• TABLE OF CONTENTS
HIDE
 Front Cover
 Effect of ultraviolet irradiation...
 Quantification of Inoculum of Colletotrichum...
 Parasitism of sclerotial bodies...
 Reaction of ten corn cultivars/lines...
 Reaction of different corn, legume...
 Temporal susceptibility of 'Carabao'...
 Purification and serological characterization...
 Symptomatology and pathogens associated...
 Back Matter
 Back Cover














Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00090520/00040
 Material Information
Title: Journal of tropical plant pathology
Series Title: Journal of tropical plant pathology.
Alternate Title: Journal of Philippine phytopathology
Philippine phytopathology
Physical Description: v. : ill. (some col.) ; 26 cm.
Language: English
Creator: Philippine Phytopathological Society
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
College Laguna
Publication Date: January-June 1997
Frequency: semiannual
regular
 Subjects
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
 Notes
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00040
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 54382605
issn - 0115-0804

Table of Contents
    Front Cover
        Front Cover 1
        Front Cover 2
    Effect of ultraviolet irradiation on banana fruit rot caused by Lasiodiplodia theobromae (pat.) Griff. and Maubl.
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
    Quantification of Inoculum of Colletotrichum gloeosporioides Penz. within a 'Carabao' mango orchard
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Parasitism of sclerotial bodies of Rhizoctonia solani Kühn by Trichoderma harzianum Rifai and Penicillium oxalicum Currie and Thom
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
    Reaction of ten corn cultivars/lines to five isolates of Erwinia carotovora var. chrysanthemi causing stalk rot
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
    Reaction of different corn, legume and rootcrop varieties to the rice root knot nematode, meloidogyne graminicola Golden and Birchfield
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
    Temporal susceptibility of 'Carabao' mango flushes to Colletotrichum gloeosporioides Penz.
        Page 45
        Page 46
        Page 47
        Page 48
    Purification and serological characterization of papaya ringspot potyvirus
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
    Symptomatology and pathogens associated with stem rot disease complex in periwinkle
        Page 68
        Page 69
        Page 70
        Page 71
    Back Matter
        Page 72
    Back Cover
        Page 73
        Page 74
Full Text

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197 Phil. Phytopath. 33(1):1-8 1


EFFECT OF ULTRAVIOLET IRRJ
ROT CAUSED BY LASIODI
(PAT.) GRIFF. A


M. G. MORTUZA


Portion of the Ph. D. dissertation of the

Respectively, former Graduate Stude
Pathology, University of the Philippines, Los B&

Key words: banana, fruit rot, Lasi
ultraviolet light

One-celled spores of Lasiodiplc
after 1 min exposure to ultraviolet 1
whereas two-celled spores were stimu
elongation proceeded even after 30
theobromae exposed to UV light wei
Disease reduction was not correlated
banana fruits to UV light. Banana fru
least rotting. Banana fruits exposed t<
with L. theobromae at the rate of 500 o
protected from rotting. Irradiated banai
exposed surface.


INTRODUCTION i
t
Banana (Musa *spp.) is the most 1
important fruit crop widely cultivated in r
tropical countries. Postharvest infection of c
banana fruits can take place before and 2
during harvest or subsequent to harvest. t
Among the postharvest diseases, fruit rots (
due to Lasiodiplodia theobromae (Pat.) I
Griff. and Maubl. and Colletotrichum r
musae (Berg. and Curt) Arx. are most r
common in the Philippines (Ilag 1977). r
Recently, there have been a number of I
effective fungicides for the control of c
postharvest diseases of fruits. Benomyl f
was extensively used in reducing I


DIATION ON BANANA FRUIT
'LODIA THEOBROMAE
4D MAUBL.


id L. L. ILAG


snior author.

: and Professor, Department of Plant
os, College, Laguna.

liplodia theobromae, physical control,


ia theobromae lost their viability
;ht (UV) (wavelength 254 nm)
ted to germinate and germ tube
nin exposure. Mycelia of L.
able to survive up to 20 min.
rith the duration of exposure of
s exposed for 10 min resulted in
UV light 12 hr after inoculation
1,000 conidia/ml were completely
fruits developed bronzing on the



s of the produce. However, benomyl is
coming less effective because
stharvest pathogens are developing
distance to it. Benomyl resistant strains
L. theobromae and C. musae have
eady been reported from Central
nerica banana plantations (Slabaugh and
ove. 1982). Similarly, Farungsang and
rungsang (1992) observed benomyl
;istant strains of C. gloeosporioides in
ngo. As a result of the development of
distant strains of pathogens and the
zard to public health and environment
e to harmful fungicides, researchers have
md ultraviolet (UV) light (wavelength
II1,.r 2fA nr\ o an aisrnahirr ,\ tn





2 1997 Phil. Phytopath. 33(1):1-8


UV light, an elicitor of resistance in
harvested crops (Wilson and others 1994),
is considered as a new technology for the
control of postharvest diseases. Unlike
vegetative plant tissue, harvested
commodities are senescing rather than
developing. The senescence process
generally reduces resistance responses in
harvested fruits.

Stevens and others (1990 and
1991) reported that UV light reduced
Botrytis rot of apple, Rhizopus and
Fusarium storage rots of sweet potato,
black rot, and bacterial soft rot of onions
and extended the shelf life of these
commodities. UV light resulted in induced
resistance to Penicillium digitatum, the
green mold pathogen of grapefruit (Droby
and others 1993), apples (Lu and others
1991) and peaches (Lu and others 1993)
against postharvest diseases and extended
their shelf life.

Mercier and others (1993) observed
that the induction of a phytoalexin in carrot
slices by UV light increased the resistance
of tissue to Botrytis cinerea and
Sclerotinia sclerotiorum infection. The
onset of UV-induced resistance coincided
with the induction of activity by
phenylalanine ammonia lyase (PAL) and
peroxidase (Droby and others 1993).

There are several advantages of
using UV light for the control. of
postharvest diseases of fruits and
vegetables. UV light does not cause
radioactivity or toxic accumulation of
chemical residues. It is easier and safer to
operate than ionizing radiation such as
gamma rays. Unlike gamma rays, it does
not cause softening of the storage roots
(Stevens and others 1991).

The purpose of this study was to
determine the effect of UV light on banana


fruit rot infection and to characterize the
activity of UV light as it is affected by
inoculum level and length of irradiation.


MATERIALS AND METHODS

Isolation of Pathogen and Preparation
of Inoculum

The banana fruit rot pathogen, L.
theobromae, was isolated from infected
banana fruits collected from the Los Bafios
public market (Laguna). The isolate was
purified and maintained in PDA medium at
room temperature. Pathogenicity tests
were carried out in ripe banana fruits (cv.
'Lakatan'). The pathogen was reisolated
from the infected fruits and purified.

Pycnidia from 15-day old cultures
of L. thoebromae were crushed over a
glass slide containing a drop of water. The
concentration of the spores was determined
using a hemacytometer following Sharvelle
(1961).

Spore Germination and Germ Tube
Elongation

Spore suspension of L. theobromae
was prepared from two-week old PDA
slant cultures and spore concentration was
adjusted to 10,000 conidia/ml. Two
loopfuls of spore suspension were spread
over clean glass slide and allowed to air
dry. The slides were exposed to ultraviolet
light by placing them under an ultraviolet
lamp which emits 400 uW/cm2 wave
energy at wave length 254 nm. Slides were
placed approximately 15 cm from the light
source. The exposure periods were 0, 5,
10, 15, 20, 25, and 30 min. Immediately
after irradiation, the slides were placed in a
plastic container (100% RH) and incubated
in darkness for. 12 hr. Non-irradiated slides
were used as control. The slides were





1997 Phil. Phytopath. 33(1):1-8 3


stained with lactophenol cotton blue to
stop further growth during the measuring
process. Spore germination was
determined and germ tube growth was
measured by randomly selecting 50
germinating spores in each treatment.

Mycelial growth

It was observed that spores and
fungal mycelia in covered glass or plastic
Petri plates were not affected at all by UV
radiation even after 30 min exposure. Thus
the mycelia of the pathogen were exposed
directly to UV light.

Agar discs from the edge of one-
day old cultures of L. theobromae were
placed on sterile glass slides. The slides
with the agar discs were exposed to UV
light for 5, 10, 15, 20, 25 and 30 min. The
slides were kept in darkness for one hour.
A bit of irradiated mycelium was
transferred into the PDA plates. The plates
were incubated in darkness for 24 hr at
room temperature. The mycelia which
failed to grow within 5 days were
considered dead. The pycnidia were
counted after 10 days of incubation.

Disease inhibition

Banana fruits of ripening grade 5
(Lizada and others 1990) were used for the
tests. Fruits were surface-disinfected by
immersion in 10% commercial bleach for 5
min, air-dried and placed in plastic trays for
one hour prior to inoculation. Two
wounds (two pin pricks) in each wound
site were made in each fruit. Five ml spore
suspension of L. theobromae at
concentration of 2,500 conidia/ml were
pipetted in each wound site. After 24 hr of
incubation, the inoculated fruits were
exposed to UV light for different duration
ranging from 2.5 min to 15 min. Irradiated
fruits were stored in the dark for 24 hr


following Bridge and Klarman (1973).
Disease development was recorded after 3
days by measuring the diameter of rotten
tissues. Control fruits were not irradiated.

Inoculum Level and Time of Irradiation

The duration of exposure was fixed
for 10 min as it was observed in the
previous study that 10 min exposure could
significantly inhibit disease development in
inoculated bananas. Fruits were wounded
as before. Three inoculum levels (500,
1,000 and 5,000 conidia/ml) of L.
theobromae were used for inoculation.
Some fruits were exposed to UV light 12
hr after inoculation while others were
treated 24 hr after inoculation. Control
fruits were not irradiated. Fruits were
stored in the dark for 24 hr at room
temperature. Disease development was
determined after 4 days as previously
described.

Statistical Analysis

Data for each set of treatments
were analyzed as a series of one way
analysis of variance designed for
randomized complete block. Whenever F
values for ANOVA were significant, the
means were compared by DMRT.


RESULTS

Spore Germination and Germ Tube
Elongation

Ultraviolet light remarkably
affected germination and germ tube
elongation of L. theobromae. The
germination of one-celled spores was
inversely related with the duration of
exposure. Spores irradiated for 15 min
were not able to germinate (Table 1). In a
separate investigation, it was observed that





4 1997 Phil. Phytopath. 33(1):1-8


Table 1. Effect of ultraviolet light exposure for
varying duration on spore germination
and germ tube elongation of
Lasiodiplodia theobromae after 12 hr
incubation.

Duration Germination Length of germ
of (%) tube (urn)
exposure one- two- one- two-
(min) celled celled celled celled

0 78.3d 48.4a 619.6d 361.4a
5 30.5c 57.1b 269.7c 609.0b
10 2.2b 56.3b 80.5b 653.8c
15 O.Oa 57.3b O.Oa 654.2c
20 O.Oa 61.6c O.Oa 709.7d
25 O.Oa 63.5d O.Oa 779.0e
30 O.Oa 66.4e O.Oa 1,025.5f

Values followed by the same letter in a column are not
significantly different (P = 0.05) by DMRT.


Table 2. Effect of duration of exposure to ultraviolet
light on mycelial growth and pycnidial
formation of Lasiodiplodia theobromae.

Duration of Mycelial Number of
exposure (min) growth pycnidia

0 + 10b
5 + 33d
10 + 13bc
15 + 16c
20 + 10b
25 -Oa
30 Oa

+ visible growth after 5 days incubation; no visible growth after 5
days. Values followed by the same letters in the column are not
significantly different (P = 0.05) by DMRT.

Table 3. Effect of duration of exposure to
ultraviolet light on Lasiodiplodia
theobromae infection in banana fruits.

Exposure Rot diameter (mm) after
time (min) 2 days 3 days 4 days

0 12.3c 23.2e 39.5d
2.5 8.0b 17.2c 36.1cd
5.0 6.5b 13.5b 28.8b
7.5 9.0b 20.3d 35.1c
10.0 1.5a 6.0a 22.2a
12.5 8.2b 17.2c 35.8cd
15.0 8.8b 18.0cd 34.7c


11 min exposure was lethal to the spores.
A minimum germination of 2.2% was
noted in spores irradiated for 10 min.
However, the germination of two-celled
spores was significantly increased by UV
light. Spores exposed for 5, 10 and 15 min
did not exhibit a significant difference in
germination but longer exposure of 20 to
30 min significantly increased germination.
The germ tube growth of two-celled spores
gradually increased with increase in
exposure period. The longest germ tube
was recorded in spores exposed for 30 min.

Mycelial growth

The mycelia of L. theobromae that
were exposed for 25 min and 30 min
appeared no longer viable (Table 2).
Growth of mycelia exposed for 10, 15 and
20 min were .visible only after two days
incubation. The fungus exposed for 5 min
produced much more pycnidia compared to
the control. The production of pycnidia
gradually decreased with increase in
duration of exposure.

Disease inhibition

Inoculated banana fruits exposed to
UV light exhibited remarkable inhibition of
disease development. All treatments
significantly reduced lesion diameter.
Fruits exposed for 10 min showed the
smallest lesions (Table 3). After a short
incubation of 2 days, there was a significant
disease reduction in irradiated fruits. This
trend in disease reduction changed after
longer incubation of 4 and 5 days. The rate
of decay development in the inoculated site
of UV irradiated banana fruits progressed
more slowly as compared to non-irradiated
control. Longer exposure (more than 10
min) was not correlated with greater
disease inhibition (Fig. 1).





1997 Phil. Phytopath. 33(1):1-8 5


All banana fruits exposed to UV
light developed blemishes in the fruit peel.
The yellow color of the fruit changed to an
unattractive bronze color. This bronzing
was more prominent in fruits treated with
higher UV doses (Fig. 2). Green banana
fruits exposed to UV light and maintained
in dark conditions for 24 hr became
blackish brown and later became deep red
when the fruits ripened.

Inoculum Level and Time of Irradiation

The effectiveness of UV light on L.
theobromae infection in inoculated banana
fruits was significantly affected by the
inoculum level of the pathogen and time of
irradiation relative to inoculation (Table 4).
Among the three inoculum levels and two
exposure times tested, L. theobromae rot
was completely suppressed at the lower
inoculum levels of 500 and 1,000
conidia/ml when the fruits were exposed to
UV light 12 hr after incubation. A slight
inhibition in rotting was noted in banana
fruits inoculated with 5,000 conidia/ml
after 4 days but the rot diameter was
almost similar to the control 5 days after
inoculation. A delay in irradiation
treatment (24 hr after inoculation) did not
show significant disease reduction.


DISCUSSION

Results revealed that one-celled
spores are more sensitive to UV irradiation
compared to two-celled spores of L.
theobromae. The sensitivity of L.
theobromae spores appeared to vary with
age. The younger one-celled conidia were
inhibited at 11 min exposure whereas the
growth of relatively older two-celled
conidia was stimulated at longer exposure.
There appears to be a differential response
at different stages of the pathogen toward
UV light. Castro and others (1971)


Table 4. Effect of ultraviolet light on Lasiodiplodia
theobromae rot in banana fruits at


various inoculum
irradiation.


levels and time of


Exposure Inoculum Rot diameter
time level (mm) after
(spores/ml) 4 days 5 days
Control 500 6.37 12.12
1,000 12.75 29.87
5,000 11.00 21.37

12 hr after 500 0.00 0.00
inoculation 1,000 0.00 0.00
5,000 7.87 23.87

24 hr after 500 8.37 20.00
inoculation 1,000 10.00 19.62
5,000 9.50 18.87











9e


Figure 1. Effect of
irradiation
inoculated
theobromae
incubation.


duration of ultraviolet
on rot in banana fruits
with Lasiodiplodia
after 2 days of


Figure 2. Effect of ultraviolet irradiation on banana
fruits after 2 days. Note bronzing of
treated bananas.





6 1997 Phil. Phytopath. 33(1):1-8


observed that the sensitivity of zoospores
of Phytophthora capsici varied at different
stages of germination. They noted higher
percentage of survivors when zoospores
were irradiated 5 min after encystment than
when irradiated 25 min after encystment.
The structure and composition of the more
mature thick-walled two-celled spores may
be quite different from the younger thin
walled one-celled spores which may
explain the varying response of the two
kinds of spores to UV light.

UV light inhibited the normal
physiology of the pathogen. When the
fungus was exposed for 10 to 20 min to
UV light, mycelial growth was visible only
2 days after irradiation. Longer exposure
of 25 min or above to UV light is
detrimental to the pathogen. The results
indicated that UV light at longer exposure
is germicidal.

The optimum exposure for
significant disease inhibition occurred in a
narrow range of 10 min and longer
exposure of fruits to UV light was not
correlated with increased disease inhibition.
Similarly, Wilson and others (1994)
reported that UV doses for maximum
induced resistance occurred in a narrow
range which was specific for each
commodity and higher induced resistance
was not correlated with the doses of UV
irradiation.

Several evidences indicate that the
resistance mechanism in irradiated fruits are
induced and active in the peel of treated
fruits. UV light is not an ionizing radiation
and does not penetrate plant tissue very
deep (Lucky 1980). The induced activity
of phenylalanine ammonia-lyase (PAL) and
peroxidase enzymes in the UV treated fruit
in plant defense mechanisms against plant
pathogens has been extensively
documented (Hadwiger and Schwochau


1971; Wilson and others 1994; Gleitz and
others 1991; Droby and others 1993).

When banana fruits were exposed
to UV light, peel blemishes developed on
the exposed peel surface. Similar
blemishes were also reported in other
commodities. Bridge and Klarman (1970)
observed that soybean cotyledons
developed bronze color when incubated in
dark conditions after UV treatment but
bronzing was reduced when incubated
under strong visible light after UV
exposure. Similarly, Droby and others
(1993) noted peel blemishes in grapefruit
exposed to higher doses of UV light than
that required for maximum response for
induced resistance. Although UV
irradiation caused blemishes to appear in
treated fruits, still there is a possibility to
use it for the protection of L. theobromae
rot in banana fruits. Storage of irradiated
commodities in darkness for some period is
necessary for UV activity and development
of induced resistance (Bridge and Klarman
1973) which resulted in bronzing on the
fruit peel. It may be possible to achieve
good protection as well as acceptable fruit
color by adjustment of dark and visible
light incubation of the irradiated fruits.
Bridge and Klarman (1973) reported that
the concentration of the phytoalexin,
hydroxyphaseollin, remained high in
irradiated soybean hypocotyls placed in
darkness for 48 hr and subsequently placed
in light for 48 hr. Further work is needed
to find out the optimum period of dark
incubation to avoid fruit blemishes.

The response of fruit to UV
treatment at different times after
'inoculation indicates the development of
resistance against L. theobromae. Induced
resistance by UV light was able to provide
protection at the lower inoculum level of
500 or 1,000 but not 5,000 conidia/ml
indicating that UV light is not effective





1997 Phil. Phytopath. 33(1):1-8


when the inoculum level is very high
Fruits treated 24 hr after inoculation
resulted in more disease than those treated
12 hr after inoculation suggesting that UN
light is effective at the beginning o
infection process but not effective where
infection is already established. The
pathogen, L. theobromae, infects through
wounds and expresses disease symptom:
only in ripe fruits. Thus, protection of th<
fruit from infection through wound!
inflicted mostly during harvesting an(
handling, may be feasible, through U\
treatment as protection is needed at th<
beginning of ripening.

Further work is needed tc
determine if a phytoalexin, in plant tissue!
in response to UV light, is involved in th<
increase in resistance of banana fruit tc
rotting by L. theobromae. The activity o
PAL and peroxidase enzymes in UV.
treated banana fruits needs to be
determined. Development of induce
resistance in UV-treated fruit peel i!


CASTRO FJ, ZENTMYER GA, BELSEF
WL. 1971. Induction of autotrophii
mutants in Phytophthora by ultraviole
light. Phytopathology 61:283-289.

DROBY S, CHALUTZ E, HOREV B
COHERNL, GABA V, WILSON CL
WISNIEWSKI MF. 1993. Factor:
affecting UV-induced resistance it
grapefruit against the green mold deca3
caused by Penicillium digitatum. PI Patho
42:418-424.

FARUNGSANG U, FARUNGSANG N
1992. Resistance to benomyl o
Colletotrichum spp. causing anthracnose o
rambutan and mango in Thailand. Act,
Horti 321:891-897.

GLEITZ J, SCHNITZLER JP, STEIMLE
D, SEITZ HU. 1991. Metabolic changes ir
carrot cells in response to simultaneou!
treatment with ultraviolet light and a funga
elicitor. Planta 184:362-367.








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LUCKY TD. 1980. Hormones with S. 1991. Ultraviolet light induce
ionizing radiation. CRC Press, Boca Raton, resistance against postharvest diseases ii
-t ,,/ -.-- I I .A it'- *i- T-- /"^ T XX T'!- -- -


rt. Lnaiuiz, eas. loiuOiglal LunuuIl I


MERCIER J,


:ance to storage painogens m carrot
Sby UV-C. J Phytopathology 137:44- WILSON CL, GHAUTH AE,
E, DROBY S, STEVENS
KHAN VA, ARUL J. 1994.
RVELLE EG. 1961. The nature and induced resistance to control
of modem fungicides. Burgess diseases of fruits and vegeta
shing Co., Minnesota, USA. 308p. 78:837-844.


8


(11:1-1





7 Phil. Phytopath. 33(1):9-16 9


QUANTIFICATION OF INOCUL1
GLOEOSPORIOIDES PENZ. I
MANGO OR(


O. S. OPINA and A.


Portion of a project funded by the
agricultural Research (DA-BAR) and Graduate 1

Respectively, Professor and Graduate S
university of the Philippines Los Bafios, College,

Key words: anthracnose, Colletotr
oculum source, mango


A study was conducted to quan
from various sources within 'Carabao' n
quantitative data to implicate infected les
inoculum. Results indicate that C. g
recovered from lesions of attached a
mummified panicles and infected branch 1
an average of 4.45 x 103 conidia per
attached leaves, 0.55 x 103 conidia per
leaves, 3.8 x 105 conidia per panicle from
8 to 108 x 102 conidia per 5-cm terry
Substantial amount of inoculum was rece
sources when subjected to moist condit
was removed. Estimates of inoculum ]
mango canopy indicated that infected
significant sources of inoculum followed
Conidia at an average of 37 conidia/m3
Kramer-Collins spore sampler above 2 ki
a spore release tower.


INTRODUCTION Fil
tri
Infected mango plant parts such as fn
aves, branch terminals, twigs, floral parts in
id mummified inflorescence are regarded Si
o inoculum sources of Colletotrichum is(
loeosporioides Penz. within mango (P
rchards (Quebral 1970; Pordesimo 1979; cc


M OF COLLETOTRICHUM
WITHIN A 'CARABAO'
'HARD


.EUSEBIO


departmentt of Agriculture Bureau of
iesis of the second author.

dent, Department of Plant Pathology,
.aguna 4031.

:hum gloeosporioides, epidemiology,



fy the amount of inoculum
mgo canopy and to provide
'litter as effective source of
oeosporioides conidia were
J detached mango leaves,
rminals. At any given time,
lesion was recovered from
lesion from detached dried
nummified inflorescence and
inal from branch terminals.
vered from all the inoculum
mn after the initial inoculum
atential within a 7-year old
leaves constitute the most
>y infected branch terminals.
vere consistently trapped by
dried leaf litter placed inside



:ell and Peak 1984). Conidia are
,ped from these plant parts but the
ority of conidia are trapped from lesions
young leaves (Fitzell and Peak 1984).
ilarly, the pathogen is consistently
ated from infected plant parts
irdesimo 1979). It is believed that the
lidia are dispersed by rain to developing





10 1997 Phil. Phytopath. 33(1):9


young flushes causing lesions on the lea,
and branch terminals or to develop
flowers and incite blossom blight wh
prevents fruit set.

Knowledge of inoculum
fundamental in disease managemi
because reduction of the amount a
efficacy of inoculum is a basic dise<
management strategy. Thorou
understanding of inoculum also provic
basis for disease forecasting that nr
indicate potential infection at a given ti
(Fry 1972). The study of inocuh
therefore, should not end at 1
identification of inoculum sourc
Quantification of initial inoculum a
relative contribution of each inoculi
source is essential in identifying whi
measures to reduce inoculum could
directed. We attempted to quantify 1
amount of inoculum from the identify
sources and used this for the estimation
inoculum potential within a mango cano]
We also attempted to provide quantitat'
data to implicate infected leaf litters
effective source of inoculum.



MATERIALS AND METHOD!

Collection of Possible Inoculum Sourc

Mango plant parts regarded
inoculum sources of C. gloeosporioih
(Quebral 1970; Pordesimo 1979; Fit2
and Peak 1984) such as leaves, leaf little
branch terminals and mummif
inflorescence were collected from the fai
maintained 20- to 40-yr old 'Carabs
mango trees from the orchard of Mei
Farmers Cooperative (MEFCO) farm
Mati, Davao Oriental. The collect
samples were securely wrapped in (
newspaper to prevent further sporulati
-_-i ~ ~ ~ ~ ~ ~ ^. -^ ^- T - x i.


es Pathology, University of the Philippir
ig Los Banos for quantification of
:h gloeosporioides inoculum.

Quantification of Inoculum
is
it Intact and shot-holed lesions cau
id by C. gloeosporioides were individually
;e from randomly collected green mat
;h leaves and dried leaf litters. Ten lesi,
es cut into 6-10 mm2 were placed in a v
ly Branch terminals were segregated i
ie three classes, namely anthracnc
m defoliated branch terminals, branch term
ie with anthracnose-infected leaves
s. apparently healthy branch terminals
id were uniformly cut into 5.0 cm. F
m terminals were placed in a vial. Randoi
-e collected mummified inflorescence w
ie individually cut into 12.5 cm long from
ie tip, chopped into small pieces and plain
:d inside a vial. Known amount of distil


y. conidia adhering the sam
re were mixed thoroughly usi


s. The v


hemacytometer.

To determine the amount
subsequent inoculum from the disease
tissues, the samples were incubated in mc
chamber for 48 hr under laborat<
Condition and new conidia were suspend
following the method described earli
SSpore measurement was done as descrit
above.
-11
Estimation of Potential Inoculum
:d
ly
Amount of mango plant parts su
as leaves, terminal branches, mummifi
inflorescence and leaf litters per tree v
Estimated from 7-yr old 'Carabao' man
*d
trees in a farm in Calamba, Laguna. T
y total number of terminals per tree v
n determined by taking the average numi





I Phil. Phytopath. 33(1):9-16 11


)1 mfe U5ass Lu
Ithy branch cos
lals with trash


d with


:h thick single-sided
was coded to precisely


Vdi i CIjal -AUlii i-i
e count was made under
450X magnification.


ND DISCUSSION


Ig 1.LC ILLIS 1UU11U ill LI;U vi .,AlLU. 11, gweospUrTNMUe IlUocUIUIII
:s were placed in a plastic container
broughtt to the laboratory. Microscopic examinations revealed





12 1997 Phil. Phytopath. 33(1):9-1


r-













Fig. 1'. Major inoculum sources of Cc
canopy: intact and shot-holi
mummified inflorescence (b); br


71







de,














etotrichum gloeosporioides within the mania
I lesions from attached mature leaves (<
ich terminals (c ) and leaf litters (d).





13


high conidial count associated on
mummified inflorescence, there was high
variation in the conidial counts. Some
mummified panicles were colonized by
saprophytes. If C gloeosporioides was
associated with the mummified
inflorescence, the conidia usually occur in
great number. More conidia were obtained
from severely infected terminals. Conidial
count of 108 x 102 and 46.6 x 102 conidia
were counted per 5 cm of defoliated and
leaf-infected branch terminals, respectively.
Surprisingly, an average of 8.0 x 102
conidia per 5 cm was obtained in the
apparently healthy terminals. Severe
infection on defoliated branch terminal may
occurred during the most susceptible stage
of the flush (few days after bud break)
causing defoliation and severe infection of
branch terminals. On branch terminals with
infected leaves, slight infection may occur
during the susceptible stage of the flush
causing few lesions in leaves and on most
succulent part of the stem hence, lesser
conidial count was observed. Ironically,
substantial amount of conidia was counted
on branch terminals with apparently healthy
leaves. It is possible that conidia from
infected leaves may have been trapped on
the leaf axil. This may also indicate cryptic
infections in the branch terminals.

Subjecting the inoculum sources in
a favorable condition for 48 hr after the
initial amount of inoculum had been
removed resulted to tremendous increase in
the amount of subsequent inoculum that
was greater than the initial amount (Table
1). This suggests that acervuli are still
capable of producing new conidia when
subjected to optimum conditions for
development. This also indicates that the
infected plant parts can provide continuous
source of inoculum if favorable weather
prevails. From these basic data, inoculum
contribution of each inoculum source and


lamle i. Comoaai counts or colletotnchum
gloeosporioides from lesions of
leaves, mummified inflorescence and
branch terminals of 'Carabao' mango
in Mati, Davao Oriental.

Inoculum Amount of Inoculum'
Source (x 10 2 conidia/unit)
Initial Subsequent Total
Leaves
Attached 4.45 5.30 9.75
Dried/Litters 0.55 2.70 3.2 0

Mummified 3,800 10,360 14,160
Inflorescence

Branch Terminal

With healthy 8.00 52.00 60.00
leaves
With infected 46.60 210.00 256.60
leaves
Defoliated 108.00 252.00 360.00
Initial inoculum = number of conidia present at the time of
sampling subsequent inoculum = conidia produced after removal
of initial inoculum; each figure represents an average number of
conidia per lesion (average of 1,650 lesions for leaves), per panicle
(average of 122 panicles for mummified inflorescence) and per 5-
cm terminal (average of 100 terminals for branch terminals)

Inoculum Potential of C gloeosporioides

The amount of inoculum from
various sources and expected inoculum
potential of C. gloeosporioides from a 7-
year old 'Carabao' mango tree were
estimated (Table 2). At the time of flower
induction, a 7-year old tree had an average
of 26 x 104 attached leaves, 1.2 x 104 dried
leaves under the canopy, 13 x 103 branch
terminals and 13 mummified and intact
inflorescence panicless). The expected
inoculum potential and relative importance
of each inoculum source were assessed on
the basis of the above information,
inoculum production capacity of each
source and observed field disease incidence
(Table 2). The estimates suggested that
infected leaves constitute the mosT
significant sources of inoculum followed by
infected branch terminals. Mummified





4 1997 Phil. Phytopath. 33(1):9-16


lum Quantity of Inoculum instar ea inslae a spore reil
ce Inoculum Potential2 containing 2 kg of infected d
Sources' (x 10o) leaves (Fig. 2). Under the lab
4,374
26 x 104 4,309 up, conidia were trapped conti


tion of each inoculum source to the Itis study provides
,culum load of C. gloeosporioides. time quantitative data to impli,
ilts show that sanitation pruning, a litters in the ground as effect
practice intended to remove inoculum and also justifi<
branch terminals and other infected standing recommendation of















70-




60-


AIM-


1997 hiL Phytopath. 33(1):9-16


_






16 1997 Phil. Phytopath. 33(1):9-16

FRY WE. 1972. Principles of Plant etiology and control Ph. D. Thesis.
Disease Management. Academic Press, University of the Philippines, Los Bafios,
Inc. Flo. USA. 378p. College, Laguna.

KRAMER CL, EVERMEYER MG, QUEBRAL FC. 1970. Anthracnose of
COLLINS TI. 1976 A new 7-day spore Mango. University of the Philippines
sampler. Phytopathology 66:60-61. College of Agriculture Extension and Plant
Disease Report 6. Department of Plant
PORDESIMO AN. 1979. Anthracnose of Pathology, UPCA.
Philippine Mango cv. 'Carabao': its








PARASITISM OF SCLEROTIA
SOLANI KUHN BY TRICHODE]
PENICILLIUM OXALICUM


C. J. R. CUMAGUI

Supported in part by the Philippine Ri
States Agency for International Development I
senior author.

Respectively, Instructor and Professor,
f the Philippines Los Bafios, College, Laguna

Key words: biocontrol, P. oxalicun
olonization, sclerotial germination, T. harzian


Trichoderma harzianum and I
vitro for their parasitism against sclei
sheath blight of rice. The most effective
pathogen was dried conidial pellets c
culture of P. oxalicum which gave tl
83.85 and 79.5%, respectively in non-s
colonize 100% of sclerotial bodies of
sterile soil. The length of time require,
to prevent the sderotial.bodies of R. so
3 and 4 days, respectively. Sclerotia
prevented by T. harzianum in soil
observed. Scanning electron microsco]
and P. oxalicum along the hyphae of j
which appeared collapsed, vacuolized a


INTRODUCTION

Rhizoctonia solani Kiihn causing
heath blight of rice is a pathogen forming
clerotia which are found in the soil and
infested crop residues and serve as
roculum for rice and succeeding crop after
ice (IRRI 1984). Trichoderma and
'enicillium are among the popular
iyperparasites used for biological control
&f crop pathogens (Kommendahl and
Vindels 1980). Baker and Cook (1974)


17

BODIES OF RHIZOCTONIA
WA HARZIANUM RIFAI AND
f CURRIE AND THOM


and L. L. ILAG

SResearch Institute (PhilRice) and United
JSAID). Portion of the M. S. thesis of the


Department of Plant Pathology, University
031.

R. solani, rice sheath blight, sclerotial



nicillium oxalicum were tested in
tia of Rhizoctonia solani causing
antagonist formulation against the
T. harzianum followed by liquid
: lowest sclerotial germination of
rile soil. T. harzianum was able to
?. solani in both sterile and non-
for T. harzianum and P. oxalicum
mi to germinate in PDA plates was
germination of R. solani was not
lates because no parasitism was
revealed coiling of T. harzianum
solani sclerotia from PDA plates
d deformed.


emphasized that they should be most
effective against survival structures of the
athogen. Understanding their mechanism
f action would greatly aid in improving
iocontrol effects. Another approach to
consider in improving their performance is
) look for at the possibility of using the
lost appropriate fungal propagules for
biological control (Kenney and Couch
981). Lewis and Papavizas (1985) found
iat young mycelial preparations of
richoderma are much better than conidial





18 1997 Phil. Phytopath. 33(1):17-2i


preparations. Papavizas and others (1984
also studied the potential o
chlamydospores as the most important
propagule produced during fermentation
and this should be explored for its survival
and proliferation in soil.

The objectives of this research ar
to determine the most appropriate
microbial structure of Trichoderma against
R. solani and to elucidate the parasitism o
Trichoderma harzianum Rifai ani
Penicillium oxalicum Currie and Thom o;
sclerotia of R. solani in media and so'
plate.


MATERIALS AND METHODS

Sclerotial Germination of R solani ani
Colonization by T. harzianum and I
oxalicum

Six-week old sclerotia of R. solar
cultured in Czapek-Dox agar [(CDA) (2
NaNO3, Ig K2HPO4*H20, 0.5g KCI
0.01g FeSO4, 30g sucrose, 20g in Ili c
distilled water autoclaved at 15 psi for 1
min)] in dark condition were placed on th
surface of sterile (pH 5.84) and non-steril
(pH 5.05) clay loam upland soil, passe
through 850p mesh sieve with moisture
adjusted to 50% (20 sclerotia/plate) an
inoculated with different types c
biocontrol formulation as follows:

Ti = fresh mycelial pellets
T2 = fresh conidial pellets
T3 = dried mycelial pellets
T4 = dried conidial pellets
T5 = granules
T6 = conidial suspension
T7 = mycelial suspension
Ts = chlamydospore suspension
T9 = mixed spore suspension of four
Trichoderma isolates
T10 = liquid culture ofP. oxalicum


TI = dried pellets ofP. oxalicum
TI2 = mixed dried pellets of T. harzianun
and P. oxalicum
T13 = control

T1 to T9 and TI2 were al
formulations of T harzianum isolate 94
016. Each treatment, added to the soil, hai
a spore density standardized at 2.84 x 10
spores/g of soil (dry weight equivalent
while T9 had 7.1 x 106 spores/g of soil a
each isolate 94-012, 94-015, 94-016 an,
94-022. Fresh mycelial pellets wer
prepared by inoculating pellets developed
by Cumagun and Lapis (1993) with spor
suspension of 7-day old culture c
Trichoderma in PDA slant prepared b
dislodging the fungal growth with 9rr
sterile distilled water. Five ml of spor
suspension were introduced to 100g steril
pellets in 500ml flask. The inoculate,
pellets were incubated for 5 days to yiel
mycelial formulation and 14 days, conidi&
formulation. For the dried pell
preparation, the inoculated pellets were air
dried for a day at room temperature after
Sand 14-day incubation. Pellets inoculate
with P. oxalicum followed the sam
procedure. Inoculum preparation fc
granules were produced by preparing
mixture of 19.69g rice bran and 0.4
glucose. This mixture was sterilized at 1
psi for Ihr and inoculated with an age
Block of Trichoderma. Incubation we
done for 10 days with regular turning c
flask following the addition of 140ml c
water and blending for 20 seconds. For th
cake preparation, a mixture of 80g whe<
flour and 20g rice bran was done an
inoculated with 52ml of the prepare
inoculum and 2 drops of Tween 80. Th
dough was kneaded and air-dried for
days. Hardened dough was then passe
through a grinder to form the granules.

Chlamydospore suspension wE
prepared by growing an agar block (





997 IPil. Phytopath. 33(1):17-26 19


irichoderma on 250ml flask containing
>otato dextrose broth (200g potato, 20g
lextrose in Ili of distilled water) for 2-3
lays in the dark. The liquid medium was
)assed through 1051 mesh sieve to remove
:onidia and the resulting inocula were
:hlamydospores along hyphal strands.
)reparation of mycelial suspension
followed the same procedure except sterile
:oconut water medium was employed with
to filtration. Preparation of liquid culture
)f Trichoderma and P. oxalicum was done
)y inoculating a 9ml spore suspension of
sach 7-day old fungus into 100ml sterile
coconutt water media and incubating for 10
lays at 300C under continuous fluorescent
ight.

After 28 days incubation at room
condition (25-350C), sclerotia were picked
rdividually with fine forceps, rinsed in tap
vater, immersed in 1.1% chlorox in 5%
thanol for 3-5 min, washed again with
terile distilled water and blotted on filter
iaper before plating out on modified
"richoderma Medium E (TME) with 10
clerotia per plate. Modified TME
contained one-half the usual amount of the
tystatin concentration of the TME medium
described by Papavizas and Lumsden in
982 (100tl g each of neomycin sulfate,
'acitracin, and penicillin G, 25mg
hlortetracycline HC1, 20 mg nystatin and
00mg sodium propionate in Ili of V-8
iice agar). The plates were exposed to
ontinuous fluorescent light and
olonization was assessed visually after 1-2
lays of incubation.

For sclerotial germination, 10
clerotia per plate were placed in PDA
containing benomyl (5.0mg active
igredient per li) to prevent growth of
richoderma on the sclerotia, and three
antibacterial antibiotics as in the original


plates were incubated at 280C in the dark.
termination of sclerotia was then assessed
visually after 24 hr and the percentage
germination was determined. Sclerotial
germination and colonization were
analyzed in CRD with two replications per
treatment while mean comparison was
lone using DMRT.

rest for Viability of Sclerotia

Two-week old sclerotial bodies of
?. solani were grown in CDA. One ml of
pore suspension (106 conidia/ml) of
rrichoderma isolate 94-016 or P. oxalicum
vas poured in PDA plates. For the test on
oil, the same density of spore suspension
>f antagonists was mixed thoroughly in
ion-sterile clay loam lowland soil (pH
i.24) at Iml/O0g soil. The moisture
-ontent of the soil was adjusted to 25%
w/w) and the mixture was placed in plates.
Twenty sclerotia of R. solani were
inoculated on both agar and soil plates and
incubated at 280C. Periodical observation
in the colonization of the sclerotia by the
antagonists was recorded. The viability of
he parasitized sclerotia was assessed by
laily plating the sclerotia in water agar
(WA) (20g agar in 1li distilled water
utoclaved at 15 psi for 15 min)] plates
after surface sterilization with 1.1% NaOCI
n ethanol for 3 min and rinsed twice in
terile distilled water.

canning Electron Microscopy (SEM)

Two kinds of specimens were
prepared for SEM studies: parasitized
clerotia of R solani and mycelial discs of
itersecting antagonists (T harzianum
isolate 94-016 and P. oxalicum) with the
athogen (R. solani). The specimens were
mixed for 2 hr in 3% formaldehyde or
lutaraldehyde and washed three times in





20 1997 Phil. Phytopath. 33(1):17-26


three times for 15 min before dehydration. mycelial preparations, but not conidia of
All specimens were substituted with 50% most isolates of Trichoderma spp and G.
and 100% isoamyl acetate for 1 hr and 30 virens, prevented damping-off of cotton,
min respectively and air-dried in a sugarbeet and radish seedlings in the
dessicator while the mycelial discs were greenhouse. This finding was not
critical point-dried. All specimens were confirmed in the experiment conducted
then ion coated using Hitachi El01 ion since almost all types of Trichoderma
sputter and viewed in a scanning electron inoculum had not much significant
microscope (Hitachi S-510). difference in reducing sclerotial
germination. Not even the mixture of
strains belonging to a single species was
RESULTS AND DISCUSSION more effective in biocontrol than single
strains. Papavizas and Lewis (1989) found
Among the different types of that several strains of G. virens and T
biocontrol inoculum tested, dried conidial harzianum used alone were equal to or
pellets of T harzianum isolate 94-016 had more effective than double and triple
a significant effect in reducing sclerotial mixtures of such strains in disease
germination of R. solani in sterile soil suppression.
(Table 1). Liquid culture of P. oxalicum
along and dried conidial pellets also Germination of sclerotial bodies of
significantly reduced sclerotial germination R solani was not reduced for almost all
in non-sterile soil. These two formulations treatments using different BCA
were later used in greenhouse test. The formulations though colonization by
effect of the kind of soil was highly Trichoderma was successful. P. oxalicum
significant only with the use of liquid was not successfully recovered from
culture of P. oxalicum lowering sclerotial sclerotia of R solani probably because of
germination from 100 to 79.5% in non- the type of medium used. Though P.
sterile soil. The rest of the treatments had oxalicum grew on TME medium, it is still a
no significant difference with respect to the non-selective medium for the organism. A
kind of soil used. All treatments with T selective medium for P. oxalicum should
harzianum isolate 94-016 had a 100% have been used. Papavizas and Lewis
colonization of sclerotial bodies while (1989) found that although strains GI-3





1997 Phil. Phytopath. 33(1):17-26 21


formulation/inoculum types of Trichoderma harzianum isolate 94-016 and
Penicillium oxalicum'.

Treatment (T) Sterile soil Non-sterile Mean Difference
soil

Fresh Mycelial Pellets 100.00a 100.00a 100.00a 0.00ns
Dried Mycelial Pellets 95.00a 100.00a 97.50ab -5.0ns
Fresh Conidial Pellets 95.00a 95.00ab 95.00abc 0.00ns
Dried Conidial Pellets 80.00b 83.72cd 81.88d -3.75ns
Granules 100.00a 100.00a 100.00a 0.00ns

Dried P. oxalicum Pellets (P) 100.00a 98.75b 99.38a 1.25ns
P+Dried T. harzianum Pellets 100.00a 100.00a 100.00a 0.00ns
Chlamydospore suspension 100.00a 100.00a 100.00a 0.00ns
Mycelial Suspension 100.00a 100.00a 100.00a 0.00ns
Mixed Suspension of T. 100.00a 100.00a 100.00a 0.00ns
harzianum isolates 2

Conidial Suspension 100.00a 100.00a 100.00a 0.00ns
Liquid culture ofP. oxalicum 100.00a 79.50d 89.75c 20.50**
Control 95.00a 90.00bc 92.50bc 5.00ns

S = Mean 97.31 95.92 96.62 1.39ns


'In a column, means followed by a common letter are not significantly different at the 5% level by DMRT;
** = significant at 1% level; ns = not significant.
2Mixed suspension containing 7.1 x 106 conidia/g soil (dry weight equivalent) of each isolate (94-012, 94-
015, 94-016 and 94-022).





22 1997 Phil. Phytopath. 33(1):17-26


Table 2. Percentage germination on Trichoi
solani after 30 days in soil wi
Trichoderma harzianum isolate 94-

Treatment (T) Kinds
Sterile

Fresh Mycelial Pellets 100.00a
Dried Mycelial Pellets 100.00a
Fresh Conidial Pellets 100.00a
Dried Conidial Pellets 100.00b
Granules 100.00a

Dried P. oxalicum Pellets (P) 0.00c
P+Dried T. harzianum Pellets 100.00a
Chlamydospore suspension 100.00a
Mycelial Suspension 100.00a
Mixed Suspension of T
harzianum isolates 2 100.00a

Conidial Suspension 100.00a
Liquid culture of P. oxalicum 15.00b
Control 0.00c

S = Mean 78.08


'In a column, means followed by a common letter ar
** = significant at 1% level; ns = not significant
2Mixed suspension containing 7.1 x 106 conidia/g so
015, 94-016 and 94-022).


erma Medium E of sclerotia of Rhizoctonia
h different formulation/inoculum types of
)16 and Penicillium oxalicum'.

if Soil (S) T-Mean Difference
Non-sterile

100.00a 100.00 0.00ns
10000a 100.00 0.00ns
100.00b 100.00 0.00ns
100.00b 100.00 0.00ns
100.00a 100.00 0.00ns

0.00b 0.00 0.00ns
100.00a 100.00 0.00ns
100.00a 100.00 0.00ns
100.00a 100.00 0.00ns

100.00a 100.00 0.00ns

100.00a 100.00 0.00ns
0.00b 7.50 15.00**
0.00b 0.00 0.00ns

76.92 77.50 1.15


not significantly different at the 5% level by DMRT;

(dry weight equivalent) of each isolate (94-012, 94-





19Y7 Mil. rPytopath. 33(1):17-26 22


germinated. T. harzianum isolate 94-016 glucanases and chitinases cell wall
and P. oxalicum began to grow on the agar degrading enzymes that may play a key role
plate around the sclerotia and colonized during the parasitic process. This
them in 5 days. Most of the sclerotia in the phenomenon might be responsible for the
aoar nlsata tPnaiad to nrmodce aerial hvnhae viability of sclerotia ofR. solani affected by


that there are species of fungi which are control against sheath blight of rice. Phil
mycoparasites on nutrient-rich media like Agri 76:251-257.
PDA but not mycoparasitic in natural soil





24 1997 Phil. Phytopath. 33(1):17-26


Figure 1. Mummified sclerotia of Rhizocta
harzianum isolate 94-016 growi
(upper right) (100x); T. harzianui
(lower left) (2000x); and coiling
(lower right) (1800x).




















Figure 2. Mummified sclerotia of Rhizoci
oxalicum growing on the surface (
spores of P. oxalicum on the surface
of P. oxalicum along the hyphae of


lia solani (upper left) (20x); Trichoderma
) on the surface of sclerotia of R. solani
conidia adhering on the surface of sclerotia
T. harzianum along the hyphae of R. solani





















nia solani (upper left) (40x); Penicillium
' sclerotia of R. solani (unner right) (100x):





1997 Phil. Phytopath. 33(1):17-26 25


LEWIS JA, PAPAVIZAS GC. 1985.
Effect of mycelial preparations of
Trichoderma and Gliocladium on
populations of Rhizoctonia solani and the
incidence of damping-off. Phytopathology
70:404-412.

MEW TW, ROSALES AM. 1984.
Relationship of soil microorganisms to rice


BUYLE LW. 1961. The ecology of
Sclerotium rolfsii with emphasis on the
role of saprophytic media. Phytopathology
51:117-119.

ELAD Y, CHET I, HENIS Y. 1982a.
Degradation of plant pathogenic fungi by
T. harzianum. Canadian Journal of
Microbiology 128:712-725.

ELAD Y, HADAR Y, HENIS Y. 1982b.





26 1997 Phil. Phytopath. 33(1):17-26

RUDAKOV OL. 1978. Physiological control. In: M. N. Burge ed., Fungi in
groups of mycophilic fungi. Mycologia Biological Control Systems. Manchester
70.130-159. University Press, Manchester pp. 161-187.

TRUTMANN P. 1983. Biological control WHIPPS JM. 1997. Developments in the
of Sclerotinia sclerotiorum with emphasis biological control of plant pathogens. In: J.
on evaluation of mycoparasites. Ph. D. A. Callow ed., Advances in Botanical
Thesis. La Troba University, Bundoora, Research Incorporating Advances in Plant
Melbourne, Australia. Pathology. California, USA 134 p.
WHIPPS JM, LEWIS K, COOKE RC.
1988. Mycoparasitism and plant disease





77I /rm. rnytopain. 33(I):L I-3b 27


ACTION OF TEN CORN CULTIP
OF ERWINIA CAROTOVORA
CAUSING S1


G. N. BASTASA am


Portion of the B. S. thesis of the senior

Respectively, Research Assistant, Fi
FARMI), and Professor and Head, Departmer
)f Agriculture (ViSCA), Baybay, Leyte.

Key words: corn, Erwinia carotovora,

The different corn cultivars/lir
the five stalk rot isolates. Stalk rot disc
time until the plants toppled and died.
IPB-UPLB, USM, Kabacan, Cotabato
more severe infection and toppling on
isolates from ViSCA, Baybay, Leyte
Among the corn cultivars/lines evaluate<
resistance to the disease.


INTRODUCTION

Corn (Zea mays L.) is one of the
nost important food crops in the country
nd ranks next to rice as staple crop.
Approximately 20.8% of the population
utilize corn as food instead of rice (PCARR
970). The presence of diseases has been
me of the causes of reduced and poor
quality yield of corn. Accounts of the
presence of the major diseases of corn have
leen documented (PCARR 1970; Gabriel
973) and one of which is the corn stalk rot
caused by Erwinia carotovora var.
hrysanthemi Dye. It is characterized by
otting of the stalks which usually starts
rom the base progressing upward and
eventually causing the leaves to wither.
infected plants either topple over or remain
standing until maturity. Dodd (1980)


ARS/ LINES TO FIVE ISOLATES
4R. CHRYSANTHEMI DYE
LLK ROT


R. M. GAPASIN


uthor, ViSCA, Baybay, Leyte.

m and Resource Management Institute
of Plant Protection, Visayas State College


resistance, stalk rot

s showed differential reactions to
se was also noted to progress with
solates from Tranca, Bay, Laguna,
nd Banga, South Cotabato caused
ie ten corn cultivars/lines than the
id were considered more virulent.
Population 30 exhibited moderate



-ported that the first sign of stalk rot is
permanent wilting of the plant. He added
iat rot generally begins at the lower most
iternode, and development increases with
sing temperatures.

An evaluation of maize germplasm
nd estimation of losses to Erwinia stalk
>t was conducted by Thind and Payak
1978). They reported that percentage
ifected plants ranged from 42.9% in CM
04 (White) to 100% in five inbred lines,
iree hybrids, one composite, and one
pen-pollinated variety. Decrease in grain
field of inoculated plants over checks
anged from 21.3% in CM 600 to 98.8% in
Ibred lines CM 300. Moreover, they
sported that an accounting of both disease
icidence and yield loss suggested that the
vo inbred lines, CM 101 and CM 110, and





28 1997 Phil. Phytopath. 33(1):27-36


the two open-pollinated varieties, Rudrapar
Local (CM 600) and Basi, possess
tolerance to this bacterial disease. Suhayda
and Goodman (1981) mentioned that the
potential grain yield of corn is lowered
because kernels on rotted plants are light
weight and some ears may be missing
during harvest.

Different plant species differ in their
reaction to Erwinia isolates. Six plant
species (Chrysanthemum morifolium,
Dieffenbachia amoena, D. maculata,
Philodendron panduriforme, P. selloum
and Syngonium podophyllum) were tested
by standardized methods for reaction to
383 strains of E. chrysanthemi from 35
plant species, sub-species or cultivars and
99 strains of other Erwinia species (Dickey
1981). Two hundred thirty nine strains of
E. chrysanthemi and 93 strains of other
Erwinia species failed to produce a positive
reaction in any of the five plant species.
Only strains of E. chrysanthemi originally
isolated from D. amoena caused a positive
reaction for D. amoena and D. maculata.
Only strains isolated from S. podophyllum
and one strain of E. carotovora subsp.
carotovora from Caladium and one strain
from banana produced positive reactions to
S. podophyllum; some Syngonium strains
and the Caladium strain of E. carotovora
subsp. cardtovora also were positive for P.
panduriforme and/or P. selloum. Positive
reactions were produced in Philodendron
plants by 126 strains of E. chrysanthemi
and four strains of E. carotovora subsp.
carotovora isolated from 22 hosts.

Five isolates of the bacterium were
collected from different parts of the
Philippines by the Department of Plant
Protection of the Visayas State College of
Agriculture. These isolates have been


yet fully known. Information from this
study will help the plant breeders for
breeding stalk rot resistance. Breeders can
incorporate genes for stalk rot resistance to
acceptable and high yielding varieties. This
study has the following objectives: a) to
determine the reaction of ten corn
cultivars/lines to five isolates of Erwinia
carotovora var. chrysanthemi causing stalk
rot; and b) to identify locally available corn
cultivars/lines with resistance to stalk rot.


MATERIALS AND METHODS

Isolation and Culture of Pathogen

Five isolates of E. carotovora var.
chrysanthemi obtained from different corn-
growing areas in the Philippines were used
in this study. The five isolates were
collected from Tranca, Bay, Laguna; IPB-
UPLB; ViSCA, Baybay, Leyte; USM,
Kabacan, Cotabato; and Banga, South
Cotabato. Maceration tests were
conducted to confirm E. carotovora. The
isolates were cultured in Petri plates
containing nutrient agar. A typical colony
of the bacterium was streaked on a nutrient
agar slant using a sterile transfer needle.
This was kept at room temperature and
served as stock culture.

Collection and Planting of Test Plants

Ten locally available corn
cultivars/lines were used in this study.
These included the following cultivars/lines:
Camotes Tiniguib, Native Pilit, Population
25, Population 30, UPLCn-2, Tropical
Early White Flint (TEWF), MIT var. 2,
Improved Tiniguib, ViSCA Composite and
Super Sweet Corn. Corn seeds were sown
in single row plots which were spaced 0.75
cm apart. Corn seeds were planted
dibblingg) in the field at a distance of 25





1997 PhiL Phytopath. 33(1):27-36 2


per hill but was thinned to one plant pe
hill. The plants were fertilized usinl
complete fertilizer 14-14-14) at the rate o
60 kg N, P205 and K20 per hectare twi
weeks after seedling emergence to ensur
vigorous growth of plants. Other culture
management like watering and spraying o
insecticide were done.

Preparation and Inoculation of Stall
Rot Isolates

Preparation of the inoculum wa
done following the standard metho<
adopted from IPB. A 48-hr bacteria
culture was used as inoculum. Thi
inoculum density was standardized usinj
Spectronic 20 (Bausch and Lomb) at 500/
transmittance and 425 wavelength. Thi
would approximate 40-50 thousand
bacterial cells per ml water. Ten-mi Tweei
80 was mixed into 1,000 ml bacteria
suspension as wetting agent. Inoculation t(
5-week old corn plants was done b,
pouring approximately 2 ml of the bacteria
suspension into the plant whorl. Sympton
development was observed starting at three
days after inoculation. Severity of damage
was evaluated at three days interval until
the 12th day. Plants showing wilting
lodging, decay, or softening of the stall
tissue with water-soaked appearance were
considered diseased and counted fo
calculating percentage diseased plants fo
each test entry.

The following scales (adopted fron
IPB) and their corresponding resistance
ratings were used to evaluate the reaction
of the plants to the different Erwinih
isolates: 1 = 0 to 25% infection (resistant)
2 = 26 to 50% infection moderatee,
resistant); 3 = 51 to 75% infection
(susceptible).


Experimental Design and Data Gatherec

A split-plot experiment witi
mainplot arranged in randomized complete
block design (RCBD) was used. Th
different stalk rot isolates were th
mainplots and the different con
cultivars/lines were the subplots replicated
three times. There were ten plants pe
cultivar/line per Erwinia isolate. The
following data were gathered: 1.) Percen
infection of stalk rot this was derived
using the formula: % infection = total no
of plants infected / total no. of plant
inoculated x 100; 2.) Resistance o
susceptibility rating the plants weri
evaluated following the scale previous,
mentioned.


RESULTS AND DISCUSSION

Symptom Development

The first symptom observed three
days after inoculation was the water-soake(
appearance along the length of the stall
starting at the point just below the base o
the whorl, progressing downward an<
followed by wilting of the youngest leaf
Severity of symptoms varied, amonj
different stalk rot isolates.

Symptoms of water-soaked area:
appearing as light brown to orange browi
were considered severe infection and wern
generally manifested by plants inoculated
with isolates from Tranca, Bay, Laguna an<
IPB-UPLB, USM, Kabacan, Cotabato an(
Banga, South Cotabato. Symptom:
appearing as yellowish green wer
considered mild infection and were
generally manifested by plants inoculated
with the ViSCA, Baybay, Leyte isolate
Both symptoms progressed with time unti
plants toppled and died.





30 1997 Phil. Phytopath. 33(1):27-36

Toppling was evident six days after showed the lowest infection even on the
inoculation and the number of toppled twelfth day after inoculation.
plants increased on the ninth day and nearly
all infected plants toppled on the twelfth Only four corn cultivars/lines
day after inoculation. High percentage showed stalk rot infection using the
toppling was observed in plants inoculated ViSCA, Baybay, Leyte isolate three days
with isolates from Bay, Laguna, USM, and after inoculation. Infection ranged from 3.3
Banga. Plants inoculated with the ViSCA to 16.7 percent and was observed on
isolate rarely toppled. ViSCA Composite (3.3%), Improved
Tiniguib (8.3%), UPLCn-2 (10.0%) and
The results of this study agree with Native Pilit (16.7%). The rest of the
Laysa and Exconde (1981) who reported cultivars/lines did not show any infection
that isolates differed in their relative three days after inoculation. However, all
aggressiveness that some caused toppling in the cultivars/lines showed infection six days
maize within 48 hrs, others after 72 hrs, after inoculation which ranged from 3.3 to
while the rest failed to cause toppling. 65.8 percent and increased thereafter until
the twelfth day. Population 30 and
Reaction of Corn Cultivars Improved Tiniguib consistently showed the
lowest and highest percent infection,
The different corn cultivar/lines respectively, 9 and 12 days after


inoculation which ranged from 46.7 to 93.3 from 30 to 83, 46.7 to 90, 46.7 to 86.7 and
percent (Table 2). On the ninth and twelfth 53.3 to 90, three, six, nine and twelve days
days from inoculation, infection ranged after inoculation (Tables 1, 2, 3 and 4).
from 43.3 to 93.3% and 50.0 to 93.3%, Population 30 consistently had the lowest
respectively (Tables 3 and 4). Population infection while Super Sweet Corn, UPLCn-
30 consistently showed the lowest disease 2, Camotes Tiniguib and Improved Tiniguib
incidence at 3, 6, 9 and 12 days after showed higher infection compared with the
inoculation, while Native Pilit showed the rest of the cultivars/lines.
highest disease incidence. Super Sweet
Corn and Improved Tiniguib reacted Percent infection of the corn
similarly to the isolate, especially during the cultivars/lines against Banga, South
third and twelfth day after inoculation. Cotabato isolate ranged from 33.3 to 76.7,
Percent infection ranged from 36.7 to 85.6, 63.3 to 90.0, three and six days after
40.0 to 83.0, 43.3 to 86.7, and 56.7 to inoculation, while ViSCA Composite had
89.6, three, six, nine and twelve days after the lowest on the sixth, ninth and twelfth
inoculation with IPB-UPLB isolate day after inoculation. The highest infection
respectively (Tables 1, 2, 3 and 4). occurred consistently until the twelfth day
Population 30 and UPLCn-2 had the lowest with Improved Tiniguib.
infection while Super Sweet Corn showed





1997 PhiL Phytopath. 33(1):27-36 31


Table 1. Percent infection of ten corn cultivars/lines inoculated with five isolates of
Erwinia carotovora var. chrysanthemi, three days after inoculation '.

Cultivar/Isolate I1 12 13 14 I5

Camotes Tiniguib 60.0 76.7 0.0 66.7 66.7
Native Pilit 80.0 66.7 16.7 63.3 60.0
Population 25 43.3 63.3 0.0 66.7 43.3
Population 30 25.8 36.7 0.0 30.0 43.3
UPLCn-2 63.3 36.7 10.0 76.7 56.7

Tropical Early White Flint 40.0 70.0 0.0 60.0 36.7
MIT var. 2 73.3 60.0 0.0 43.3 33.3
Improved Tiniguib 70.0 70.0 8.3 83.3 76.7
ViSCA Composite 63.3 76.7 3.3 53.3 53.3
Super Sweet Corn 80.0 85.6 0.0 76.7 73.3

1 I, = Tranca, Bay, Laguna; 12 = IPB-UPLB; I3 = ViSCA, Baybay, Leyte; I4 = USM, Kabacan, Cotabato; Is
= Banga, South Cotabato. Mean of 10 plants per cultivar/line per isolate.


Erwinia carotovora var. chrysanthemi, six days after inoculation 1

Cultivar/Isolate I1 12 13. L4 5

Camotes Tiniguib 80.0 76.7 14.1 90.0 90.0
Native Pilit 93.3 66.7 40.4 80.0 73.3
Population 25 60.0 63.3 13.7 73.3 63.3
Population 30 46.7 40.0 3.3 46.7 73.3
UPLCn-2 70.0 40.0 20.0 86.7 66.7

Tropical Early White Flint 7.67 80.0 26.7 63.3 70.0
MIT var. 2 80.0 73.3 51.1 66.7 76.7
Improved Tiniguib 90.0 80.0 65.8 86.7 93.3
ViSCA Composite 73.3 80.0 10.0 73.3 63.3
Super Sweet Corn 86.7 83.0 21.5 83.3 76.7

1 I, = Tranca, Bay, Laguna; I2 = IPB-UPLB; 13 = ViSCA, Baybay, Leyte; I4 = USM, Kabacan, Cotabato; Is5
= Banga, South Cotabato. Mean of 10 plants per cultivar/line per isolate.





32 1997 PhiL Phytopath. 33(1):27-3t


Table 3. Percent infection of ten corn
Erwinia carotovora var. chrysan

Cultivar/Isolate I,

Camotes Tiniguib 83.3
Native Pilit 93.3
Population 25 60.0
Population 30 43.3
UPLCn-2 73.3

Tropical Early White Flint 76.7
MIT var. 2 80.0
Improved Tiniguib 90.0
ViSCA Composite 73.3
Super Sweet Corn 86.7

SIi = Tranca, Bay, Laguna; 12 = IPB-UPLB; 13 = N
= Banga, South Cotabato. Mean of 10 plants per c




Fable 4. Percent infection of ten corn cultiva
carotovora var. chrysanthemi, twelve

Cultivar/Isolate Ii

Camotes Tiniguib 83.3
Native Pilit 93.3
Population 25 60.0
Population 30 50.0
UPLCn-2 73.3

Tropical Early White Flint 86.7
MIT var. 2 80.0
Improved Tiniguib 93.3
ViSCA Composite 76.7
Super Sweet Corn 90.0

SI = Tranca, Bay, Laguna; 12 = IPB-UPLB; 13 = V
= Banga, South Cotabato. Mean of 10 plants per a


iltivars/lines inoculated with five isolates ol
remi, nine days after inoculation 1

12 13 14 15

80.0 61.9 86.7 86.7
76.7 44.1 83.3 70.0
70.0 52.2 73.3 66.7
46.7 36.7 46.7 73.3
43.3 63.3 86.7 70.0

86.7 46.7 70.0 70.0
73.3 47.8 61.9 73.3
83.3 82.5 86.7 90.0
80.0 56.7 76.7 63.3
83.0 43.3 86.7 83.3

iCA, Baybay, Leyte; 14 = USM, Kabacan, Cotabato; Il
tivar/line per isolate.




/lines inoculated with five isolates of Erwinia


12 13 14

83.3 69.3 90.0


I5


73.3 65.9 73.3 76.7
56.7 41.4 53.3 80.0
63.3 76.7 86.7 76.7

86.7 63.3 73.3 73.3
80.0 61.9 73.3 73.3
83.3 82.5 90.0 90.0
86.7 56.7 76.7 63.3
89.6 68.5 90.0 83.3

!CA, Baybay, Leyte; 4 = USM, Kabacan, Cotabato; I
ivar/line per isolate.





irfy/ mnu. rnytopatu. iJq1):Z7-M6 33


such that some would exhibit symptoms observed.
earlier than others. These variations among
the isolate capability to induce symptoms Resistance/susceptibility rating
pectolytic enzyme they produce. Pectolytic (Table 7) shows that only Population 30
enzymes such as pectin methyl was rated moderately resistant to Tranca
transeliminase and the polygalacturonases and ViSCA isolates and is moderately
are the primary enzymes acting on the susceptible to susceptible against IPB-
pectic substances of the middle lamella UPLB, USM and Banga isolates. Other
(Laysa and Exconde 1981). corn cultivars/lines fall under moderately
susceptible to susceptible to the five stalk
Regardless of variety, the isolate rot isolates.
IPB-UPLB gave higher percent infection,
three days after inoculation but infection Population 30 possesses some
was insignificant compared with isolates degree of resistance to most of the stalk rot
from USM, Tranca and Banga (Table 5). isolates, hereafter, can be used as a
These isolates were significantly different reference or material in the development of
from the ViSCA isolate until the ninth day; resistant corn cultivars with desirable
on the twelfth day, differences among agronomic characters. Undoubtedly, this is


pectolytic enzymes produced by the
different isolates vary is not known. LITERATURE CITED
However, they clearly play an important
role in the initiation of infection. CLARK RL, FOLEY DC. 1985. Stalk rot
resistance and strength of maize from the
Population 30 consistently had plant introduction collection. PI Dis
lower mean infection even until the twelfth 69:419-421.
day after inoculation and this was
significantly different from the other DICKEY RS. 1981. Reaction of eight plant
cultivars/lines (Table 6). This clearly species to strains from several hosts and to
indicates that Population 30 may have strains of other Erwinia species.
played an important role in minimizing Phytopathology 71 23-24.
infection by the isolate.
DODD JL. 1980. The role of plant stresses
Resistance/Susceptibility in the development of corn stalk rots. P1
Dis 64:533-537.
Although the mean percent infection
of stalk rot on the ten corn cultivars/lines GABRIEL BP. 1973. Insects and mites
was taken at different period of time (3, 6, injurious to Philippine crop plants. UPLB,
9 and 12 days after inoculation), College, Laguna. pp. 12-16.





34 1997 hil. Phytopath. 33(1):27-3


ViSCA, Baybay, Leyte 3.8b 26.7b 53.5b 65.3"

USM, Kabacan, Cotabato 62.0a 75.0" 75.9a 79.3a

Banga, South Cotabato 53.3" 74.7" 74.7" 78.7a

l Means within the same column having a common letter are not significantly different at 5% level usin
Duncan's Multiple Range Test (DMRT).




Table 6. Percent infection .regardless of isolate (mainplot) of the ten corn cultivars/line
(subplot) at different days after inoculation '.

Cultivar Observations After Inoculation (days)
3 6 9 12

Camotes Tiniguib 54.0"b 70.2bc 79.9"b 83.2"b





97 Phil. Phytopath. 33(1):27-36 35





56 1997 Phil. Phytopath. 33(1):27-3(

LAYSA FD, EXCONDE OR. 1981. subsequent xylem occlusion by Erwinih
Virulence and aggressiveness of Erwinia amylovora and the fate of its extracellula
carotovora var. chrysanthemi Dye isolates polysaccharide in apple shoots
in maize. Phil Agric 64:307-322. Phytopathology 71:697-698.

PCARR. 1970. The Philippine THIND BS, PAYAK MM. 1978
Recommends for Corn. pp. 24-25. Evaluation of maize germplasm anm
estimation of losses to Erwinia stalk rot. P
SUHAYDA CC, GOODMAN RN. 1981. Dis Reptr 62:319-322.
Early proliferation and migration and





1997 Phil. Phytopath. 33(1):37-44 3'


REACTION OF DIFFERENT CORN,
TO THE RICE ROOT-KNOT NEMA
GOLDEN ANI


O. B. ZAMORA, R. M


Respectively, Undergraduate Stu4
Protection, Visayas State College of Agricu

Key words: corn, Meloidogyne gra


This study was conducted t<
of different corn, legume and roc
nematode, Meloidogyne graminicoa
of the nematode that can affect th
sweetpotato varieties evaluated N
nematode. No galls were develop<
were non-host of the nematode. TI
the taro variety PSB-VG-2 (Iniito)
Mg-9 and GO-049 varieties of m
found susceptible. The rest of the
Pathogenicity test using different in
mungbean variety Mg-9 showed
herbage yield and number of pods e
Likewise, no significant effects wa
petiole and number of leaves except
GO-049.


INTRODUCTION

Corn (Zea mays L.) is an important
cereal crop in the Philippines and other
countries because it is utilized for human
food, ingredient for livestock and poultry
feeds and as raw materials in the
manufacture of industrial products like
starch, oil, glucose and alcohol (Raquiline
1982).

In the Philippines, mungbean
[Vigna radiata (L.) Wilczek] locally


EGUME AND ROOTCROP VARIETIES
DDE, MELOIDOGYNE GR4MINICOLA
BIRCHFIELD


;APASIN and J. L. LIM


nt and Professors, Department of Plani
ire (ViSCA), Baybay, Leyte, Philippines.

inicola, mungbean, peanut, sweetpotato, tare


determine the resistance/susceptibility
:rop varieties to the rice root-knot
and determine the inoculum density
Growth. All the corn, peanut and
re resistant to the rice root-knot
>n these varieties suggesting that they
mungbean variety, Taiwan green and
ere found moderately resistant while
igbean and taro, respectively, were
trieties were moderately susceptible.
ulum densities of M graminicola on
I significant effect on plant height,
;ept on the weight of roots and pods.
observed on plant height, length of
a top and root weights of taro variety



field legumes because they are very rich in
protein and vitamins, calcium and sodium.
They can also be used as a substitute for
soybean meal in the poultry ration (Agarcio
1985). Worldwide, peanut is mainly an
oilseed crop.

Rootcrops like sweetpotato
(Ipomoea batatas L.), and taro [Colocasia
esculenta (L.) Schott] are grown mostly
for their roots largely utilized as
carbohydrate source in human diet and





58 1997 Phil. Phytopath. 33(1):37-44


itional suoolements. Their nutrition


(Pardales 1981). The corm!
have also been found to cor
which can also be utilized


Sand cormels
tain mucilaee


biodegradable plastics and in the
production of fuel alcohol.

These crops mentioned earlier are
commonly used as rotation crops after rice
to reduce pest population and also increase
farmer's income in the uplands. Since
many of the most commonly used
pesticides are expensive and
environmentally unsafe efforts are now
geared towards the integration of cultural
and biological methods in controlling pest
problems. Plant parasitic nematodes like
the root-knot nematodes, Meloidogyne
spp. are among the most important and
widely distributed throughout the world
and ranked high in the list of animate
pathogens that affect production of
economic crops (Sasser 1977). The host
range of this nematodes is extremely wide
and includes over 2,000 kinds of plants
(Shutleff 1980). M. incognita (Kofoid and
White) Chitwood and M. graminicola
Golden and Birchfield are important
nematodes (Fofie and Raymundo 1979)
infesting lowland and upland rice. M.
graminicola is found in about 40% of the
18 provinces surveyed (Soriano and Prot
1992; Villanueva and Prot 1992).

Castillo and Litsinger (1978)
reported that Rotylenchulus reniformis
Linford and Oliviera and Meloidogyne spp
are the only important nematode pests of
mungbean in the Philippines. In the upland
areas, mixed population of these two
genera were found in almost any field
where mungbean is grown, and heavy root
infection were frequently associated with


crop decline (Catibog and Castillo 1975).

Record as early as 1911 showed
that sweetpotato is a host of root knot
nematode (Bessey 1911). In a survey
conducted by Castillo and Maranan (1974)
in 25 provinces in the Philippines and
Gapasin (1979), Meloidogyne spp. was
cited as one of the most prevalent
nematode found associated with
sweetpotato. Pratylenchus sp.,
Helicotylenchus sp., H. dihystera (Cobb)
Sher, Rotylenchus reniformis,
Meloidogyne sp., M javanica, Longidorus
sylphus Thorne, Tylechorhynchus sp. and
Aphelenchoides sp. were reported on taro
and dasheen in Hawaii and the Philippines
(Conners 1980; Paris 1940; Byars 1917;
Nirula 1959; Gapasin 1978).

The use of resistant varieties is an
important component in the management
of nematodes. For example, of the 24
cultivars of green mungbean inoculated
with M incognita, ML62 and ML80 were
observed to be resistant (Darekar and
Bhosale 1985). Gapasin (1984) found W-
86, LA-89, BPA-4 and Senibastian
sweetpotato cultivars resistant to the root-
knot nematode, M. incognita based on egg
mass index.

M graminicola has not been
reported infecting other crops except rice
and some weed species. Its presence in
cultivated fields throughout the cropping
season might affect crops planted after rice
or they may act as alternate hosts, thus
increasing nematode population in the area.
This study therefore aims to determine the
resistance/susceptibility of the different
corn, legume and root crop varieties to M
graminicola and the inoculum density of
the nematode that can affect their growth.





1771 rnu. nuyropain. 33a1):3/-44 39


MATERIALS AND METHODS

Resistance/Susceptibility of the
Different Crop Varieties to the Rice
Root-Knot Nematode.

Field soil was sterilized for 2 to 4
hr in a steel plate baking pan. The
sterilized soil was placed in 6-inch diameter
clay pots. Eggs ofM. graminicola were
used as inoculum in the experiment and
were extracted using the method of Hussey
and Barker (1973) from the susceptible
rice (UPLRi-5) plant. A day before
planting, 1,000 eggs of the nematode were
inoculated into the surface of each potted
sterilized soil by pippetting out the
appropriate volume of egg suspension into
each pot. After inoculation, the inoculum
was covered with a layer of soil to prevent
them from dessication.

Planting was done a day after
inoculation. There were twelve corn (IES
E02,IES Glut#2, IES 89-06, USM var. 2,
USM var. 4, USM var. 8, USM var.
10,VM2 X USM var. 6, V9374, Bisaya,
Takoro and Sweet corn), six mungbean
(Taiwan green, Mg-5, Mg-7, Mg-9, Mg-50
and Pag-asa-7), two peanut (UPL-Pn2 and
Spanish), ten sweetpotato (Ifugao,
Miracle, Siete Flores, VSP-2, VSP-6,
Binoras, Inubi Zambales, Binicol, Tres
Colores and Tinagimtim) and eight taro
(PRG-337, PRG-063, PRG-066, GO-049,
GO-104, PSB-VG-1 (Kalapo), BSP-VG-2
(Iniito) and PSB-VG-3 (Dalwangan)
varieties used in this experiment. Five
plants were used per variety and pots were
arranged in a completely randomized
design outside the screenhouse.

The same care and management
practices were given to all crops.
Handweeding was done regularly until the
crops were about 40 days old. Spraying of


and fungicide benomyll) at the rate of 3
tbsp/5 gal and 2 tbsp/5 gal of water,
respectively. First spraying was done 2
weeks after planting, and was continued up
to the 4h week before harvest to prevent
any possible serious damage caused by
insect pests and infection by fungal
pathogens. Fertilization was done by
applying complete fertilizer two weeks
after planting.

The plants were harvested 60 days
after planting. Roots were collected from
each pot and washed with tap water and
the galls were counted. The gall index
rating of Taylor and Sasser (1978) was
used: 1 = 1-2 galls/root; 2 = 3-10
galls/root; 3 = 11-30 galls/root; 4 = 31-100
galls/root and 5 = more than 100
galls/root. Plants were rated for their
resistance based on the gall index using the
following scale by Gapasin (1984): 0-1.9 =
resistant (R); 2.0-2.9 = moderately
resistant (MR); 3.0-3.9 = moderately
susceptible (MS) and 4.0-5.0 = susceptible
(S).

Reaction of Mungbean (Mg-9) and Taro
(GO-049) to Different Inoculum Levels
of Meloidogyne graminicola

Mungbean (Mg-9) and taro (GO-
049) which were found susceptible in
experiment 1 were further evaluated as to
their reaction to different inoculum
densities of the nematode. Soil
sterilization, inoculum preparation, care
and management of the crops were the
same as in experiment 1 but inoculum
densities used were 1;000, 3,000, 5,000
and 10,000 eggs.

This experiment was laid out in a
completely randomized design (CRD). A
space of 25 cm between rows and 20 cm





40 1997 Phil. Phytopath. 33(1):37-44

mungbean and taro were as follows: To were either moderately susceptible or
untreated (check), variety of mungbean or susceptible with Mg-9, the most
taro; Ti variety of mungbean or taro + susceptible having a gall index of 4.0.
1,000 eggs ofM. graminicola; T2 variety Among the taro varieties, PSB-VG-2
of mungbean or taro + 3,000 eggs ofM. (Iniito) was moderately resistant to the
graminicola; T3 variety of mungbean or nematode while the rest were either
taro + 5,000 eggs ofM. graminicola; T4 moderately susceptible to susceptible. GO-
variety of mungbean or taro + 5,000 eggs 049 was the most susceptible exhibiting a
ofM. graminicola. gall index rating of 4.0.

In mungbean, the following data Mungbean is usually used as a
were gathered: plant height (cm), number rotation crop or as an intercrop by farmers.
of pods (g), weight of pods (g), fresh Similarly, taro which is usually planted as
herbage yield (g), root weight (g), number an intercrop or relay crop or planted in the
of galls in the roots per plant and nematode lowland near rice fields, was also found
population in the soil from 300g soil host of the nematode. Our results suggest
sample per pot. Plant height (cm), length that these susceptible varieties should not
of petiole (cm), number of leaves per plant, be planted in nematode-infected areas as
top weight (g), root weight (g), number of they would continuously harbor the
galls in the roots per plant and nematode nematode providing inoculum for the next
population in the soil from 300g soil crop such as rice or other susceptible
sample per pot were gathered in taro. crops.

Reaction of Mungbean (Mg-9) and Taro
RESULTS AND DISCUSSION (GO-049) to Different Inoculum Levels
ofMeloidogyne graminicola
Resistance/Susceptibility of the
Different Crop Varieties to the Rice Table 2 shows the effect of
Root-Knot Nematode different inoculum densities of M.
graminicola on growth parameters of
Evaluation of the mungbean (Mg-9) and on the number of
resistance/susceptibility of the different galls produced in the roots and nematode
crop varieties 60 days after inoculation population in the soil. The different
with 1,000 eggs of the rice root-knot inoculum densities of the nematode did not
nematode, M. graminicola revealed that all significantly affect plant height, herbage
the corn, peanut and sweetpotato were weight and number of pods of mungbean.
resistant to rice root-knot nematode (Table An increasing inoculum density was found
1). This implies that the varieties evaluated to significantly affect the weight of pods
were non-host and therefore, can be used and root weight of mungbean. The weight
by farmers as rotation crops after rice of pods generally decrease with increasing
(Barsalote and Gapasin 1995). The nematode density. This result clearly
varieties of mungbean and taro were found indicates that greater damage was inflicted
to be host of the nematode since galls were on the crop at high inoculum density. The
observed on them. Among the mungbean galls apparently have cause disruption on
varieties evaluated, Taiwan green was nutrient uptake thus reducing weight of
rated moderately resistant while the rest





1997 Phil. Phytopath. 33(1):37-44 4


VT HIll, lUIll
IES E02 0.00 0 R
IES Glu#2 0.00 0 R
IES 89-06 0.00 0 R
USM var. 2 0.00 0 R
USM var. 4 0.00 0 R
USM var. 8 0.00 0 R
USM var. 10 0.00 0 R
VM2 X USM var. 6 0.00 0 R
V9374 0.00 0 R
Bisaya 0.00 0 R
Takoro 0.00 0 R
Sweet corn 0.00 0 R
Mungbean
Taiwan green 8.00 2 MR
Mg-5 12.20 3 MS
Mg-7 17.80 3 MS
Mg-9 33.60 4 S
Mg-50 17.80 3 MS
Pag-asa-7 30.40 3 MS
Peanut
UPL-pn2 0.00 0 R
Spanish 0.00 0 R
Sweetpotato
Ifugao 0.00 0 R
Miracle 0.00 0 R
Sietes flores 0.00 0 R
VSP-2 0.00 0 R
VSP-6 0.00 0 R
Binoras 0.00 0 R
Inubi Zambales 0.00 0 R
Binicol 0.00 0 R
Tres colors 0.00 0 R
Tinagimtim 0.00 0 R






42 1997 Phil. PhvtoDath. 33(1):37-44


Table 2. Effect of Meloidogyne graminicola at d
mungbean (MG-9), and on the number
soil.'

Inoculum Plant Herbage No. of
Density (eggs) Ht (cm) Yield (g) Pods

Noninoculated
Control 61.76 55.60 16.80
1,000 66.40 39.90 11.00
3,000 62.80 52.16 14.80
5,000 62.40 48.30 11.00
10,000 63.00 44.94 10.60

CV (%) 10.59 37.49 49.52
'Means with the same letters are not significantly different at 5% lev





Table 3. Effect of Meloidogyne graminicola al
of taro (GO-049) and on the number (
soil.1

Inoculum Plant Length of No. of
Density (eggs) Ht (cm) Petiole(cm) leaves

Noninoculated
Control 63.30 46.70 3.40
1,000 61.60 45.80 3.80
3,000 64.50 48.10 3.40
5,000 59.10 43.40 3.80
10,000 60.80 45.10 3.40

CV (%) 7.19 8.49 16.85
'Means with the same alers are not significantly different at 5% le


:rent inoculum densities on growth parameters of
galls in the roots and nematode population in the


Vt of Root No. of Nematode pop
A- -\ 117 -: --. 1 --% --- Inn --.I


8.94ab 14.10ab 27.00bc 168.80bc
.1:10b 15.50a 43.00b 246.00ab
1.08b 10.24bc 93.40a 369.60a

19.68 24.38 57.02 55.04
based on DMRT.





different ioculum density on growth parameters
galls in the roots and nematode population in the



Top Root No. of Nematode pop
Wt. (g) Weight (g) galls/root per 300-g soil


20.10ab 104.66a O.OOc O.OOc
10.12ab 85.68ab 3.00c 139.60c
128.76a 102.28a 7.00bc 294.60b
01.OOab 3.82ab 25.80b 321.20b
91.10b 62.18b 54.80a 602.80a

20.00 24.67 89.50 42.80
based on DMRT.


1) sRal- n ndn





1997 Phil. Phytopath. 33(1):37-44 4.


pods and roots. According to Taylor and
Sasser (1978), galls and giant cell
formation changes the physiologic
activities of the plant system. The number
of galls produced was highest using 10,000
inoculum density of the nematode. This
significantly differed from the rest.
Likewise, population density in the soil
was highest with the same inoculum
density. Significant differences were also
observed in the nematode population in the
soil.

Noninoculated mungbean plants
had comparable height with inoculated
plants using different nematode inoculum
densities per plant. However, in citrus
plants (cv. Ladu) Davide and de la Rosa
(1974) reported stimulation of the top
growth when inoculated with 20,000
individuals of Tylenchulus semipenetrans,
but was reduced when inoculated with
30,000 individuals.

In taro, M. graminicola did not
significantly affect plant height, length of
petiole, and number of leaves (Table 3).
Significant differences were found on top
and root weights and on the number of
galls and nematode population in the soil.
Plants inoculated with 10,000 eggs had the
lowest top and root weights at 91.0 g and
62.18 g, respectively. This could be due to
the high number of galls and nematode
population (54.8 and 602.8/300g soil,
respectively) associated with these
treatments.

In mungbean, the herbage yield,
weight of pods and root weights decreased
as the nematode density was increased.
Likewise, in taro top and root weights
decreased with increasing nematode
inoculum density. This implies that M.
graminicola could affect some of the
growth parameters of mungbean and taro


These findings corroborate the
observations of Catibog and Castillo
(1975) on mungbean; on ampalaya, and
Olasiman (1981) on sweetpotato. Total
fruit yield of ampalaya at 2- and 4-week
old plants inoculated with 1,000 and 5,000
eggs were reduced by 68% to 70% and
further reduced to 81% and 82% when
inoculum density was increased to 10,000
and 20,000, respectively.


LITERATURE CITED

AGARCIO BC. 1985. Let's produce
mungo. Dept of Agro and Soil Science,
ViSCA, Baybay, Leyte 4 p.

BARSALOTE EB, GAPASIN RM. 1995.
Pathogenicity of the rice root-knot
nematode (Meloidogyne graminicola)
Golden and Birchfield on upland rice. Phil
Phytopath 31:95-102.

BESSEY EA. 1911. Root knot and its
control. US Dept of Agr Bur Plant
Industry Bull 217:89p.

BYARS LP. 1917. A nematode disease of
dasheen and its control by hot water
treatment. Phytopathology 7:66.

CASTILLO MB, MARANAN LR. 1974.
Plant parasitic nematodes associated with
sweetpotato and cassava in the Philippines.
J Nematol 10:56-58.

CASTILLO MB, LITSINGER JA. 1978.
Plant parasitic nematodes of mungbean in
the Philippines. In: The First International
Mungbean Symposium. Shanhua, Taiwan:
AVRDC. pp. 195-200.

CATIBOG CS, CASTILLO MB. 1975.
Pathogenicity ofMeloidogyne javanica on





4 1997 Phil. Phytopath. 33(1):37-44

'ONNERS I. 1980. Updated checklist of NIRULA NK. 1959. Root-knot nematode on
microorganisms and viruses found in Colocasia Curr Sci (Banglore) 28:125-126.
Iawaii. Unpublished Manuscript. Dept of
'It Pathol, Univ of Hawaii, USA. OLASIMAN AS. 1981. Susceptibility of
BNAS-51 sweetpotato variety to root-knot
)AREKAR KS, BHOSALE DJ. 1985. nematode. Unpublished BS Thesis. ViSCA,
Reaction of some green gram cultivars to Baybay, Leyte 46 p.
oot-knot nematode. Journal of
4aharashtra Agricultural University PARDALES JR. 1981. How to increase
0:227. gabi corm yields. PRCRTC, ViSCA, Baybay,
Leyte 6p.
)AVIDE RG, DELA ROSA AG. 1974.
effects of Tylenchulus semipenetrans and PARIS GK. 1940. A check list of fungi,
theirr pathogens on citrus in the bacteria, nematodes and viruses occurring in
'hilippines. Phil Phytopath 15:9-22. Hawaii and their hosts. P1 Dis Rep Suppl
121:11-12.
OFIE AS, RAYMUNDO SA.1979.
'arasitic nematode in continuously RAQUILINE JM. 1982. Adaptability test of
dropped uplands. Int Rice Res Newsl 4:17. the different recommended varieties of corn
in Leyte. BS Thesis. ViSCA, Baybay, Leyte
iAPASIN RM. 1978. Survey, 32 p.
athogenicity and control of plant parasitic
ematodes associated with maior root SASSER JN. 1977. Worldwide


iAPASIN RM, VALDEZ RB. 1979.
'athogenicity of Meloidogyne spp. And
!otylenchulus reniformis on sweetpotato. (
unn Trop Res 1:20-25. ]
1
jAPASIN RM. 1979. .Survey and
lentification of plant parasitic nematodes
associated with sweetpotato and cassava.
Lnn Trop Res 1(2):120-124.

iAPASIN RM. 1984. Resistance of fifty
wo sweetpotato Upomoea batatas (L.) i
sam.] cultivars to Meloidogyne incognita 1
nd M. javanica. Ann Trop Res 61-19. ]

RUSSEY RS, BARKER KR. 1973. A
comparison. of methods of collecting
locula of Meloidogyne spp. including a i
ew technique. P1 Dis Reptr 57:1025-
028.


ematol 9:26-29.

IUTLEFF MC. 1980. Compendium of
rn diseases. American Phytopath Society
ept of P1 Path, University of Illinois,
rbana 2:70-75.

)RIANO IRS, PROT JC. 1992.Plant
Lrasitic nematodes associated with irrigated
:e in the Philippines. Phil J Crop Sci 17:28.

LYLOR DP, SASSER. JN. 1978. Biology,
entification and control of root-knot
matodes (Meloidogyne sp.). Dept of Plant
ithology, N. C. State University, Raleigh.

[LLANUEVA LM and PROT JC. 1992.
arietal testing of upland rice cultivars for
distance to Meloidogyne graminicola. Phil
Crop Sci 17:47.







TEMPORAL SUSCEPTIBI]
FLUSHES TO COLLETOTRICJ


O. S. OPINA, A. A. EUV


Portion of a project funded by Dept
Research (DA-BAR).

Respectively, Professor, University
Student, Department of Plant Pathology, Ui
Laguna 4031.

Key words: anthracnose, Colletotri


A study was conducted to es
of 'Carabao' mango flushes against
younger leaves were found more si
resistant as they grew older. The
short duration. The pathogen can ir
15 days after bud break (DABB) an(


INTRODUCTION

Mango anthracnose caused by C.
gloeosporioides Penz. is the most prevalent
and destructive disease infecting mango
(Mangifera indica L.) (Dangan 1997;
Jeffries and others 1990). All infectible
parts of mango cultivars grown in the
Philippines are susceptible to the pathogen .
The pathogen infects leaves particularly at
the bronze to light green stages. Leaf
petioles may also be infected resulting in
premature leaf fall. In severe cases, the
entire shoot can become blighted and
dieback often result if wet weather prevails.
The pathogen also infects the inflorescence
and prevents fruit set. Infection can also
occur in the fruits during fruit development
but it remains latent until fruits begin to
ripen (Jeffries and others 1990; Pordesimo
1979; Clara 1927).


4!

ITY OF 'CARABAO' MANGO
rUM GLOEOSPORIOIDES PENZ.


EBIO and N. A. M. BASIO


tment of Agriculture Bureau of Agricultura


researchh Associate and former Undergraduate
versity of the Philippines Los Bafios, College.


*hmm gloeosporioides, host resistance, mango


iblish the duration of susceptible stage
Colletotrichum gloeosporioides. The
sceptible to the pathogen and became
susceptibility of young leaves was of
ect detached young leaves only up to
10 DABB for attached leaves.


Control of mango anthracnose is
normally done by repeated application ol
protectant and systemic fungicides. An
average of 6-7 fungicide sprays is done to
protect inflorescence and developing fruits
from anthracnose infection (Estrada,


is essential in the developer
disease management
Phenology and growth 1


susceptibilit
attributes ar
Knowledge
amount of si
also provide
control. S
continuity o
canopy by
inoculum, e
stage of flush


lent of effective
alternatives
khoA,-.r ,.+ +h~


y and other umque nos
e of important considerations
of the temporal availability an(
susceptible parts of the host car
Sthe initial target for disease
ince leaf infection provide!
if infection within the mangc
providing initial source o:
establishment of the vulnerable
,hes will provide basis for theii





-V 1J.'.7 I 1 UU. 1 11. LUIPIIAl. J.1/A.Y T-'J"W


. I Ij I \ N- I
)btain flushes of different ages. 6 DABB showed more resistance to the
nnthnaon with ;Qo/. rlicPac cp avritv 11


process. Three mango terminals for each 16 0 0
DABB = days after bud break
leaf stage were selected, coded and were 2Avage ofthreetrials
inoculated simultaneously by spraying the





1997 Phil. Phytopath. 33(1):45-48 47
























1A ISH 13 D)BB 15 DA5B

Figure 1. Phenological development of 'Carabao' mango flushes from bud break up to 15
days after bud break (DABB).




















r15 ilDA ll
II U












D L 8 IABB015 L I)A BB

Figure 2. Detached mango leaves inoculated with virulent isolate of Colletotrichum





48 1997 Phil Phvtnnnth t 1-4-lAR


Assessment of the reaction of
detached and attached young leaves of
'Carabao' mango to C. gloeosporioides
showed a very short duration of susceptible
stages. Complete resistance to the
pathogen was achieved at 16 DABB for
detached leaves and 12 DABB for intact
leaves. This difference could be attributed
to the maintenance of optimum conditions
for infection for the detached leaves. It is
also possible that the pre-formed and
induced resistance factors in the leaves have
diminished after they are detected. Despite
difference, the data show a relatively small
window for leaf infection which could be
taken advantage for the management of the
disease. Since lesions from infected leaves
provide inoculum for the infection of
susceptible plant parts such as flushes,
succulent stem terminals, inflorescence and
fruits, protecting the leaves during their
susceptible stage from the pathogen will
significantly reduce subsequent initial
noculum from the leaves thereby breaking
:he continuity of the disease cycles.
Anthracnose infection on susceptible leaves
:an be prevented by protective fungicides
or by cultural methods. Cultural practices
such as fertilization and irrigation could be
manipulated so that flushing time coincide
when conditions are unfavorable for
mthracnose development.


LITERATURE CITED

CLARA, FM. 1927. The anthracnose
disease of mango in the Philippines. Phil
Agr Rev 20:271-271.

DANGAN, JM. 1997. Compendium of
mango diseases in the Philippines. Phil.
Phytopath Soc. University of the
Philippines Los Baiios, College, Laguna.

DODDS JS, JEFFRIES P, JEGER MJ.
1989. Management strategies to control
latent infection in tropical fruit. Aspects of
Applied Biology 20:49-56.

ESTRADA, AB. 1994. Epidemiology and
controll of mango anthracnose. University
f Canterbury, Kent. 170p.

rEFFRIES P, DODD, JC, JEGER MJ,
PLUMBLEY RA. 1990. The biology and
controll of Colletotrichum species on
tropicall fruits and vegetables. P1 Path
39:344-366.

PORDESIMO. 1979. Anthracnose of
Philippine mango cv. 'Carabao': Its
etiology and control. Ph. D. Thesis, Univ.
)f the Philippines, Los Bailos.





i yI run. rnyropamn. 33j():4Y-o/ 45


FURIFICAMTION AND SEROL(
OF PAPAYA RIN(


A. A. EUSEBIO1, L. D. VALENCIA


Supported in part by projects funde(
Resources Research and Development
Technology (DOST) and Australian Cet
(ACIAR).

Respectively, 'Research Associates,
Plant Breeding (IPB), 2Research Associate
Department of Plant Pathology, University (

Key words: Carica papaya, papa!
virus


Papaya ringspot potyvirus
transmission of diseased papaya e
zucchini squash cv. Blackjack. The
in zucchini using the infected zucc
symptomatic leaves of zucchini wet
was extracted and clarified using
involved extraction with 0.5M p
containing sodium sulfite and clarif
was precipitated in polyethylene
centrifugation. After resuspension,
30% sucrose cushion and the pellet
protocol used 0.5M borate buffer, p
clarification using Triton X-100 folb
cushion. The pellet was resuspel
differential centrifugation follow
centrifugation. Virus yield was app
either procedure. Antiserum (As)
rabbits by a series of intramuscular
immunosorbent assay (ELISA) the
reacted strongly to PRSV-infected ]
virus preparations at dilution of 1:1
probe the protein band corresponding
PRSV and infected zucchini in wes
papaya disease surveys, screening fo
hybrids and inbreds, and in indexing


WItAL CHAKAC EKRILZA'ION
SPOT POTYVIRUS


V. N. VILLEGAS2 and N. B. BAJETV


by the Philippine Council for Agriculture and
PCARRD), Department of Science and
-r for International Agricultural Research


department of Plant Pathology and Institute of
Professor, IPB and 3Associate Professor of
the Philippines Los Bafios, College, Laguna.

Sringspot, potyvirus, purification, serology,



)RSV) was isolated by mechanical
ract to both papaya cv. 'Solo' and
late was propagated and maintained
ini as inoculum. After 21-30 days,
collected, homogenized and the sap
wo protocols. The first protocol
tassium phosphate buffer, pH 8.4
ation using Triton X-10. The virus
glycol and sodium chloride by
virus suspension was centrifuged in
suspended in the buffer. The other
[ 6.8 containing mercaptoethanol and
ved by centrifugation in 30% sucrose
led and subjected to one cycle of
d by cesium sulfate gradient
)ximately 4-12 mg/kg of tissue using
against the isolate was prepared in
injections. Using the enzyme-linked
As, especially when cross-absorbed,
paya and zucchini, and with purified
100 to 1:5000. The As was able to
r to the viral coat protein in purified
,rn blots. The As has been used for
PRSV resistance of promising papaya
F tissue-cultured papaya.









Papaya (Carica papaya L.) is one
of the most widely grown fruits in the
tropics and subtropics. In the Philippines,
it is the 6th most important cultivated fruil
crop. Papaya is not only well known as a
relished food item and a source of vitamin
C but also as a primary source of papair
and some other materials foi
pharmaceutical purposes (PCARRD 1977).

Papaya production in the
Philippines faces a number of problems
including pests and diseases (Bajet and
others 1992). A viral disease of papaya,
the papaya ringspot caused by the papaya
ringspot potyvirus (PRSV), attacks papaya
wherever it is grown. PRSV has two
strains, the papaya or P strain which infects
papaya and cucurbits and the watermelon
or W strain which infects only cucurbits
(Milne and Grogan 1969). The PRSV was
first detected in Silang, Cavite in 1982 bul
it was not till 1984 that an outbreak
occurred at an epidemic level. After its
detection in Cavite, it has spread to the
neighboring provinces of Batangas,
Laguna, Rizal and Quezon (Opina 1986).
Survey revealed that PRSV has reached
Bulacan, Pampanga, Nueva Ecija,
Pangasinan, La Union, Ilocos Sur, Ilocos
Norte, Nueva Vizcaya, Cagayan and as fai
as Albay, Camarines Norte and Sur,
Sorsogon, Mindoro and Palawan. Very
recent reports also confirm the presence ol
PRSV in Marinduque, Negros Oriental,
Aklan and Leyte (Eusebio 1992; Eusebic
and others 1994). Based on past
experiences, it is very difficult to eradicate
the disease once it has established in an
area.

The exact origin of PRSV is
difficult to ascertain. It was first reported
in Hawaii in the 1940s (Gonsalves and Ishii
1980), and in Puerto Rico in the 1946


(Adsuar 1946). Similar disease appeared ii
Florida and Cuba in 1964 (Conover 1964
Cook 1972) and in some parts of Centra
and South America including El Salvador
Colombia, Venezuela (De la Rosa an<
Lastra 1983; Purcifull and others 1984) an<
then later in the African continent and ii
the Indian subcontinent (Purcifull anm
others 1984). In Asia, Taiwan reported thi
presence of PRSV in 1975 (Chang 1979)
the Philippines in 1984 (Opina 1986)
Thailand in 1986, and Malaysia in 1992
Australia reported it in 1992 (Thomas an<
Dodman 1993). It is now widespread ii
most countries where it occurs (Purciful
and others 1984; Brunt and others 1990).

Methods for the identification o
infected plants rely mostly on symptom an<
this is rather late as the infected plant ha
already served as source of inoculum
Efficient eradication to lower if not tc
eliminate totally the source of inoculun
needs efficient and effective identification
of diseased plants early so that they could(
also be rouged out early. In addition, thi
development of cultivars with mor
desirable reactions to the disease can be
hastened if a more sensitive detection
system is available.

Serology is a very popular metho<
to detect and identify the causes o
diseased plants. Since the technique i;
based on the specific reaction of the
antibody and the pathogen (antigen)
positive serological reaction mean!
detection and alsodefinitive identification
of the pathogen. This procedure has beet
used for PRSV detection in the Philippine!
but the antisera used were sourced fron
abroad (Eusebio 1992; Eusebio and other:
1994; Villegas and others 1995). Thi;
paper reports successful isolation
propagation, purification and the
production of antiserum to an isolate of th<
papaya strain of PRSV in the Philippine!





1997 Phil. Phytopath. 33(1):49-67 51


(PRSV-P). Other local researchers have
also reported purification and antibody
production (Exconde 1987).


MATERIAL AND METHODS

Isolation and Maintenance of PRSV

Papaya ringspot potyvirus (PRSV)
was isolated by mechanical inoculation of
extract of diseased papaya from the
Institute of Plant Breeding, University of
the Philippines, Los Banos campus (IPB
isolate) to both papaya cv. 'Solo' and
zucchini squash cv. Blackjack. The extract
was confirmed to be infected with PRSV
by ELISA. The isolate was designated as
PRSV-P (Philippine isolate) and
propagated further in zucchini using the
infected seedlings to confirm the identity of
the strain. Twenty one to 30 days after
inoculation, the symptomatic leaves of the
propagation host were harvested and used
in the subsequent experiments.

PRSV Purification

Two extraction and clarification
protocols of the sap expressed from
infected zucchini were tested to purify the
putative PRSV. The first procedure used
was that of Hammond and Lawson (1988)
for the purification of potyvirus and
cytoplasmic inclusion proteins from
zucchini with some modifications. Briefly,
200g of freshly harvested infected zucchini
tissues (3 weeks after inoculation) were cut
into small pieces and homogenized in a pre-
cooled blender in the presence of cold
0.5M potassium phosphate buffer, pH 8.4
with 0.5% sodium sulfite at Ig tissue per
5ml. After passing through the
homogenate in several layers of gauze
cloth, the resulting filtrate was centrifuged
at 14 000 rom for 15 min in Beckman Tvoe


added with 2% (v/v) Triton X-100. After
15 min stirring, 0.1M sodium chloride and
4% polyethylene glycol (PEG 6,000) were
added and further stirred for 2 hr or
overnight at 4-60C. The mixture was
centrifuged at 14,000 rpm (Type 16 rotor)
for 1 hr and this PEG pellet was
resuspended with the addition of cold 0.1M
borate buffer, pH 8.0 (BK buffer) using a
glass tissue homogenizer. The homogenate
was stirred for 1.5 hr at 4-60C, then
centrifuged at 14,000 rpm for 10 min
(Type 16 rotor). The supernatant was
underlaid with 30% sucrose made in BK
buffer and centrifuged at 32,000 rpm for 2
hr in Beckman 45 Ti rotor. The pellet was
recovered and resuspended with the tissue
homogenizer in minimal amount of BK
buffer and kept at 4-60C for further
purification. Further purification by cesium
sulfate (Cs2SO4) gradient centrifugation
method was carried out by adding 3.3g of
Cs2SO4 in the partially purified PRSV
preparation and the volume adjusted to a
total of 10ml. The tube was gently
inverted to dissolve the cesium sulfate.
The preparation was centrifuged for 18-20
hr in a Beckman SW 41 rotor at 32,000
rpm at 40C. After the run, opalescent virus
band was collected using a syringe and the
virus was freed of the cesium salt by
resuspending it in a small volume of BK
buffer and a small portion of it was
examined for light scattering at 260 nm
using UV spectrophotometer. The
remaining purified virus was stored at 200C
for antiserum production.

The other method essentially
followed the protocol supplied by Dr. J.
Thomas and Mr. D. Persley, Queensland
Department of Primary Industries,
Indooroopilly, Australia. The same amount
of starting material (200g) was extracted in
Ili of 0.5M borate buffer, pH 6.8 with 2%
mercaptoethanol. The homogenate was
naeeoa thrniloh fnilr Inv1re nf oaln1P rlAth





52 1997 Phil. Phytopath. 33(1):49-67


and the filtrate was centrifuged at around
8,000 rpm in a Sorvall GSA rotor for 15
min at 40C. The supernatant was collected
and underlaid with 30% sucrose prepared
in 0.25M borate buffer, pH 6.8 but without
mercaptoethanol (resuspending buffer, RB)
and centrifuged at 40,000 rpm for 2 hr in
Beckman 45 Ti rotor. The pellet was
allowed to soak in a small volume of cold
RB on ice for 1 hr or longer then
resuspended using the glass tissue
homogenizer and the suspension was left
overnight at 40C with slight shaking or
stirring. The suspension was collected and
subjected to another cycle of differential
centrifugation. The pellet was resuspended
in cold RB and final purification was done
by cesium sulfate gradient centrifugation as
above. The virus band was collected,
resuspended in RB and centrifuged as
above and the virus pellet was resuspended
in a small volume of RB. Virus yield was
calculated using the Potyvirus group mean
extinction coefficient of 2.35 (Brunt and
others 1990).

Antiserum Production

One mg of the purified PRSV per
ml was added with equal volume of
complete Freund's adjuvant (FA) and the
mixture was emulsified by passing it
through two glass syringes equipped with a
needle with double male Luer locks.
Emulsification was checked by allowing a
small portion of the mixture to drop on a
water sample. A desirable emulsion was
achieved when the drop failed to disperse
in the water. The virus emulsion was
injected equally into hindleg of a New
Zealand rabbit. Succeeding injections
(every 7 days for 3 consecutive weeks)
were with the same virus concentration but
emulsified with complete FA. Three to 5
ml blood samples were collected before the
first (preimmune serum) and the
succeeding injections. Seven to 9 days


after the last booster injection, the rabbit
was sacrificed and the blood collected.
About 35 ml of antiserum (As) was
obtained after centrifugation of the blood at
10,000 rpm in a Sorvall SS34 rotor for 30
min at 40C. The As was stored in 50%
glycerol at -200C.

Characterization of PRSV-P Antiserum

Membrane-immunoblot assay
(MIBA). The procedure followed was
originally for the rice tungro viruses as
supplied by Prof. Roger Hull, John Innes
Institute, United Kingdom (UK) and used
for PRSV by Robles (1995). Antigens,
approximately 3-5 pl (healthy and infected
zucchini and papaya, purified PRSV-P and
buffer) were blotted separately onto the
nitrocellulose membrane (NCM); after
which, the NCM was reincubated for 2 hr
with shaking at room temperature in
1/1000 volume of goat anti-rabbit serum
conjugated with alkaline phosphatase
diluted with wash solution. Final washing
was done in 100 mM Tris HC1 (pH 9.5) for
3 min and the NCM was incubated in the
color development buffer containing 0.1
mg/ml nitroblue tetrazolium (NBT) and
0.05 mg/ml bromochloroindoyl phosphate
(BCIP) dissolved in 4 mM MgCl2 and 70%
dimethylformamide (DMF), respectively.
The NCM was incubated with shaking until
the colors have fully developed. The
membrane was washed with distilled water
and blotted dry on a Whatman filter paper.

Sodium Dodecyl Sulphate
Polyacrylamide Gel Electrophoresis
(SDS-PAGE) and Western Blot (WB)
Analyses. Proteins in the various samples
(healthy papaya and zucchini, infected
papaya and zucchini) and the purified
PRSV-P were analyzed in SDS-PAGE
(Suzuki and others 1989) using a BioRad
mini-eel electrophoresis system. The





1997 Phil. Phytopath. 33(1):49-67 53


ouea onto a [NUM usmg a BloKad Application of I
tsblot cell system and the probing
roteins using the As (i.e: western Detectiol


is done
by the r
r from Th
)r compare

V*nlAVmun,


111LU l1
md inci
Swas
hnifi&rd


infect
.---'-.


)n.

SV-P
San


s uuul uy ui
a within an<
I


(-r Antiserum

SPRSV in p
les. Testing i
ificity of the PI
cting field saml
outside the
Id collected s&


of healthy
id zucchini
ISA plate at
i overnight al


d saline containing 1%


anuserum at i:l;uu onuuon was men resisance. rrenimnary sucrviung iui
added and incubated for 2-3 hr at 370C or PRSV-P resistance was conducted using
overnight at 40C. Cross absorption was one-month old seedlings of interspecific
done by diluting the PRSV antiserum with hybrids and other Carica species. Initial
an equal volume of an extract of healthy assessment of the plants was undertaken by
1. I I I .-r' A T-'T A _-_ __ L_ T- T IOX T A- -. A -


evaluated to compare their serological Lawson's protocol, considerably large
relationship with the Philippine PRSV As. amounts of major but diffused protein
bands with molecular weights between the





i4 1997 Phil. Phytopath. 33(1):49-67


(PRSV-W) capsid protein,
Ia. The differences could be


conditionss in conducting SDS-PAGE. the rabbits was assessed using MIBA. A
Hames and Rickwood (1994) reported that positive result is indicated by a blue-violet
some proteins behave anomalously in SDS- color reaction around the blot which is
PAGE in some instances. In addition to brought by the precipitation of the color
:hese capsid proteins, proteins of higher development reagents (NBT and BCIP).
molecular weights were also resolved and Figure 3 illustrates the reactions of varying
identified with the infected samples. These dilutions (1:10, 1:100. 1:1000 and
were likely the cytoplasmic inclusion 1:10000) of different batches of sample of
rntPrinf nf PRURV Acnnrdina tn




1997 Phil. Phytopath. 33(1):49-67 5!


*


f I


Figure 2. Cesium sulfate profile of the zucchini extract containing papaya ringspc
potyvirus (PRSV). Note the sharp UV-absorbing band which contains th
PRSV articles.


m









1:1000 1




*



.* ..
Aus1










0 -




e .






ghil PI


Legend: A = Healthy C. pepo
B = Infected C. pepo

Blank 1:10A 1:1C
Buffer 1:10A 1:1C
Buffer 1:10B 1:1C
(+) Control 1:10B 1:1C

Figure 3. Reactions of healthy (A) and infe,
locally produced antiserum to i
dilutions in membrane immunob
two antisera produced).


i000 1:10,000








PRSV


















,




3V





A 1:1,000A 1:10,000A
A 1:1,000A 1:10,000A
B 1:1,000B 1:10,000B
B 1:1,000B 1:10,000B

ed Cucurbitapepo cv. Blackjack (B) with the
paya ringspot potyvirus (PRSV) at various
t assay (MIBA). (Shown here is one of the


__ _







PRSV-P As to different test samples Western blot assay. The purified
(healthy and infected zucchini and purified PRSV-P obtained through Hammond and
PRSV). Strong reactions were exhibited Lawson's protocol showed a major
by the purified PRSV at As dilution 1:1000 protein band that reacted or probed by the
and the inoculated samples for all the As PRSV-P As (A2) (Fig. 4). The same
batches (1", 2nd, 3rd and 4h) at sample antiserum also detected a protein of similar
antigen dilution 1:10. The same samples size with the infected sample (lane B),
gave only slight reaction at Ag dilution partially purified preparation (lane F) and
1:100. Diluted preparations of infected pellet obtained after extracting the virus
zucchini (1:1000 and 1:10000) were not band from the Cs2SO4 suspension (lane E)
recognized by all As batches. Healthy and diluted preparations of the purified
zucchini preparations were all negative for PRSV (lanes C and D) (Fig. 4a). This
all As samples at any dilution. The same protein band was similar to the PRSV viral
reactions were basically observed at higher coat protein because of its size and mobility
dilution of As (1:50,000) where the in the gel which were similar to those
reactions between the control and the reported else where (Suzuki and others
infected zucchini at Ag dilution of 1:100 1989). Additionally, aside from this major
can still be recognized. At the most diluted protein band, the As was able to recognize
As preparation (1:10,000), antigens gave some high. and low molecular weight
only positive reaction up to 1:10 Ag proteins which are likely to be the inclusion
dilution. The corresponding healthy body proteins and their degradation
controls at all dilutions were negative for products. There was no reaction with the
all the As batches, corresponding healthy zucchini sample
(lane A). The Thailand PRSV As, our
Between the two sets of As reference As, was also able to recognize
obtained from two rabbits (A1 .and A2), the same viral proteins efficiently (Fig 4b).
more intense reactions were given by As This result suggests that the locally
set A2 for all batches and dilutions tested. produced As worked comparatively well
It is not surprising to have this type of with the As obtained from Thailand.
observation because rabbits, despite from
the same breed/colony, may vary in their Comparison of PRSV-P
reaction to an antigen as earlier reported Antiserum with Other PRSV Antisera.
(Van Regenmortel 1982). Furthermore, it The locally produced PRSV As was
was observed that the color reaction evaluated along with the different PRSV
1L - -_ - is S Ath IL_ _. _I P A A - - / I I -


wan


- u -lS.b 1 LFVuJ


) ass


intPneQ until thp rnlnr r'an nn 1nnaPr hP t.hp A titpr





-.77y I 1111. KliyLUIpaUtl. jjklj;i7-v I


r'- 'V-.7C ~"'






1997 Phil. Phytopath. 33(1):49-67 59

Table 1. Absorbance at 405nm of different samples at varying dilutions against PRSV-P
antisera in the Philippines (A1 and A2), Australia, Taiwan and Thailand in PTA-
ELISA.

Antiserum Test Absorbance (405nm)
Dilution Sample' Philippines Australia Taiwan Thailand
A A2


1:100







1:1000







1:10000







1:20000







1:50000


0.00
0.27
1.32
1.48
0.00
2.36

0.00
0.00
0.34
0.50
0.00
1.82

0.00
0.00
0.04
0.10
0.00
0.59

0.00
0.00
0.02
0.07
0.00
0.28

0.02
0.02
0.04
0.01
0.00
0.16


0.00
0.34
1.44
1.40
0.00
2.12

0.00
0.00
0.42
0.53
0.00
1.64

0.00
0.00
0.06
0.07
0.00
0.63

0.00
0.00
0.02
0.03
0.00
0.41

0.01
0.00
0.01
0.00
0.00
0.20


-- I ________ _______ L ______


0.00
0.00
0.41
0.83
0.00
2.08

0.00
0.00
0.00
0.11
0.00
1.49

0.00
0.00
0.01
0.02
0.00
0.57

0.00
0.00
0.01
0.03
0.00
0.35

0.00
0.00
0.01
0.00
0'.00
0.20


'HP = Healthy papaya; HZ = Healthy zucchini; IP = Infected papaya; IZ =
Pc = Positive control, purified PRSV.


0.00
0,01
1.47
1.57
0.00
1.94

0.00
0.00
0.64
1.30
1.13
2.20

0.00
0.00
0.33
0.36
0.00
1.82

0.00
0.00
0.23
0.23
0.00
1.60

0.00
0.00
0.10
0.11
0.01
1.22


0.00
0.00
1.35
1.40
0.00
1.90

0.00
0.00
0.37
0.80
0.00
2.03

0.00
0.00
0.27
0.21
0.04
1.59

0.00
0.00
0.19
0.13
0.00
1.35

0.00
0.00
0.08
0.03
0.00
0.87


Infected zucchini; B = Buffer;








Using the locally produced PRSV
As (A1 and A2) absorbance readings of the
different samples were comparable to the
absorbance readings using the anti-PRSV
obtained from different countries. Among
the antiserum dilution tested, locally
produced PRSV As can detect infected
tissues with absorbance comparably high
compared to healthy samples at dilution
1:10,000. The least concentrated
preparation (1:50,000) can detect purified
PRSV preparation. For detection purposes,
the optimum As dilution for Philippine
PRSV is 1:1,000. At this dilution, the
absorbance readings of the infected
samples were several times higher than the
absorbance readings of the healthy samples.

For the remaining sera tested, the
absorbance readings of the purified PRSV-
P were comparatively high for all the
dilutions tested. Infected samples can still
be detected at As dilution 1:50,000 using
Taiwan and Thailand PRSV As.

Detection of PRSV in Field
Collected Samples. Using the locally
produced PRSV-P As (A1 and A2),
different reactions were obtained among
the field infected papaya samples. There
were symptomless samples (samples 4, 11,
24-25 and 27) that showed positive
reaction to PRSV-P As and there were
samples that manifested the typical PRSV
symptoms but the absorbance readings
were too low to consider their reaction as
positive like those from samples 2, 4-5, 7,
15-16, 22 and 25 (Table 2).

These symptomatic field samples
that showed low absorbance readings are
infected already but the viral antigen
concentration in the plant part that was
processed for the assay were not efficiently
released and reacted to the antibody. Kado


following a typical growth curve after
infection and the appearance of symptoms
follows the logarithmic phase. Thus, the
symptomatic field samples reacted but with
quite low absorbances, hence a negative
reaction to the As could mean also that the
virus concentration was at a declining
phase of the curve. It is also possible that
other agents unrelated to the As caused
those symptoms, hence the negative
reaction.

Both leaf samples from tissue-
cultured plants and infected leaves of
papaya hybrids and inbreds collected in the
field gave consistent results when assayed
by PTA-ELISA using the locally produced
PRSV-P As as well as from Australia
PRSV As (data not shown). No virus was
detected from samples obtained from the
tissue-cultured plants while all PRSV
infected leaf samples collected in the field
gave positive reaction to both antisera
(Table 3).

Screening for PRSV-P Resistance
of Promising C papaya Inbreds and
Hybrids. All interspecific Fl hybrids
reacted negatively to the antisera before
they were inoculated with the PRSV-P.
Thirty days after inoculation with the
PRSV-P, 11 hybrid plants reacted
positively to the locally produced
antiserum. Out of the 11 hybrids, four
reacted positively with the Australia serum
(Table 4). This indicate that the locally
produced antiserum is more specific to our
local PRSV-P isolate.

Typical PRSV symptoms, like
malformation of the leaves, mosaic and few
oily streaks on upper stem petioles were
already evident on the leaf of samples
obtained from hybrids SE 5 (4XI), SE
19(4XI) and 2H 63 and expectedly also
reacted positively to the As. Samples from
.o .. .* *


Ln Inn-F IML.l 2-211't-An CMAn r









Table 2. Absorbance at 405nm of field c,
PRSV-P As (A, and A2) in PTA-I

Sample Remarks
No.

1 Healthy papaya (Symptomless)
2 Diseased papaya (reduced lamina)
3 Healthy papaya (Symptomless)
4 Healthy papaya (Symptomless)
5 Diseased papaya (chlorotic/mosaic
6 Diseased papaya (reduced lamina)
7 Diseased papaya (chlorotic/mosaic
8 Healthy papaya (Symptomless)
9 Diseased papaya (reduced lamina)
10 Healthy papaya (Symptomless)
11 Healthy papaya (Symptomless)
12 Healthy papaya (Symptomless)
13 Healthy papaya (Symptomless)
14 Healthy papaya (Symptomless)
15 Diseased papaya (reduced lamina)
16 Diseased papaya (reduced lamina)
17 Diseased papaya (reduced lamina)
18 Healthy papaya (Symptomless)
19 Healthy papaya (Symptomless)
20 Healthy papaya (Symptomless)
21 Healthy papaya (Symptomless)
22 Diseased papaya (reduced lamina)
23 Diseased papaya (chlorotic/mosaic
24 Healthy papaya (Symptomless)
25 Healthy papaya (Symptomless)
26 Diseased papaya (reduced lamina)
27 Healthy papaya (Symptomless)

Threshold Value


6


Elected papaya samples against the Philippin
LISA.

PRSV A1 PRSV A2
Absorbance/Reaction Absorbance/Reaction

0.00 0.00
0.02 0.59 +
0.00 0.00
0.00 0.16 +
0.00 0.02
0.06 + 0.38 +
0.00 -0.15 +
0.00 -0.01
0.09 + 0.49 +
0.00 0.00
0.00 0.08 +
0.00 0.00 -
0.00 0.00 -
0.00 0.00 -
0.00 0.00 -
0.00 0.01 -
0.21 + 1.19 +
0.00 0.00 -
0.00 0.00 -
0.00 0.00 -
0.00 0.00 -
0.00 0.06 +
0.05 + 0.35 +
0.00 0.03 +
0.00 0.05 +
0.11 + 0.18 +
0.00 0.08 +

n A n (13






62 1997 Phil. Phytopath. 33(1):49-67






Table 3. Absorbance at 405 nm of symptomatic papaya leaves collected in Tranca, Bay,
T 2aolna x~th DP C1_D Ao /A-1 aA Ane.+mlt. i; DTA CT TO A





1997 Phil. Phytopath. 33(1):49-67 63



Table 4. Absorbance at 405 nin of interspecific F1 hybrids with PRSV antisera from
Philippines (A2) and Australia in PTA-ELISA'.

Entries Before inoculation 30 days after inoculation
A2 Australia A2 Australia

SE 2 (4 X 1) 0.00/- 0.00/- 0.02/- 0.00/-
SE 2 (4 X 1) G/S 0.00/- 0.00/- 0.00/- 0.00/-
SE 3 (4 X 1) 0.00/- 0.00/- 0.03/- 0.00/-
SE 4 (4 X 1) 0.00/- 0.00/- 0.03/- 0.00/-

SE 4 (4 X 1) G/S 0.00/- 0.00/- 0.09/+ 0.00/-
SE 5 (4 X 1) 0.00/- 0.00/- 0.12/+ 0.16/+
SE 7 (4 X 1) 0.00/- 0.00/- 0.69/+ 0.67/+
SE 7 (4 X 1) G/S 0.00/- 0.00/- 0.00/- 0.00/-

SE 8 (4 X 1) 0.00/- 0.00/- 0.08/+ 0.03/-
SE 9 (4 X 1) 0.00/- 0.00/- 0.00/- 0.00/-
SE 12 (4 X 1) 0.00/- 0.00/- 0.00/- 0.00/-
SE 13 (4 X 1) 0.00/- 0.00/- 0.00/- 0.00/-

SE 15 (4 X 1) 0.00/- 0.00/- 0.00/- 0.00/-
SE 17 (4 X 1) 0.00/- 0.00/- 0.00/- 0.00/-
SE 19 (4 X 1) 0.00/- 0.00/- 0.64/+ 0.55/+
ME 1 (4 X 1) 0.00/- 0.00/- 0.01/- 0.00/-


ME 9 (4 X 1) 0.00/-
ME 10 (4 X 1) 0.00/-

ME 11 (4 X 1) 0.00/-
ME 12 (4 X 1) 0.00/-
ME 15 (4 X 1) 0.00/-
ME 18 (4 X 1) 0.00/-

ME 19 (4 X 1) 0.00/-
2 H 63 0.00/-

Threshold Value 0.02

Each figure is a mean absorbance of two wells per


).00/- 0.00/- 0.00/-
).00/- 0.00/- 0.00/-

).00/- 0.00/- 0.00/-
).00/- 0.15/+ 0.00/-
).00/- 0.07/+ 0.00/-
).00/- 0.12/+ 0.00/-

).00/- 0.00/- 0.00/-
).00/- 0.37/+ 0.10/+

0.02 0.05 0.04





64 1997 Phil. Phytopath. 33(1):49-67


rilA-tLLA out nave not yet developed
symptoms at the time of the assay. A few
days later, these same plants developed the
typical symptoms of PRSV. The same
results were noted with the same set of
samples tested with the antiserum from
Australia (Table 5) where eight of the 25
hybrids tested reacted positively to the two
antisera. Of the eight plants four had the
typical symptoms of PRSV and the rest did
not. These results demonstrate that this
serological assay was sensitive enough to
detect the virus even in infected but still
non-symptomatic plants or before the
manifestation of the disease, thus
supporting its use in the early identification
of diseased plants. Additionally, the
hybrids that were positive for the virus but
were not yet showing symptoms were
probably exerting same effect on the virus.
While they were infectible, these plants
showed delayed symptom expression and
delayed symptom expression may be a
reflection of a suppressed virus
multiplication or movement in the plant
(Matthews 1991; Walkey 1985). This has
:o be confirmed with more studies,
however.


ACKNOWLEDGMENT

We would like to thank the
Australian Center for International
Agricultural Research (ACIAR), the
Philippine Council for Agriculture and
Resources Research and Development
PCARRD)-Department of Science and
technology (DOST) for the financial
support, Dr. Asuncion K. Raymundo,
institute of Biological Sciences, UPLB-
"AS and Dr. Evelyn Mae T. Mendoza,
JPLB-IPB for allowing us the use of the
ultracentrifuge and SW40 Dr. Ossmat
kzzam for availing IRRI


LITERATURE CITED

ADSUAR J. 1946. Studies on virus
diseases of papaya (Carica papaya L.) in
Puerto Rico. Transmission of papaya
mosaic Puerto Rico Agr Exp Sta Paper
number 1-86.

AGUILAR MG. 1992. Isolation and gel
electrophoresis of protein from ringspot-
infected papaya (Carica papaya L.) BS
Biology Thesis, UP Los Bafios, College,
Laguna. 33p.

BAJET NB, FABELLAR NG, DIZON TO,
TALENS ACD, ROPEROS NI. 1992.
Papaya ringspot and other diseases of
papaya in the Philippines: Proc. Symp.
Workshop. The Phil Phytopath Soc, Dept
of Pit Path, UP Los Bafios, 4031 College,
Laguna.

BRUNT AK, CRABTREE, GIBBS A.
(eds.) 1990. Viruses of tropical plants.
Cab. Int., Wallingford, Oxon, UK. pp. 475-
479.

CHANG CA. 1979. Isolation and
comparison of two isolates of papaya
ringspot virus in Taiwan. J Agr Res China.
28:207-216.

CONOVER RA. 1964. Distortion ringspot,
a severe virus disease of papaya in Florida.
Proc Fla Hortic Soc 77:440-444.

COOK AA. 1972. Virus diseases of
papaya. Florida Agr Exp Sta Tech Bull
750, 19p.

DE LA ROSA M, LASTRA R. 1983.
Purification and partial characterization of
papaya ringspot virus. Phytopathol Zeitsch
106:329-336.





1997 Phil. Phytopath. 33(1):49-67 6!


Table 5. Absorbance at 405 nm of interspi
PRSV antisera from Philippines (/


ENTRIES
A


ific F1 hybrid and other Carica species with
) and Australia in PTA-ELISA.


Absorbance (405nm)1
Reaction Australia/ Reaction




































visions from -the same tissue. J Virol 1989. The use of SDS-polyacrylamide gel
h 20:203-217. electrophoresis to assess purification





1997 Phil. Phytopath. 33(1):49-67 67

Annual Report, ACIAR and IPB, UP Los YEH S-D, GONSALVES D. 1984.
Bafios, College, Laguna. Purification and immunological analysis
of cylindrical inclusion protein induced
WALKEY DGA. 1985. Applied plant by papaya ringspot virus and watermelon
virology. W. Heinemann, London, UK. 329 mosaic virus 1. Phytopathology 74:1273-
p. 1278.












T. O. DIZON S. V. SIAR and L. A. REYES


Respectively, Research Associate Professor, University Researcher and Laborator)
Technician, Institute of Plant Breeding, University of the Philippines, Los Bafios, College
Laguna 4031 Philippines.

Key words: dieback, Fusarium solani, F. moniliforme, periwinkle, Phomopsis sp.,
Phytophthora parasitica, Sclerotium rolfsii, stem rot


This is the first report of stem rot or dieback of periwinkle in the
Philippines. It is a complex disease caused by Sclerotium rolfsii,
Phytophthora parasitica, Fusarium solani, F. moniliforme and Phomopsis
sp. The symptoms of the disease were characterized by wilting followed by
browning, drying and death of the stem or part of the stem or branch of the
plant. Infected plants seldom recover. Plants infected with S. rolfsii
produced white mycelial growth on the base of the stem. Later production
of round, smooth, brown sclerotial bodies of the fungus was observed. P.
parasitica caused water-soaking of affected stem which later turned brown
to black and dried. Fusarium solani and F. moniliforme produced white
mycelial growth at the surface of the affected stem. Phomopsis produced
numerous, minute black fruiting structures at the surface of the affected
stem.


INTRODUCTION To date very few diseases infec
periwinkle in the Philippines. Baniquec
Periwinkle or Vinca [Catharanthus (1990) mentioned stem rot caused b,
roseus (L) Don], locally known as Rhizoctonia solani Kuihn, whilf
chichirica, belongs to the Family Divinagracia (1985) and Troung and other!
Apocynaceae. It is a. trailing evergreen (1987) cited mosaic. However, like other
perennial herb grown as ornamentals in crops, diseases become a serious problem
baskets and beds and extensively as ground of periwinkle.
cover. Presently, it is gaining popularity
among ornamental plants being sold as This study was done to describe thi
flowering bedding plant. It can be symptoms of stem rot or dieback and t<
propagated easily using cuttings. It bears identify the pathogens associated with thi
small, simple flowers of varying color and disease complex in periwinkle.


lUOLUY AINU) PAlHUGEtLNS AS:





1997 Phil. Phytopath. 33(1):68-71 69

MATERIALS AND METHODS wilting and drying of the stem and leaves.
Infected plants seldom recovered. A single
Collection of Infected Samples plant may be infected by one pathogen or a
combination of any of the five pathogens.
Whole plant or stem tissues infected
with the disease were collected from the Identification of the Pathogens
UPLB-Institute of Plant Breeding
Ornamental Pot area. Symptoms were It was observed that about five
described and tissues exhibiting the common pathogens have been causing stem
symptoms were brought to the laboratory rot or dieback in periwinkle. These include
for isolation. Sclerotium rolfsii Sacc., Phytophthora
parasitica Dast., Fusarium solani Appel
Isolation of the Pathogens and Wr., F. moniliforme Sheldon and
Phomopsis sp.
Isolation was carried out using
tissue planting technique in potato dextrose Sclerotium rolfsii caused formation
agar (PDA). Tissues were cut into small of white mycelial growth at the base of the
sections, disinfected using 20% commercial stem close to the soil. Later, production of
bleach and rinsed twice with sterile distilled minute, round, brown sclerotial bodies was
water. Tissues were plated in PDA and noted. P. parasitica caused initial water-
incubated at 28-300C for several days until soaking of the stem or branch of affected
growth of the fungus was evident, plant. Later, affected stem turned brown to
Purification and identification of the black; dry and died. F. solani and F.
organisms were done. moniliforme produced white mycelial
growth at the surface of the affected stem
Pathogenicity tests especially at high relative humidity.
Phomopsis produced numerous, minute,
Isolated organisms were inoculated black fruiting structure on the surface of the
into the stem of the plant using mycelia of affected stem. Previous works from other
5- or 7-day old cultures. Inoculated plants countries revealed that P. parasitica and
were incubated for 24 hr under moist Phomopsis are the pathogens of stem rot
plastic bag and observed for symptom and dieback, respectively. They cause
expression, wilting and collapse of infected tissues.

Pathogenicity test
RESULTS
Mycelial inoculation of the S.
Symptomatology rolfsii, P. parasitica and Phomopsis caused
wilting of the plant 2 to 3 days after
Generally stem rot caused rapid inoculation. On the other hand, F. solani
wilting of the whole plant or part of the and F. moniliforme produced the symptoms
stem or branch being infected. Affected 7 days after inoculation. Symptoms
stem or branch became brown, dried and developed on inoculated plants were similar
soon died. In case of dieback, upper to naturally infected plants.
portion of the infected stem exhibited





70 1997 Phil. Phytopath. 33(1):68-71

LITERATURE CITED DIVINAGRACIA GG. 1985. Diseases of
important foliage and flowering ornamental
BANIQUED NC. 1990. Survey and plants. NRCP-UPLB Terminal Reptr.
identification of diseases attacking
ornamental and medicinal plants. BPI-CES. TROUNG HX, BENIGNO MA, FAVALI-
HEDAYAT, CALILUNG VJ. 1987.
In PCARRD Res. Highlights '89, Los Ordinary and yellow strains of cucumber
Bafios, Laguna, Abstr. mosaic virus isolated from tomato and
periwinkle. Phil Agr 70:143-157.









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