodes. There have been several
attempts, however, to estimate bacterial wilt losses on tomato in the field
without considering the influence of nematodes and other pathogens present.
But the data obtained do not reflect the actual loss due to bacterial wilt.
It has been well demonstrated abroad that bacterial wilt and Fusarium
wilt become more severe in the presence of root-knot nematodes. For
instance, Johnson and Powell (1967) showed that the presence of root-knot
nematode (M. incognita) greatly increases the severity of the bacterial wilt
on flue-cured tobacco. Likewise, Fusarium wilt on tobacco (Poster &: Powell,
1967), cotton (Minton & Minton, 1966), carnations (Schindler, et al, 1'961)
and mimosa (Gill, 1958) can become severe in the presence of nematodes.
In some cases, wilt resistant varieties became susceptible after being infected
with root-knot nematodes (Lucas, et al. 1955; Melendez and Powell, 1967).
In the Philippines the involvement of nematodes in disease complexes
has never been investigated, although it is commonly observed that nema-
todes are often associated with other pathogens in a number of root-rot
diseases. This study was conducted primarily to determine the influence of
the root-knot nematode (M. incognita) on the development of bacterial wilt
and Fusarium wilt of tomato.
MATERIALS AND METHODS
Three varieties of tomatoes, viz., Marglobe and two native varieties, one
with round fruits and the other with oblong fruits were used as test plants.
These were used to evaluate and compare the reactions of the plants be-
tween varieties to the single and combined effects of the three pathogens
mentioned below. They were grown in 6-inch clay pots filled with decon-
taminated soil. The plants, 1 ft. in height, were arranged into 8 groups
consisting of 4 pots each. Each group represented a treatment. The treat-
DAVIDE ROOT-KNOT NEMATODES
ments consisted of 1) control, 2) bacteria alone, 3) nematode alone, 4) Fusa-
rium alone, 5) nematode-bacteria, 6) nematode-Fusarium, 7) bacteria-Fusarium,
and 8) nematode-bacteria-Fusarium combination.
Inoculations were done at the same time for all of the above treatments.
For the treatment using nematode alone, each plant was inoculated with ap-
proximately 2,000 larvae of Meloidogyne incognita; for the bacteria, 5-day old
cultures of Pseudomonas solanacearum were made in water suspension and
10 cc of this was used as inoculum for each plant; and for the Fusarium
treatment, 2-week old culture of Fusarium oxysporum f. lycopersici was sus-
pended in water and 10 cc was used to inoculate a plant. Evaluation on the
severity of the disease was based on the percentage of plant wilted within 7
and 14 days after inoculation.
RESULTS AND DISCUSSION
The data obtained are summarized in Tables 1 and 2. The results
showed that the percentage of plants wilted seven days after inoculation was
relatively lower than that at 14 days. There is evidence that the three test
varieties of tomato reacted invariably to the different treatments. Plants
inoculated with either nematode or Fusarium alone showed no symptoms of
wilting 7 days after inoculation. However, there was 25% wilting of the
Marglobe and native (oblong fruit) varieties treated with bacteria alone.
There is evidence of increased wilt severity in the combined treatment with
bacteria and nematode, in that there was 75% wilting of Marglobe plants
and 25% in native (round fruit) variety. With the nematode-Fusarium
treatment, only plants of the native variety with round fruits showed 25%
wilting. Likewise, in the bacteria-Fusarium combination there was 25%
wilting in both native varieties and none in Marglobe. In the nematode-
bacteria-Fusarium treatment there was 50% wilting in the native (round
fruit) variety, 25% in the native (oblong fruit) variety, and none in Mar-
globe 7 days after inoculation.
TABLE 1. Percentage wilting in 3 varieties of tomato 7 days after inoculation
Treatment Native Native
Marelobe (Round (oblong
Control 0 0 0
Nematode alone 0 0 0
Bacteria alone 25 0 25
Fusarium alone 0 0 0
Nematode-bacteria 75 25 0
Nematode-Fusarium 0 25 0
Bacteria-Fusarium 0 25 25
Nematode-bacteria-Fusarium 0 50 25
Nos. 1 & 2]
80 PHILIPPINE PHYTOPATHOLOGY [VOL. 8
TABLE 2. Percentage wilting in 3 varieties of tomato 14 days after inoculation
Treatment Native Native
Marglobe (round) (oblong)
Control 0 0 0
Nematode 0 0 0
Bacteria alone 75 75 75
Fusarium alone 0 0 0
Nematode-bacteria 100 100 50
Nematode-Fusarium 0 25 0
Bacteria-Fusarium 100 75 75
Nematode-bacteria-Fusarium 100 100 100
a Vascular discoloration observed in stems of the plants.
Fig. 1. The influence of root-knot nematodes on the severity of the wilt disease
on tomato. A) Inoculated with bacteria (P. solanacearum, B) bacteria + nematode
(M. incognita), and C) bacteria + nematode + Fusarium oxysporum f. lycopersici.
Fourteen days after inoculation, a more severe wilting was observed in
plants with the nematode-bacteria-Fusarium combination than in plants inocu-
lated with any one of these pathogens alone. In this treatment there was
100% wilting in all plants of the three varieties (Fig. 1). In the bacteria-
Fusarium treatment, wilting was 100% in Marglobe and 75% in the two
native varieties. On the other hand, in the nematode-Fusarium treatment
there was only 25% wilting in the native (round) variety and none in other
Marglobe and native (round) variety and 50% in native (oblong) variety.
With bacteria alone wilting was 75% in Marglobe and native (round) varie-
ties and 25% in native (oblong) variety. None of the varieties used showed
wilting in the Fusarium treatments 14 days after inoculation although the
plants were already showing vascular discoloration of stems, the diagnostic
symptom of the disease.
The three pathogens (M. incognita, P. solanacearum and F. oxysporum
f. lycopersici) were evidently growing in the host tissues. The nematodes
developed considerable galling in the roots. Reisolations of both P. solana-
cearum and F. oxysporum f. lycopersici from the diseased tissues were made
The results of this study clearly indicate that root-knot nematodes in-
fluence the expression of bacteria wilt and Fusarium wilt on tomatoes. The
three pathogens can interact together to cause more damage to the host plants.
This information may also be true for other host crops in this country.
GILL, D. L. 1958. Effect of root-knot nematodes on Fusarium wilt of mimosa. Pl.
Dis. Reptr. 42: 587-590.
JOHNSON, H. A. and N. T. POWELL. 1969. Influence of root-knot nematodes on bac-
terial wilt development in flue-cured tobacco. Phytopathology 59: 486-419.
LUCAS, G. B., J. N. SASSER and A. KELMAN. 1955. The relationship of root-knot nema-
todes to Granville wilt resistance in tobacco. Phytopathology 45 537-540.
MELENDEZ, P. L. and N. T. POWELL. 1967. Histological aspects of the Fusarium wilt-
root knot complex in flue-cured tobacco. Phytopathology 57 286-292.
MINTON, N. A. and E. B. MINTON. 1966. Effect of root-knot and sting nematodes on
expression of Fusarium wilt of cotton in three soils. Phytopathology 56: 319-322.
PORTER, D. M. and N. T. POWEL.. 1967. Influence of certain Meloidogyne species on
Fusarium wilt development in flue-cured tobacco. Phytopathology 57: 282-285.
SCHINDLER, A. F., R. N. STEWARD and P. SEMENIUK. 1961. A synergistic Fusarium-
nematode interaction in carnations. Phytopathology 51: 143-146.
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The undersigned, SILVINO MERCA, business aaeo HLPIEPYO
PATHOLOGY, (title of publication), publshed seianuly(rqecofis),n
English (language in which printed), at UPCA ColgLun.(fiefpbictn)
after having been duly sworn in accordance witlahrbsumstefoown
statement of ownership, management, circulation, ecwihi eurdb c 50
as amended by Commonwealth Act No. 201.
Editor. IRINEO DOGMkA, JR ............. PAColgLun
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In case of public cation other than daily, total nubro oispine n icltd
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1. Sent,. to paid subscribers ... . . ... .... ................. .120
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Doc. No. 707
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Book No. IV
Series of 1972
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