BOARD OF DIRECTORS 1995-1996
President RIZALDO G. BAYOT
Vice-President NENITA L. OPINA
Secretary TEODORA O. DIZON
Treasurer LINA C. LAPITAN
Auditor FRANCISCO A. ELAZEGUI
Business Manager CEFERINO A. BANIQUED
Board Member AVELINO D. RAYMUNDO
Board Member ANGELITA D. TALENS
Board Member GERARD V. MANINGAS
Board Member LUCIANA M. VILLANUEVA
Board Member VIVENCIO R. MAMARIL
Immediate Past President RUSTICO A. ZORILLA
EDITORIAL BOARD 1995 1996
OSCAR S. OPINA Editor-in-Chief
CHRISTIAN JOSEPH R. CUMAGUN Associate Editor
TEODORA O. DIZON Associate Editor
.- AeV -I. -~ - ^
Philipp. Phytopathol. 1995, Vol. 31(1): 9-19 1
EFFECT OF TEMPERATURE AND HUMIDITY ON THE
GERMINATION AND GROWTH OF LASIODIPLODIA
THEOBROMAE (PAT.) GRIFF. & MAUBL.,
CAUSE OF STEM-END ROT OF MANGO
(MANGIFERA INDICA L.)
M.G.MORTUZA anJ LINA L. ILAG
Portion of the MS thesis of the senior author submitted to the University of the
Philippines Los Banos (UPLB), College, Laguna 4031.
Respectively, Graduate student and Professor, Department of Plant Pathology,
University of the Philippines Los Banos, College, Laguna.
Keywords: environmental factors, germ tube growth, Lasiodiplodia theobromac,
mango, spore germination, stem-end rot
The effects of temperature and relative humidity on the germination and
growth of Iasiodiplodia theobromae were studied in vitro. Spore germination
started at 15"C and significantly dropped at 40"C. A positive correlation of
germination with temperature was observed up to 35"C. One-celled spores
germinated more rapidly than the two-celled spores in all temperatures tested
except at 15 and 40"C. A higher percentage germination was generally
accompanied by longer growth of the germ tubes. The longest germ tubes
were produced by the one-celled spores incubated at 300C.
There was no variation in the thermal death point (TDP) of three isolates of
the fungus collected from three different areas in Laguna and Batangas. The
TDP of spores of two-week old cultures was 53 and 54C from three-week old
The germination of spores and growth of the germ tubes significantly
increased with the increasing relative humidity (RH). The minimum RH
required for germination was 95%. Maximum germination and germ tube
growth were recorded at 100% RH. The percentage germination and growth rate
of one-celled spores were higher than the two-celled spores in all RH tested. A
longer incubation period always produced higher germination and germ tube
2 Philipp. Phytopathol. 1995, Vol. 31(1): 1-8
Mango (Mangifera indica L.) is one of
the most important fruits in the
Philippines (Mendoza and Wills, 1986).
It is a delicious and nutritious fruit as it
contains high amounts of total solids,
proteins, starch and vitamins. The damage
caused by pests and diseases during the
postharvest period, along with poor
handling of fruits in transit has resulted in
enormous losses. At present, fruit diseases
are still regarded as a major problem in
the mango industry.
Stem-end rot is a major postharvest
disease. It causes considerable losses in
almost all mango growing countries of
the world (Sangchote, 1991). The highest
disease incidence was reported from
Bangladesh, with an average of 12.5%
(Quroshi and Meah, 1991). Stem-end rot is
characterized by the appearance of violet
discoloration, usually starting at the
peduncle of the ripe fruit which may
entirely rot within 3 4 days (Alicbusan and
Schafer, 1958). Stem-end rot is caused
by Lasiodiplodia thcobromae (Pat.) Griff. &
Maubl. (Syn. Botryodiplodia theobromae
Pat.). The perfect stage is Physalospora
rhodina (Berk. & Curt.) Cke.
The diagnostic discoloration or decay
at the stem-end of infected fruits impairs
the market quality of the fruits rendering
them unfit for consumption and
eventually causes total loss of the mango
fruits. The pathogen is known to cause
die-back, leaf blight, withering of tips,
blossom blight, twig blight and fruit rot
(Sabalpara et al., 1991).
Temperature and humidity are the
most important environmental factors
which greatly influence the growth and
development of the fungus (Verma and
Singh, 1970; Prasad and Sinha, 1981). It is
therefore important to know the
pathogens minimum, optimum and
maximum_ temperature and humidity
requirements for the efficient and effective
management of-lie disease. This study
determmeTthe effect of these two physical
factors affecting the stem-end rot fungus
which may influence the postharvest
occurrence of the disease. The objectives
of this study are to determine the cardinal
temperatures for germination and germ
tube growth, the thermal death point of
the fungus as well as the minimum and
optimum humidity requirements.
MATERIALS AND METHODS
Collection and Isolation of the pathogen
Isolates of L. theobroinae were obtained
from infected mango fruits collected from
Laguna (Santa Cruz and Los Bafios) and
from Batangas. The collected mangoes
were surface-sterilized. Small tissues from
the infected mesocarp were placed in a
petri dish plated with potato dextrose agar
(PDA) and exposed continuously under a
fluorescent light at room temperature. The
pathogenicity of the isolates was tested
in ripe mangoes. The Los Baios isolate was
selected for temperature and humidity
studies as it was the most virulent isolate.
Effect of temperature
Dry spores from two-week old
cultures were placed on clean, dry glass
slide by crushing pycnidia on the slide.
The slides were placed inside glass bottles
-of 3.5 liter capacity. The bottles were filled
with 1 1 water to provide high humidity
inside the bottle. A wire net was fitted
Philipp. Phytopathol. 1995, Vol. 31(1): 1-8 3
inside each bottle 2.5 cm above the water
level to house the slides. The bottles were
closedd and incubated at 10, 15, 20, 25, 30,
35 and 40"C. The incubated slides were
removed from the bottles after 8, 16 and 24
hr and were mounted with lactophenol
cotton blue for microscopic studies.
The number of germinating one-celled
and two-celled spores were counted and
the growth of the germ tube was measured
by randomly selecting 100 germinated
spores at each temperature. The" thermal
death point of spores as determined in
this study is the temperature which kills
the spores of the fungus when exposed
to it for 5 min. The procedure as adopted
by Quimio and Quimio (1974) was
Effect of humidity
RESULTS AND DISCUSSION
Effect of temperature
Germination of the spores. The
;ermination of spores of L. theobronae was
significantlyy influenced by different
temperatures and incubation periods.
Jone of the spores germinated at 10"C
ven after 24 hr of incubation. The lowest
temperature required for germination was
15"C (Fig 1). The spores were able to
;erminate after 16 hr of incubation. The
ige of the spores significantly affected the
germination. The germination of older two-
:elled spores was higher than the younger
one-celled spores at both incubation
i 1"L r rr~- ^ _ /
4 Philipp. Phytopathol. 1995, Vol. 31(1): 1-8
10 15 20 25 30 35
Figure 1. Germination of spores of Lasio-
diplodia thcoblvinra after 8,16 and
24 hr of incubation at different
temperatures and 100'% relative
Length of germ tube(Pm)
200 2T i Li
. . . . .
.t:, ^ .; .:
20 25 30 35
Figure 2. Germ tube elongation of germi-
nated spores of Lasiodiplodia
thcobronmac after 8, 16 and 24 hr
at different temperatures and
100% relative humidity.
Philipp. Phytopathol. 1995, Vol. 31(1): 1-8 5
30"C. One-celled spores germinated more
rapidly than the two-celled spores in all
The spores of L. thlobronmat germinated
faster at 35"C even after a short incubation
of8hr. Among the temperatures tested, the
highest germination was recorded at this
temperature. One-celled spores always
germinated better than the two-celled
spores. The germination of one-celled
spores significantly increased with
increasing incubation period whereas
germination of two-celled spores did not
The percentage spore germination
decreased significantly at 40'C. Generally,
at this elevated temperature, the two-celled
spores germinated earlier than the one-
celled spores after a short incubation
period but after a longer incubation period
of 24 hr, the percentage germination of
one-celled spores was almost similar to
that of two-celled spores.
The results revealed that the
percentage germination gradually
increased with increasing temperature
from 15 to 35"C. There was a drop in the
number of germinating spores at 40"C.
The probable cause may be that hot and
high humid conditions may have some
adverse effect on the growth of the fungus.
Warm air can hold more moisture than
normal air (Hidore and Oliver, 1993) so at
high humidity levels, spores/germ tubes
absorbed more warm moisture from the
surroundings which had a detrimental
effect on the fungus.
Growth of germ tubes. Temperature
significantly affected the growth of germ
tubes of spores of L. thcobromac (Fig. 2).
The longest germ tube was recorded at
15'C in two-celled spores in all incubation
periods tested. The germ tube length
was not much after 16 hr but this
increased several fold after 24 hr of
incubation. Two-celled spores incubated
at 20"C produced longer germ tubes after
short incubation period of 8 hr whereas
one-celled spores surpassed the growth
of two-celled spores at the longer
incubation of 16 and 24 hr. The growth of
germ tubes of both spore types was very
poor after 8 hr but it increased rapidly after
The growth of germ tubes of
L. theobroinac was longer at 25"C than
at the lower temperatures. Vigorous
growth was observed after 8 hr of
incubation. One-celled spores always
produced longer germ tubes than the two-
Among the temperatures tested, the
fungus produced the longest germ tubes
at 30"C. Growth of the germ tubes was
affected by age of the spores. The
younger one-celled spores produced
longer germ tubes than the older two-
celled spores, although the difference was
insignificant at 8 hr of incubation.
Spores incubated at 35"C exhibited
vigorous growth when incubated for
8 and 16 hr and produced longer germ
tubes than at 30"C after the same
length of incubation. Germ tube elongation
rapidly increased with increasing
incubation period. Although the
fungus produced longer germ tubes at
shorter incubation, this failed to exceed
the growth at 30"C after a longer
incubation period. One-celled spores
produced longer germ tubes than the
two-celled spores in all incubation period
6 Philipp. Phytopathol. 1995, Vol. 31(1): 9-19
Figure 3. Germination of spores of Lasio-
diplodia theobromac after 8, 16 and
24 hr of incubation at different
relative humidity and 25"C.
85 90 95
Figure 4. Germ tube elongation of germi-
nation spores of Lasiodiplodia
theobromae after 8, 16 and 24 hr
,at different relative humidity
IIpp. Phytopathol. 1995, Vol. 31(1): 1-8 7
The pathogen was able to grow at Effect of humidity
C but produced short germ fubes. Two-
led spores produced slightly longer Germination of spores. The germi-
m tubes than the one-celled spores in nation of spores of L. theobromae was
'incubation periods. The results significantly affected by. different levels of
)ported the findings of Pathak and relative humidity (RH). The minimum RH
vastava (1969). They noted that the required for germination of both types of
:imum temperature for growth of the spores was 95% (Fig. 3). Percentage germi-
:hogen was 30"C. Verma and Singh nation of one-celled spores was higher than
70) also reported that germ tube growth the two-celled spores in all incubation
one-celled spores was best at 30"C and period tested. Two-celled spores
,se of two-celled spores at 33oC. germinated only after 16 hr of incubation.
It was observed that two-celled spores Increased germination of spores was
minated and grew faster than the one- observed at 97.5% RH. Although the
ed spores under stress conditions such germination of one-celled spores was
ow (15C) and high (40C) temperature, higher than the two-celled spores in all
! probable cause may be that the thicker incubation periods, the difference after 24
Ils of two-celled spores are more hr incubation was not significant.
distant to stress than the thin walls of Maximum germination of the spores of L.
!-celled spores. At ambient or favorable theobromae was recorded at 100% RH. The
tperatures one-celled spores germinated germinations of one- and two-celled
I grew better. An unusual frequency of spores were significantly different. One-
nching of some germ tubes was noted celled spores germinated earlier than the
35 and 40oC. Sometimes several germ two-celled spores. The germinationof both
es originated from a single spore. The types of spores was totally inhibited at 85
ure of spores to germinate at 10"C was and 90% RH due to lack of sufficient
to unfavorable temperature. moisture needed for germination.
Thermal death point. The thermal Growth of germ tubes. Different
ith point (TDP) of the spores of levels of relative humidity and incubation
heobromae was between 53 and 54"C. period significantly affected the growth of
ire was no variation in TDP amoig the fungus as it affected the germination.
ee isolates collected from various places. At 95% RH, growth of the germ tube was
ores from two-week old cultures very poor after 8 hr but it increased
staining 5% two-celled spores were significantly after longer incubation of 24
sitiveto53"C while spores from three- hr (Fig. 4). Longer germ tubes were
ek old cultures containing 20% two- recorded for one-celled spores .in all
ed spores were sensitive to 54"C. The incubation period. A similar trend was
.se may be that the thick-walled two- also noted at 97.5% RH. One-celled spores
ed spores are more resistant to stress exceeded the growth of two-celled spores
editions. Quimio and Quimio (1974) in all incubation period tested.
de a similar observation when they
orted that the TDP of 5 out of 6 isolates Among the RH tested, the pathogen
n three-week old cultures was 54"C. produced the longest germ tubes at 100%
8 Philipp. Phytopathol. 1995, Vol. 31(1):
RH. Growth of the germ tubes was affect
by the age of the spores. As in lower lF
one-celled spores produced longer ge
tubes than the two-celled spores. 1
difference in growth between the t
types of spores was more prominent at t
It was observed that high hun
conditions favored the germination a
growth of the fungus. Generally, higi
spore germination and better growth of
pathogen was recorded after an extend
There was a close relations
between germination and growth of
fungus. A higher percentage germinat
was generally accompanied by ]on;
growth of the germ tubes at all te
peratures. When the germination of o
celled spores was higher than the tv
celled spores in a given condition, ge
tubes were also longer.
It was also observed that spo
incubated at lower temperatures (15 a
20"C) were normal in size and the ge
tubes produced by these spores w
thin while spores incubated at higl
temperatures (35 and 40"C) appeal
larger and the germ tubes were thicker a
with more branching. Incubati
temperature therefore appeared to aff
not only germination and germ tL
growth but also the morphology of
ALICBUSAN,R.V. and L.A.SCHAFI
1958. Diplodia rot of mango. The P
Agric. 42: 319-322.
DOBERSKI,J.W. 1981. Comparat
laboratory studies of three fun
pathogens of Elm bark beetle Scoly
scolutuis:Effect of temperature a
humidity on infection by Beau'vcria I
siana, Metarhiziuin anlisopli
Paccilomiyces farinosus. J. of Inve
Pathol. 37: 195-200.
HIDORE, J.J. and J.E. OLIVER. 19(
Climatology : an atmospheric Scien
MacMillan Publ. Co., New York. 43:
MENDOZA,D.B. and R.B.H. WILLS (e
1986. Mango: fruit developme
postharvest physiology and market
in ASEAN. ASEAN Food Handli
Bureau. Malaysia, 111p.
PATHAK, V.N. and D.N. SRIVASTA\
1969. Epidemiology and prevention
Diplodia stem-end rot of mango fru
Phytopathol. Z. 65(2): 164-175.
QUIMIO,A.J. and T.H. QUIMIO. 19
Control of Diplodia rot of mango
hot water treatment. Pl
Phytopathol. 10(1-2): 16-18.
QUROSHI, S.U. and M.B. MEAH. 19
Postharvest loss in mango owing
stem-end rot. International J. of Tr
Agric. 9(2): 98-105.
PRASAD, S.S. and A.K. SINHA. 19
Effect of temperatu re and humidity
fruit rot of mango. Nat'l. Acad. E
Letters 4(9): 345-347.
SABALPARA, A.N., D.G. VALA and K
SALANKY. 1991. Morphologi,
variation in Botryodiplodia theobrol
Pat. causing twig-blight and die- bz
of mango. Acta Horticulturae 2
SANGCHOTE, S. 1991. Botryodiplodia
of mango and its control. A<
Horticulturae 291: 296-303.
VERMA,O.P. and R.D. SINGH. 19:
Epidemiology of mango die-ba
caused by Botryodiplodia tlheobron
T -L -T J T A L- '- A/f. 01 o) 0
ilipp. Phytopathol. 1995, Vol. 31(1): 9-19 9
INFECTIOUSNESS OF AB
INFECTED WITH CERCOSPC
(BERK. AND CL
Respectively, Professor, Department of
agriculture, Baybay, Leyte 6521-A, Philippine
ant Pathology, University of the Philippines
Keywords: Cercospora arachidicola, Cercospo
'ISSED PEANUT LEAVES
I ARACHIDICOLA HORI
id O.S. OPINA
-t Protection, Visayas State College of
nd Associate Professor, Department of
,os Bafios, College, Laguna, Philippines.
uJil mhl'r ,l1ntlhJll infporl-imini ,norinc nm1on
10 Philipp. Phytopathol. 1995, Vol. 31(1): 9-1
and severity of early and late leafspot being more predominant (Paningbatai
diseases, Backman and Crawford (1984) 1980). The knowledge of the lock
found that a Florunner peanut cultivar's epidemiology of the early and late leafspo
yield potential of about 4,400 kg ha-' was should provide a satisfactory
reduced by an average of 57 kg ha- for each understanding of the population dynamic
percent defoliation or infection. They of the two pathogens in the Philippine
found that the yield-reducing potential of Teng (1985) laments that even in wel
C. arachidicola was similar to that of researched crops, the more fundament,
C. personatum. pest biology such as life cycles is incomple
which causes the comprehensive
Recognizing leafspots as the major management of yield constraints i
threat to peanut production, the cropping system more difficult.
development of effective management
strategies to reduce the primary source of The disease cycles for early and la
leafspot inoculum or retard the rate of leafspot pathogens have been concisely
disease increase is necessary to improve described (APS, 1984; Paningbatan an
peanut yields. Opina, 1990). Although ascospores an
chlamydospores could act as potent.
The development and relative severity inocula, conidia from intact and abscisse
of the two leafspots depend on the complex infected leaflets serve as the main initi
interaction among the host, the pathogen inoculum. Conidia may be detached frol
and the environment. In the southeastern lesions at any time, but peak dispers
part of the United States, there has been a period occurs in the morning when de
gradual shift from predominantly. early dries off and at the onset of rainfall (Smil
leafspot to a substantial late leafspot and Crosby, 1973). The quality an
incidence (Smith and Littrell, 1980). No quantity of initial inoculum contributed I
definite explanation was indicated, but infected and defoliated leaflets have nevi
II11UtLLt ItL UI b ~tc HUliL t.lI LU I IUI i Ll ut jnlu t
practices. Temperatures from 25"C to 30'
and high relative humidities equal to
more than 95% favor disease developme
(Jensen and Boyle, 1966). Depending up(
the host variety and environment
conditions, both leafspots appear in 8-
days (APS, 1984; Paningbatan and Ila
1981). Hemingway (1955) found that
personatumn had much greater epiderr
potential than C. arachidicola based <
incremental lesions during a growii
season of peanut.
Inr a regional peanut disease survey
the Philippines, both early and la
leafspots were found present with the lati
This study therefore aimed I
determine the contribution of infected ar
defoliated peanut leaves in the epidem
development of the early and late leafspo
MATERIALS AND METHODS
Infectiousness of infected and absciss(
One-month old plants of BPI P9 pean
cultivar were inoculated with conidia of
arachidicola and C. personatuni conidia tak(
from 3-week old onion agar cultures. TI
p 1 cui.. 1 1jr .
-idia ml-. Five weeks after inoculation,
icissed infected leaflets were collected,
shed with sterile distilled water and
-dried for 24 hr in the laboratory. The
flets were then subjected to the following
editions of the unsterilized sandy loam
1: leaflets placed on the surface of the dry
1; leaflets on the surface of the wet soil
watering it to field capacity at 2-day
ervals; leaflets buried at 3-inch below the
face of the dry soil; and leaflets buried
3-inch below the surface of the wet soil.
e ambient air temperature and relative
midity ranged from 21 to 35"C and 70
The infectiousness of the two
thogens after leaflet abscission was
Imined at 5-day intervals with a
reomicroscope until the lesions' ability
produce conidia had stopped. At each
npling, 5-10 random leaflets were
shed with sterile distilled water, blotted
r and placed in sterile petri dishes lined
:h moist filter paper. After two days of
ubation at 25-30"C, 25 random lesions
- treatment were examined for conidial
)duction from which the percentage of
ectious lesions was calculated. The
;ree of conidial production per lesion
s also rated as 0 = no conidial
iduction; 1 = 1-25% of lesion area showed
tidial production; 2 = 26-50%; 3 = 51 to
to and 4 = > 75%. Analysis of variance
I comparison of means were made.
ect of temperature on conidial viability
Twenty five days after inoculatihig
arachidicola and C. personatum on a
ceptible peanut cultivar, cv. BPI P9, the
ected leaflets were collected, surface-
-ilized with 5% NaOCI for 2 minutes,
;ed with sterile distilled water, blotted
SCIALLk. FlcICLt 1L i Llllct: iii T: F LC l. 1ilUC VVW LI
)ist filter paper. A crop of conidia of
form age was produced by incubating
lesions for 48 hr in a continuously
hted 25"C-chamber. The leaves
re then air-dried at 28-32"C for 48 hr.
led leaflets were stored at 0, 15, 20, 25,
Conidial viability was examined at
ekly intervals until viability had been
t. With a 10-ul automatic micropipette,
lidia were collected from lesions and
;pended in 10 microliters on sterile
pression slides. After 24 hr of incubation
petri dishes lined with moist filter
pers, germinated conidia were counted.
e treatments were replicated five times
I each replicate consisted of 50-100
ectiousness and sporulation of lesions
Data presented in Table 1 and plotted
Fig. 1 clearly indicate that both
thogens were still infectious by
iducing conidia after removal of infected
ves from the plant. One hundred
cent of lesions of both pathogens within
Says after defoliation remained
)rulating when leaflets were placed on
buried in dry sandy loam soil.
ereafter, the proportion of infectious
ions gradually declined until
ectiousness was totally lost at 60 days
;. 2, Table 2). In wet soil, lesions of both
hogens stopped producing conidia at 20
's after leaflets were placed on the soil
face. However, sporulation of C.
,hidicola and C. personatum drastically
now -a i in 1 r; 4, i --- ---/.,-r.,
Mean 100 a 73 b 56 c 44 ef 43 f 26g
Philipp. Phytopathol. 1995, Vol. 31(1): 9-19 13
2 l UD
0 t UI,
0 10 20 30 40 50 60 0 10 20 30 40 50 60
C. arachidicola C. personatumn
Days of storage
Figure 1. Infectiousness and degree of sporulation of Cercospora arachidicola and
C. personatumn lesions after defoliation as influenced by soil moisture: SD,
placement of leaflets on the surface of the dry soil; SW, placement on the surface
of the dry soil; SW, placement on the surface of the wet soil; UD, placement 3-
inch below the surface of the dry soil; and UW, placement 3-inch below the
surface of wet soil.
Philipp. Phytopathol. 1995, Vol. 31(1): 9-19
The index of sporulation of both
pathogens based on percentage of lesions
area exhibiting sporulation gradually
declined with infected leaflets placed on the
surface or buried in the dry soil (Fig. 2).
Rapid decline in the degree of sporulation
was evident with lesion on leaflets placed
either on the surface or buried in the wet
Effect.of temperature on viability of
Thlp ffpft rf f rliffprpnt ctnrncp
Results show that lesions of both
pathogens could remain infectious ever
after defoliation over certain periods oj
time depending upon the moisture
content of the soil. The 50-day infectious
period of lesions in leaflets deposited or
the surface or buried in the dry sandy loamr
soil represents the maximum length ol
time during which infected leaflets could
serve as potential sources of primary
inoculum or could enable the pathogen tc
tide over peanut-free periods in the
C. 1J TL, 1C r_ -- r_ A _
16 Philipp. Phytopathol. 1995, Vol. 31(1): 9-19
Percent conidial germination
Figure 2. Viability of conidia of Cercospora arachidicola and C. personatum as influenced
by storage temperatures.
Table 3. Degree of sporulation of Cercospora arachidicola and C. personatum lesions fom abcissed infected leaflets of BPI peanut cv. P9 stored
at different conditions in and on the sandy loan soil'
Weeks of Storage
Pathogen Temperature lemperaturi Pathogen
(.C) 0 1 2 3 4 5 6 7 8 9 10 11 Mean Mean
Ca 0 100 96 73 72 71 34 29 3 3 2 I 0 40 a
15 100 97 42 35 26 18 14 0.2 0 0 0 0 28 f
20 100 94 57 46 48 9 7 0 0 0 0 0 30 e
25 100 95 85 53 29 17 1 0 0 0 0 0 32 ef
30 100 80 30 17 1 0 0 0 0 0 0 0 19 g
35 100 32 19 1 0 0 0 0 0 0 0 0 13 i
Mean 100 a 82 b 51 d 37 e 29 g 12 i 8.4 k lo 0.4 op .4 op 0.2 op 0 p 27 a
Cp 0 100 83 56 50 42 22 7 4 3 3 1 0 31 c
15 100 85 65 43 13 11 8 7 6 6 2 0 29 d
20 100 78 66 48 40 32 27 25 12 4 2 0 36 h
25 100 93 64 32 21 16 10 4 1 0 0 0 28 ef
30 100 80 21 10 4 0 0 0 0 0 0 0 18 h
35 100 23 17 3 0 0 0 0 0 0 0 0 12h
Mean 100a 73 c 48 d 31 f 20 h 13 i 9j 7k 41 2m In Op 25 b
'Values, transformed to arc sine for analysis, are means of five replicates. In a column or in a row, means with the same letter are not significantly different at 5,, level by DI)Ml1; (a stands
for Cervospora arachidicila, Cp for C. personatihm.
18 Philipp. Phytopathol. 1995, Vol. 31(1): 9-19
In examining the effect of temperature
on viability, conidia of both pathogens were
found to survive until 10 weeks at 0"C.
Survival of conidia of C. arachidicola and
C. personatun for 6-7 weeks and 8-10 weeks
at 15-25CC designates wider range of
temperature tolerance by the conidia of the
latter. This indicates that in places where
temperature fluctuates within this range,
population of C. personatumn persists better
than that of C. arachidicola, a plausible
explanation why late leafspots is relatively
predominant disease in certain peanut
fields in the Philippines (Paningbatan,
ALLEN, D.J. 1983. Pathology of tropical
legumes. Wiley, N.Y. USA. 488 p.
SOCIETY (APS). 1984. Compendium
of peanut diseases. D.M. Porter, D.H.
Smith and R. Rodriguez-Kabana, eds.
Minnesota, USA. 73 p.
BACKMAN, P.A. and M.H. CRAWFORD.
1984. Relationships between yield loss
and severity of early and late leafspot
diseases of peanut. Phytopathology
FEAKINS, S.D. (Ed.). 1973. Pest control
in groundnuts. PANS Manual No. 2.
London, U.K. Center for Overseas Pest
Research, 3rd ed. 196 p.
GARREN, K.H. and C.R. JACKSON. 1973.
Peanut diseases. Pages 429-494. In:
Peanuts: Culture and Uses. Amer.
Peanut Res. Educ. Assoc., Inc.
Oklahoma State University,
GIBBONS, R.W. 1980. Peanut improvement
research technology for semi-arid
tropics, Pages 27-73. In: Proceedings
of the International Symposium on
Development and Transfer of
Technology for Rainfed Agriculture
and the SAT Farmer. International
Crops Research Institute for the Semi-
Arid Tropics (ICRISAT). Patancheru,
HEMINGWAY, J.S. 1955. The prevalence
of two species of Cercospora on
groundnuts. Trans. Brit. Mycol. Soc.
HUANG, H.C. and G.C. KOZUB. 1993.
Survival of mycelia of Sclerotinia
sclerotiorum in infected stems of dry
bean, sunflower and cafola.
Phytopathology 83: 937-940.
JACKSON, C.R. and D.K. BELL. 1969.
Diseases of peanut (groundnut)
caused by fungi. University of Georgia,
College of Agriculture Experiment
Station Research Bulletin No. 56. p. 7-
JENSEN, R.E. and L.W. BOYLE. 1966. A
technique for forecasting leafspot on
peanuts. Plant Dis. Reptr. 50: 810-814.
KNUDSEN, G.R., H.W. SPURR and C.S.
JOHNSON. 1987. A computer
simulation model for Cercospora
leafspot of peanut. Phytopathology
PANINGBATAN, R.A. 1980. Culture,
morphology and pathogenic
variation of Cercospora species causing
leafspots in peanut (Arachis hypogaea
L.). M.S. Thesis. University of the
Philippines at Los Bafos, College,
Laguna. The Philippines. 116 p.
Philipp. .Phytopathol. 1995, Vol. 31(1): 9-19
rAINIIN\I(AIAIN, K.A. and L.L. ILA(
1981. Two leafspots of peanut in th
Philippines: etiology and hos
response to infection. Phil. Agric. 6'
PANINGBATAN, R.A. and O.S. OPINA
1990. Comparative cohort life an,
reproductivity table analysis c
Cercospora arachidicola Hori an
Phaeoisariopsis personata (Berk. an,
Curt.) von Arx on peanut. Trans
National Academy Sci. Techno
SMITH, U.H. and F.L. CROSBY. 197
Aerobiology of two peanut leafsp
fungi. Phytopathology 63: 703-707.
SMITH, D.H. and R.H. LITTRELL. 198
Management of peanut foliar disease
with fungicides. Plant Dis. 64: 356-36
TENG, P.S. 1985. Integrating crop and pe
management: the need for moi
comprehensive management of yie.
constraints in cropping systems. J. I
Prrf Trnnirc 9(1?. 1 -9i
20 Philipp. Phytopathol. 1995, Vol. 31(1): 20-26
CULTURAL REQUIREMENTS FOR MAXIMUM CONIDIAL
PRODUCTION OF CERCOSPORA KIKUCHII, THE
CAUSE OF PURPLE SEED STAIN OF SOYBEAN
FE M. DELA CUEVA, MARINA P. NATURAL and R. A. HAUTEA
Respectively, University Researcher, Institute of Plant Breeding, Associate Professor,
departmentt of Plant Pathology, and University Researcher, Institute of Plant Breeding,
University of the Philipppines at Los Bafios, College, Laguna.
This research was partly supported by the Department of Science and Technology
mnd the Institute of Plant Breeding, University of the Phlippines at Los Bafos, College,
Keywords: Cercospora kikuchii, conidial production, purple seed stain, soybean
The effect of different types of medium, pH, exposure to light and temperature
on conidial production of Ccrcospora kikuchii was studied. Abundant in vitro
sporulation of the fungus was recorded in mungbean seed decoction agar
followed by soybean leaf decoction agar and V-8 juice agar after 14 days of
Maximum sporulation was noted at pH 6.0 6.6, at 25"C, exposure to
12-hr light and 12-hr darkness daily and lowest sporulation in soybean seed
decoction agar, pH 7.5 at 25'C and at 35"C at pH 6.5 under continuous light.
INTRODUCTION planting material, can result in reduced
germination, weakened seedlings and can
Purple seed stain of soybean [Glycine serve as sources of primary inoculum
inax (L) Merr.] caused by Cercospora kikuchii (Wilcox and Abney, 1973).'
(Matsumoto and Tomoyasu, 1925) is now
an important disease of soybean in the Studies of C. kikuchii in the
Philippines The disease, which is very Philippines are limited due to the difficulty
prevalent during the wet season plantings in isolating and maintaining the fungus
(May October), reduces the quality of in vitro. Varying information on the cultural
harvested seeds (Sinclair and Backman, requirements of the fungus has been
1989). Infected seeds, when used as published. Crane and Crittenden (1967)
Phillpp. Phytopathol. .1995, Vol. 31(1): 20-26 21
reported that C. kikuchii produced mycelial
growth when grown in potato dextrose
agar (PDA), Czapek-dox, and Bacto agars.
Matsumoto and Tomoyasu (1925) and
Murakishi (1951) claimed the fungus to
sporulate only in living host tissues while
Vathakos and Walters (1979) obtained
abundant conidia on dead soybean plant
This study was conducted at the Plant
Pathology Laboratory, Institute of Plant
Breeding, University of the Philippines at
Los Banos from June 1993 to March 1995
to isolate the causal organism of purple
seed stain and determine the best culture
medium, pH, temperature and period of
exposure to light that can induce
maximum in vitro sporulation of the
MATERIALS AND METHODS
Isolation of the fungus
Seeds with purple seed stain were
collected from the newly harvested
soybeans. The isolation of the fungus was
done in three batches. The first batch
consisted of whole seeds, seeds without
seedcoat and seedcoat alone for the second
and third batches, respectively. All the
materials were disinfected by immersing
in 10% sodium hypochlorite for 1 min,
rinsed in three changes of sterile distilled
water, washed in 70% ethyl alcohol and
rinsed in three changes of sterile distilled
water. The seeds were planted in acidified
PDA and incubated at 25"C for 7 days.
Seeds showing signs of Cercospora growth
were examined; fungal growth were
scraped from the seed surface, suspended
in sterile distilled water and examined
microscopically. Conidial suspensions of
C. kikuchii were streaked on agar slants and
colonies were allowed to develop. Pure
cultures were obtained by picking a colony,
suspending in sterile distilled water and re-
streaking on agar slants.
Effect of agar media on growth and
Sporulation of C. kikuchii in various
media was compared using V-8 juice agar
(V-8A), mungbean seed decoction agar
(MSDA), soybean seed decoction agar
(SSDA), soybean leaf decoction agar
(SLDA) and PDA.
MSDA was prepared by boiling 100
g of mungbean seeds, collecting the
decoction and restoring the volume to 1000
ml. Twenty grams of agar was added as
solidifying agent. Similarly, SSDA was
prepared by boiling 100 g of soybean seeds
and SLDA by boiling 300 g soybean leaf.
V-8A and PDA were prepared according
to the method described by Tuite (1969).
A loopful of conidial suspension
standardized at 10; spores/ml from 14-
day culture of C. kikuchii in PDA slants was
streaked on the various media slants. The
cultures were incubated at room
temperature (28 300C). Spore count was
done at 7-day intervals until after 21 days
of incubation. Two ml sterile distilled water
was added per slant to dislodge the conidia.
Conidia were counted using a
Effect of pH on sporulation
SLDA medium was adjusted to 4.0,
4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 pH
levels. The. pH of the media was
determined after sterilization. Conidial
suspensions from 14-day cultures of
C. kikuchii were seeded on the culture
slants and incubated at 25oC.
S"'' .i- .'-.,U"' -, --.. -VI .1 .,. --
0tJIjL/U LIXC.U I VV AZ3 V AILA.LCt. L LCy YJ U3 1
Effect of temperature on sporulation
Conidial suspensions from 14-day
cultures of C. kikuchii were uniformly
seeded on SLDA slants, pH 6.5 and stored
at 20, 25, 350C and room temperature
(28 -30"C ). Sporulation was determined
14 days after incubation.
Effect of light period on sporulation
Five varying light periods were used
to determine the effect of light on
sporulation, viz. 15-hr light with 9-hr
darkness and vice-versa, 12-hr light and
12-hr darkness, 24-hr continuous light, and
9A-hr rrcnfiniiniic i nrknpec A Innnfull of
lypicaL colonies oi L. ALIucLri wete
visible 5-7 days after streaking conidial
suspensions in PDA slants. The pure
culture exhibited white to gray colonies
with no pigmentation on culture media,
and produced conidia ranging from 2.23 -
3.6 um x 50 -100 um.
Effect of media on the growth and
The results showed that MSDA gave
the highest conidial production and
yielded 3.54 x 10' and 4.71 x 10' conidia/
ml after 7 and 14 days of incubation,
respectively (Table 1). This was followed
by SLDA yielding an average of 1.75 x 10M
conidia/ml after 7 days and 2.22 x 10s
conidia/ml after 14 days. No significant
diffprpnce in funcnal -nnrulatiinn was
slants, pH 6.5 and incubated at 25"C. Spore 8A, SSDA and PDA. T
Li L.L.. 1 1 VVW tJ 3 i C tL t. t t.&CI A tILtI -r 1 A LU J y 3 J
incubation and no significant difference
RESULTS AND DISCUSSION was noted between V-8A and SLDA.
Isolation of the fungus The results showed that sporulation ol
C. kikuchii was highly affected by the type
The pure culture of C. kikuchi was of media. All the five media used
readily isolated. from seeds without supported sporulation of C. kikuchii. This
seedcoat after 4-7 days of incubation, contradicts the report of Crane and
The fungus was also observed from the Crittenden (1967) who obtained only
whole seeds and from seedcoats and mycelial-growth of the fungus onPDA. The
associated with Colletotrichum sp., Alternaria isolate used by Matsumoto and Tomoyasu
sp., Phonma sp., Aspergillus sp., Eurotiumn sp., (1925) sporulated on raisin decoction again
Monilia sp., and Fusarium sp. These but not on 13 other decoction agars or on
fungi delayed the growth of C. kikuchii three defined liquid media. Roy and Abney
making isolation more difficult. This result (1977) reported that their C. kikuchii isolates
does not conform with that of Murakishi sporulated on PDA and V-8A while
(1951) who reported that typical mycelial Vathakos and Walters (1979) could not
growth and the purple coloration of the obtain sporulationonthese two media. Yeh
agar characteristic of C. kikuchii were and Sinclair (1980) observed that V-8Abest
produced from all of the seed coats, supported conidial production ol
however, plated cotyledons rarely yielded C. kikuchii while our results showed that
the fungus. the conidial yield in SLDA did not differ
Philipp. Phytopathol. 1995, Vol. 31(1): 20-26 23
significantly with that of V-8A. MSDA gave
the highest conidial yield but was also
favorable for the growth and sporulation
of C. canescens, the causal organism of
mungbean leafspot, hence, SLDA was used
in the subsequent studies. In addition,
SLDA is much cheaper and easier to
formulate than MSDA.
Effect of hydrogen ion concentration (pH)
The results showed that conidial
production was greatly influenced by the
pH of the medium (Table 2). Sporulation
was noted from pH 4.0 to 8.2, with pH 6.6
as the optimum yielding 4.90 x 10' conidia/
ml. The lowest conidial yield, 0.77 x 10'
conidia/ml, was recorded at pH 7.5 but this
was not significantly different from pH 4.0,
4.5, 5.0. 7.0 and 8.0. This result does not
corroborate with that of Murakishi's (1951)
wherein growth of the fungus was
observed at a pH range of 3.0 to 8.6 but no
spores were produced. He reported,
however, that changes in hydrogen-ion
concentration only affects the pigment
produced by the fungus.
Effect of temperature on sporulation
Twenty five degrees centigrade (25"C)
favored conidial production (Table 3). It
yielded an average of 5.29 x 10' conidia/
ml. Cultures incubated at room
temperature (28-30'C) gave an average
conidial count of 3.13 x 10' conidia/ml of
suspension. Cultures of C. kikichii which
were stored at 35"C produced very small
colonies but they failed to grow and
spQrulate even after 14 days of incubation.
The result agrees with Matsumoto and
Tomdyasu (1925) who observed the
occurrence of conidia in cultures stored at
25"C on raisin agar. Nagel (1934) found
temperature between 23 and 27'C favorable
for spore formation of Cercospora beticola
and several other species of Cercospora.
Murakishi (1951), on the other hand, noted
that the the fungus grew very little at 12"C
and failed to grow at 40"C but optimum
temperature for growth was 270C.
Effect of light period
Exposure to 12-hr light and 12-hr dark
period induced the greatest conidial
production (1.60 x 10' ), followed by 15-hr
dark and 9-hr light period (0.56 x 10') while
cultures incubated under continuous light
period yielded the least (0.056 x 10 .)
(Table 4). It was also observed that longer
daily light exposure (15-hr light and 9-hr
dark) brought about significant decrease in
spore production compared to shorter daily
light exposure. Conidial production in this
study was quite low compared to other
studies like effect of media, pH and
temperature because the culture used in
this experiment was just a subculture of the
original isolate. The results obtained,
however, partly conformed with Yeh and
Sinclair's (1980) study which obtained
abundant spore yield at 12-hr of alternating
light and dark and contradicted with
Vathakos and Walters (1979) which yielded
abundant conidial production under
alternating 8-hr of light and 16-hr of dark
but they both reported that conidial
production under continuous dark was
sparse. Lyda et al. (1979), however, found
no significant difference in sporulation of
the fungus between continuous light and
12-hr of alternating light and dark at 20"C
and obtained abundant conidial production
.on V-8A in both complete light and dark.
The results of this study show that
sporulation of C. kikuchii was highly
affected by the type of media. MSDA,
SLDA and V-8A favor maximum conidial
production of the fungus after 14 days of
24 Philipp. Phytopathol. 1995, Vol. 31(1): 20-26
Table 1. Sporulation of Cercospora kikuchii on various agar media at different incubation
Medium' 7 days 14 days 21 days
V-8A 0.52 c 2.12 b 0.29 a
MSDA 3.54 a 4.71 a 0.36 a
SSDA 0.76 c 1.43 d 0.73 b
SLDA 1.75 b 2.22 b 0.70 b
PDA 0.91 c 0.71 b
'V-8A=V-8 Juice Agar; MSDA= Mungbean seed decoction agar; SSDA=Soybean seed decoction agar;
SLDA= Soybean leaf decoction agar; PDA=Potato dextrose agar.
2Mean of 6 replications; means followed by the same letter in a column are not significantly different at 5%
level with DMRT.
Table 2. Sporulation of Cercospora kikuchii 14 days after seeding on soybean leaf decoction
agar adjusted to various pH and incubated at 25C and at 140 foot-candles
pH Before pH After Conidia/ml1
Sterilization Sterilization (x105)
4.0 4.4 1.01 cd
4.5 4.9 2.30 bcd
5.0 5.2 1.26 cd
5.5 5.7 2.53 bc
6.0 6.2 3.48 ab
6.5 6.6 4.90 a
7.0 6.9 2.43 bcd
7.5 7.6 0.77 d
8.0 8.2 2.02 bcd
Means of 6 replications; means with a common letter in a column are not significantly different at 5 % level
IIIpI. rIlyUlpadlmu. IVU VOu. 3 ItI). zu-Lo ILJ
ible 3. Effect of varying temperatures on length of exposure to light and dark period
sporulation of Cercospora kikuchii The highest conidial production was noted
seeded in soybean leaf decoction from cultures incubated under 12-hr of
agar and incubated for 14 days alternating light and dark period.
in continuous darkness
The results of this study suggest that
temperature Conidia/mlP SLDA adjusted to pH 6.0 6.6 incubated
("C) (x10 ) at 25uC at 12-hr of alternating light and dark
periods are the optimum requirements for
20 .0.14c the growth and sporulation of C. kikuchii
.25 5.29a in the Philippines. It was, however,
28-30 3.13b observed that high sporulation cannot be
35 0 sustained after 4 or 5 transfers under these
conditions as the cultures become mycelial
ean of 6 replications; means followed with in nature, hence, it is suggested that to
imon letter are not significantly different at 5% obtain abundant supply of conidia
el with DMRT. sufficient for screening purposes and other
laboratory studies, newly isolated cultures
should be used.
ible 4. Effect of exposure to varying light
periods on sporulation of
Ccrcospora kikuchii seeded in LITERATURE CITED
soybean leaf decoction agar CRITTENDEN.
CRANE, J.L. and H. W. CRITTENDEN.
Light Condition' Conidia/ml 1967. Growth of Cercospora kikuchii
on various media. Plant Dis. Reptr.
12-hr L,12-hr D 1.60 a 51:112-114.
15-hr D, 9-hr L 0.56 b
S0.3 LYDA, S.D., M.D. CHEN and R.H.
rL, 9r D 0.16 d HALLIWELL. 1979. Light-temperaLur"
SL 0.106 d interactions in growth and sporulation
of Cercospora kikuchii. Phytopathology
icubated at 25"C for 14 days; 12 -hr L, 12- hr D =67: 1-A7 (str.).
- hr light and 12- hr darkness daily; 15-hr L, 9-
D = 15-hr light and 4-hr darkness daily; 15-hr MATSUMOTO, T. and R. TOMOYASU.
9-hr 1. = 15-hr darkness and 9-hr light period 1925. Studies on purple speck of
ily; CI, = continuous light; CD = continuous soybean seed. Ann. Phytopathol. Soc.
rkness. Japan 1: 1-14.
Acan of 6 replications; means followed by the
me letter in a column are not significantly MURAKISHI, H.H. 1951. Purple seed
Terent at 5",, level with DMRT. stain of soybean. Phytopathology
cubation. It was also observed that 41:305-308.
nidial production of the fungus NAGEL, C. M. 1934. Conidial production
creased towards either alkaline or acidic
in species of Cercospora in pure culture.
i of the medium. Sporulation was also Phytopathology 24:1101-1 '3.
'served to be greatly influenced by the
26 Philipp. Phytopathol. 1995, Vol. 31(1): 20-2E
ROY, K. W. and T.S. ABNEY. 1977.
Antagonism between Cercospora
kikuchii and other seed-borne fungi of
soybean. Phytopathology 67: 1062-
SINCLAIR, J. B. and P. A. BACKMAN, eds.
1989. Compendium of Soybean
Diseases. The American Phyto-
pathological Society Inc. St. Paul. MN.
TUITE, J. 1969. Plant Pathological Methods.
Burgess Publ. Co. USA. 239p.
VATHAKOS, M. G. and H. J. WALTERS.
1979. Production of conidia by
Cercospora kikuchii in culture.
Phytopathology 69: 832-833.
WILCOX, J. R. and T. S. ABNEY. 1973.
Effects of Cercospora kikuchii on
soybeans. Phytopathology 63:796-797.
YEH, C. C. and J. B. SINCLAIR..1980.
Sporulation and variation in size of
conidia and conidiophores among
five isolates of Cercospora kikuchii. Plant
Dis. 64:373 -374.
hilipp. Phytopathol. 1995; Vol. 31(1): 27-31 27
T. O. DIZON anc
Respectively, University Researcher, Ih
'hilippines at Los Bafos, College, Laguna, PI
)f Tsukuba, Tsukuba, Ibaraki, Japan.
Key words: Aecidium mori, mulberry, n
The occurrence of mulberry rust i.
Zamboanga City. Symptoms of the dise
MULBERRY RUST AND
OF ITS PATHOGEN,
1. M. KAKISHIMA
tute of Plant Breeding, University of the
,pines and Associate Professor, University
!ported to occur for the first time in
consist of formation of distinct bright
Forest Nursery, Baguio City on Sta. Rita, Zamboanga City causing
28 Philipp. Phytopathol. 1995, Vol. 31(1): 27-31
about 80 to 90% infection (personal
This study was conducted to
describe the symptoms of the disease
and the morphology of the causal
MATERIALS AND METHODS
Collection of specimen
Mulberry stems, leaves and petioles
heavily infected with rust were collected
from a mulberry nursery and brought to
the laboratory for examination. The
symptoms were described.
Leaf and stem diseased tissues
were cut into lcm2 and 0.1cm in
length, respectively, and fixed in formalin-
acetic acid-alcohol (FAA) prior to
microscopic examination. The morpho-
logy of the fruiting structures
was described. The size of the
aeciospores was measured using filar
Light microscopy (LM). Fixed
leaves and stem tissues were dehydrated
and rehvdcrated using alcohol series,
embedded in paraffin, sectioned in
microtome .and stained with safranin
and fast ~rieen combination. The
method of lensen (1962) was
Scanning cLlctron microscopy (SEM).
ILeatf pieces were critically-point
d:' 1 d in liquid C02, mounted in
aluminum stubs, coated with gold
i: a sputter .',t.:; .and observed
Bright yellowish orange aecia
were found along the veins and lamina
clearly distinguishable on the underside
of leaf surface (Fig. 1). When aecia
are ruptured, the affected portion
shows a powdery appearance, bright
yellowish orange in color. Severely
infected leaves turn yellow, become
wrinkled, rolled and fall-off.
On young stem, affected portion
become hypertrophied. One or more aecia
are produced forming hypertrophied
lesions (Fig. 2). Severely infected
stems may cease to grow, shrivel
Besides leaves and stems, petioles,
peduncles and flowers are likewise affected
by the fungus. Hypertrophied lesions
producing numerous aecia are commonly
observed. The same symptoms on plant
parts were reported by Hiura (1931).
Morphology of the fungus
Aecia hypophyllous, erumpent,
sub-epidermal, scattered or aggregate,
densely formed on evoluted veins or on
elongated veins, profoundly immmersed,
yellowish; peridia well-developed,
peridial cells loosely conjunct, easily
splitted, oblong, walls thin (Fig. 3 A,B);
aeciospores angularly globose to
ellipsoid, densely verrucose, subhyaline,
catenulate, verrucae distinct on spore
surface, 10-15 x 7-13u (Fig. 3 C,D).
Aecia and aeciospores formed on leaves
and stems were morphologically
Philipp. Phytopathol. 1995, Vol. 311): 27-31 29
Philipp. Phytopathol. 1995, Vol. 31(1): 27-31 29
Figure 1. Aecia of Acciuiiiim iori Barclay on the underside
of leaf surface of mulberry.
Figure 2. Hypertrophied lesion on stem of mulberry
showing four aecia of Accidium iiori.
30 Philipp. Phytopathol. 1995, Vol. 31(1): 27-31
Figure 3. Aecidium mori Barclay. Aecia, surface view and vertical section
showing a peridium (p) under SEM (A,B); aeciospores under
SEM (C) and LM (D).
ipp. Phytopathol. 1995, Vol. 31(1): 27-31 31
RCLAY, A. 1980. A descriptive list of
the Uredinae occurring in the
neighborhood in Simla (Western
Himalayas). Pt. 3 J. Asiatic Soc.,
Bengal Pt. 2, 97p.
RCLAY, A. 1981. Additional Uredinae
from the neighborhoods of Simla. Jour.
Asiatic Soc. Bengal Pt. 2, 225-226.
URA, M. 1931. Observations and
experiments on the mulberry rust
caused by Accidium mori Barclay.
Japanese J. of Botany 5: 253-257.
JSEN, W.A. 1962. Botanical
histochemistry. W. H. Freeman and
Co., London 408p.
'BAYASHI, T. and E. DE GUZMAN.
1988. Monograph of tree diseases
in the Philippines with taxonomic
notes on their associated micro-
organisms. Bull. For. and For. Prod.
Inst., No. 351.
32 Philipp. Phytopathol. 1995, Vol. 31(1): 32
LIALCFJL Wt1 I JVV j- U-1, VVIL-A- --V -%.Y rV .- V .
ak periods of both nematodes occurred on the 12th week. Th
populations from the peak periods until the 16th week could b
petition and crop senescence. The marked increase in pop
matodes from the 16th to the 18th week was probably due
avy rains that occurred during these periods, which could ha(
'~f~"' ""'~'"' ''"-"~~"~~~"
ipp. Phytopathol. 1995, Vol. 31(1): 32-39 33
The build up of plant parasitic
natode populations in the soil is usually
ociated with the incidence of crop injury
I thus forms the basis of advisory work
cropping programs. Nematode
pulation increase and crop yield
trctions are not only influenced by the.
ial density of nematodes in the soil but
> by the plant and nematode species and
pping season (Castillo et al., 1978).
Very few studies on population
tamics of plant parasitic nematodes
'e been conducted in the tropics. In
ia, crop species and growing season
re reported as factors determining
)ulation build up of Tylenchorhynchus
ukhopadyaya and Prasad, 1968).
damba (1974) studied the population
id of R. reniformis in multiple cropping
nts in the Philippines. Castillo et al.
76) reported the changes in population
R. reniformis on seven successively
nocultured crops and observed that
ile susceptible crops favored population
Id up with densities increasing with
nber of croppings, resistant crops
Studies on nematode build up during
dropping period were also conducted.
gh (1976) reported the monthly
:tuations in field populations of
conemoidcs spp., Helicotylenchus
tstera, M. incognita and Pratylenchus zea
corn, tobacco, and tomato in the West
ies. He reported higher densities ofM.
,gnita on susceptible tobacco and tomato
n on the resistant corn. Calinga and
stillo (1978) related the fornightly
tuations in R. reniformis population on
h sitao with root growth and nematode
Generally, density levels of plant
asitic nematodes on susceptible crops
iain either relatively constant or change
y slightly during 'r.e first month of
pping, then rapidly increase thereafter
il they peak at 2 to 3 months after
citing (Ferris and Bernard, 1961; Singh,
6; Castillo et al., 1978).
Studies on population ch .1ges of
ceniformis and M. incognita within a
pping period of tomato, cowpea, squash
lacking. Thus, this study.
MATERIALS AND METHODS
Experimental area and cropping
iod. The experimental area was 10 x 21)
located in San Francisco, San Pablo City.
! soil was loamy with pH 4.9. Soil
lysis showed 2.8% organic matter, 0.15%
191.30 ppm P, and 1.72 meq./110 g K.
s area was planted to tomato, eggplant
I cowpea prior to the conduct of
experiment. These previous crops
-e heavily invaded by the root-knot (M.
)gnita) and reniform (R. rcniformis)
natodes. The experiment was
ducted during the November to March
Preparation of the experimental field.
experimental area was 390 sq. m. Field
paration was made by a native plow and
row. In RCBD, five 5 x 10 m blocks,
rated by a 1.0 alleyway, with five 5 x
m plots each, were prepared. The
tments, replicated five times, were the
crops, namely, tomato, sweet pepper,
ermelon, cowpea and squash, grown
irately in each of the five blocks.
Test plants. The crop species and
eties used and their specifications are
34 Philipp. Phytopathol. 1995, Vol. 31(1): 32-:
shown in Table 1. made at. the 4th week after transplanting
All crops, except cowpea, were started
in seed boxes containing baked composed All crops were further treated with tv
soil and transferred to 9 x 10 cm plastic bags formula of liquid fertilizer (Wonder Grov
containing baked soil about 7 to 10 days namely, 12-6-6 and 6-12-6. The former w
after seedling emergence. applied at fortnightly intervals aft
transplanting/planting until the flowerii
Tomato, sweet pepper, watermelon, stage of tomato, at the rate of 30 ml/
and squash were transplanted in' the liters of water per block. The latt
furrows of the plots of each block. The was applied at the same rate after t]
tomato seedlings were about 1 month old, fruiting stage until the first harvest
while sweet pepper, watermelon and tomato.
squash were about 15 days old when
transplanted. Cowpea was directly seeded. Weeds, insect pests and fung
Basal application of complete inorganic diseases were controlled, when needed, 1
fertilizer (14-14-14) was made at the rate of handweeding, spraying with monocr
49 kg NPK/ha. Side dressing with urea at tophos and spraying with Benom)
the rate of 91 kg N/ha for each crop was respectively, at recommended rates.
Table 1. Crop species u!
Savi ex Hassk.)
d their spec
-90 0.30 x 0.75
-100 0.30 x 0.75
-90 1.0 x 1.50
-90 1.0 x 1.50
"rr r -~r~ ."" \I"~Y'
S */* t __" I l C "-(4 1 J LU L
parasitic nematodes were monitored at
irtnightly intervals after transplanting/
planting until the last harvesting of
ie late maturing crops (squash, sweet
pepper and watermelon). Soil sampling
insistedd of randomly collecting ten 50-cm'
)il and 1 g root samples from each plot at
depth of 7 to 15 cm. These subsamples
ere pooled and a composite 300 cm'
id Ig root sample obtained. Nematodes
om the soil were extracted by the
iutinary sieving, using the 45-um pore
eve (325-mesh) as the terminal sieve
)mbined with the Baermann funnel
chnique. The presence of nematodes in
ie roots was determined by dissecting
iem from roots stained in acid-fuchsin
ctophenol (McBeth et al., 1941).
Identification of nematode species.
species identification of Meloidogyne was
ised on the descriptions provided by
'hitehead (1968). This was done by
:aminations of perineal patterns of 20 egg-
ying females randomly collected from the
ots of each of five crops at the termination
the experiment. Species identification
Rotylenchulus was based on the descrip-
ons of Dasgupta et al., (1968). The Rotylen-
iulis specimens identified were randomly
illected from the nematode samples from
ich crop. They consisted of 10 specimens
Ich of second stage juveniles, adult males,
mature females and adult females.
Nematode populations in each plot
hiring the different sampling periods were
The most abundant nematode in the
perimental area was the reniform nematode,
I II- ,rl Il lt. I i L & V | AIUL- J L ILCII LUItLtJ L I
hloidogync spp. ranked second inabundance.
re species identified on tomato, sweet pepper,
iwpea and squash was M. incognita. On
atermelon, 90% was identified as
. incognita and 10% as M. arenaria.
,cause of the difficulty of separating these
io species during each of the nine
mpling periods and the possibility that
e specimens identified as M. arcnaria were
st variants of M. incognita, all the root-
lot nematodes identified on watermelon
ere considered as M. incognita.
)pulations of the other plant parasitic
,matode genera, namely, Helicotyl'nchus,
,lienchorh ynchus, Pratylenchus,
'phinema were very low (total
changes in nematode populations on
le five crops during the sampling
R. renifornis population on tomato was
latively low during the 2nd, 4th and 6th
eeks after transplanting (39, 6 and 55/
simple, respectively) (Fig. 1). On the 8th
eek, the number increased considerably
,83/sample), then declined (343/sample)
ightly during the 10th week. The
population peaked (948/sample) on the
2th week, afterwhich it drastically
creased starting from the 14th,(352/
simple) to the 16th week (326/sample), the
me the crop was harvested. The
population rose. up (543/sample) again on
ie 18th week.
On sweet pepper and watermelon, the
apulations were consistently low
roughout the experimental period (less than
36 Philipp. Phytopathol. 1995, Vol. 31(1): 32-39
2 4 6 8 10 12 14 16 18
2 4 6 8 10 12 14 16 J1
SM. incognita COWPEA
rLn rL I
2 4 6 8 10 12 14- 16 8
Week after planting
Fig. 1. Population changes of Rotylenchulus renifornnis and Mcloidogyne incognita in soils
and roots of five crops. Underlined sampling periods indicate the last harvesting
of the crops.
40/sample for the former and less
than 100/sample for the latter) (Fig. 2).
These remained somewhat constant
until the last harvests of both crops (18th
Lower populations were noted on
cowpea and squash than on tomato
(Fig. 1). However, the trends in population
changes on the three crops were generally
similar, with peak populations being
consistently observed on the 12th week.
The peak populations on squash and
cowpea were 420 and 750/sample,
M. incognita population on tomato was
also relatively low but tended to increase
gradually starting from the 2nd (13/sample)
up to the 8th week (50/sample) after
transplanting (Fig. 1). On the 10th week, the
number increased considerably (257/sample),
thenpeaked (639/sample) during the 12th week.
The population started to decline from the 14th
(424/sample) to the 16th (304/sample) week,
the time when the crop was last harvested.
The population rose up (429/sample) again on
the 18th week.
On sweet pepper and watermelon, the
populations were likewise consistently low
(less than 301 /sample for R. renifornis) until
the 16th week after transplanting,
afterwhich the population slightly
increased on the 18th week (57/sample for
sweet pepper and 34/sample for
watennelon) (Fig. 2).
Similar to R. rcniformis populations,
populations of M. incognita were lower on
"~L"L~~" """ "T~ ~"'-"~'"'- '-'- '-'-'-'-'- ~-
Philipp. Phytopathol. 1995, Vol. 31(1): 32-39
Week after planting
Figue2. Population im.-inges of
Rotylenchulus reniformnis and
Mcloidogyne incognita in soils and
roots of two crops. Underlined
sampling period indicate the last
harvesting of the crops.
cowpea and squash than on tomato
(Fig. 1). However, the trends in population
changes on the three crops were generally
similar, except that on cowpea the
population peaked on the 14th week while
on squash and tomato, the density peaks
were reached on the 12th week.
Table 2 shows the comparative
susceptibility/resistance of tomato,
cowpea, squash, sweet pepper, and
watermelon, based on mean counts of
R. reniformis and M. incognita 18 weeks after
Mean recoveries of R. rcnifonnis from
tomato, cowpea and squash were not different,
but higher than those from sweet pepper and
2 4 6 8 10 1214 16 -i
watermelon. Recoveries of M. incognita were
highest on tomato and cowpea, followed by
recovery from squash. Lowest recovery was
from watermelon. Recovery from sweet pepper
did not differ from that of recovery from squash
Based on R. reniformnis and M. incognita
recoveries at the termination of the experiment
Ind on the peak populations, tomato, cowpea
.lnd squash were susceptible toboth nematodes,
w while sweet pepper and watermelon were
resistant. Higher recoveries were obtained
rom the susceptible than the resistant crops.
, n the latter, the populations of the nematodes
remained constantly low throughout the
dropping periods. Observations of slight
callingg and presence of a few eggmasses of
both nematodes in roots indicated some degree
of nematode feeding and reproduction on the
resistant crops. Examination of stained roots
also revealed a few R. reniformis and M.
incognita females in feeding position on three
crops. Montalvo and Ennard (1994) reported
watermelon cv. Sugar Baby, the same variety
used in this study, as the least susceptible to
the Puerto Rican population of M. incognita
among the other varieties tested.
In the susceptible crops, the initial
population of R. reniformis and M. incognita
in the experimental area remained relatively
low and changed only slightly during the first 6
weeks of cropping. The drastic increases in
R. renifornis population starting from the 8th
week and in M. incognita starting from the
10th week could probably be attributed to more
root development and nematode reproduction.
Except on cowpea, where the density of M.
incognita peaked on the 14th week, the peak
periods of both nematodes occurred on 12th
week or about 3'months of cropping. These
38 Philipp. Phytopathol. 1995, Vol. 31(1): 32-39
Table 2. Mean numbers of Rotylenchulus renirfonris and Meloidogyne incognita in plots
grown to five crops during the termination of the experiment (18 weeks
Nematode recoveries/300 cm" soil and 1 g roots
Crop Species R. rcnifoniis M. incognita
Tomato 542 b 429 c
Cowpea 414 b 479 c
Squash 270 b 87 b
Sweet pepper 30 a 57 ab
Watermelon 27 a 34 a
Data are means of five replicates. Among crops, means followed by the same letters are not significantly
different at the 5% level with DMRT.
findings concurred with previous general
observations (Ferris and Bernard, 1961; Singh,
1976; Castillo et al., 1978). The consistent
decline in nematode populations even on the
susceptible crops from the peak periods until
the 16th week could probably be related to
competition for food and crop senescence. This
agreed with the report of Calinga and Castillo
(1978) on bush sitao. The marked increases in
R. reniformnis and M. incognita on the
susceptible crops from the 16th week up to the
18th week or even after the last harvest of
tomato (16th week) and cowpea (14th week)
could probably be due to the unusual
heavy rains that occurred during these
periods. Abundant moisture in the soil
could had stimulated hatching of nematode
In controlled pot experiments, where
newly hatched juveniles or eggs were placed
close to roots, one life cycle of R. renifonnis
and M. incognit. on susceptible crops were
reported to be less than 30 days (Birchfield,
1962; Sivakumar and Seshadii, 1971) and less
than 50 days, (Lim and C astillo, 1978; Perez
and Castillo, 1973), respectively. Under field
conditions, where the nematodes still have to
locate and reach the roots before they could
feed, the life cycle takes longer, as indicated in
the present study. For instance, on the
susceptible tomato and cowpea, completion of
the first life cycle of R. reifornnis appeared to
be within the 6th to 8th week; that of M.
incognita, within the 8th to 10th week. This
information is useful when susceptible trap
crops, like Colopogniumn and Centrosema, are
used to manage nematode populations.
BIRCHFIELD, W. 1962. Host-parasite
relations of Rotylenchuilus reniformis on
Gossypiuin hirsutium. Phytopathology
CALINGA, R.H. and M.B. CASTILLO. 1978.
Population dynamics of plant parasitic
nematodes. II. Rotiylenchulus rnifornnis
Philipp. Phytopathol. 1995, Vol. 31(1): 32-39 39
onmonoculturedbushsitaoinnemacur- McBETH, C.W., A.L. TAYLOR and A.L.
treated and non-treated farmer's field. SMITH. 1941. Note on staining
Phil. Phytopathol. 14: 93-98. nematodes in root tissues. Helminthol.
Soc. Wash. Proc. 8: 26.
CASTILLO, M.B., M.B. ARCEO and J.A.
LITSINGER. 1978. Population MONTALVO, A.E. and J. ENNARD. 1994.
dynamics of plant parasitic nematodes. I. Reaction of ten cultivars of watermelon
Rotylcnchulus rcnifonnis in -a poorly (Citrullus lannatus) to a Puerto Rican
drained soil and its effect on yield of population of Meloidogyne incognita.
field legumes. Phil. Agric. 61:238-252. Ann. Appl. Nematol., J. Nematol. 26:
CASTILLO, M.B., N.B. BAJET and R.R.
HARWOOD. 1976. Nematodes in MUKHOPADHYAYA, M.C. and S.K.
cropping patterns. I. Population of PRASAD. -1968. Population dynamics
Rotylenchulus rcnifornis on successively of Tylenchorhynchus. Nematologica 14:
monocultured crops. Phil. Agric. 59: 404-418.
PEREZ, R.A. and M.B. CASTILLO. 1973.
DASGUPTA, D.R., D.T. RASKI and S.A. Development and histopathology of
Rotiylenchulus Lintord and Uliveira, 1W4U
(Nematoda: Tylenchidae). Helminthol.
Soc. Wash. Proc. 35: 169-191.
FERRIS, V.R. and R.L. BERNARD. 1961.
Seasonable variations of nematode
populations in soybean field soil. Plant
Dis. Reptr. 45: 789-793.
LIM,B.K.andM.B.CASTILLO. 1978. Interactionsof
Meloidogyne incognita and Rotylnchnulus
rcnifonris with selected soybean varieties.
Kalikasan, Phil. J. Biol. 7: 165-176.
MADAMBA, C.P. 1974. Population
dynamics of plant parasitic nematodes
under various cropping sequences. Adv.
in Food, Agr., For. and Comm. Res.
and Dev. II. p. 116-123. UPLB-CA,
rnytopatnoi. v: 0o-0/.
SINGH, N.D. 1976. Studies on the
population dynamics of selected plant
nematodes on three crops. Plant Dis.
Reptr. 60: 783-786.
SIVAKUMAR, L.V. and A.R. SESHADII.
1971. Life history of the reniform
nematode, Rotylcnchulus reniformis
Linford and Oliveira. 1940. Indian J.
Nematol. 1: 7-20.
WHITEHEAD, A.G. 1968. Taxonomy of
Meloidogyne (Nematoda: Hetero-
deridae) with descriptions of four new
species. Trans. Zool. Soc. Lond. 31:
40 Philipp. Phytopathol. 1995, Vol. 31(1): 40-51
ISOLATION AND TRANSMIS
VIRUS IN TH
LOLITA M. DOLO
Respectively, University Researcher,
Professor, Department of Plant Pathology
A portion of MS Thesis of the senior at
at Los Bafios Graduate School.
Keywords: Bemisia tabaci, isolation, g
A virus causing the leaf curl
maintained in tomato. The putativ
transmitted by whiteflies in a circulati
after 2 hr acquisition access period
has a latent period of 24 hr in the vec
symptoms 2-3 weeks after inoculation
implantation method but not mechanic<
virus include Datura stranmoniium, I
N. sylvestris. Different tomato entries
.urls, crinkling, leaf size reduction a
The TLCV isolates positively react
radioactive DNA probes specific for
coi ntries. The results confirm the c
gemmivirus TLCV in the Philippines.
Tomato yellow leaf curl is a severe viral
disease of tomato. It has been commonly
observed in natural plantings of tomato in
several countries including Thailand
(Atthatom and Sutabutra, 1986), Indonesia,
Taiwan (Green et al., 1987) and has been
reported to be serious in tomato in the
ON OF TOMATO LEAF CURL
S and N. B. BAJET
stitute of Plant Breeding, and Assistant
university of the Philippines at Los Bafios,
or submitted to University of the Philippines
iniviruses, tomato leaf curl virus, tomato,
disease in tomato was isolated and
omato leaf curl virus (TLCV) wai
manner. The virus was transmitted
I 1 hour inoculation access period. It
and inoculated plants developed the
'LCV can be transmitted by the tissue
y transmissible. The host range of the
otiana glutinosa, N. tabacum, and
acted with varying degrees of leaf
to monoclonal antibodies and to non-
iato yellow leaf curl virus from other
urrence of the B. tabaci transmitted
Eastern Mediterranean countries and the
Middle East (Czosnek et al., 1988).
Tomatoes showing similar symptoms have
also been described in North and Central
Africa (Makkouk and Laterot, 1983) and
in the Philippines (Retuerma et al., 1971).
The virus could infect tomatoes at all
Philipp. Phytopathol. 1995, Vol. 31(1): 40-51 41
reductions ranging from about 17 to 100%
depending on the occurrence of the
whitefly vector, Bernisia tabaci Genn. (Saikia
and Muniyapa, 1989; Castellani et al., 1984).
The incidence of the disease was reported
to be high in summer at Southern India
(Saikia and Muniyapa, 1989), during
autumn in Israel (Berlingen et al., 1988)
while Al-Musa (1982) has observed
tremendous increase of leaf curl incidence
from 13.2% of spring grown tomatoes to as
high as 100% in fall grown tomatoes.
A lot of work is known about tomato
yellow leaf curl virus (TYLCV) including
its relation to B. tabaci (Cohen, 1967;
Cohen and Nitzani, 1966), isolation and
purification from tomatoes (Supat et al.,
1990; Czosnek et al., 1988), serological
(Pissawan et al., 1991), physical (Hassain
and Ahmed, 1989) and biochemical
characteristics (Brunt et al., 1990). TYLCV
belongs to a group of bipartite
geminiviruses characterized by genomes
consisting of two 2.7 kb, circular, single
stranded DNA components designated
A and B (Stanley and Gay, 1983;
Hamilton et al., 1983; Howarth et al., 1985).
In the Philippines, beside the report
on the occurrence and transmission of a
putative tomato leaf curl virus, by B. tabaci
(Retuerma et al., 1971; Benigno, 1976), no
other study was conducted on this virus.
The importance of leaf curl disease in
This study was conducted to
understand the nature of the tomato leaf
curl by performing isolation and
transmission studies using sap,
whiteflies and the tissue implantation
MATERIALS AND METHODS
Isolation and transmission
Tomato plants with leaf curl symptoms
collected from the field were used as
sources of inoculum for mechanical
inoculation and isolation using whiteflies,
B. tabaci identified by Dr. A. C. Sumalde,
Department of Entomology, UPLB.
The inoculum for mechanical
transmission was prepared by grinding
young tomato leaves in 0.1 M phosphate
buffer, pH 7.5 containing 1% sodium sulfite
and carborundum. Inoculations were
made by rubbing the leaves of test plants
with cotton swab soaked in the crude sap.
The test plants were Nicotiana glutinosa L.,
N. tabacumn L., N. sylvestris L., Chenopodiumi
arnaranticolor Coste and Reyn, Datura
stramnoniuin L. and the three cultivars of
Lycopersicon esculenturn Mill., viz. Seeda,
Marikit and VC 11-1.
42 Philipp. Phytopathol. 1995, Vol. 31(1): 40-51
and maintained in tomato cv. Seeda inside allowed for 48 hr AAP on TLCV-infected
insect proof screencages. tomato plants. After AAP, 1, 5 and 20
whiteflies were transferred to each healthy
Transmission experiments were tomato seedling and allowed for 24-hr IAP.
further conducted using the isolate as the Each treatment consisted of 10 seedlings
virus source to confirm for the presence of and replicated two times. The whiteflies
other viruses. The different indicator hosts were killed with insecticide and test plants
and test plants previously specified were were kept in Mylar cages for symptom
used -in sap inoculation, whitefly development.
transmission and by tissue implantation
method. The first two methods were done Acquisition access period AAP was
as previously described. In the tissue determined by allowing non-viruliferous
implantation method, a piece of 12-15 mm whiteflies to feed on TLCV-infected plants
bud was cut from the middle portion of the for 2, 4, 8 and 24 hr AAP. Twenty viruli-
stem of a 5-week old infected tomato plant ferous whiteflies were then transferred to
and was inserted to a previously slitted healthy tomato seedlings for 24 hr IAP and
bark in the stem (15-20 mm long) of a 6- were killed with insecticide spray.
week old healthy tomato seedling. The
inoculated plants were incubated in insect Inoculation access period After 48-
proof screencages for symptom hr AAP, viruliferous whiteflies were
development, allowed to feed on healthy tomato
seedlings for periods of 1, 2, 4 and 24 hr
The host range of the TLCV was
determined by infesting 10-15 seedlings
of C. amaranticolor, C. quinon, D. stramonium,
D. metel, N. tabacuin, N. glutinosa, N.
sylvcstris, Capsicini anuumi, L. esculentum
cv. Marikit, VC 11-1 and Seeda, Goinphrena
globosa and Ciecumis sativus with
20-30 virus-exposed whiteflies per
seedling for 24 hr. Forty five days after
inoculation, non-viruliferous whiteflies
were allowed to feed on all the plants
that showed symptoms for back
inoculation of the virus to healthy tomato
Number of whiteflies Transmission
tests were made to determine the minimum
number of whiteflies that can transmit
TLCV.' Non-viruliferous whiteflies were
i-lr ana were Kinea Dy Insectlclae spray.
Latent period The latent period of
TLCV in the whitefly was determined by
allowing the whiteflies to feed on the TLCV
source for a 48 hr AAP. Simultaneously,
all the virus-exposed B. tabaci individuals
were transferred to a healthy tomato
plants. Groups of 20-25 whiteflies were
collected from this tomato plant after
2, 8, 20, 24 and 48 hr and each group
was then transferred to healthy tomato
seedlings and allowed 24 hr IAP. After
this 24 IAP, the inoculated plants
were sprayed with insecticide. Plants
inoculated by each group were
incubated separately for symptom
development. Incubation period in the
plant was studied by determining
the number of days that leaf curl
symptoms appeared on six commercial
varieties of tomato after inoculation with
Philipp. Phytopathol. 1995, VoL 31(1): 40-51 43
Monoclonal antibody test/non-radioactive
The isolates showing a range of leaf
curl symptoms (Fig. 1) were sent to
Prof. B. D. Harrison, Scottish Crop
Research Institute (SCRI), United
Kingdom. These samples were tested for
the presence of the leaf curl virus using
the monoclonal antibodies produced
against isolates of TYLCV at SCRI. The
results were sent back to us. Another batch
of samples exhibiting leaf curl and
yellowing symptoms were likewise sent to
Dr. Sylvia K. Green, Asian Vegetable
Research and Development Center
(AVRDC), Taiwan. The samples were
tested for their reactions to the different
TYLCV isolates using the non-radioactive
succeeding tests / assays ana
44 Philipp. Phytopathol. 1995, Vol. 31(1): 40-51
Transmission of TLCV Host range and symptomatology
Varying degrees of leaf curl and The reaction of the different virus
stunting were observed on tomato cv. indicator hosts and test plants are shown
Seeda, Marikit and VC 11-1 2-3 weeks after in Table 3. Only D: stramonium, N. tabacum,
exposure with viruliferous whiteflies. N glutinosa, N. sylvestris and the three
Back inoculation of TLCV to healthy cultivars of L. esculentum developed
N. glutinosa, N. tabacum, D. stramonium, symptoms 2-3 weeks after inoculation
C. amaranticolor and the three cultivars of using whiteflies. The virus induced
L.cscul'entum was successful using whitefly chlorosis and leaf curl symptoms on
vector, but failed by mechanical inoculation D. stramonium, while leaf puckering, leaf
[Table 1). About 60% of the leaf curl curl and crinkling symptoms were
infection was transmitted to healthy tomato exhibited in N. tabacum, N. sylvestris and
seedlings by tissue implantation N. glutinosa, respectively (Fig. 3a-d). On
method. Whitefly transmission was more the tomato cv. Seeda, interveinal yellowing,
efficient than tissue implantation method crinkling and stunting developed while the
(Table 2). cv. Marikit and VC 11-1 showed severe
upward leaf curls, reduction of leaf size and
The results of the transmission study severe plant stunting (Fig. 2). The other
were consistent with the findings of other species namely, C. amaranticolor, C. quinoa,
researchers (Czosnek et al., 1988; Atthatom D. metel, C. anuum and C. sativus failed to
and Sutabutra, 1986; Green, 1986). They develop any symptoms of the virus. Back
have successfully transmitted TYLCV from inoculation to healthy tomato seedlings
tomato by using whiteflies and grafting but using whiteflies yielded negative results
not by sap inoculation, implying their immunity to the virus
Tahle 1 Trnnmis'inn of the nutative tomato leaf curl virus to different indicator hosts
Indicator Host Tissue
Nicotiana glutinosa +
N. tabacum +
N. sylvestris +
Datura stramonium +
I NT--f4-t f-% /\ 1 rn Mn yr a nt Plct- I'vtro (+ rn -A
ith virus symptoms.
Philipp. Phytopatnol. 1995, Vol. ,1(1): 40-51 45
figure 2. Reactions of some tc
a) Marikit b) Marilag
Figure 3. Symptoms of the pui
(TLCV) in some hosts:
in Dnatiira strlnTlll ni l
glutiuosa (B), downward
(C) pucket ing in N. si/L
ito cultivars to TLCV:
eeeda d) VC 11-1.
e tomato leaf curl virus
n chlorosis and leaf curl
), leaf curl in Nicotiana
aaf curling in N. tabacunt
46 Philipp. Phytopathol. 1995, Vol. 31(1): 40-51
The host range of TLVC was confined
to solanaceous plants. Similarly, th<
host range of TYLCV as reported bI
Atthatom and Sutabutra (1986) was als(
limited to solanaceous plants. However
Green (1986) observed that the Taiwar
isolate of TLCV could be experimental]
transmitted to D. stramoniumn, Pctunim
hybrida, Physalis floridana, Solanuni
t-iberosurn and Lonicera japonica. Tho
putative TLCV as described by Retuerm;
et al. (1971) known to infect tomato bu
not other plant species including
N. glutinosa, D. stramonium, C. anuum
Gossypiumn, hirsutuin, Canna indica, Vinc
rosea and N. tabacun. Results suggest tha
the putative TLCV might be different fron
the isolate used by Retuerma et al. (1971
Transmission Efficiency. Result:
showed that 100% transmission of TLCO
can be attained by using 20 whiteflies pe
plant. Transmission efficiency was reduce
to 75% and 10% when the number o
whiteflies was reduced to five and one pe
plant, respectively (Table 4). Green (1986
observed that one whitefly was sufficient
to cause an effective transmission rate o
the leaf curl virus.
Acquisition Access Period Thi
putative TLCV can be acquired in.2 h
feeding but 100% transmission wa:
achieved only if acquisition period wa:
extended to 24 hr. This means that as the
acquisition feeding increased, the rate o
transmission also increased (Table 5)
Atthatom and Sutabutra (1986) noted i
shorter period of acquisition feeding whicd
is 15-30 min while Green (1986) reported i
1 hr acquisition feeding for TYLC\
Inoculation Access Period TLCV car
be transmitted after 1 hr IAP while 24 hi
IAP resulted to 100% transmission
Increasing IAP improved the efficiency o0
transmission (Table 6).
Latent Period The TLCV wa,
transmitted after 24 hr after AAI
(Table 7). This suggests that the virus
required an incubation of 24 hr in th(
whitefly before it could be efficiently
transmitted. The presence of latent perioc
indicated TLCV as a circulative virus
Circulative viruses have latent period in the
insect vector of about 4-48 hr (Matthews
1991; Walkey, 1985). It is consistent witl
the other geminiviruses (Bird anc
Maramorosch, 1978; Cohen and Nitzany
1966; Goodman et al., 1977; and Russo e
al., 1980). The latent period of TLCV is 2.
hr and the symptoms developed-2-3 week,
after IAP. Similar findings were observec
on the Thailand isolate (TYLCV) (Atthatorr
and Sutabutra, 1986). Cohen (1967
observed 21 hr latent period of the TYLC\
in the vector in Israel. This was contrary tc
the Taiwan leaf curl virus and the JapanesE
tomato yellow dwarf virus that were
reported to have a short latent period of
hr (Green, 1986; Osaki and Inouye, 1978)
Retuerma et al. (1971) also noted one hi
acquisition feeding and 3 min inoculatior
feeding periods for TLCV (Philippine
isolate) that were shorter than the finding,
Philipp. Phytopathol. 1995, Vol. 31(1): 40-51 47
with 10 plants per replication.
of some test plants to the putative tomato le.
tticolor no reaction
vein chlorosis; leal
downward leaf cu
48 Philipp. Phytopathol. 1995, Vol. 31(1): 40-51
Table 4. Transmission efficiency of the Table 7. Acquisition access period of the
putative tomato leaf curl virus on putative tomato leaf curl virus by
tomato cv. Seeda using Bemisia Bemisia tabaci.
Latent Period Transmission
Number of Transmission (Hours) Efficiency (%)
Insect/Plant Efficiency (%)
1 5 8 0
5 75 20 0
20 95 24 55
showed severe symptoms 3 weeks after
Table 5. Acquisition access period of the inoculation.
putative tomato leaf curl virus by
Beinisia tabaci Relationships with other TYLCV isolates
Acquisition Transmission All the five samples sent to SCRI
Feeding Efficiency reacted strongly with monoclonal
(Hours) (%) antibodies against few isolates of TYLCV
(B. D. Harrison, personal communication).
2 10 Similarly, the fresh tomato samples with
4 20 leaf curl and yellowing symptoms which
were sent to Dr. Sylvia K. Green of AVRDC,
Taiwan hybridized positively with the
24 100 Thailand yellow leaf curl virus isolate and
the S'outh Indian leaf curl virus but
negatively to the Taiwan isolate using the
non-radioactive labelled probes (S. K.
Table 6. Inoculation access period of the Green, personal communication)
putative tomato leaf curl virus by
Bemisia tabaci. The putative TLCV can be transmitted
by tissue implantation method, but not
Acquisition Transmission using sap transmission. The host range is
Feeding (Hours) Efficiency (%) limited only to some solanaceous species
including D. strainonium, N. glutinosa,
1 25 N. tabacumn, N. sylvestri, and L. esculentum.
2 30 TLCV can be acquired by B. tabaci
after 2 hr and transmitted after 1 hr IAP.
4 55 The latent period of T LCV in the vector
24 100 was 24 hr and the symptoms developed
)L.. U LIltt t-ULL-LIVIT L UlIIlLcLUL IC11 LU]L
tomato % Transmission'
:ultivar 7 Days 14 Days 21 1
trikit 0 60 11
: 11-1 0 65 11
3 0 10 9
tigaya 0 10 11
allow Plum 0 90 11
proved Pope 0 10
1 of 2 replication; with 10 plants per replication.
ioculated seedlings 2-3 weeks after LITERATURE CITED
t feeding. TLCV reacted strongly with
nonoclonal antibodies and DNA AL-MUSA, A. 1982. Incidence, e(
es specific for TYLCV from other importance and control of
tries, yellow leaf curl virus in Jorda
Disease 66: 581-583.
The results provide evidence that
'LCV, a gemirnivirus and belonging ATTHATOM, S. and T. SUTAI
ie group of B. tabaci transmitted 1986. Tomato yellow leaf curl
,es reported elsewhere (Cohen, 1967; Thailand. In: Proc. Plan
itom and Sutabutra, 1986; Green, 1986) Diseases of Horticultural
r in the Philippines and it is related in the Tropics and Subtropics
the TYLCV isolates from other Book Series no. 33, Taipei,
Tries. Further studies should be pp.61-64.
ucted to establish the physical and
iemical characteristics and probable BENIGNO, D. A. 1976. Plant virus
ability. Likewise, disease management in the Philippines. In: Pro
igh host plant resistance should be .posium on Virus Diseases of
er pursued and developed as tomato Crops. Tropical Agriculture I
var gave different reactions to the Center Ministry of Agricult
V (Dolores, 1995). Forestry, Kitanakazuma Yata
Tsukuba-gun, Ibarakiken, 300-
50 Philipp. Phytopathol. 1995, Vol. 31(1): 40-51
BERLINGEN, M. J. R. DAHAN and S.
MORDECHI, 1988. Integrated pest
management of organically grown
greenhouse tomatoes in Israel. Appl.
Agric. Res. 3 (5) 233-238.
BIRD; J. and K. MARAMOROSCH. 1978.
Viruses and virus diseases associated
with whiteflies. Adv. Virus Res. 22:55-
BRUNT, A., K. CRABTREE and A. GIBBS.
1990. Viruses of tropical crops. CAB
International, Wallingford Oxon OXO
8DE UK, p 29-30.
CASTELLANI, E., A. M. NUR, and M. I.
MOHAMED. 1984. Tomato leaf curl
disease in Somalia. Annalidelia-Facolta-
degli-Studi Italy, 12 pp. 145-161.
COHEN, S., and F. E. NITZANI. 1966.
Transmission and host range of the
tomato yellow leaf curl virus.
Phytopathology 56: 1127-1131
COHEN, S. 1967. The occurrence in the
body of Biemisia tabaci of a factor
apparently related to the phenomenon
of "periodic acquisition of tomato
yellow leaf curl virus". Virology 31:
CZOSNEK, H., P. BER, Y. ANTIGNUS, S.
COHEN, N. NAVOT and D. ZAMIR.
1988. Isolation of tomato yellow leaf
curl virus, a geminivirus.
DOLORES, L. M. 1995. Isolation and
characterization of a virus causing
the leaf curl disease of tomato,
Lycopcrsicon csculentumn Mill. in the
Philippines. M.S. Thesis, UPLB,
GOODMAN, R.M., J. BIRD and P.
THONGMEEARKOM. 1977. An
unusual virus-like particle associated
with golden mosaic of beans.
Phytopathology 67: 37-42.
GREEN, S. K. 1986. Virus disease of tomato
and Chinese cabbage in Taiwan and
source of resistance. In: Plant Virus
Diseases of Horticultural Crops in
the Tropics and Subtropics, FFTC,
Taiwan, Republic of China. March
GREEN, S. K., Y. SULYO and D. E.
LESEMANN. 1987. Leaf curl virus
on tomato in Taiwan Province. FAO
Plant Prot. Bull. 35:62.
HAMILTON, W. W., Q. D. M. BISARO,
R. H. A. COUTTS, and K. W. BUIK.
1983. Demonstration of the bipartite
nature of the genome of a single
stranded DNA plant virus by infection
with the cloned DNA components.
Nucleic Acid Res. 11, 7387-7396.
HASSAIN. M. and K. M. AHMED. 1989.
Electron microscopy of tomato leaf
curl virus. Bangladesh Botanical
Society, Dhaka Chittagong Univ. In:
Proc. 6th National Botanical
Conference, Chittagong, Bangladesh,
BBS 1989. p. 11.
HOWARTH, A. J., J. CATON, M.
BOSSERT, and R. M. GOODMAN.
1985. Nucleotide sequence of
bean golden mosaic virus and model
for gene regulation in gemini
viruses. Proc. Nat'l. Acad. Sci., USA
IPB Annual Report. 1986. Institute of Plant
Philipp. Phytopathol. 1995, Vol. 31(1): 40-51 51
MAKKOUK, K. M. and H. LATEROT.
1983. Epidemiology and control of
tomato yellow leaf curl virus. In: Plant
Virus Epidemiology, R. T. Plumb and
J. M. Thresh, eds. Blackwell, Oxford.
MATTHEWS, R. F. 1991. Plant Virology.
3rd ed. Academic Press. New York.
OSAKI, T, and T. INOUYE. 1978.
Resemblance in morphology and
intranuclear appearance of virus
isolated from yellow dwarf diseased
tomato and leaf curl diseased tobacco.
Ann. Phytopathological Soc. Japan 44:
PISSAWAN, C., A. MURAYAMI and M.
IKEGAMI. 1991. Tomato yellow leaf
curl in Thailand and tobacco leaf curl
virus in Japan are serologically
identical. Ann. Phytopathol. Soc.
Japan 57: 595-597.
RETUERMA, M. L., G. O. PABLEO and
W.C. PRICE. 1971. Preliminary
study of the transmission of Philippine
tomato leaf curl virus by Bemisia tabaci.
Philipp. Phytopathol. 7: 29-34.
RUSSO, M., S. COHEN, and MARTELLI,
G. P. 1980. Virus like particles in
tomatoplants affected by yellow leaf
curl disease. J. Gen. Virol. 49 (1): 209-
SAIKIA, A. K. and V. MUNIYAPA. 1989.
Epidemiology and control of tomato
leaf curl virus in Southern India. Trop.
Agric. 66 (4):350-354.
STANLEY, J. and M. R. GAY. 1983. The
nucleotide sequence of cassava
latent virus DNA. Nature (London)
SUPAT, A., C. PISSAWAN, T.
SUTABUTRA, and R.
Characterization of nucleic acid of
tomato yellow leaf curl virus. Kasetsart
J. (Nat. Sci. Suppl.) Vol. 24: 1-5.
WALKEY, D. G. A. 1985. Applied Plant
Virology. William Heineman Ltd.,
London. 329 p.
We would like to thank Drs. Brian
D. Harrison and Sylvia K. Green for
preparing monoclonal antibody and
nucleic acid hybridization analyses,
respectively ,of the isolates.
52 Philipp. Phytopathol. 1995, Vol. 31(1): 52-53
RHIZOCTONIA SOLANI CAUSING COLLAR ROT OF
KAMANTIGUE (IMPATIENS BALSAMINA L)
E. H. NATOC and NAOMI G. TANGONAN
Respectively, Former Undergraduate Student and Professor, Division of Plant
Pathology, College of Agriculture and Director for Research, University of Southern
Mindanao, Kabacan, Cotabato.
Keywords: collar rot, Inpatiens balsainina, kamantigue, Rhizoctonia solani
Collar rot disease of kamantigue (Impaticns balsamina) was reported and
described. Infected plant showed yellowing or chlorosis of the leaves, stunted
growth, and wilting followed by drying or death of plant. The causal organism
was reported as Rhizoctonia solani.
Symptoms of Collar Rot in
Kamantigue or touch-me-not Kamantigue
(Inpaticns balsamina L.) is a sun-loving,
very floriferous annual herb, 1-2 feet high, Initial symptoms of collar rot disease
with narrow pointed toothed leaves observed in kamantigue is the yellowing
(Steiner, 1960). It is an attractive border or chlorosis of the leaves, stunted growth
plant that produces abundant flowers and wilting followed by drying or death of
of different colors-red, pink, violet, fuchsia the plant. On the collar region or base of
or tangerine. Diseases such as powdery the plant above ground, symptoms of
mildew (Banigued, 1990), rust water-soaked discoloration, rotting of
(Divinagracia, 1985), and mosaic (Ocfemia, tissues, and necrosis followed by girdling
1924) have been reported to attack this the affected region were noted. The
ornamental plant. presence of white to brownish mycelial
threads and round, dark brown sclerotial
Rhizoctonia solani Kuhn., an ubiquitous bodies are apparent on the rotting region
fungal pathogen, is known to cause (Fig. 1).
various diseases of about 34 economically
important crop-plants (Tangonan and Causal Pathogen of Collar Rot of
Quebral, 1992). A cursory look at some Kamantigue
local kamantigue plants grown in
USM revealed a heretofore unreported In potato sucrose agar (20 g potato,
collar rot disease caused by the same 15 g sugar, 15 g agar, and 1,000 ml water)
t. .ngus. culture, the fungus produced thick mycelial
Philipp. Phytopathol. 1995, Vol. 31(1): 52-53 53
growth, creamy white or light brown to
dark brown after a week, and globose
sclerotial bodies. The characteristic right
angle branching of its hyphae when viewed
through the microscope is shown in Fig. 2.
Pathogenicity test on healthy
kamantigue plant showed characteristic
symptoms described above in less than two
weeks after inoculation. The fungus was
identified as Rhizoctonia solani.
This is the first report on the occurrence
of Rhizoctonia solani causing collar rot
disease of kamantigue or touch-me-not.
BANIQUED, N.C. 1990. Survey and
identification of diseases attacking
ornamental and medicinal plants. BPI-
CES in PCARRD Research Highlights
'89, Los Bafos, Laguna (Abstr.).
DIVINAGRACIA, G.G. 1985. Diseases of
important foliage and flowering
ornamental plants. NRCP-UPLB
Terminal Rept. (Mimeogr.).
OCFEMIA, G.O. 1924. Notes on some
economic plant diseases new in the
Philippine islands. Phil. Agric. 13:163-
STEINER; M.L. 1960. Philippine ornamen-
tal plants and their care. 2nd ed.,
Carmelo and Bauermann, Inc., 233 p.
TANGONAN, N.G. and F.C. QUEBRAL.
1992. Host index of plant diseases in
the Philippines. 2nd ed., 273p.
Figure 1. Collar rot disease of kamantigue
or touch-me-not caused by
Rhizoctonia solani. Note girdled
portion on the base of the stem.
I gue' 2.
Characteristic right angle hyphae
of Rhizoctonia solani.
54 Philipp. Phytopathol. 1995, Vol. 31(1): 54-57
INOCULATION TECHNIQUES FOR SCREENING RESISTANCE
AGAINST LEAF DISEASES OF BANANA UNDER
LORNA E. HERRADURA and C.R. CARREON
This research was supported by the IDRC-PCARRD Banana (Philippines) project.
Respectively, Agriculturist II and Research Assistant, Davao National Crop Research
and Development Center, Bago-Oshiro, Davao City, Philippines.
Keywords: banana, black cross, cordona leaf spot, freckle, inoculation techniques,
screening, resistance, yellow sigatoka
Inoculation methods namely, clipping for black cross, swabbing of spore
suspension for cordana leaf spot and yellow sigatoka and the combination of the
two for freckle were developed for four banana diseases. Removal of the waxy
bloom prior to inoculation period favored the development of leaf diseases. The
inoculation techniques developed were used in the standard greenhouse
assessment of resistance of different banana cultivars in the genebank to the above
banana leaf diseases.
INTRODUCTION diseases. Screening under greenhouse
condition is necessary for greater control
There are several leaf diseases of some factors affecting results. The study
affecting banana throughout its growing was aimed to develop proper inoculation
period. The yellow sigatoka and black leaf procedure for the screening of resistance of
streak are considered economically various banana cultivars against some
important in commercial plantations, while banana leaf disease in the regional banana
banana freckle, cordana leaf spot, rust, germplasm bank.
black cross.and chloridium leaf speckle are
common diseases affecting native varieties.
Reports have shown that the number of MATERIALS AND METHODS
functional leaves affects the quality and
quantity of fruit harvests. Thus, it is Banana leaf samples with infected
important to evaluate the resistance of the portion of yellow sigatoka, cordana leaf
different cultivars of banana to these leaf spot, freckle and black cross were collected
rly in the morning. Leaves of about 1-2 produced symptoms on inoculated leaves
a were placed in petri dishes lined with (Fig. 1). Brown oval spots with necrotic
oist tissue paper for the organisms to center were observed on inoculated plants
orulate. These were incubated for 48 hr kept inside the screen house 12-13 days
d examined under the stereomicroscope after inoculation while symptoms appeared
ed by using a sterile fine i
ked into potato dextrose
its. Colony growth of sport
; of different leaf disease
I to PDA slants to maintain
Various inoculation proce
d to initiate disease develop
:use. The early s'
ament observed on pla
he screenhouse can be attr
e to the plants.
-inkling of carborundur
oculation did not increase i
Suggesting that injuries
ry for successful infection
a musae .e
ae scraped from 17-day culture
Preparation of Inoculum
sion of 10-to 2(
icola on banana 1
a ;, t.rf^^,w-t/v^i,-, 1 n
puIe CUILUIC 01 ~ mUutC i/l -uay the lear lamma or the moculatea plants. At
old) or M. musicola (10- to 20-day the surface, brownish rust-like flecks
old) which eventually aggregated to form a
Standardize spore count lesion with a yellow margin and a black
Add Tween 20 (0.5 ml/100 ml
spore suspension) necrotic center were the typical symptoms
observed. Sigatoka symptoms developed
Preparation of. Test Plants when plants were placed inside a screen
Wash leaves with soap and water house with the morning dew provided to
Rinse the plants. Exposure of the experimental
Inoculation plants to dew may facilitate leaf spot
Inoculation development since dew lasting 2-3 hr may
*- Swab spore suspension on leaves results to maximal conidia formation
Cover with plastic bag for 15 hr (Wardlaw, 1972).
Remove plastic cover and keep
plants in screenhouse for Freckle [Phyllostictina musarum (Cooke)
Symptoms appear 2-4 weeks after
The initial symptom is the appearance
;ure 1. Outline of inoculationprocedure of fine black dots on the upper surface of
for cordona leaf spot and yellow inoculated leaves. After 46 days, these
56 Philipp. Phytopathol. 1995, Vol. 31(1): 54-4
progress to typical freckle symptoms as Black Cross [Phyllaclora musicola B & S
numerous, minutes, grayish brown or
dark-brown raised, more or less rounded Black streaks on the nether leaf surface
spots (Wardlaw, 1972). In the screenhouse, of Cardaba and Madurangga cultivars wei
inoculation was done by cotton swabbing observed on the 6th day when the te,
with spore suspension followed by plants were covered with plastic bags fc
clipping the infected leaves on the healthy about 15 hr. Infection appeared on the their
leaf for 6 days (Fig. 2). This inoculation to the fifth leaf, although black streaks o
procedure gave favorable results the first and second leaf were recorded o
when clipped infected leaves were already one Cardaba test plant. Brown dots on th
dry before removal of the inoculum. nether surface of the leaves were observe
Disease symptom was slight on plants kept on the other banana entries, Abuhor
inside the screenhouse and was observed Latundan, Lakatan and Umalag. It wa
only after 2 months. observed that streaks develop
horizontally,thus, forming a cross on the 9t
day, especially on Cardaba an
Preparation of Inoculum Madurangga. Initial symptoms on other
SColl n of ne l s cultivars did not progress except fc
Clctiong of feeees Latundan with longer brown streaks. Th
Scraping of spores
Spores stored in distilled water experimental plants were maintain
overnight (for spore germination) inside the screenhouse for 1 to 2 month
Spore count for further disease development wit]
Add Tween 20 at 0.5 ml/100 ml, occasional moisture provided.
distilled water + 2 g agar
Cut infected leaves for clipping Inoculation with P. musicola usin,
the clipping method with shortened
Preparation of Test Plants incubation period gave positive results afte
the first trial (Fig. 3).
Wash leaves with soap and water
WARDLAW, C.W. 1972. Banana Diseases
Swab spore suspension William Clowes and Sons, Limite(
Clip infected leaves London. 878 p.
Cover with plastic bags for 15 hr
Moisture provided with rotary
Remove infected leaves after 6
Transfer to screenhouse
Spots appear 7 weeks after
Figure2. Inoculation procedure for
banana freckle by leaf clipping
nlus cotton swnhhinm with ,nnrp
lipp. Phytopathol. 1995, Vol. 31(1): 54-57 57
Collection of infect
Cutting infected leave
entire leaf area
Wash with tap wati
Wash leaves with soc
Swab Tween 20 at 0.
Clip infected leaves
Cover with plastic b
Moisture provided w
Remove inoculum al
Keep inside glasshoi
Figure 3. Inoculation prc
es suitable to cover the
f Test Plants
ap and water
5/75-100 ml water
bags for 15 hr
vith rotary sprinkler
after 6 days
use for observation
ar 3-4 weeks after
ocedure for black cross by
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