• TABLE OF CONTENTS
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 Front Cover
 Biological control of sheath blight...
 Detection of banana bract mosaic...
 Cassia alata L. plants ("Akapulko")...
 Note: Spatial structure of natural...
 Note: Rust of gendarussa caused...
 Information for contributors














Title: Journal of Tropical Plant Pathology
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Title: Journal of Tropical Plant Pathology
Series Title: Journal of Tropical Plant Pathology
Physical Description: Serial
Language: English
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
Publication Date: 1998
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Bibliographic ID: UF00090520
Volume ID: VID00002
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electronic_oclc - 54382605
issn - 0115-0804

Table of Contents
    Front Cover
        Front Cover 1
        Front Cover 2
    Biological control of sheath blight of upland rice with Trichoderma species
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
    Detection of banana bract mosaic virus (BBrMV) in Musa spp. by serological and molecular techniques
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
    Cassia alata L. plants ("Akapulko") with mosaic is infected with a possible tobamovirus
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
    Note: Spatial structure of natural epidemics of sheath blight rice
        Page 25
        Page 26
        Page 27
        Page 28
    Note: Rust of gendarussa caused by puccinia thwaitesii B. & Br.
        Page 29
        Page 30
        Page 31
        Page 32
    Information for contributors
        Page 33
        Page 34
Full Text






PHILIPPINE PHYTOPATHOLOGY
Official Publication of the Philippine Phytopathological Society, Inc.

PHILIPPINE PHYTOPATHOLOGICAL SOCIETY, INC.
BOARD OF DIRECTORS 1998-1999


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PHILIPPINE PHYTOPATHOLOGY
EDITORIAL BOARD 1998-1999


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Biological Control of Sheath Blight of Upland Rice
with Trichoderma Species1

Alfredo M. Sinohin1 and Aleli Cornelia R. Plete2

1Supported by the Philippine Rice Research Institute (PhilRice)
2University Researcher, Department of Plant Pathology, University of the
Philippines Los Bafios and Science Research Analyst, PhilRice Los Bafios, College,
Laguna, respectively

ABSTRACT

The potential of Trichoderma isolates against Rhizoctonia solani
Kuhn causing rice sheath blight was evaluated. CES 4 isolate was observed
to be most antagonistic against R. solani in the laboratory. Similarly, CES
4 showed potential in greenhouse tests using unsterilized soil with different
application methods.

Improved technique of pelleting using rice bran (B-l) was employed
in mass production of Trichoderma. Favorable growth, sporulation and
an tagonism were observed in the laboratory while significantly lower disease
se erity was obtained in the greenhouse. No appreciable differences were
exhibited in the field. Pelletized Trichoderma applied through soil
incorporation + broadcast (35 DAS) showed a comparable or better effect
th an chemical treatment.

Key Words: biological control, inoculum carrier, pellet, Rhizoctonia solani,
sheath blight, inoculum substrate, Trichoderma


INTRODUCTION

Su cessful commercial production of
rice in the Philippines is still far from being
realized. Several constraints plague this
venture and diseases are one of
them. Sh sath blight caused by Rhizoctonia
solani Ku in is one of the major diseases in
upland anld lowland rice culture where no
resistant varieties have been found.

Chemical control has been
considered as the first line of defense against
sheath bli ght but is quite expensive and has
negative effects on the environment and
human 'eings. Breeding for disease


resistance has been resorted to but is time-
consuming and resistance gene sources are
still unavailable (Lapis 1994). If biological
control in upland rice culture can be
developed, as in other crops, using
Trichoderma, Gliocladium, Chaetomium
and Paecilomyces, production constraints
may be alleviated, if not minimized (Rosales
1985; Soytong 1988; Davide and Zorilla
1987).

Rosales and Mew (1982) found that
sheath blight of rice in dryland can be
controlled using Trichoderma that
decomposes infected rice straw and
parasitizes the mycelium of the pathogen.






Further studies about the effect of
antagonists on the survival of R. solani on
straw and substrate have implicated T
harzianum in sheath blight control in
dryland rice (Rosales, 1985).

The use of natural substrates for mass
production of Trichoderma is needed
against pathogens with high competitive
saprophytic ability. Hadar et al. (1979)
tried different substrates for growing
Trichoderma. Gonzales (1982) and Castro
(1990) found rice hull-rice bran to favor
fast growth and abundant sporulation of
antagonists. However, much work is yet to
be done not only in perfecting product
formulation but also in improving the
procedure for delivering the mycoparasite
to the soil. The nature and concentration
of the substrate is important to
mycoparasitism, competition, and survival
of antagonists (Stack et al., 1988).

This study aimed to (1) harness the
potential of Trichoderma sp. under upland
condition (2) identify among locally
available agricultural waste materials the
best inoculum carrier/substrate and (3)
determine the most suitable application
method of biocontrol agents.

MATERIALS AND METHODS

Screening of Biocontrol Agents.

Five isolates of Trichoderma
(T. harzianum, T. viride, T glaucum,
T. pseudokoningii, T aureoviride), 11
Trichoderma sp. coded (CES 1, CES 2, CES
3, CES 4, UPC 1, UPC 2, UPC 3, IPB 1,
IPB 2, IPB 3 and IPB 5), one each of
Chaetomium sp., Gliocladium sp. and
Paecilomyces lilacinus, were obtained from
the Field Crops Disease Laboratory


(FCDL), Department of Plant Pathology, UP
Los Bafios, College, Laguna. These were
revived and maintained including a
pathogenic R. solani isolate in Potato
Dextrose Agar (PDA) and tested for
antagonistic properties against R. solani
using dual culture test, hyphal parasitism
and sclerotial germination.

Identification of the inoculum
carrier and test for sporulation.

Fungal isolates that showed
promise were used in the screening for the
most suitable inoculum carrier/substrate.
Eleven substrates were considered, namely:
white clay, coir dust, rice bran, rice hull,
rice, ipil-ipil, white clay + rice bran (1:1),
ipil-ipil + coir dust (1:1), ipil-ipil + rice
(1:1), ipil-ipil + rice bran (1:1) and ipil-ipil
+ white clay + rice hull (1:1:1). Equal
amount of substrates was placed and
spread evenly on petri plates, added with
enough water, to provide moisture, and
sterilized at 15 psi for 30 minutes. After
cooling, 10-ml suspension of Trichoderma
was poured in each plate and then incubated
at room temperature for 3 days. Growth
of Trichoderma was evaluated based on
mycelial growth on the substrate..

Greenhouse Screening of
Antagonists.

Three antagonists selected from the
laboratory test were mass produced in rice
bran and ipil-ipil substrate and evaluated
for antagonism in the greenhouse using
different application methods. Plastic trays
with sterilized and unsterilized soil were
used with the following treatments:
Simultaneous application, Trichoderma
followed by Rhizoctonia, Rhizoctonia
followed by Trichoderma, seed soaking,


2 Philippine Phytopathology






seed coating, and R. solani alone

Mass Production by Pelleting.

Pelletizing as described by Cumagun
and Lapis (1993) was followed with some
modifications such as the addition of
molasses and complete fertilizer, removal
of "gaw-gaw", and processes like soaking
and cooking. First class rice bran (B-l) was
used as substrate for easy pelleting.

Pellets were prepared by mixing rice
bran substrate in a solution containing 5%
molasses (w/v), 1% complete fertilizer (w/
w) and 30% water (w/v), blended
thoroughly and then processed to form the
desired pellets thru a meat grinder to form
the desired pellets. Three hundred grams
of pellets were placed in a polypropylene
bag and then sterilized at 15 psi for 30
minutes.

Inoculation was done after the pellets
have cooled down and shaken using a 20-
ml spore suspension of 7-day-old culture
of Trichoderma poured into the bag,
incubated under continuous light at room
temperature and shaken from time to time
to avoid aggregation of pellets.

Laboratory Evaluation of Pelletized
Antagonists.

The sporulation of the antagonists on
the pellets was evaluated by counting the
total number of spores per pellet with the
aicLof a hemacytometer after 3, 5, 7, 12,
14 and 21 days incubation. Likewise, dual
culture tests using mycelial agar plug of
Trichoderma and its pelletized form were
done to compare the effectiveness of
Trichoderma in pellet form.


Greenhouse Evaluation for
Antagonistic Activity of Pelletized
Trichoderma.

This study was conducted at the
Department of Plant Pathology, UP Los
Bafios. The experiment was setup in a
completely randomized design with three
replications and with six treatments and
three rates as follows:

Treatments:
T1 = soil incorporation of palletized
Trichoderma
T2 = soil incorporation + broadcast (35 DAS)
T3 = soil incorporation + broadcast
(35 & 55 DAS)
T4 = broadcast at planting
T5 = chemical control with Benlate
T6 = R. solani


Rates:
Al = 100 kg/ha
A2 = 200 kg/ha
A3 = 300 kg/ha


Field Evaluation for Antagonistic
Activity of Pelletized Trichoderma.

This study was conducted under
upland conditions at the UPLB Central
Experiment Station (CES). The experiment
was set-up in a randomized complete block
design with six treatments and three
replications. The rice variety C22 was used.
Seeds were sown in a plot with a size of 12
m2 with 12 rows at the rate of 15 g/row.

Pelletized Trichoderma were applied
at the rate of 50g/sq. m. or 500 kg/ha.
Artificial inoculation of R. solani grown in
rice grain-rice hull substrate was done by
incorporating the inoculum into the soil at
the rate of 10 g/sq. m. before seeding.


Philippine Phytopathology 3







The treatments were T1 = soil
incorporation of palletized Trichoderma, T2
= soil incorporation + broadcast (35
DAS), T3 = soil incorporation +
broadcast (35 & 55 DAS), T4 = broadcast
at planting, T5 = chemical control, and
T6 = R. solani alone

Data collected were disease severity
using IRRI's Standard Evaluation System
(SES, 1998), plant height, yield
components (productive tillers, filled and
unfilled grain, 1,000 seed weight), and grain
yield.


RESULTS AND DISCUSSION

Out of 19 isolates tested, only seven
showed antagonistic activity against
R. solani in dual culture test (Table 1).
T. glaucum, T viride, and five coded
Trichoderma sp. i.e. CES 2, CES 4, UPC
2, UPC 3 and IPB 3 inhibited growth and
eventually completely colonized the
pathogen after a few days. Microscopic
examination revealed coiling, adhesion and
direct penetration except CES 2 (Table 2).
In 1932, Weindling first reported
Trichoderma as a potential mycoparasite


Table 1. Antagonism of 19 fungal antagonists against Rhizoctonia solani in a dual culture test.

Reaction Type' Test Organism

Chaetomium sp., Gliocladium sp., Paecilomyces lilacinus,
Trichoderma pseudokoningii
+ Trichoderma harzianum, Trichoderma aureoviride
++ CES 1, CES 3, UPC 1, IPB 1, IPB 2, IPB 5 (coded Trichoderma sp.)
+ + + Trichoderma glaucum, Trichoderma viride, CES 2, CES 4,
UPC 2, UPC 3, IPB 3

'+++ = Pathogen growth inhibited and antagonist was able to grow over the pathogen
++ = Pathogen growth inhibited but antagonist was not able to grow over the pathogen
+ = Mutual inhibition initially, but antagonist was overgrown by pathogen
= Pathogen growth not inhibited, antagonist was overgrown by pathogen


Table 2. Mode of action of seven Trichoderma isolates on Rhizoctonia solani.

Antagonist Mode of Action

Trichoderma viride Coils around hypha of pathogen
CES 4 Coils around hypha of pathogen
Trichoderma glaucum Coils around and adheres onto the pathogen's hypha
UPC 3 Coils around and adheres onto the pathogen's hypha
UPC 2 Very few coil around, most adhere onto pathogen's hypha
IPB 3 Coils around, adheres onto and directly penetrates into the
hypha of the pathogen
CES 2 No adhering, no coiling around and no direct
penetration; hypha of pathogen near the antagonist appear
segmented and distorted



4 Philippine Phytopathology






in biological control of plant pathogens. He
observed the parasitic nature of T lignorum
against a number of soilborne plant
pathogens. Ultrastructurally, Benhamon
and Chet (1993) noted that coiling appeared
to be mediated by a fine galactose rich
external matrix originating from R. solani
hyphae. The presence of galactose residues
indicates that there are receptors with
galactose-binding affinity at the cell surface
of Trichoderma, which maybe responsible
for the binding of the galactose-rich matrix
of R. solani. Furthermore, several studies
indicated that lectin-sugar interactions


might trigger adhesion, coiling and
penetration of the host by the antagonist
(Inbar and Chet 1992; Nordbring-Hertz and
Chet 1986).

Among the seven antagonists,
only three (T viride, T glaucum and CES
4) were able to suppress the germination
of the pathogen's sclerotial bodies
(Table 3). The inhibition of the sclerotial
growth of R. solani was probably due to
the penetration of the hyphae of
Trichoderma into the rind and cortex of
the sclerotia, as has been observed by


Table 3. Antagonistic ability of Trichoderma isolates on sclerotial bodies of Rhizoctonia solani

Antagonistic Reaction' Isolate

+ T glaucum, T viride, Trichoderma sp CES 4

CES 2, CES 3, IPB 3


* (+) = with antagonistic ability


(-) = without antagonistic activity


Table 4. Differential growth of Trichoderma isolates on sterilized substrates, 3 days after inoculation.

Antagonist
Substrate
Trichoderma viride Trichoderma glaucum Trichoderma sp. (CES 4)

White clay
Coir dust +
Rice bran +++ ++ ++
Rice hull ++ +
Rice ++ ++ ++
Ipil-ipil + + +
White clay + rice bran (1:1) -
Ipil-ipil + coir dust (1:1) -
Ipil-ipil + rice bran (1:1) +++ +++ +++
Ipil-ipil + rice (1:1) ++ + +
Ipil-ipil + white clay + rice (1:1:1) +

*+++ = excellent growth ++ = good growth + = fair = poor growth


Philippine Phytopathology 5






Henis and his associates (1983) in S. rolfsii.
In degraded sclerotia, the cortex was
replaced by Trichoderma hyphae, which
grew out of the rind.

The three isolates that were used to
screen the most suitable inoculum carrier/
substrate showed excellent growth on
sterilized rice bran alone and rice bran +
ipil-ipil (1:1, v/v) substrate while poor
growth was noted in unsterilized substrate
(Table 4). Minimal growth may be
attributed to the presence of saprophytes
that liberate extracellular products harmful
for the antagonist, or the substrate itself
contains chemicals that are inhibitory to the
organism.

Greenhouse screening of the best
antagonist showed that T viride performed
well in sterilized soil while CES 4 had the
best result in unsterilized soil (Table 5). As
for the method of application, simultaneous
and seed coating methods provided the
highest disease control with 59.79% and
54.86%, respectively using CES 4 in
unsterilized soil. However, the result from
field application of the antagonist mass-


produced in rice bran + ipil-ipil substrate
was non-significant It was found that the
substrate tended to dissociate into the soil
after application thereby losing its
foodbase. With this observation, the study
opted to use pelleting by Cumagun and
Lapis (1993) with modifications.

Pelletized Trichoderma (CES 4)
showed favorable growth, sporulation and
antagonistic ability in laboratory tests.
Sporulation was highest until 7 to 14 days.
This indicates that pellets should be
incubated not more than two weeks prior
to inoculation. Faull (1988) pointed out
that the possession of a substrate is vital to
the establishment of a biological control
agent in any environment.

In the greenhouse, sheath blight
severity ratings, across rates, were lowest
with soil incorporation and broadcast with
4.82 and 5.60 percent, respectively (Table
6). Ratewise, soil incorporation and
broadcast alone or in combination reduced
sheath blight severity at 300 kg/ha as
compared to the control. Henis (1984)
commented that Trichoderma is an


Table 5. Percent sheath blight control of 15-day-old rice seedlings as affected by different application
methods of three Trichoderma sp. under sterilized and unsterilized soil

Application Sterilized Unsterilized
Method
Trichoderma Trichoderma Trichoderma Trichoderma Trichoderma Trichoderma
glaucum viride sp CES 4 glaucum viride sp CES 4
Simultaneous 11.13 61.28 41.84 46.75 10.18 59.79
Trichoderma followed
by Rhizoctonia 6.02 74.51 17.17 32.91 22.26 33.86
Rhizoctonia followed
by Trchoderma 9.42 74.99 61.79 48.55 326 44.62
Seed soaking 13.62 24.61 42.68 47.11 11.77 27.33
Seed coating 24.23 41.11 44.26 43.84 8.74 54.86

Control Treated
% Control = ------------------------------- x 100
Control


6 Philippine Phytopathology






opportunistic rather than an obligate
parasite, which requires a foodbase to
attack living hyphae in the soil. Moreover,
Knudsen and his colleagues (1991)
observed that PEG (polyethylene glycol)
treatment of T harzianum in alginate pellets
enhanced hyphal extension of the fungus
in soil, although it did not enhance
production of conidia on pellet surfaces.

In the field, broadcast alone or
combined with soil incorporation (at 35
DAS) gave the highest disease control with
75.67% and 70.59%, respectively (Table 6).
These results confirmed that inoculum
preparation of Trichoderma using wheat
bran substrate lowered disease severity,
increased yield and decreased the pathogen
(Sivan et al., 1984; Hadar et al., 1979; Chet
et al., 1979). They likewise confirmed that
incorporation of hyphal biomass or spores
in a pellet is a promising development in
formulation of fungal biocontrol agents for
soil application (Lewis and Papavisas
1985; 1987).

Considering plant height, soil
incorporation + broadcast at 35 DAS


induced the growth of plants with 105.6 cm.,
which is significantly taller than control
(97.2 cm) (Table 6). Lindsey and Baker
(1967) reported that Trichoderma could
stimulate plant growth directly by the
suppression of minor pathogens around the
roots.

No appreciable difference was
obtained on the yield components of C22
rice plant using the different treatments.
Using yield as an index, chemical control
had the highest yield of 2.78 t/ha followed
by SI + B at 35 DAS with 2.72 t/ha which
gave a 19.31% and 16.74% increase,
respectively (Table 6). However, no
significant yield differences were observed
among the treatments.

CONCLUSION

Trichoderma can be mass-produced
by pelleting using available materials such
as rice bran. Pelleting provided a condition
necessary for the competence and survival
of the antagonist. Soil incorporation +
broadcast at 35 DAS is apparently the best
method of application.


Table 6. Effect of different application methods of pelletized Trichoderma on sheath blight severity,
plant height, and grain yield of rice cultiuar C22.

Treatments % Disease Plant Grain Yield
Severity1 Height (ton/ha)1
(cm.)2
soil incorporation 17.03 23.67 99.8 ab 1.95- 16.31
SI + broadcast at 35 DAS 4.05 (70.59) 105.6 b 2.72 (16.74)
SI + B at 35 and 55 DAS 9.48 (31.15) 105.3 b 2.50 (7.30)
broadcast 3.35 (75.67) 105.0 b 2.45(5.15)
chemical control 6.48 (52.94) 103.1 ab 2.78 (19.31)
control 13.77 97.2 a 2.33

'Means are not significantly different at the 5% level by DMRT. Numbers in parenthesis are percent control.
2Means followed by a common letter (s) are not significantly different at the 5% level by DMRT.


Philippine Phytopathology 7







A non-appreciable difference
between treatments obtained in the study
does not mean that the use of biocontrol
agents as an alternative disease
management strategy is not effective.
Biological control still has a lot to offer. With
several approaches now being undertaken
to solve problems in biological control as
well as to improve techniques in aspects of
inoculum production and formulation, it is
likely that effective and economical
biological control methods will soon become
available.

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Philipp. Phytopathol. 21:60 (Abstr.)

STACK JP KENERLY CM PETTIT RE. 1988.
Application of biological control agents, pp.
43-54. In Mukerji, K. G. and K. L. Garg
(Eds.). Biological Control of Plant Diseases,
Vol. II, CRC Press, Boca Raton, Florida

WEINDLING R. 1932. Trichoderma lignorum
as a parasite of other soil fungi.
Phytopathology 22: 837-845.


Philippine Phytopathology 9

























































10 Philippine Phytopathology






Detection of Banana Bract Mosaic Virus (BBrMV)
In Musa spp. by Serological and Molecular Techniques

Lorna E. Herradura1 and Lydia V. Magnaye2

'Senior Plant Pathologist and Head, Plant Disease Laboratory, and
2Former Head, Plant Disease Laboratory, Bureau of Plant Industry-Davao National
Crops Research and Development Center, Bago-Oshiro, Davao City, Philippines

ABSTRACT

Banana bract mosaic virus (BBrMV) isolates from different parts
of the country were transmitted to different Musa spp. by sap and aphid
inoculation. PCR products (DNA) of the different isolates exhibiting varied
symptom types on different varieties were tested by Southern blot to
determine differences in isolates. Detection using serology and molecular
techniques were also used on the different isolates to test the efficiency of
the two techniques. The different isolates of BBrMV from sap and aphid
transmission using two aphid species did not show any differences as
supported by Southern blot test. On symptomatic plants, routine detection
can be done by enzyme linked immunosorbent assay (ELISA).
Immunocapture reverse transcriptase polymerase chain reaction (IC-RT-
PCR) would be more sensitive in detecting low concentration of the virions.


INTRODUCTION

Banana bract mosaic was first
observed in 1979 in Davao, Mindanao,
Philippines on a number of local banana
cultivars. It reached epidemic proportions
in 1988 in Mindanao where several
thousand mats of Cavendish banana were
affected (Magnaye 1994). The disease has
spread all over the country on almost all
banana cultivars except in Northern Luzon
where only a few occurrences were
recorded in some varieties (Magnaye and
Herradura 1997). It has also been reported
to occur in banana plants in Southern India
based on symptomatology, morphology,
sequence homology and nucleic acid
hybridization assays (Rodoni 1997) and in
Walgolla, Sri Lanka where it was observed
on 'Embul' (AAB, syn. 'Mysore'); plants
(Anon., 1995).


The putative causal virus, banana
bract mosaic virus (BBrMV), has been
shown to be transmitted by Rhopalosiphum
maidis and Aphis gossypii (Magnaye and
Espino 1990) and by Pentalonia
nigronervosa (Mufiez 1992) in a non-
persistent manner. Purification of BBrMV
done by Mufiez (1992) in the Philippines
showed that the virus had flexuous rod
shaped particles with-modal length of about
750 nm. He suggested that BBrMV could
be a potyvirus. Bateson & Dale (1995) and
Thomas et al. (1997) similarly and
independently worked on plant materials
from the Philippines and results of their
studies provided additional evidence that
BBrMV is indeed a member of the
Potyviridae. Bateson & Dale (1995)
reported that BBrMV is a potyvirus based
on particle morphology of flexuous rods
measuring 660 to 760 nm and that


Philippine Phytopathology 11






potyvirus degenerate primers were able to
amplify the viral RNA. Likewise, a 38-kDa
coat protein reacted with a general
potyvirus antiserum (Thomas et al 1997).

BBrMV infected plants show
continuous greenish to brownish spindle-
shaped streaks or long continuous and
discontinuous stripes irregularly scattered
along the petiole and leaf lamina. It was
also observed that leaf symptoms might or
may not be shown (Magnaye and Espino,
1990). With the survey of virus and
viruslike diseases of Musa spp. conducted
in 1997, it was observed that symptoms
quite similar to banana bract mosaic were
quite common. For instance, symptoms on
Cavendish are red spindle streaks on
pseudostem. These same symptoms are
also easily observed on Lakatan. On
Latundan, chimera or yellow blotches on
the lamina were observed. This is atypical
of the symptoms observed on other
varieties. On Cardaba, symptoms on the
lamina are yellow green spindle streaks.
With these varied symptoms, it is not
known if these are expressions of the same
BBrMV. Thus, this study was undertaken
to determine by molecular and serological
techniques whether or not BBrMV was
associated with these symptoms and to
determine which tests could be used in the
routine detection of BBrMV in the country.
The study further aimed to determine if
BBrMV transmitted by two different aphid
vectors are the same based on a BBrMV
specific probe.

MATERIALS AND METHODS

Collection of Isolates. Suckers
of banana showing symptoms of banana
bract mosaic (BBrMV) were brought to the
Davao National Crop Research and
Development Center, Bureau of Plant
Industry, Davao City. These were planted


inside screenhouses and the plants reared
and maintained as isolates for the
transmission work.

Rearing of Aphids. Three aphid
species, P nigronervosa, A. gossypii and
R. maidis were collected in the field and
transferred to different host plants which
were kept in different insect cages.
P nigronervosa was reared on healthy
banana plants, A. gossypii on Euphorbia
hirta and R. maidis on Echinocloa colonum.
The aphids were transferred to another
batch of healthy plants before they were
cultured and reared for the transmission
experiments.

Vector Transmission. Non-
viruliferous aphids previously reared on
healthy plants were allowed to feed on
BBRMV-infected leaf pieces from the
different collected isolates. Transmission
was done singly for the different aphid
species using six test plants per trial. Parallel
tests were done using three aphid species
to determine transmission efficiency.

Virus Isolates. Suckers of
different banana cultivars showing different
symptoms were brought to the screenhouse
facilities of BPI-DNCRDC. Sap and aphid
transmissions were done to isolate the
putative BBrMV. Leaf samples of the test
plants that developed symptoms after the
transmission and leaf samples of banana
suckers collected from the field were
brought to the Queensland Department of
Primary Industries (QDPI) Virology
Laboratory, Australia under Australia
Quarantine and Inspection Service License
(AQIS).

Enzyme-linked
Immunosorbent Assay (ELISA). Plant
sources used in the transmission
experiment were subjected to triple antibody


12 Philippine Phytopathology
12 Philippine Phytopathology






sandwich-ELISA (TAS-ELISA) using
monoclonal antibody against BBrMV
supplied by Dr. J. E. Thoma, QDPI. The
ELISA plates were coated with BBrMV
polyclonal IgG from 3d bleed antiserum
diluted in carbonate coating buffer to 3 iAg/
ml at 100 pl/well. The plates were incubated
for 2-3 hrs at room temperature after which
they were washed with PBS-T 3 times at 3-
minute intervals. BBrMV samples and
controls were ground in mortar and pestle
with extraction buffer (0.2M K phosphate
pH 7.0 + 15mM EDTA + 2%PVP +
2%PEG + 0.4%NA2SO,) at a dilution rate
of Ig/lOml. Healthy controls were
Cavendish cv. Williams also from QDPI
which have been regularly indexed for the
different banana viruses. The extracted sap
samples were added at 100 pl/well and
incubated at 50C overnight. After
incubation, the plates were washed as
above. BBrMV monoclonal tissue culture
supernatant 1G11 was the detecting
antibody used. This was diluted in 9ml
PBST + 1.0g of skim milk powder. The
mixture was added at 100 Al/well and
incubated at room temperature.- After
washing as above, the sheep anti-mouse
conjugate diluted at 1:1000 in PBST was
added in all wells. The plate was incubated
for 3 hrs at room temperature and washed
as above. Finally the substrate (p-
nitrophenyl phosphate at lmg/ml in
diethanolamine buffer) was added at 100
pl/well and the plate incubated for 1 hour
at room temperature. ELISA reading was
taken after 30 minutes.

Immunocapture Reverse
Transcriptase Polymerase Chain
Reaction (IC-RT-PCR). Symptomatic
samples that were negative by ELISA were
tested using IC-RT-PCR following the
protocol at QDPI using degenerate primers
(M. Sharmam, pers. comm.). Briefly, IC-
RT-PCR was done by coating eppendorf


tubes with the BBrMV antibody stock and
incubated for 2-3 hr at room temperature.
The tubes were washed with phosphate
buffered saline-Tween 20 (PBST) after
which the extracted sap was incubated in
the previously coated tubes for 4 hr at room
temperature. Same tubes were washed
with PBST as above. After these steps,
complementary DNA synthesis was done
using Pharmacia complementary DNA
(cDNA) kit for IC-PCR. Briefly, 8 ul of
diethyl pyrocarbonate (DEPC) treated
water was added to each tube and 1.0 ul
of 20 uM Poty 1/U341 degenerate primers
added. The tubes were heated at 800C for
10 min and immediately chilled on ice.
After which, 5 pl of bulk 1"t strand cDNA
mix and 1 Al of dithiothreitol (DTT) solution
were added. These were again spun briefly
and incubated for 1 hr at 370C. After
complementary DNA synthesis, DNA
amplification was done in a PCR machine
(Hybaid Omnigene Thermal Cycler). The
PCR cycling parameters used were an
initial denaturation of 940C for 1 min,
followed by 35 cycles of 940C for 20 s, 600C
for 1 min and, 720C for 1 min, an extension
of 720C for 3 min and finally a hold of 30C
for 3 min.

The PCR product was
electrophoresed in a 1% TBE agarose gel
for about 2 hr. About 8 ul of PCR product
was loaded in each well with 5 ul of 100 bp
marker used. Staining was done using
ethidium bromide for 20-30 min before UV
visualization and the gel photographed.

Southern blot hybridization.
The RT-PCR products were analyzed by
Southern blot following the procedures of
Sambrook et al. (1989). The samples were
electrophoresed in 1% agarose TBE gel and
stained with ethidium bromide. The gel
was rinsed in distilled water after which it
was washed with denaturation buffer (1.5M


Philippine Phytopathology 13







NaC1, 0.5M NaOH) for 30 min in a shaker
at room temperature followed by washing
with neutralizing solution (1.5M NaC1, 0.5M
Iris HCI pH 7.2, 0.001M EDTA) for 15 min
two times at room temperature.

The products were capillary-blotted
to Hybond-N nylon membranes
(Amersham International). Capillary blot
was set up using 20x SSC solution (3M
NaCI, 0.3M NA citrate) for transfer for 2
hours. The blot was rinsed in 2X SSC and
air-dried for 30 min after which it was
cross-linked by a UV for 5 min.

Pre-hybridization of the blot was
done for 30 min at 450C in 30 ml
digoxigenin (DIG) Easy-hyb solution
(Boehringer Mannmeim, Germany). Pre-
hybridization solution was removed and the
hybridization solution added (1.5 ul
digoxigenin-labeled probes of 341 bp
prepared from cloned inserts of BBrMV -
509 using a PCR labeling kit) (Boehringer
Mannmeim, Germany) added in 5.25 ml
Easy-hyb solution pre-warmed at 45C.
Hybridization was done overnight at 450C.
The hybridization solution was removed
and the blot was washed two times for 15
min at room temperature using 2X SSC,
0.1% SDS followed by two times for 15 min
washing at 680C using 0.1X SSC, 0.1%
SDS (high stringency).

For detection, the blot was washed
in wash buffer (0.1M maleic acid + 0.1M
NaCI pH 7.5) + 0.3% Tween 20 at room
temperature for 5 min followed by 30 min
incubation at room temperature in 2%
blocking solution. One ul of DIG-antibody
was then added to the blocking solution and
incubated for another 30 min at room
temperature. To remove the unbound
antibody, the blot was washed in wash
buffer (2 times for 15 min at room
temperature). The blot was equilibrated in


100mM Tris-HCI pH 9.5 + 100mM NaCI
+ 50mM MgCI2) for 5 min at room
temperature before adding the
chemiluminescehce substrate dropwise to
cover the entire blot. It was then incubated
at room temperature for 5 min. The
membrane was again blotted dry to remove
excess lumigen and sealed in plastic before
incubation at 370C for 10 min, After
incubation, the blot was placed together
with the film. This was exposed at room
temperature approximately for 2 hr and then
developed.

RESULTS AND DISCUSSION

Symptoms of Banana Bract
Mosaic Virus. Symptoms observed on
the inoculated plants are not totally
characteristic of the first reported symptoms
of BBrMV which are continuous greenish
to brownish spindle-shaped streaks or long
continuous and discontinuous stripes
irregularly scattered along the petiole
(Magnaye and Espino, 1990). Initial
symptoms observed on the lamina of
inoculated plants of abaca and Lakatan are
spindle streaks which would sometimes
appear as plain chlorotic and some having
green centers. On the other inoculated
plants of the same cultivar, which have
chimera symptoms, some leaves have faint
chlorotic bands that would eventually
progress to chlorotic blotches. Faint to dark
green mottle and pinkish streaks were
observed on the pseudostem. On Butuhan,
which is a seeded banana, the initial
symptoms are spindle streaks that have been
observed on the pseudostem and petiole.
Leaf symptoms can eventually be observed
on the youngest expanded leaf having faint
chlorotic spindle streaks. Magnaye and
Espino (1990) reported that the
characteristic symptoms of BBrMV could
initially be observed on the petiole and that
leaf symptoms may or may not appear. On


14 Philippine Phytopathology





inoculated Butahan plants, the first initial
symptom was observed on the pseudostem
and petiole and then similar symptoms
developed on leaves. Other inoculated
Musa spp. like abaca and Lakatan exhibited
initial symptoms on the leaf. Thus,
depending on the Musa spp. and cultivar,
relying only on symptoms for BBrMV
diagnosis can be misleading.

Detection of BBrMV by
ELISA. The symptomatic Lakatan,
Butuhan and abaca plants reacted to the
BBrMV antisera in TAS-ELISA. All the
symptomatic plants were positive for
BBrMV. However, on some aphid
transmitted isolates showing symptoms,
ELISA test using monoclonal antibody
IgG11 were negative (Table 1). Our results
provide further evidence and support the
claim of Thomas et al. (1997) that the
polyclonal antiserum he produced detects


all isolates tested but some individual
monoclonal antibodies do not react with
all isolates (Thomas and Iskra-Caruana,
Brisbane and Montpellier 1996, personal
communication).

There were also some inoculated
plants, which remained symptomless in the
transmission experiment that gave negative
results in TAS-ELISA. After a month or
more, these same plants showed symptoms
and were positive for BBrMV using the
same test. With these plants, the incubation
period was longer and that the virus titer in
the plant might not be sufficient for it to be
detected by TAS-ELISA or the antibody
used was too specific to detect the virus
present. Jordan (1992) observed that on
potyviruses, negative results maybe
attributed to a low concentration of the
virions in the samples or alternatively, there
exist some epitope variation among isolates.


Table 1. Absorbance values at 405 nm in TAS-ELISA of BBrMV infected banana and abaca.

Samples/Symptoms Absorbance
(A405nm) Remarks
Healthy .02
Positive .09 +
Butuhan (faint chlorotic spindle streaks) .10 +
Butuhan (streaks on petiole and pseudostem .06
Butuhan (streaks on lamina) .07
Lakatan (mild mottle) .12 +
Lakatan (chlorotic veins) .68 +
Lakatan (faint chlorotic spindle streaks) .29 +
Lakatan (chlorotic spindle streaks with green center) .08
Lakatan (enlarged) veins .06
Lakatan i(chlprotic streaks) .09
Lakatan (numerous faint chlorotic streaks) .06
Lakatan (numerous faint chlorotic spindle streaks) .06
Abaca (enlarged veins; chlorotic blotch) .03
Abaca (chlorotic spindle streaks with dark green centers) .08
Abaca (Mottle on pseudostem) .09
Abaca (whitish to faint chlorotic blotch along veins) .06
Lakatan (chlorotic spindle streaks with green center) .02
Abaca (field collected) .01
Abaca (field collected) .03-

Philippine Phytopathology 15






All the symptomatic isolates not
detected by:ELISA were positive using IC-
RT-PCR (Table 2). This indicates that IC-
RT-PCR was more sensitive than ELISA.
Thomas et al. (1997) showed that both
ELISA and RT-PCR could detect BBrMV
in field-infected and glasshouse banana.
The use of IC-RT-PCR would be useful in
detecting low concentrations of virions in
the sample since the test would be more
sensitive. The extreme sensitivity and high
specificity of RT-PCR make it a suitable
technique for the detection of viral infections
that are difficult to detect or diagnose by
serology (Seal, pers. Comm.). In the case
of the BBrMV IC-RT-PCR, the primer used
can detect the core of the coat protein to
the polyA tail at the 3' end. The efficiency
of immunocapture (IC) was also shown by
Harper et al. (1999) in detecting BSV. He
suggested that the use of IC-RT-PCR is at
least as sensitive as standard PCR probably
due to the removal of inhibitory substances
by the washing steps during IC-PCR.

Aphid transmitted isolates used in
the experiment coming from different parts
of the country were detected using the
BBrMV-specific probe for the Philippine
isolate. No PCR products were amplified
from healthy banana plants. The PCR


products were Southern blotted onto the
Hybond N and hybridized with a DIG-
labeled BBrMV specific probe. Thomas et
al. (1997) have shown that BBrMV-specific
probe gave positive reaction on all isolates
tested except one; which came from India.
Positive reaction obtained in the experiment
on the different banana varieties collected
from several provinces of the country shows
that there are no differences among the
isolates of BBrMV. Positive reactions by
Southern blot on isolates transmitted using
two aphid species P nigronervosa, and R.
maidis further confirm the findings of
Magnaye and Espino (1990) and Munez
(1992) that P nigronervosa and R. maidis
transmit BBrMV.

CONCLUSION

Our findings showed that the
various symptoms we suspected to be
associated with BBrMV were indeed
caused by BBrMV. Although we did not
rule out the presence of other viruses from
the initial field samples, results of field
transmission experiments proved that we
isolated BBrMV as this is the only banana-
infecting virus that can be non-persistently
transmitted by both P nigronervosa and
R. maidis and one that is also mechanically
transmitted.


Table 2. Detection of BBrMV by ELISA and IC-RT-PCR on Musa spp. samples.

Sample/Symptom ELISA IC-RT-PCR

Abaca (enlarged veins, chlorotic blotch) +
Abaca (numerous faint chlorotic spindle streaks) +
Abaca (light green streaks along the veins) +
Butuhan (streaks on petioles and pseudostem) +
Lakatan (numerous faint chlorotic streaks) +
Abaca (field-collected) +
Lakatan (field-collected yellow spindle streaks) +
Lakatan (field-collected) +

16 Philippine Phytopathology






Routine detection of BBrMV can be
done by ELSA. However, a disadvantage
in the use of a monoclonal antibody to
BBrMV developed at QDPI is its high
specificity, which has limited sites for
reaction. This specific antibody cannot
detect field samples, which may have
different epitopes. For low concentration
of virions, IC-RT-PCR can be used especially
when testing valuable materials.


LITERATURE CITED


ANONYMOUS. 1995. MusaNews. Infomusa
4 (2), 26-30.

BATESON MF, DALE JL. 1995. Banana
bract mosaic virus: Characterization
using potyvirus specific degenerate PCR
primer. Arch. Virol. 140: 515-527.

HARPER G, DAHAL G, THOTTAPPILLY
G, HULL R. 1999. Detection of
episomal banana streak badnavirus by
IC-PCR. J Virol Methods 79: 1-8.

JORDAN R. 1992. Potyviruses, monoclonal
antibodies, and antigenic sites. Pages
81-95 in: Potyvirus Taxonomy (Arch Virol
Supply 5). Bamett OW (ed). Springer-
Verlag. Vienna.

MAGNAYE LV. 1994. Virus Disease of Banana
and Current Studies to Eliminate the
Virus by Tissue Culture. Proc. 1" PPS-
SMD National Symposium on Pests and
Diseases of Banana in the Philippines.
Apr 23-24, 1993. Davao City.

MAGNAYE LV, ESPINO RRC. 1990. Note:
Banana bract mosaic, a new disease of
banana. I. Symptomatology. Philipp.
Agric 73: 55-59.

MAGNAYE LV, HERRADURA LE. 1997.
Rescue and Conservation of Southeast
Asian Banana Germplasm, INIBAP and


IPGRI Terminal Report.

MUNEZ AR.' 1992. Symptomatology,
transmission and purification of banana
bract mosaic virus (BBrMV) in 'Giant
Cavendish' banana. Unpublished M.Sc
Thesis. University of the Philippines Los
Baios, College, Laguna, Philippines.

RODONI BC, AHLAWAT YS, VARMA A,
DALE JI, HARDING. 1997.
Identification and characterization of
banana bract mosiac virus in India.
Plant Dis.. 81:669-672.

SAMBROOK J, FRITSCH EF, MANIATIS
T. 1989. Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor
Laboratory Press, Cold Spring, N.Y.

THOMAS JE, GEERING ADW, GAMBLEY
CF, KESSLING AF, WHITE M.
1997. Purification, properties and
diagnosis of banana bract mosaic
potyvirus and its distinction from abaca
mosaic potyvirus. Phytopathology 87:
698-705.


ACKNOWLEDGEMENT

This work was done as part of a
collaborative project between Queensland
Department of Primary Industries, Australia
and Bureau of Plant Industry, Philippines.
We are grateful for the technical support of
Dr. John Thomas and Cherie Gambley
during the laboratory work in Australia.


Philippine Phytopathology 17





























































18 Philippine Phytopathology






CASSIA ALATA L. PLANTS ("AKAPULKO") WITH MOSAIC IS INFECTED
WITH A POSSIBLE TOBAMOVIRUS1

Walter Timothy D. Bidad and Narceo B. Bajet 2

1 Portion of thesis by the senior author.
2 Former undergraduate student and Associate Professor, Department of
Plant Pathology, University of the Philippines Los Banos, College, Laguna.

ABSTRACT

Cassia alata L. plants (locally called "akapulko") that showed mosaic
symptoms typical of virus infection were homogenized and the sap me-
chanically transmitted to healthy C. alata, Nicotiana glutinosa L. and N.
tabacum. C. alata developed mosaic symptoms while the N. glutinosa and
N. tabacum plants developed necrotic local lesions. Planting of seeds col-
lected from symptomatic plants showed that two seedlings out of 50 or 4%
(100 % germination) developed early symptoms of mosaic indicating a
possible seed transmission of the putative virus in C. alata. The virus is
possibly a tobamovirus.


INTRODUCTION

Cassia alata L. locally known as
"akapulko" belongs to family Leguminosae.
This plant is found abundant throughout
the Philippines thriving on wastelands near
water and is known to have medicinal prop-
erties. The leaf juice is used for treatment
of skin diseases such as herpes, eczema,
ringworm, and insect bites. Tincture from
the leaves is purgative while a decoction of
leaves and flowers is abortifacient, astrin-
gent and works as expectorant (De Padua,
et al.1987).

A number of the C. alata plants in
the herbal garden being maintained
near the Institute of Biological Sciences
(IBS), UPLB show mosaic symptoms
typical of virus infection. These symptoms
include vein clearing, yellowing of leaves,
leaf curling and severe leaf mottling or
mosaic.


There have been very few reported
studies on mosaic infection of Cassia spp.
but none on C. alata. Van Velsen (1961)
reported the first recorded mosaic
symptoms in C. occidentalis and C. tora
growing wild at Ulaveo coconut plantation
at Kokopo, in Papua, New Guinea.
Diseased Cassia plants have since been
located widespread on the Gazelle
Peninsula among ornamental cassias
growing in the New Guinea. The other
reported virus diseases of Cassia spp. are
enumerated by Brunt et al. (1996) and this
include tobacco mosaic tobamovirus-O
(TMV-O or odontoglossum ringspsot
tobamovirus, ORSV) and cymbidium
mosaic potexvirus (CymMV) (Lawson and
Ali, 1975).

In the Philippines, there are also no
studies that have been conducted on
diseases of akapulko (Tangonan and
Quebral, 1992) although there were a


Phytopathological Journal 19







number that attempted at it being an ex-
perimental host of certain viruses like to-
bacco mosaic virus strain 0 (TMV-O) iso-
lated from an orchid Laeliocattleya 'Randy'
(Rillo, 1975) and papaya ringspot virus
'(PRSV) (Malabanan, 1989). There were
more studies conducted on
C. occidentalis, a related species, and the
plant was found to be infected with tobacco
mosaic virus (Aquino, 1981) and other
mosaic virus isolates in the Philippines
(Paguio and del Rosario, 1965).

Thus, this study was conducted to
determine whether the mosaic on these
C. alata plants is due to a virus or viruses
and possibly identify to which group the
virus-belongs to. Knowledge on the disease,
including its nature and possible mode of
transmission is important because the leaf
juice has a medicinal property. Partial
results have been reported earlier (Bidad,
1992).

MATERIALS AND METHODS

Collection, Preparation of Plants
and Mechanical Transmission

Infected young leaves were harvested
from the UPLB, IBS Herbal Garden. These
were macerated in a sterile mortar and
pestle with 0.02 M phosphate buffer, pH
7.2, in 1:1 (wt/vol) proportion. The sapwas
extracted by squeezing it through three
layers of gauze cloth with Carborundum
#320 powder mixed with the sap extract
prior to inoculation. The inoculum was
rubbed slightly on the upper leaf surface of
the test plants using the forefinger dipped
in the inoculum while the other hand
supported the lower part of the leaf.
Inoculated leaves were washed with tap
water minutes after inoculation to remove
excess Carborundum.


The test plants were seedlings of
N. glutinosa and N. tabacum. The C. alata
seeds were also planted similarly. Plants
were covered with Mylar cages after
germination until the termination of the
experiment.

Seed Transmission

Fifty seeds from mosaic infected
C. alata plants were collected and planted
in pots with sterilized soil. Plants were
watered daily and complete fertilizer was
applied. Upon germination the plants were
covered with Mylar cages. The plants were
monitored for any symptoms that may
develop.

RESULTS AND DISCUSSION

The symptoms observed on C. alata
plants are typical mosaic with slight
distortion on the edges of the younger
leaflets. These symptoms persisted on these
young leaflets but faded as they matured
and those at the bottom developed less
discernible symptoms.

Inoculation of N. glutinosa and
N. tabacum with the sap extracted from
these symptomatic leaflets resulted to infec-
tion with percentage of infection of 50%
and 80%, respectively (Table 1). Both host
species developed discreet water soaked
lesions about 3-4 days after inoculation and
these lesions eventually became necrotic
with size of about (1 mm-3 mm).

There were fewer necrotic lesions
observed in N. glutinosa than inN. tabacum
and more of the N. tabacum seedlings
developed the symptoms. This apparent
difference between the two host plants could
have been the result of a reduction in light
intensity or of different wavelengths and


20 Phytopathological Journal





Table 1. Mechanical transmission of the putative tobamoviru in mosaic infected Cassia alata to Nicotiana
tabacum and N. glutinosa seedlings.

HOSTS PLANTS INFECTED % INFECTED REMARKS
PLANTS
INOCULATED

N. tabacum' 8/10 80.0 Necrotic lesions

N. glutinosa 5/10 50.0 Necrotic lesions


photoperiods. Likewise, it could also be due
to the variation in the optimum amount of
rubbing and pressure applied during inocu-
lation (Bawden and Roberts, 1947;
Kassanis, 1952). Another reason could be
the differences in the number of passes
during inoculation. Holmes (1964)
mentioned that more than one or two
passes over a leaf area will result in
successively fewer lesions probably because
of increasing injury. It could have been also
affected by initial concentration of the
putative virus in the diseased C. alata or
this plant has some components that
inhibited the virus (Gibbs and Harrison,
1976; Matthews, 1991).

./There were two seedlings or 4.0%
that showed symptoms of infection out of
the 50 matured seeds collected from pods
of symptomatic C. alata (Table 2). The
seedlings initially showed vein clearing
followed by severe mottling and leaf curling
after 6 wk of planting. The 4.0% infection
was low and this could have been due to
the fact that, at the time of seed formation,
the temperature was very high. A smaller
proportion of seed is infected at very high
or low temperatures than at intermediate
temperatures. For seed transmission to oc-
cur, plants in general, may have been in-
fected before the ovules are fertilized (Gibbs
and Harrison, 1976; Matthews, 1991).

Eugenio and Del Rosario (1962)
did a study on the host range of a TMV


isolate in the country and found out that
the isolate was able to infect 24 species of
plants belonging to 16 families, confined to
the most important crops and the most
common weeds growing in tobacco fields,
except for seven introduced species. These
seven species were: Trianthema
portulacastrum L., Datura alba L.,
Corchorus olitorius L., Ipomoea trilgba L.,
Amaranthus viridis L., A. spinosus L., and
Los Banos bush sitao, a hybrid between
Vigna sinensis x V sesquipedalis). In 1976,
Retuerma described a mosaic in another
related Cassia sp., C. podocarpa L. The
early symptom was vein clearing wherein
areas between the veins turned yellow with-
out distinct borders and progressed towards
the leaf margin. However, the yellowing of
the areas between the veins did not cover
the whole leaf. These yellowed or chlorotic
areas caused ruffling of the leaves and such
symptoms disappeared as the leaves be-
came older.

This present study has shown that
C. alata is another host of and thus extends
the host range of a tobamovirus in the
country (Eugenio and Del Rosario, 1962;
Tangonan and Quebral, 1992) and those
reported abroad for the same virus group
(Brunt et al. 1996). Recently, we also
showed that a tobamovirus infects
Mussaenda spp. in the country (Siar and
Bajet, 1996). This is the first report, as far
as we know, that C. alata is shown to have
any disease at all, especially of what appears


Phytopathological Journal 21






Table 2.Mechanical and seed transmission of the putative tobamovirus in mosaic infected
Cassia alata L.

NO. OF PLANTS
WITH SYMPTOMS
METHOD PERCENT
TOTAL PLANTS INFECTION REMARKS
TESTED

Seed Transmission 2/50 4.0* Vein clearing and
mottling of leaves

Sap Transmission 4/10 40.0** Severe curling and
mottling of leaves

2 out of 50 seedlings of C. alata showed symptoms
** 4 out of 10 C. alata plants showed symptoms


to be a natural infection of a virus. A
number of researchers have shown that the
plant was not infectible with papaya
ringspot potyvirus (Malabanan, 1989) and
tobacco mosaic tobamovirus, orchid strain
or (TMV-O) (Rillo, 1975) now known to be
odontoglossum ringspot tobamovirus
(ORSV) (Brunt etal. 1996). However, other
Cassia spp,, like C. occidentalis and C. tora
have been shown to be infectible by a
number of plant viruses (Brunt et al. 1996;
Paguio and del Rosario,1965; Van Velsen,
1961) and that C. podocarpa L. was also
shown to be infected by a virus (Retuerma,
1976).

We believe that the virus causing
the mosaic in C. alata is a tobamovirus
because the mosaic symptoms were similar
to those commonly observed with TMV-
infected plants, it was mechanically
transmissible to N glutinosa and
N. tabacum, had a thermal inactivation
point of about 85-90 C, and showed slight
but positive reaction in enzyme-linked
immunosorbent assay with antisera to TMV
and ORSV, both tobamoviruses but not
with antiserum to potato virus Y (PVY),
potato virus X (PVX), bean yellow mosaic
potyvirus (BYMV), papaya ringspot


potyvirus (PRSV), peanut mottle (PMV)
and peanut stripe potyviruses (PStV). More
significantly, however, is the production of
necrotic local lesions in N. glutinosa.
Holmes (1964) showed that N. glutinosa
and other tobacco cultivars with the TMV
necrotic gene produce necrotic local lesions
which are the most common symptom of
infection by TMV and most of the
tobamoviruses (Brunt et al. 1996). The N.
tabacum plants that also developed
necrotic local lesions similar to those on
N. glutinosa were one of those tobacco
cultivars that have the TMV necrotic gene.
Although very seldom, there are, however,
other virus groups that induce local lesions
on both N. glutinosa and N. tabacum.
These are cymbidium ringspot tombusvirus,
tobacco ringspot nepovirus, eggplant
chlorotic dwarf nucleorhabdovirus and
orchid fleck rhabdovirus (Brunt et al. 1996).

We did not determine if seed
transmission was a result of the virus being
in or on the seed. It was possible that the
akapulco seeds were carrying extra-
embryonic virus (i.e. on the seed coat) and
the seedlings became infected by contact
with the virus during germination. Most of
the tobamoviruses that have been shown


22 Phytopathological Journal





to be seed transmitted were seed contami-
nants as transmission was eliminated when
the seeds were soaked in acidic conditions
prior to planting (Noordam, 1972) and
none has been reported so far as
tobamovirus in embryos are concerned.
Among host plants, species of the
Leguminosae, to which C. alata belongs,
seem to be prone to infection through the
seed (Brunt, et al. 1996; Corbett and Sisler,
1964; Fulton, 1964).

Diseases with a wide host range are
more difficult to control and are capable of
causing explosive epidemics (Gibbs and
Harrison, 1976; Matthews, 1991).
Considering akapulko's medicinal
properties, the plant could be very popular
and thus, more households may want to
plant it in their backyards or some
enterprising individuals may commercially
exploit it. It was observed that a number of
people who visited the Herbal Garden
usually collect seeds from this plant (Bidad,
1992). Since some of the seeds are infected
with the virus, planting them would cause
possible early disease spread not only in
C. alata but also to other economically
important crops as well (Eugenio and del
Rosario, 1962; Tangonan and Quebral,
1992). The tobamoviruses are highly stable
and may persist on the seed for long
periods so that commercial distribution of
seeds with seed-borne virus may be a very
effective means of spreading the disease.
Seed transmission provides a very effective


means of introducing virus into a crop at
an early stage with random foci or infec-
tion throughout a plantation (Gibbs and
Harrison, 1976; Matthews, 1991).

Likewise, it is not known what
effect, if any, the tobamovirus infection does
on the medicinal properties or components
of akapulko. It is known, however that
virus infection induce many changes,
though probably secondary consequences,
in the metabolism of the host (Matthews,
1991),

SUMMARY AND CONCLUSION

Sap of C. alata plants maintained
at the UPLB Herbal Garden, Institute of
Biological Sciences with mosaic symptoms
was mechanically inoculated to C. alata,
N. glutinosa and N. tabacum. Inoculated
C. alata seedlings developed mottling, leaf
curling and were stunted. Necrotic local
lesions developed on N. tabacum and N.
glutinosa. Fifty seeds collected from pods
of infected C. alata L. were planted and
two seedlings showed symptoms of
infection like vein clearing and then the
leaves developed severe mosaic or mottling
and leaf curling after 6 wk. Extracts of the
field collected and symptomatic C. alata
reacd slightly positive but not with extracts
of leaves of symptomless or apparently
healthy plants to antisera of TMV and
ORSV. The virus is probably a
tobamovirus.


Phytopathological Journal 23






LITERATURE CITED


AQUINO VM. 1981. Inclusion bodies and
some virus infected plants in the
Philippines. M.S. Thesis. UPLB,
College, Laguna.

BAWDEN FC FM ROBERTS. 1947. The
influence of light intensity on the
susceptibility of plants to infection
with certain viruses. Ann. Appl. Biol.,
26:105-115.

BIDAD WTD. 1992. Transmission studies of
mosaic disease of Cassia alata
(Leguminosae). B.S. Thesis. UPLB,
College, Laguna.

BRUNT AA K CRABTREE MJ DALLWITZ
AJ GIBBS L WATSON. 1996.
Viruses of plants. CAB International,
NY USA

CORBETT MK HD SISLER. 1964. Plant
Virology. University of Florida Press.
pp. 17-21.

DE PADUA SL. et al. 1987. Handbook on
Philippine Medicinal Plants, Vol. 1:
UPLB, College, Laguna. p. 37.

EUGENIO CP MS DEL ROSARIO. 1962.
Host range of tobacco mosaic virus
in the Philippines. Philipp. Agr. 46:
175-197.

FULTON RW 1949. Properties of certain
mechanically transmitted viruses of
prunes. Phytopathology 47:683.

GIBBS AJ BD HARRISON. 1976. Plant
Virology; the Principles. Butter and
Tanner Ltd., UK.

HOLMES FO. 1964. Symptomatology of viral
diseases in plants. In Plant Virology.
University of Florida Press, Gainesville.
pp. 17-38.


KASSANIS B 1952. Some effects of high
temperature on the susceptibility of
plants to infection with viruses. Ann.
Appl. Biol. 39: 58-369.

MALABANAN JC 1989. Host range of
papaya ringspot virus (PRSV). B.S.
Thesis. UPLB, Laguna.

MATTHEWS REF 1991. Plant Virology. 3rd
ed. Academic Press Inc., San Diego,
CA, USA.

NOORDAM D 1973. Identification of plant
viruses; methods and experiment.
Cent. Agr. Pub. Doc., Wageningen,
The Netherlands.

PAGUIO OR MS DEL ROSARIO. 1965.
Range and symptoms of two virus
isolates from Crotolaria saltiana Andr.
and Cassia occidentalis L. Philipp.
Phytopathol. 1: 38-39.

RETUERMA ML 1976. Identification of
Philippine plant viruses by electron
microscopy. M.S. Thesis. UPLB,
College, Laguna.

RILLO EP. 1978. Isolation and identification
of the viruses causing mosaic disease
on Laeliocattleya 'Randy' and
chlorotic leaf streak on Oncidium
"Golden Shower" in the Philippines.
M.S. Thesis, UPLB, College, Laguna.

SIAR SV NB BAJET. 1996. Symptomatology,
transmissibility and serological
identification of Mussaenda virus.
Philipp. Phytopathol. 32:35-40.

TANGONAN NG FC QUEBRAL. 1992. Host
index of paint diseases in the
Philippines. Department of Science
and Technology. Taguig, Metro Manila.

VAN VELSEN RJ. 1961 Cassia mosaic. The
Papua New Guinea Agr. J. 14: 124.


24 Phytopathological Journal






Note: Spatial Structure of Natural Epidemics
of Sheath Blight of Rice

Avelino D. Raymundo1, Marizaldy R. Pantua2, and Paul S. Teng3

'Associate Professor, Department of Plant Pathology, University of the Philippines
Los Bafios, College, Laguna; 2Former Research Assistant, Entomology and Plant Pathology
Laboratory, International Rice Research Institute (IRRI), Los Baios, Laguna; and 3Former
Plant Pathologist, IRRI


INTRODUCTION

Sheath blight, caused by
Rhizoctonia solani Kuhn, has become a
major disease of rice (Parmeter, 1970. It
has spread rapidly and can be destructive
in areas where intensive cropping is
practiced (Ou, 1984; Lapis and Peralta,
1987). Since R. solani is soilborne
(Papavisas et al., 1975; Naiki and Ui, 1978),
inoculum, particularly sclerotial bodies,
persists in the soil and serves as source of
infection during the ensuing season. The
magnitude of this inoculum in the soil and
its behavior in relation to spatial dimension
of sheath blight epidemics has yet to be
determined accurately.

The objective of the study was to
ascertain the spatial structure of naturally
occurring epidemics of sheath blight of rice
in farmer's fields.

METHODOLOGY

Two farmer's fields in Pila, Laguna,
planted to rice cultivar PSB Rc2, were
selected for study during the wet season.
Areas comprising 25 x 25 hills in each
location were monitored for sheath blight
incidence 8 times at weekly intervals. In
site 1 in Barangay San Antonio initial
reading was done close to flowering while
in site 2 in Barangay Pansol data gathering
started at maximum tillering stage.


RESULTS AND DISCUSSION

Sheath blight was common in both
fields. In the first site in Barangay San
Antonio where disease distribution was
uniform, approximately 25 percent of the
hills showed symptoms during the first
reading at 55 days after transplanting. Six
weeks after, infection was observed in the
whole plot. In the second site in Barangay
Pansol, an aggregate pattern of disease
dispersion was observed. A focus of
expansion was observed emanating close
to an open irrigation canal indicating that
inoculum was transported by irrigation
water. During the initial reading at 35 days
after transplanting in this site, symptoms
were seen in 15 percent of the hills. Eight
weeks later, disease spread was observed
in 80 percent of the hills (Fig.l).
Geostatistical analysis indicates that
variability in patterns among diseased plant
is random and not influenced by spatial
dependence among neighboring hills.
R. solani inoculum is abundant in
the soil as manifested by high sheath blight
infection that can easily be observed in
uniform or random aggregate pattern.
Sclerotial bodies are the primary structures
that survive in the soil and are the principal
sources of inoculum in the ensuing
cropping season (Palo, 1926; Baker and
Martinson, 1970). In lowland rice cropping
systems, sclerotia may float on water
surface and upon contact with any part of


Phytopathological Journal 25






the host, which very likely is the leaf sheath,
initiate infection by producing germ tubes
(Than, 1990). The characteristic buoyancy
of sclerotial bodies facilitates this contact
with the rice host (Hashiba and Mogi,
1975). This buoyancy could have hastened
the observed rapid spatial intensification of
the disease during the season as irrigation
water carrying sclerotia can increase
disease foci significantly.

Soil inoculum appears to be readily
available for infection as shown by the


20



.c




10:




:


finding that disease spread was not influ-
enced by spatial dependence among ad-
joining hills. Yin (1985) has calculated that
during a rice-growing season in the Philip-
pines, the number of sclerotial bodies
formed in a hectare of rice field is 2.62
million. Many of these sclerotia are left in
the field after harvest and may survive for
several months as earlier reported by Palo
(1926). Their survival is further enhanced
when farmers plant other crops, which are
also hosts of R. solani (Baker, 1970).


SHEATH BLIGHT INCIDENCE
WET SEASON 1992
San Antonio, Pila, Laguna

+ x + x x n + + x x x x + + + +
+x+x~ xD++x~ixxx++0 ++


++++x x
KxxxxxlIO+
1- + x x x + Li +
++xx'l+0+

*+ + + X+



x++**Oxx
x + x *
XX + + +
XXX++XD
. + + + 0 0 + +
+x+x++x
xxx+xx U X
x 7 I x 7


4


k c
+ x

4- x
+ +
x +


+ x
* O++
* D + +
+ + + x
++xx
+ + x x
+* X + +


x x + + D + U + + + OD
S.-0 F x x D * 7 * l
* x * * * x * + *
+ xx * * x +
+ + x + 0D + + +
S x x + x x x n"* x +
+ D * xx D * x + x
i[! x x 7; x + x I * * + +
*x+x+**[OO+x+x
* x + x D + * D 1 + x + x
* * x x + x x
* + D + + xx + + + +
x Dx x x x x D x + x
+ + x + * x x + x OD *
Si + * + + + C]
x * + * O + + x
x O * * xxx
+ + * x + xx + x
+ + x + + + + + x + x x +
+ + x + + + + x + + x +
S0 [ Dx OOLN xx x
+ x O D x + + x + + + + + +
+xxDx++x++ ++++
+XX+X*L+L++X++
X7 ZS7 ~ L I S 5, .1 5, ,5


AS AS 4&S C


20


1.00 < + < 1.000
1:00 < x < 14.00
14.00 < .< 21.00
21.00 < < 43.00


Fig. 1. Rice sheath blight incidence in Pansol, Pila, Laguna, wet season, 1992.

26 Phytopathological Journal


1st Quartile
2nd Quartile:
3rd Quartile:
4th Quartile:


rr


II IX ^






LITERATURE CITED

BAKER KE 1970. Types of Rhizoctonia dis-
eases and their occurrence, pp.125-148. In
Parmeter, J. R. Jr. (Ed.). Rhizoctonia solani,
Biology and Pathology. Univ. California
Press. Los Angeles. 255pp.

BAKER KF MARTINSON CA. 1970. Epi-
demiology of diseases caused by
Rhizoctonia solani, pp. 172-188. In
Parmeter, J. R. Jr. (Ed.). Rhizoctonia solani,
Biology and Pathology. Univ. California
Press. Los Angeles. 255pp.

HASHIBA T MOGI S. 1975. Developmental
changes in sclerotia of the rice sheath blight
fungus. Phytopathology 65: 159-162.

Lapis DB Peralta GA. 1987. Disease control
in rice: Biological control of sheath blight
of rice caused by Rhizoctoniasolani Khun.
Central Experiment Station Ann. Rept.,
Univ. Philipp. Los Bafios, College, Laguna

NAIKI T UI T 1978. Ecological and morpho-
logical characteristics of the sclerotia of
Rhizoctonia solani Kuhn produced in soil.
Soil Bio. Biochem. 10: 471-478.


OU SH. 1984. Rice Diseases. 2"n Ed. Com-
monwealth Mycological Institute, Kew, Sur-
rey. England. 290pp.

PALO MA. 1926. Rhizoctonia diseases of
rice. 1. A study of the disease and of the
influence of certain conditions on viability
of the sclerotial bodies of the causal fun-
gus. Philipp. Agric. 15:361-376.

PAPAVISAS GC ADAMS PB LUMSDEN
RD LEWIS JA. 1975. Ecology and epi-
demiology of Rhizoctonia soloni in field soil.
Phytopathology 65: 871-877.

PARMETER JR JR. (Ed.). 1970. Rhizoctonia
solani, Biology and Pathology. Univ. Cali-
foria Press. Los Angeles. 255pp.

THAN H. 1990. Characterization of
Rhizoctonia solani Kuhn in rice-based crop-
ping systems. Unpublished MS Thesis, Univ.
Philipp. Los Bafios, College, Laguna

YIN S. 1985. Inoculum distribution pattern in
relation to rice sheath blight. Unpublished
MS Thesis, Univ. Philipp. Los Bafios,
College, Laguna


Phytopathological Journal 27




























































28 Phytopathological Journal






Note: Rust of Gendarussa Caused by
Puccinia Thwaitesii B. & Br.

Naomi G. Tangonan & Makoto Kakishima

Respectively, Professors, Dept. of Plant Pathology, College of Agriculture, University
of Southern Mindanao, Kabacan, Cotabato, Philippines and Institute of Agriculture and
Forestry, University of Tsukuba, Ibaraki 305, Japan.


ABSTRACT

Puccinia thwaitesii B. & Br. Is hereby reported as causing rust disease
of Justicia gendarussa Burm. E (=Gendarussa vulgaris Nees), locally known
as "malabulak", "tuhod-manok" and "kapanitulot" (Tagalog), or "bonlaw"
(Bisaya or Cebuano, Ilongo). Infected leaves show enlarged yellow blotches
on the upper surface; dark brown to black pustules of powdery masses
teliaa and teliospores) that protrude in a circular fashion surrounded by a
yellow halo are evident on the undersurface. Teliospores measure 24 x 30
microns.


INTRODUCTION

Justicia gendarussa Burm. F is a
shrub that grows to 2 m tall. Stems are
branching semi-woody, reddish when
young, turning green at maturity. Leaves
are opposite, lanceolate, acute or
acuminate, 10 cm long, green or variegated
with short petioles (Madulid, 1995). Locals
use its leaves for medication of flu and fever
and as antiseptic bath for mothers who just
gave birth. It is also a popular ornamental
cultivated by stem cuttings.

Tangonan and Quebral (1992) listed
three diseases of gendarussa, namely: algal
spot, leafspot, and rust. The latter was
identified only to the generic level as caused
by Puccinia sp. (Divinagracia, 1985). This
report gives its species name, P thwaitesii.

This study aimed to identify the cause
of rust disease affecting leaves of gendarussa
at USM, Kabacan, Cotabato.


METHODOLOGY

Infected leaves were collected
from the Medicinal Plant Collection of
USM located at the College of Arts and
Sciences. Direct microscopic examination
was done and teliospores were measured
and photographed. Some of the specimens
were sent to the co-author, Dr. M. Kakishima
of the University of Tsukuba's Institute
of Agriculture and Forestry, Japan for
identification/confirmation.

IDENTIFICATION

Based on measurements of
teliospores and using the keys and species
descriptions of Lohsomboon et al (1986);
Sydow P & H (1904); Arthur and Cummins
(1936); Boedijn (1959); the rust disease of
gendarussa is identified as caused by
Puccinia thwaitesii Berk. & Br.


Phytopathological Journal 29






Symptoms.

Infected plants (leaves) show large,
stark yellowish blotches on the surface that
are irregular in shape. A slight dark
discoloration is noticeable on the middle
area. When examined on its undersurface,
however, numerous enlarged circular dark
brown to black powdery masses of the
causal fungus are apparent. These powdery
pustules teliaa and teliospores) protrude and
easily come off when rubbed. A yellow halo
radiates from the circular mass of the dark


pustules. Slight to sever puckering of these
infected parts may be observed until the
leaves eventually shrink and dry up (Fig 1
and 2). In severe cases, the stems and
branches also get infected.

SIGNS

On microscopic examination,
binucleate teliospores are abundant which
are noticeably thick-walled and orange to
dark brown in color. Teliospores measure
an average of 24 x 30 microns (Fig 3).


Fig. 1. Rust-infected gendarussa
caused by Puccinia thwaitesii B. &
Br. Note lower two leaves showing
characteristic symptoms: masses of
black teliospores protrude on the
lower surface of the leaves which
later become shrunken and dry up.


Fig. 2. Upper leaf surface symptom
(left), lower leaf symptom (middle),
of rust-infected gendarussa and
healthy leaf (right).


30 Phytopathological Journal
30 Phytopathological Journal











Fig. 3. Two-celled teliospores of
P thwaitesii B. & Br. Causing rust
of gendarussa.


LITERATURE CITED


ARTHUR, JC CUMMINS, GB. 1936.
Philippine rusts in the Clemens collection
1923-1926. II. Philipp. J. Sci. 61:463-488.
BOEDIJN KB. 1959. The Uredinales of
Indonesia. Nova Hedwigia 1:463-496.
DIVINAGRACIA GG. 1985. Diseases of
important foliage and'flowering ornamental
plants. NRCP-UPLB Term. Rpt., Mimeog.
LOHSOMBOON P MANOCH L
VISARATHANONTH, N KAKISHIMA
M ONO Y SATO S. 1986. Materials for
the rust flora in Thailand II. Trans. Mycol.
Soc. Japan 27:271-281.


MADULID DA. 1995. A pictorial cyclopedia
of Philippine ornamental plants. Bookmark,
Inc., 388 p.
SYDOW P SYDOW H. 1904. Monographia
Uredinearum Vol. 1, Fratres Borntrager,
Lipsiae. 972 p.
TANGONAN NG QUEBRAL FC. 1992.
Host index of plant diseases in the
Philippines, 2nd ed., 273 p.


Phytopathological Journal 31






INFORMATION FOR CONTRIBUTORS


1. Membership in the Philippine Phytopathological Society is prerequisite to publishing in
Philippine Phytopathology or at least one author must be a member of this society. The
Editorial Board, however, may relax this rule for contributions of exceptional merit. It may
also invite distinguished scientists to contribute articles of interest to the Society.

2. Manuscripts must be reports of original research, excerpt of meritorious reviews, and should
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manuscript is final.

3. The manuscript should be typed on one side of 8 '2 x 11 inch paper, double spaced throughout.

4. Papers other than Notes may be organized conveniently under: Title, Author(s), Author's
position and address, Key words, Abstract, Introduction, Materials and Methods, Results,
Discussion, (or Results and Discussion) and Literature Cited.

5. Acknowledgments should be placed at the end of the articles i.e. after Literature Cited.

6. In the text, citations should be by name-and-year system, e.g. Molina (1996), Ou and Nuque
(1980). With three or more authors, use and others (e.g. Ou, Nuque and Silva (1981) should
appear Ou and others (1981).

7. Literature citation should be in alphabetical order. Do riot cite unpublished work; it should
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be consulted in abbreviating the names of journals. Examples of abbreviations : Phil
Phytopath, J Mol Biol, P1 Dis Rep J Agr Res, Amer J Bot

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