Front Cover
 Front Matter
 Enhancing the efficacy of Trichoderma...
 Effect of potato X potexvirus (PXV)...
 Restriction mapping of rDNA internal...
 Modeling Philippine downy mildew-yield...
 Host range and pathogenic race...
 Root rot of avacado caused by Phytophthora...
 Phytopathological note: Leaf blight...
 Abstracts of paper presented and...
 Back Cover

Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00090520/00041
 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: July-December 1997
Frequency: semiannual
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
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: VID00041
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
    Front Matter
        Front Matter
    Enhancing the efficacy of Trichoderma harzianum Rifai by chitin amendment against sheath blight of rice
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
    Effect of potato X potexvirus (PXV) on the development of late blight in selected potato (Solanum tuberosum L.) genotypes
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
    Restriction mapping of rDNA internal trasncribed spacer region of Rhizoctonia solani AG1-1A
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
    Modeling Philippine downy mildew-yield loss relationship in corn
        Page 110
        Page 111
        Page 112
        Page 113
        Page 114
    Host range and pathogenic race of Fusarium oxysporum f. sp. vasinfectum causing Fusarium wilt of cotton
        Page 115
        Page 116
        Page 117
        Page 118
        Page 119
        Page 120
        Page 121
    Root rot of avacado caused by Phytophthora cinnamomi Rands in the Philippines
        Page 122
        Page 123
        Page 124
        Page 125
        Page 126
        Page 127
        Page 128
        Page 129
    Phytopathological note: Leaf blight disease of durian caused by Rhizoctonia solani Kühn
        Page 130
        Page 131
        Page 132
        Page 133
    Abstracts of paper presented and submitted during the 28th anniversary of the pest management council of the Philippines, Inc. and annual scientific meeting of the Philippine phytopathological society, Inc. in Iloilo City, May 20-23, 1997
        Page 134
        Page 135
        Page 136
        Page 137
        Page 138
        Page 139
        Page 140
    Back Cover
        Page 141
        Page 142
Full Text

I I II, ' r~

Official Publication of The Philij


Business Manager
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Immediate Past President





Subscrnpons: Coamamiam ~aoo be a"
LTLB, CoOeI, Latpg 4031. Ptiippna Phaytp~e i,
Phdaplogpal Soidy, kc It is sea fr to m~bmu i paod
(domaic) md $25.00 per cpy dentm poa f- -d pi
Soce): Inc.. hfoamat reading maa. wi be ltpl

pine Phytopathlogical Socety, Ic.

ECTORS 1997-1998


DARD 1997-1998

N Associate Editor
Associate Editor


ed to n TREASL tE, P. P. S. co Dqt*mOf df l Mba p,
iod m. i 11 d p"=bsOf d F M-i
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72 1997 Phil. PhytoDath. 33(2):72-8



Supported by Philippine Rice Rese,
thesis of the senior author.

Respectively, Instructor and Profe:
of the Philippines Los Baios, College, Lag

Key words: biocontrol, chitin, Pen
sheath blight, Trichoderma harzianum

Four isolates of Trichodi
Penicillium oxalicum were test
Rhizoctonia solani causing she
harzianum completely overgrew I
entire medium surface. Parasitism i
around the hyphae of the pathogen
vacuolation. P. oxalicum also sho
but did not inhibit the growth of R.
the presence of volatile compound.
antibiosis was detected using T.
shared promising cellulolysis adeqi
ability. Chitin as a suspected ei
increased the ability of isolate 94
56.67% compared to 19.34% inhil
P. oxalicum prevented R. solani
and granules stored at 4, 20 and 3'
of T harzianum with time. The i
and granules was 200C. The Ic
21.30% with sheath blight, respe
granules with chitin applied simulta


Trichoderma has been extensively
studied for biological control of plar
diseases (Papavizas 1985; Chet 1987) bu


JN and L.L. ILAG

ch Institute (PhilRice) and portion of the M. S

or, Department of Plant Pathology, Universit)
la, Philippines 4031.

illium oxalicum, Rhizoctonia solani, rice, rice

ma harzianum and an isolate of
I for their biocontrol abilities against
h blight of rice. All isolates of T.
e culture of R. solani covering the
is revealed by coiling of T harzianum
using distortion, broken segments and
'ed a similar mechanism of parasitism,
olani in dual culture test. The test for
; was negative, but enzyme-mediated
arzianum isolate 94-016 which also
,cy index and competitive saprophytic
ancer of biocontrol by Trichoderma
)16 to inhibit growth of R. solani by
tion using the biocontrol agent alone.
*owth 24 hr after inoculation. Pellets
'C decreased in colony forming units
eal temperature for storage of pellets
iest real area infected of 25.22 and
ively were obtained using pellets and
sously with the pathogen in non-sterile

its efficiency in a competitive environment
like the soil is non- appealing because of it
passive nature of mycoparasitism i.e. onl,
when applied to special ecological niche
such as sterile or fumigated soils or soilles

- tn/ --I-- J .h -------b---- -- ---
onditinn, In sheath light of rice chitin in enhancing the biocontrol ability of

- studies done locally by (
y studies done locally by (
l / on

--u11aUiIu cuiu LapJia ki _7;j aII VVVU LA1L CL UIIILA -LIL Lk11jJQLUI0 k. 'V ...l lFl_ -
potential of Trichoderma as an antagonist performance of the granular and pelletized
of Rhizoctonia solani KOhn but failure in formulation of T. harzianum, and the effect
biocontrol ability was also reported of chitin in enhancing its competitiveness
especially in soil, whether sterile or non- including liquid culture of P. oxalicum
sterile. (with reference to timing of application and
kind of soil) in controlling sheath blight
To enhance growth, survival and under greenhouse condition.
competitiveness of Trichoderma in natural
soil requires substrates as source of
nutrients (Lewis and Papavizas 1985). A MATERIALS AND METHODS
substance known as chitin was postulated
by the authors as a means to increase the Dual Culture Test
chitinolytic enzymes reported to be
important in biocontrol mechanisms. Chitin Four T. harzianum isolates (94-012,
is a structural component of many fungi 94-015, 94-016, 94-022), an isolate of P.
including plant pathogens and of insect oxalicum and R.. solani were grown
pests of higher plants. Trichoderma individually on PDA plates (90 mm diam)
harzianum Rifai produces chitinases when for 5-6 days at 280C under continuous
grown on chitin (Elad and others 1982) and fluorescent light. Using a cork borer, a 5
these enzymes are effective in inhibiting mm mycelial disc of each isolate was
chitin-containing fungi. obtained and planted on the surface of Malt
Extract Agar (MEA) plate with the
The use of another soil fungus, biocontrol agent and pathogen positioned
Penicillium oxalicum Currie and Thom for 60 mm diametrically opposite each other.
the biological control of plant parasitic Control set-up consisted of the pathogen
nematodes (Davide, personal alone. Treatments were replicated five
communication) was also tested for control times. Plates were incubated under the

'ncPrn T hkrrizymjm Oip nnnflafinn duvnnrninv n

74 1997 Phil. Phytopath. 33(2):72-86

covered the entire medium incubated for 24 hr at the same
surface. temperature. The plate cover lid were
removed and the bottom plate with newly
Class 2 = Trichoderma overgrew the transferred mycelial disc (5mm diameter) of
pathogen at least two-thirds of R. solani was inverted over on a bottom
the medium surface. plate containing each of the Trichoderma
isolates and sealed with parafilm wax.
Class 3 = Trichoderma and the pathogen Agar plate containing mycelial disc of R.
each colonized one-half of the solani and inverted over a non-inoculated
medium surface and neither MEA plate served as control. Each test
organism appeared to dominate antagonist including the control were
the other. replicated three times. The plates were
incubated in the dark at 28C. Radial
Class 4 = Pathogen overgrew Trichoderma growth was measured after two days and
at least two-thirds of the medium analyzed by Completely Randomized
surface and appeared to Design (CRD). Means were compared
withstand invasion by using Duncan's Multiple Range Test
Trichoderma. (DMRT).

Class 5 = Pathogen completely overgrew Effect of Chitin on the Biocontrol
Trichoderma and occupied the Activity of T. harzianum and Test for
entire medium surface. Antibiosis of P. oxalicum

An isolate of Trichoderma was A 9-ml spore suspension of a 7-day-
considered antagonistic to R solani if the old T harzianum isolate 94-016 was
mean score for a given comparison (when inoculated into 95ml sterile coconut water
rounded to the nearest whole class number) media containing 5 g ground crab shells in
was < or = 2 but not highly antagonistic if 500ml Erlenmeyer flask. Isolate 94-016
the number was > or = 3. Microscopic was employed because past studies
interaction between the antagonist and the conducted on crop residue decomposition
pathogen was observed using Nikon showed that it possessed the highest
Photographic Equipment H-III (Mode C). cellulolysis adequacy index (CAI) and
This experiment was done twice. competitive saprophytic ability (CSA)
*Uh r-f : I A 9 CA

among t e our states use

'est for the Presence of Volatil

ky UU L IIVIu~uUUIII. JJikj; I&-0O U

Fsltvvered throh, sre -sutio An AVl.AI
filtered through sterile suction flask
!-.*+. 4I+A- -* ... I-,-& 4 )rr %n

fi;%i yL yUj l. lflll l iU VIJ LRIt' OiLU~L/ lllILJIi
vas mixed with 8 ml melted PDA (l:l,v/v)
md then poured into plates. Eight ml of
sterile distilled water on 8 ml melted PDA
a plates, served as control. A mycelial disc
)fR. solani was placed at the center of the
late exposed to continuous fluorescent
ight at 300C and its linear growth rate was
determined 24 hr after inoculation. The
ame technique was used to determine the
effect of culture filtrate of.P. oxalicum on
he linear growth of Trichoderma and R.
olani. The experiment was arranged in
.RD with five replicates and means were
compared using DMRT.

Population Dynamics of T. harianum
n Stored Pellets and Granules

A 9-ml spore suspension of a 7-day-
ild culture of T. harzianum isolate 94-016
vas inoculated into 500 ml flask containing
OOg fresh sterile pellets and grown for 14
lays. Granules were prepared according to
:umagun and lag (1997). Colony forming
inits (CFU)/g of pellets and granules were
determined at 14 days using Trichoderma

sing sowing at five seedlings per pot with two
t -! # =v F P, x ..

FAll J. I 7Y) iday IUIII -Uj1IIiIu ll'.' au1.
ive types of formulation were used: dried
onidial pellets of T. harzianum isolate 94-
16 with and without chitin, granules of T.
arzianum isolate 94-016 with and without
hitin and liquid culture of P. oxalicum
rown in coconut water media for 2 wk.
'he solid formulations were applied by
broadcast at 2.94 x 107 CFU/g of soil while
ie liquid formulation containing mycelial
iat was blended for 2 min in an osterizer
before drenching at 100 ml/kg in the soil.
'he liquid formulation contained
approximately 4.48 x 106 spores/ml. At
maximum tillering stage, plants were top
ressed with 40 kg/ha urea. Inoculation of
heath blight i'ocul,'n (grown for 2 wk in
:3 ratio of sterile rice grain-rice hull
moistened with water in dextrose bottles)
tas done 40 days after transplanting at the
ite of 5 g/kg of soil. The timing of
application of biological control agents
BCA) was varied as follows: (1)
simultaneous application of antagonist and
athogen; (2) application of antagonist
even days before the pathogen; (3)

Disease infection was assessed 1 revealed vacuolation and broken hypha
wk after the last inoculation with R. solani segments of the pathogen and coiling o
using % RAI (Percent Real Area Infected), hyphae of T. harzianum along the hypha,
the most reliable method because it of R. solani (Fig. 1). These microscopi,
represents actual infected tissue (Sharma observations indicate the possibility that cel
and others 1990). This is the estimate of wall degrading enzymes are excreted b,
plant tissue affected by disease expressed as Trichoderma. This finding started witl
a proportion of the total leaf or sheath area. Weindling's classic discovery on the
The formula developed for this method mycoparasitic activity of Trichoderm(
was, RAI % = 100 x (length x width of lignorum in 1932, a historical step in the
lesion)/[(length x width of leaf or sheath) x development of biological control, but the
C.F.] where C.F. = Correction Factor (0.67 overriding concern remains to be thi
for leaf and 0.83 for sheath). The leaf reproducibility of the laboratory results t<
correction factor was that of Gomez (1972) natural field conditions. Garrett (1944
while the sheath correction factor was described the natural soil as an environment
developed by taking the length x width of where microflora is far more complex an(
sheath versus real areas of the sheath the concentration of free nutrients is ver
measured using an automatic leaf area much lower. The rationale behind thi
meter (Li-cor, USA). The experiment on addition of soil amendments like rice bral
the effect of type of formulation, kind of in the pellets and granules and chitin is tc
soil, and timing of application was increase the nutrient status of the soil an
analyzed in a split-split plot factorial design possibly for mycoparasitism to occur due tc
while the effect of chitin on the two the fact that Trichoderma is a facultative o
Trichoderma formulation in a split-split- opportunistic mycoparasite which mean
split plot design with three replications that the fungus attacks living myceliun
each. Mean comparison was done using when furnished with an external food bas(
DMRT. (Henis, 1983). Coiling of P. oxalicum or
hyphae of R. solani causing distortion
vacuolation and collapse of hyphal cell wal
RESULTS AND DISCUSSION was also observed (Fig. 2). This is the firs
reported study of parasitism of P. oxalicun
The four isolates of T. harzianum, on the rice sheath blight pathogen.
having the same rating of Class 1 showed
no significant variation in their antagonistic Test for the presence of volatile
ability to R. solani in vitro. They all compounds of the four T. harzianun
completely overgrew the pathogen and isolates and P. oxalicum was negative (dati
covered the entire medium surface after 4 not shown). However, antibiosis wa!
days of incubation. Hyphae of R. solani demonstrated where P. oxalicum was fount
ceased to grow upon contact with the to be far better than the four isolates of T
hyphae of any of, the Trichoderma harzianum in this area of defense since i
isolates. No rating was done on the completely inhibited R. solani. Burg(
interaction of P. oxalicum and R. solani (1988) noted that the production anc
because the system was not applicable, release of a wide range of compound!
Their interaction showed no inhibition of including antibiotics is widespread among
growth of the pathogen. Light microscopy the members of the Fungi Imperfecti.whert

LW7 r l. ruvtopath. 33(2l:72-86 77

Figure 1. Effect of Trichoderma harzianuj
mycelia of R. solani (left); vacuolation, brol
and coiling of Trichoderma isolate 94-016

Figure 2. Effect of Penicillium oxalicum
mycelia ofR solani (left); Coiling of P. c

on hyphae ofRhizoctonia solani. Healthy
-n hyphal segments of R. solani (middle);
around the hyphae of R. solani. (right)

n hyphae of Kthizocionlia tsolani; healthy
valicum on hyphae of R. solani (middle)

78 1997 Phil. Phytopath. 33(2):72-86

Figure 3. Effect of culture filtrate of Trichoderma
harzianum isolate 94-016 added to PDA (l:l,v/v)
on the linear growth of Rhizoctonia solani; means
followed by the same letter are not significantly
different at 5% level by DMRT.

Figure 4. Effect of culture filtrate of Penicilliumn
oxr/icumn added to PDA (l:1.v/v) on the linear
growth of Rhizoctoina solani and Trichoderma
lharzianum isolate 94-016 24 hr after inoculation:
means followed by the same letter are not
significantly different at 5% level by DMRT.
Legend: 94-016 = isolate of 7T harzianum: CF =
culture filtrate.

these two organisms belong. The effects of
T: harzian lm isolate 94-016, with and

R .o~h CF R nl 94-016+CF 04016e

without chitin, in inhibiting growth of R.
solani are significantly different from the
control (Fig. 3). Chitin enhanced T.
harzianum isolate 94-016 by inhibiting
growth of R. solani by 56.67% compared
to 19.34% inhibition using the biocontrol
agent alone. Biot;ontrol activity of T.
harzianum isolate 94-016 was enhanced
with the addition of ground crab shells as
source of chitin of the fungus thus
providing greater growth inhibition of R.
solani than the set-up without the
amendment. Chet (1990) and Elad and
others (1982) observed that T. harzianum
excreted chitinase into the medium when
grown on chitin. Lytic enzymes like
chitinases from Trichoderma spp. were
suggested as important in the biocontrol
action (Lynch 1990) and such hypothesis
called enzyme-mediated antibiosis was
verified in the experiment. Faull (1988)
commented that antibiosis can have
advantages over other types of antagonism
for biological control. Unnecessary contact
between the antagonist and the pathogen
for one, is an example where antibiotic
substance may freely diffuse in water, air or
substrate to other microorganisms. The
inhibitory effect of P. oxalicum in the
growth of R. solani and T. harzianum
isolate 94-016 was significantly different
from their treatments without the culture
filtrate (Fig. 4). The growth of T.
harzianum isolate 94-016 and R. solani
was inhibited 95.93 and 100%,
respectively. There was complete growth
prevention ofR. solani by P. oxalicum 24hr
after inoculation. One can infer from this
study that it is not advisable to apply T.
harzianum and P. oxalicum together
because of their inhibitory effects.

The agar test showed that the
members of the genus Trichoderma are
active both as hyperparasites and as

94- 016 94-016 5% chtm Control

1997 Phil. Phvtnnath 33t(2!72-R6f 7

antibiotic producers (Hadar and others
1979). However, the validity of the agar
test for antibiosis and parasitism can be
questioned, especially if it is used as the
only primary screen for the obvious reason
that it will not select organisms which act
by competition. The selection of the four
T harzianum isolates was actually taken
out of 56 isolates based from its first
screening using Garrett's method on test
for CAI (unpublished results). Parasitism,
antibiosis and competition are mechanisms
reported to occur in Trichoderma-
pathogen interaction. Such condition was
emphasized by Cook and Baker (1983) in
their conclusion that the greater the
number and diversity of methods used by
an antagonist to inhibit a pathogen, the
more successful it will be in biological

Cumagun and Lapis (1993) plated
pellets of Trichoderma in water agar and
found them to be viable for three months.
This is not a reliable test -for viability
because the method did not take into
account the CFU of the biocontrol agent.
This problem was overcome in the .present
experiment. The best storage temperature
for both pellet and granule formulations ol
T. harzianum isolate 94-016 is at 200C.
Storage at 35C is not recommended
because of contamination problem. The
decreasing trend in CFU of stored pellets
and granules through time for 8 wk (Fig.
5) implies that BCA formulations should be
used preferably while they are newly
prepared. The use of old formulation
stored for a long time is not advisable.

Past studies on Trichoderma pellets
by Lapis and Cumagun (1994) showed
inconclusive result when tested in non-
sterile soil under greenhouse condition. A
nnrc;hlp arrnr itlintifipd i the wrnno timing

*- ~ v



-, -

*- --- -
,z ^\_--.-----

Figure 5. Population dynamics of Trichoderma
harzianum isolate 94-016 in pellets and granules
stored at 4, 20 and 350C.

of application of R. solani in the soil.
Inoculation of R. solani in the soil in this
experiment was done at maximum tillering
stage instead of at planting to provide
favorable microclimate for infection of the
rice plants. The test plants were further
watered three times a day and enclosed
with plastic cages during night time for 7
days to hasten and increase sheath blight
infection. Sterile soil was also employed
for comparison with non-sterile soil. Stack
and others (1988) proposed that
pasteurizing or sterilizing the soil prior to
the addition of the biocontrol agent might
permit a higher population of the agent to
develop and perhaps result in a higher level
of disease control. In the experiment
however, this hypothesis did not hold true.

go) 1007 Phil Phvfnnsth 1(i.77..R;

The rice plants grown in sterile soil were
more vigorous allowing crowding in
between plants and increasing relative
humidity beneath the canopy than those in
non-sterile soil because competition for
nutrients with other organisms was
removed, thus disease infection by R.
solani was more favorable.

Disease control will focus more in
non-sterile soil because this is the real
status of the rice ecosystem. Table 1 shows
the percent real area infected (% RAI) by R
solani using various types of BCA
formulation applied a week before the
pathogen, simultaneously with, and a week
after the pathogen in sterile and non-sterile
soil. There was a significant difference in
the reduction of sheath blight infection
between sterile and non-sterile soil by
simultaneous application of the pathogen
and BCA regardless of the type of BCA
formulation. No differences in disease
infection were observed when each
formulation was applied after the pathogen
in non-sterile soil. The liquid culture of P.
oxalicum, in comparison with Trichoderma
formulation without chitin, had the lowest
%RAI of 39.29% in non-sterile soil only
when applied simultaneously with R solani.
The lowest %RAI of 25.22 and 21.30%,
respectively, were obtained from pellets
and granules of T harzianum isolate 94-
016 with chitin in non-sterile soil, the
former having a significant difference as
compared to pellets without chitin. The
other treatment with significant difference
on the effect of chitin from the rest of the
treatments was granules applied after the
pathogen in non-sterile soil (Table 2).

Simultaneous application appears to
be the most effective timing of application
for all types of formulation. Elad and
others (1980) found this to be true when

their results suggested that a biocontrol
preparation can effectively reduce survival
of R. solani in organic debris (beet seed)
when it is added with or soon after
pathogen. They showed that the longer the
pathogen was in contact with the biocontrol
fungus before a susceptible crop, the more
successful was the biocontrol. Lapis and
Plete (1994) also observed that
simultaneous application of BCA and
pathogen caused the highest sheath blight
disease control of 59.71% using a
Trichoderma isolate CES 4 in a non-sterile
soil. Likewise, Galapon-Gonzales (1982)
recommended that simultaneous infestation
of previously sterilized and unsterilized soil
with each of the pathogen and with either
Trichoderma or Penicillium a week before
seeding, provided good protection and
effective control against post-emergence
damping-off of greenhouse-grown tomato.
A possible explanation for this can be
referred to the previous study on sclerotial
germination and colonization obtained in
both sterile and non-sterile soil plates
(Cumagun 1998; Cumagun and lag 1997).
T. harzianum isolate 94-016 grew
prolifically 24 hr after inoculation as
manifested by the presence of green
pigment on the surface of the soil plate.
Fungal growth subsided and was no longer
visible a week after inoculation which could
mean decline in the active phase of the
BCA. This hypothesis could be applied in
the greenhouse in which the BCA was most
effective when simultaneously applied with
the pathogen than a week before the
pathogen since a week incubation period of
the antagonist could be at. its peak of active
metabolism and perhaps critical in
biocontrol ability. Application of BCA a
week after the pathogen was obviously not
effective because sclerotial bodies of R.
solani were observed to cause visible
infection in rice sheath in a span-of 5 to 7

1997 Phil. Phytopath. 33(2):72-86 81

Table 1. Percent real area infected (%RAI
time of application, type of fort
condition. '

Time of Application and I
Type of Formulation Steril

Before the pathogen
pellets 74.3
granules 63.3
liquid cultures 68.9
treated control 89.8
untreated control 0.0

Simultaneous with the pathogen
pellets 76.7
granules- 80.3
liquid cultures 64.3
treated control 89.8
untreated control 0.0

After the pathogen
pellets 56.7
granules 70.5
liquid cultures 61.2
treated control 89.8
untreated control 0.C

Mean 59.C
S** = significant at 1% level, = significant at
timing of application, means followed by a comm

I by Rhizoctonia solani in rice as affected by
kulation and kind of soil under greenhouse

ind of Soil F-mean Difference
S Non-sterile

tab 44.64b 59.48 59.68**
jb 48.49b 55.90 14.81ns
ib 58.64ab 63.80 10.31ns
Ia 70.72a 80.28 19.12*
)c 0.00c 0.00 0.00ns

)ab 56.77ab 66.74 19.94*
)ab 43.21b 61.75 37.09**
Ib 39.29b 51.81 25.05**
la 70.72a 80.28 19.12*
)c 0.OOc 0.00 0.00ns

ib 72.22a 64.49 -15.47ns
3b 58.42a 64.48 12.11ns
5b 65.13a 63.19 -3.87ns
la 70.72a 80.28 19.12*
)c 0.OOc 0.00 0.00ns

5 46.60 52.83 12.47
Vo level, ns = not significant; in a column under each
a letter are not significantly different at the 5% level

82 1997 Phil. Phytopath. 33(2):72-86

Table 2. Percent real area infected (%RAI) by Rhizoctonia solani on rice as affected by
time of application, type of formulation, kind of soil and chitin amendment under
greenhouse condition.

Formulation and Time of % RAI Mean Difference
Application With Chitin Without Chitin
Pellets in Sterile Soil
before the pathogen 59.31a 74.32a 66.81 -15.01ns
simultaneous with the 70.25a 76.70a 73.48 -6.45ns
after the pathogen 49.53a 56.75a 53.14 -7.22ns
Pellets in non-sterile soil
before the pathogen 55.52a 44.64b 50.08 10.88ns
simultaneous with the 25.22b 56.77ab 40.99 -31.55*
after the pathogen 61.28a 72.22a 66.75 -10.94ns
Granules in sterile soil
before the pathogen 47.97a 63.30a 55.64 -15.33ns
simultaneous with the 60.54a 80.30a 70.42 -19.77ns
after the pathogen 51.83a 70.53a 61.18 -18.70ns
Granules in non-sterile soil
before the pathogen 42.42a 48.49a 45.45 -6.07ns
simultaneous with the 21.30a 43.21a 32.26 -21.91ns
after the pathogen 32.80a 58.42 45.61 -25.63*
Mean 48.16 62.14 55.15 -13.97
** = significant at 1% level, = significant at 5% level. ns = not significant; in a column under each

type of formulation and kind of soil,
the 5% level DMRT.

means followed by a common letter are not significantly different at

niologicai coniroi requires not omy me amendment to increase tne ctntmolytic
addition of antagonists but also the enzymes of the fungus which is important in
introduction of biocontrol agent in an biocontrol activity. However, this result in
environment that favors antagonistic ability the greenhouse must also be tested under
(Nelson and others 1983). The authors different conditions in the field. Though an
believe that enzyme-mediated antibiosis environmentally friendly approach, the only
could also be operating in the natural soil constraint identified in this kind of practice
environment. Addition of chitin induces T. is the economics of control of rice sheath
harziamnum to produce chitinase (Chet blight under field conditions but it can be
1990). The enzyme was not identified- in integrated as a component in plant disease
this study but a few workers like Harman management to reduce fungicide use.
(1992) had made extensive examination of
the separate chitinases produced by T
harzianum strain PI with at least 3 separate LITERATURE CITED
kinds of activity. Some of the enzymes are
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presumably cleave monomer from the end mycoparasites for biological control of
of the chitin polymer, others cleave the plant diseases. Ann Rev Phytopathol 38:
dimer (chitobases) and still others .cleave 59-72.
the polymer randomly (endochitinases).
These enzymes are effective in inhibiting BELL DK, WELLS HD, MARKHAM
chitin-containing fungi. The influence of CR. 1982. In vitro antagonism of
chitin may also result from an increase in Trichoderma species against six fungal
the number of mycolytic microorganisms plant pathogens. Phytopathology 72:379-
such as the actinomycete capable of 382.
digesting mycelium of soil-borne pathogens
(Mitchell and Alexander 1961). They BAKER R. 1988. Trichoderma spp. as
found that soil treatment with either of 2 plant growth regulators. CR Critical

1~~1 ~ ~L

84 1997 Phil. Phvtonath. 33(21:72-9

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mode of action and potential as a production of Trichoderma spp. as a mean=
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University of the Philippines Los Bafios.
105D. FAULL JL. 1988. Competitive antagonism

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Philippines. 154p.

GARRETT SD. 1944. Root disease fungi.
Chronic Botanica Company.
Massachusetts, USA. 177 p.

GOMEZ KA. 1972. Techniques for field
experiments with rice. International Rice
Research Institute, Los Bafios, Laguna,
Phil. 37p.

Biological control of Rhizoctonia solani
damping-off with wheat bran culture of
Trichoderma harzianum. Phytopathology

HARMAN GE. 1992. Methods of genetic
manipulation for the production of
improved bioprotectant fungi. In D.F.
Jensen and others ed., New Approaches in
Biological Control of Soil-Borne Diseases.
IOBC/WPRS Bulletin 15(1):181-187.

HENIS Y. 1983. Ecological principles of
biocontrol of soil-borne plant pathogens:
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A. Reddy ed., American Society for
Microbiology. Washington, D.C. pp. 353-

1980. Introduction of microbial antagonists
to specific courts of infection: seeds,
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Allanheld and Osmun.- Totowa, N.J.

Improved use of Trichoderma as a means
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Bogor, Indonesia. pp. 169-177.

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upland and lowland rice Phase II. In 7th
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Gliocladium and their effect on the
proliferation of the fungi in soil. PI Pathol
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LYNCH JM. 1990. Fungi as antagonists.
In P.E. Dumm ed., New Directions in
Biological Control. Alan R. Liss, Inc. New
York. pp. 243-253.

The mycolytic phenomenon and biological
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Microbiological processes associated with
the use of chitin for biological control. Soil
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damping-off in container media amended

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Panama 21:960-969. agents. In K.G. Mukerji and K.L. Garg.
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harzianum and Fusarium oxysporum and

1997 Phil PhvtoDath 33(2):87-102 81



Portion of the Ph.D. dissertation of t

Respectively, Senior Scientific Office
Agricultural Research Institute (BARI), Ga
Plant Pathology, University of the Philippine;
Seed Specialist/Virologist, International Pot
Department of Plant Pathology, UPLB, Coll

Key words: epidemiology, potato la

Experiments to determine the
on the development of late blight
conducted at La Trinidad, Benguet I
genotypes were inoculated artificial
inoculation and the data on the disea:
latent period, sporulation period, lesi.
cuticle thickness and peroxidase ac
development of Phytophthora infest
infection with PXV delayed the onse
the level of zoospore penetration, i
development. PXV infection redu,
infection rating in some genotypes.
peroxidase activity in the genotype
Slightly thicker cuticle was observed
was higher in genotypes with prior P
late blight alone.


Potato (Solanum tuberosum L.) is
an important vegetable as well as food crop
that grows well in many countries including
the Philippines. Potato is basically a
starchy food and is used as a staple in many



Senior author.

r, Tuber Crops Research Center, Bangladesh
ipur, Bangladesh; Professor, Department of
(UPLB), College, Laguna, Philippines 4031;
to Center, Baguio and Associate Professor,
ge, Laguna, Philippines 4031.

e blight, potexvirus

effect of potato X potexvirus (PXV)
in selected potato genotypes were
>r two seasons. The selected potato
ly with PXV prior to late blight
e development like incubation period,
in size, infection rating and change of
vity were taken. PXV affected the
ns in some potato genotypes. Prior
of the potato leaf blight and reduced
capacity to sporulate, size of lesions
ed the percent blight infection and
There was no significant trend of
with virus or late blight infection.
in virus-infected plants. Tuber yield
'V infection than those infected with

countries, mostly in the developed world.
Boiled potatoes are almost similar in food
value with cooked rice, but production of
proteins and calorie per unit area and unit
time by potato is higher than those by rice
and wheat (CIP 1982).

W5 1997 Phil. Phvtopath. 33(2):87-102

Among me Imngai diseases or
potato, late blight caused by Phytophthora
infestans (Mont.) de Bary is the most
important, and also found worldwide where
potato grows. Late blight affects the
leaves, stems, petioles, and tubers and
causes serious yield losses (Alexopoulos
and Mims 1979; Anonymous 1990). Among
the viral diseases of potato, PLRV, PYV
and PXV are the most important. In the
field conditions, double infection by both
fungal and viral diseases are common in
attacking single potato plant. When more
than one pathogen attack a single plant, in
most cases the interaction between them
causes additive effects.

As multiple diseases are common in
field conditions, it is necessary to develop a
variety tolerant or resistant to combined
effects of pathogens. Resistance of a host
against a pathogen may be changed by the
presence of or interaction with another
pathogen. Due to this reason the
International Potato Center (CIP) has given
more emphasis on developing varieties with
combined resistance to viruses and fungal
pathogens (Anonymous 1992). Reports on
the interaction between viral and late blight
of potato are available (Darsow and
Wulfert 1989; Kalra and others 1989a and
1989b). Mukhopadhyay and Sengupta
(1967) reported that pre-infection with
viruses that cause mild mosaics may have
synergistic effect on the natural
susceptibility of potato to the late blight in
the field. Kalra and others (1990) reported
that the effect of PYV infection inhibited
the liberation and germination of zoospores
ofP. infestans. '

It was hypothesized that the virus
infection can increase resistance in the host
to subsequent P. infestans infection, the
increase in resistance if any is greater in

plants more resistant to blight or to PXV;
and the change in reaction of potato to late
blight is influenced by PXV through the
production of chemical substances
detrimental for the fungal pathogen. Thus,
the objectives of this study were: (1) to
determine the peroxidase activity of potato
as influenced by PXV and late blight and
the changes, if any, of thickness of cuticle,
(2) to quantify the effect of PXV to late
blight development in relation to yield of
some potato genotypes.


The experiments were conducted at
the Northern Philippines Root Crops
Research and Training Center
(NPRCRTC), La Trinidad, Benguet for dry
(January-April, 1996) and wet season (May-
August, 1996) both in the field and
greenhouse conditions. Laboratory
experiments were conducted at the
Department of Plant Pathology, University
of the Philippines Los Bafios (UPLB),
Laguna and NPRCRTC, La Trinidad,

Source/Collection and Propagation of
the Potato Genotypes

Potato genotypes were received
from the International Potato Center (CIP),
Lima, Peru as in vitro plantlets through the
courtesy of Dr. Oscar Hidalgo, CIP, with
known reactions to virus diseases and
potato late blight (Perez and others 1992;
Chien and others 1990; Gayao and others
1991; CIP 1994). The.genotypes used in
the study were multiplied through
successive apical stem cuttings (5-6) to
achieve around 500 seedlings per genotype
and allowed to grow roots for 21 days in

1997 hil. Phvtopath 33(2):87-102 89

rreparauon oI imoculum ot r. nmetians

Late blight infected potato leaves
with typical symptoms (new lesion) were
collected from the field at La Trinidad,
Benguet, washed with distilled water and
inserted inside a sliced potato tuber which
was previously surface sterilized with
burning alcohol. They were kept in the
cool chamber at 180C in plastic bags with
sufficient moisture for 24 hr to enhance
sporulation. The sporangiospores were
scraped and suspended in distilled water
and placed in the refrigerator (about 6C)
for 2 hr for zoospore release. The amount
of zoospore in the suspension was
standardized to about 3000 motile
zoospore/ml by counting 10 ml aliquot from
the suspension under microscope (Diccion

Collection, Isolation and Propagation of
PXV Isolate

Isolates of PXV were collected
from the field at Benguet and Institute of
Plant Breeding (IPB), UPLB, Laguna and
identified through host range, ELISA test
and through EM studies (Hossain 1997).
The ringspot strain of PXV used in the
study was propagated in Nicotiana
glutinosa and N. tabacum var. White

Artificial Inoculation of Test Plants with
P. infestans

The test plants were inoculated with
a zoospore suspension of 3000 zoospore/ml
using a plastic hand sprayer following the
procedure of Chien (1989) with some
modifications. The plants were totally
wetted with the zoospore suspension at
5:00 PM on March 11, 1996 and covered
with polyethylene sheets for 15 hr until 8:00

AMm in e iollwing morning. ne plants
were also sprayed with water until 7 days
after inoculation to maintain high humidity.
Similarly, the plants were-inoculated by P.
infestans in wet season on June 14, 1996.

Artificial inoculation with P.
infestans was done at 5:00-6:00 PM using
a hand sprayer at 45 days after planting
(DAP) during both dry and wet seasons.
For maintaining high relative humidity, the
field was irrigated before and after
inoculation followed by spraying with water
using hand sprayer until 7 days after

Effect of PXV on the Development of
Late Blight in Selected Potato Genotypes

Nine potato genotypes with known
reactions to PXV and late blight (resistant,
moderately resistant and susceptible or
immune) were used on the experiment in
dry season. In the wet season, 6 genotypes
were included in the field experiment.

Recommended doses of fertilizers
like complete fertilizer (14-14-14) at the
rate of 120-120-120 kg/ha of N,P205 and
K20. One-half of complete fertilizer and
chicken manure at the rate of 4 tons/ha
were basally applied in the soil and one-half
complete fertilizer and urea at the rate of 50
kg/ha were side dressed one month after
planting during ridging. Diagrans 5G at the
rate of 15 kg/ha was basally applied with the
complete fertilizer. The control plots and
PXV alone plots were sprayed with
fungicide (Dithane M-45 and Ridomil MZ-
58) and insecticide (Tamaron) was sprayed
in all plots for controlling late blight
infections and vectors of the viruses,
respectively. Irrigation and other cultural
practices were performed whenever

Each plot comprised 21 plants with c
30 cm spacing. Under greenhouse t
editions, 3 plants per genotype were used t
each treatment. The design of the r
eriment was split plot with three '
locations. The treatments (main plots) f
*e: 1) inoculated potato plants with PXV a
days after planting; 2) inoculated potato e
its with P. infestans 45 days after a
citing; 3) inoculated potato plants with r
V 30 days after planting and followed by a
infestans 15 days after inoculation of ii
V; and 4) control.
Late blight development was
armined using standard rating scale r
rnfling 1982). Data on zoospore
etration, incubation period, latent
iod, size of lesions, sporulation, plant f
opy development, peroxidase activity f
cuticle thickness, tuber yield per plant, t
*e gathered. v
oxidasee Assay s
Peroxidase activity (PA) was g
lyed following the procedures of Kar c
others (1985). Briefly, discs (12mm c
neter) of each leaf blade (3rd leaf from ii
collected from 5 different plants of each n
tment category were pooled together 1
kept at 40C for 30min within 10min of c
ipling before using them for enzyme 1
-action. Chilled leaves were ti
logenized with cold 0.1M phosphate d
Fer, pH 6.0 (100mg leaf in 2ml buffer) in
)re-chilled mortar and pestle. The
logenate was centrifuged at 4,250rpm
20min, and the supernatant was used for

fllu Elq ^ U F" AFl" l Va-sj,, *'J.**Z jI ^
:tract and 0.5ml of 0.1M phosphate
iffer, pH 6.0). The reaction was allowed
proceed for 3min at 270C after which the
action was stopped by adding 0.5ml of
4 H2S04. The amount of purpurogallin
rmed was determined by determining the
isorbance at 420nm. Enzyme activity was
:pressed in units where one unit is the
nount of purpurogallin formed which
ised the absorbance by 0.1/min under
say conditions. PA was tested before
oculation and 7 days after inoculation
ith PXV and 7 and 15 days after
oculation with P. infestans.

[easurement of Cuticle Thickness

The leaf samples were collected
om the upper third compound leaf (third
>m tip) from 5 plants for each group for
e cuticle thickness measurement. After
fishing with distilled water, the leaf
mple was placed inside a potato tuber and
ced with fine razor blade by hand. The
actions were stained with lactophenol and
ycerine (50%) for one minute and placed
I glass slides. The specimens were
vered with coverslips, and a drop of oil
mnersion and observed under compound
microscope at 400x magnification.
sickness was measured with calibrated
;ular micrometer and expressed in mm.
sickness of cuticle was measured three
nes: at the time of virus inoculation, 7
ys and 21 days after virus inoculation.

1997 Phil. Phytopath 33(2):87-102!

PXV and P. infestans appeared observed on same genotypes pre-infect,
longer (3.92 days) than those inoculated with PXV (59.33%) (Table 2). Among tl
with P. infestans (3.22 days). The same genotypes pre-inoculated with PXV, la
trend was observed during the wet season blight infection efficiency was high
as there was 4.17 days forthe symptoms of reduced in the genotype CIP 384112.1
late blight to appear on the genotypes with 68.67% and the least (43.33%) was
inoculated with both PXV and P. infestans CIP 676025. In case of genotypes infect
while there was about 3.5 days with the with late blight alone, infection efficiency
genotypes inoculated with P. infestans. P. infestans was highest (89.33%) in C]
Among those inoculated with P. infestans 375335.1 and lowest (61.33%) in C]
alone during the dry season, the range of 676025..
incubation period was from 3-5 days while
the range of incubation period with the Sporulation Period
genotypes inoculated with both virus and P.
infestans was from 3-6.3 days (Table 1). Significant variations of mes
sporulation periods were observed betwet
Significant variation was observed those inoculated with P. infestans aloi
between the mean latent periods with and those with PXV+P. infestans. TI
different genotypes between late blight average sporulation periods of differe:
alone and PXV+late blight infection (Table genotypes with late blight and those wil
1). Increased latent period was noted in PXV+late blight were 8.57 days and 7.2
virus inoculated plants compared to those days, respectively. Among the differed
inoculated with P. infestans alone. The genotypes infected with late blight alon
average latent periods for different the highest sporulation period was 10.8
genotypes under field conditions during dry days in CIP 800953 and lowest was 7

mtection, respectively. In tne dry season
greenhouse study, the range of latent perio(
with the genotypes inoculated with P
infestans alone was from 5 to 7 days whih
it was 5 to 8 days with both virus and P
infestans inoculated genotypes (Table 1)
In wet season greenhouse study, th<
average latent periods of P. infestans fo]
different genotypes were 5.44 days anc
5.93 days for late blight alone an(
PXV+late blight infection, respectively
(Table 1).

Infection Efficiency

The mean infection efficiency

sporulation penoa ot 9.zu days m U
800983 was significantly higher than th
recorded in CIP 384112.14 (7.60 day!
The lowest (6 days) was observed in C
375335.1 (Table 2).

Sporulation Capacity

The mean sporulation capacity of.
infestans varied significantly between la
blight alone (697.59 sporangia/125mm
and PXV+late blight infection (423.1
sporangia/125mm2) and among tl
genotypes (Table 2). Among tl
genotypes, the fungus sporulated the mo
rwT fnnof2'i -A;k A And (

92 1997 Phil. Phytopath. 33(2):87-102

Table 1. Incubation and latent periods of late blight on selected potato genotypes as
influenced by potato X potexvirus (PXV) infection in La Trinidad, Benguet'.

Treatment/ Incubation period (day) Latent period (day)
Genotype Dry Season Wet Season Dry Season Wet Season
Field GH Field GH Field GH Field GH

LB alone
CIP 375335.1 3 3 3 3 6 5 5 5
CIP 384112.8 3 3 4 4 6 5 7 7
CIP 384112.14 3 3 3 3 5 5 5 4
CIP 384724.13 3 4 3 4 6 6 5 6
CIP 390353.3 3 4 4 5 6 6
CIP 676025 3 3 3 5 5 5
CIP 720090 3 3 3 5 5 4
CIP 800953 3 3 3 3 5 5 5 5
CIP 800983 5 4 5 4 8 7 7 7
Mean 3.22 3.33 3.5 3.44 5.67 5.4 5.67 5.44

CIP 375335.1 3 3 4 4 6 5 6 6
CIP 384112.8 4 4 4 4 7 7 7 7.3
CIP 384112.14 4 4 3 4 6 6 5 6
CIP 384724.13 4 4 4 4 8 7 6 6
CIP 390353.3 4 4 4 6 6 6
CIP 676025 4 3 3 6 5 5
CIP 720090 3 3 3 5 5 4
CIP 800953 3 3 4 3 6 5 6 5
CIP 800983 6.3 5 6 5 10 8 8 8
Mean 3.92 3.67 4.17 3 78 6.67 6.00 6 33 5.93

CV (%) (main plot) 10.1 6.7 19.9 6.5 7.6 4.1 4.8 2.4
LSD (5%) (main plot) 0.42 0.27 NS 0.27 0.55 0.27 0 41 0.16

= Not included in the study: LB = Late blight: PXV+LB = Inoculated with PXV prior to late blight;
GH = Greenhouse

OOPi 'D.l 1

'able 2. Infection efficiency (%), sporulatii
(sporangia/125mm2) of Phytophthor
influenced by potato X potexvirus (F

Genotype efficiency(%)
LB alone PXV+LB L

IP 375335.1 89.33a 67.67a
IP 384112.8 69.67b 50.00c
IP 384112.14 86.33a 68.67a
IP 384724.13 74.67b 60.67b
IP 390353.3 83.67a 65.63ab
IP 676025 61.33c 43.33b
IP 720090
IP 800953
IP 800983

lean 77.50 59.33
V (%) (main plot) 3.5
SD (1%) (main plot) 6.51

n a column means followed by a common letter are n
egend: LB = Late blight; = Not included in the stud]

n period (days) and sporulation capacity
r infestans in different potato genotypes as
CV) infection in La Trinidad, Benguet'.

Iporulation period Sporulation capacity
(day) (sporangia/125mm2)
3 alone PXV+LB LB alone PXV+LB

7.20e 6.00d 258.33e 217.33e
8.20c 7.00c 887.00d 564.00c
7.80d 7.60b 1133.33c 351.67d
7.40e 6.73c 319.00e 194.67ef
10.80a 7.07c 1308.67b 864.00b
10.00b 9.20a 57.00f 38.67g
S 55.67f 64.00g
S .133.00f 109.67fg
2126.33a 1404.00b

8.57 7.27 697.59 423.11
1.3 4.1
0.33 61.63

t significantly different at the 5% level by DMRT.

ILl~~~~cL ??I?\.Qrl In~ nr

genotypes inoculated with

lesions in all the genotypes was significantly 676025 (3.25mm) while the thinnest was
lower with prior PXV infection than the that of CIP 375335.1 (2.79mm) at 21 DAI.
same genotypes without the virus at 6, 8 With the non-inoculated genotypes at the
and 10 days after inoculation with mean same age, the thickest (3.13mm) and
lesion diameter of 5.9mm, 10.5mm and thinnest (2.75mm) cuticle was in CIP
17.1mm, respectively. The corresponding 676025 and CIP 357335.1, respectively. In
means of lesions in those infected with late the greenhouse during the dry season, the
blight alone were 7.2mm, 14.9mm and mean cuticle thickness of the non-
24.9mm, respectively (Table 3). inoculated genotypes was 2.83mm while
the virus inoculated ones had a mean of
Late Blight Infection Rating 2.89mm at 21 DAI but the difference was
not significant (Table 5).
PXV infection prior to late blight
inoculation significantly reduced the rating Peroxidase Activity
of late blight infection both in the field and
greenhouse experiments. In the field, the There was no consistent trend of
mean late blight ratings with PXV were peroxidase activity in the genotypes
1.48, 2.81, 3.07, 3.33 and 3.96 and 1.67, infected with PXV and late blight or both at
3.44, 3.67. 3.93 and 4.59 with late blight various stages of plant development (Table

light, the range of late blight rating was for non-inoculated genotypes.
om 1.00 (CIP 800983) to 8.67 (CIP
20090) and those with late blight alone Yield
lowed range of late blight rating 1.67
IP 800983) to 9.0 (CIP 720090) at 82 The yields of the potato genotypes
'AP. significantly differed when inoculated alone
with PXV and P. infestans or both with the
In the greenhouse experiment same pathogens. The highest mean yield
during the dry season, the mean late blight (343.52 g/plant) was obtained in the non-
iting of plants with PXV and late blight inoculated genotypes, but was not
ifection was lower than the mean late significantly different from the yield
light rating of the genotypes infected with obtained with the genotypes infected with
.te blight alone at 56, 63, 70, 77 and 84 PXV alone (336.48 g/plant) in the dry
>AP (Table 4). season (Table 7). However, yields of the

between the thickness of the cuticle of inoculated and inoculated with PXV alone.
XV-inoculated and non-inoculated potato The highest yield (701.33 g/plant) was


'able 3. Diameter (mm) of late blight lesi
different days after inoculation (E
(PXV) infection in the field during \

Treatment/ Late 1
Genotype 6 DAI 7 DAI

,B alone
CIP 375335.1 9.7 13.0
CIP 384112.8 6.7 13.0
CIP 384112.14 10.0 13.0
CIP 384724.13 6.7 10.3
CIP 800953 7.7 11.7
CIP 800983 2.3 3.3
Mean 7.2 10.7

,XV + LB
CIP 375335.1 8.0 11.3
CIP 384112.8 4.7 5.3
CIP 384112.14 8.0 12.7
CIP 384724.13 6.7 9.3
CIP 800953 6.3 7.0
CIP 800983 1.7 2.0
Mean 5.9 7.9

'V(%) (main plot) 2.6 1.9
.SD (1%) (main plot) 0.60 0.60

ns (LB) in different potato genotypes at
J) as influenced by potato X potexvirus
-t season (May-August 1996).

ight Lesion Diameter (mm)
8 DAI 9 DAI 10 DAI 11 DAI

18.0 23.7 30.0 36.0
18.7 21.0 23.0 25.7
19.3 24.3 31.7 39.0
14.3 31.0 33.7 35.7
15.3 21.7 27.0 32.0
4.0 4.3 4.3 5.0
14.9 21.0 24.9 28.9

15.0 19.0 23.0 28.0
6.0 7.0 11.3 13.7
17.0 21.7 27.0 34.0
14.0 24.0 24.7 25.3
8.0 10.0 13.7 16.0
3.0 3.0 3.0 3.3
10.5 14.1 17.1 20.1

4.7 2.5 1.4 4.1
2.00 1.44 0.95 3.31

96(, 1997 Phil. Phytopath. 33(2):87-102

Table 4. Severity of late blight (LB) in some potato genotypes as influenced by potato X
potexvirus (PXV) infection during dry season (January-April 1996)'

Late blight rating


Field Greenhouse
Days after planting Days after planting
54 61 69 76 82 56 63 70 77 84

LB alone
CIP 720090
CIP 384112.14
CIP 800953
CIP 375335.1
CIP 676025
CIP 384724.13
CIP 384112.8
CIP 390353.3
CIP 800983

CIP 720090
CIP 384112.14
ICIP 800953
CIP 375335.1
CIP 676025
CIP 384724.13
CIP 384112.8
CIP 390353.3
CIP 800983





8.33 9.00 9.00
5.00 5.00 6.67
3.67 4.00 6.33
5.33 5.33 5.33
2.33 3.00 4.00
2.67 3.00 3.33
3.00 3.00 3.00
1.67 1.67 2.00
1.00 1.33 1.67
3.67 3.93 4.59

7.67 8.33 8.67
4.00 4.00 6.00
3.00 3.33 5.33
4.33 4.33 4.33
1.67 2.00 3.00
2.67 3.00 3.00
2.33 2.67 2.67
1.00 1.33 1.67
1.00 1.00 1.00
3.07 3.33 3.96





5.67 6.33 7.33
3.00 4.00 4.67
3.33 4.00 4.67
4.00 5.00 5.00
2.33 3.00 3.33
3.00 3.33 3.67
2.00 2.33 2.67
2.67 3.33 3.67
1.33 1.33 1.67
3.04 3.63 4.07

5.00 5.67 6.67
2.33 2.67 3.67
3.00 3.33 3.33
2.67 3.00 3.67
2.00 2.33 2.33
2.00 3.00 3.00
1.33 1.67 2.00
1.67 20.00 2.33
1.00 1.00 1.00
2.33 2.74 3.11

CV(%) 10.7 9.3 11.1 14.1 10.4 9.4 16.1 6.7 7.1 8.4
LSD (5%) ns 0.09 0.12 0.15 0.12 0.08 0.06 0.06 0.07 0.07
LSD (1%) 0.14 0.17 0.22 0.17 0.11 0.15 0.10 0.11 0.10
SStandard late blight rating scale of 1-9 (Henfling 1982).

1997 Phil. Phytopath 33(2):87-102 97

Table 5. Thickness of cuticle (rnm) of different potato genotypes as influenced by potato X
potexvirus (PXV) infection (dry season)'.

Cuticle thickness (pm)
Genotype Field Greenhouse
30 DAP 51 DAP 30 DAP 51 DAP
Before Inoc. 21 DAI Control Before Inoc. 21 DAI Control

CIP 375335.1 2.67 2.79f 2.75d 2.57 2.70c 2.71bc
CIP 384112.8 2.88 3.13abc 2.96abc 2.63 2.92abc 2.78abc
CIP 384112.14 2.67 2.84ef 2.79cd 2.57 2.71c 2.63c
CIP 384724.13 2.92 3.05bcd 3.05ab 2.78 2.85bc 2.84abc
CIP 390353.3 2.83 3.01cde 2.96abc 2.63 2.78c 2.79abc
CIP 676025 3.04 3.25a 3.13a 3.00 3.13ab 3.00ab
CIP 720090 2.79 2.92def 2.84cd 2.70 2.85bc 2.78abc
CIP 800953 2.80 2.92def 2.92bcd 2.78 2.85bc 2.84abc
C1P 800983 3.05 3.21ab 3.05ab 3.00 3.20a 3.06a

Mean 2.85 3.01 2.94 2.74 2.89 2.83
CV (%) (main plot) 4.6 9.4
Sin a column. means followed by a common letter are not significantly different at the 5% level by DMRT:
DAP = Days after planting: DAI = Days after inoculation (PXV).

Table 6. Peroxidase activity in the different potato genotypes as influenced by potato X
potexvirus (PXV) infection, during dry season, 19961.

Peroxidase activity (unit/g fresh wt)
Genotype 30 DAP 37 DAP 53 DAP 62 DAP
T4 T1 T4 TI T2 T3 T4 TI T2 T3 T4

CIP 375335.1 137 149 126 78 167 111 87 111 80 106 108
CIP 384112.8 140 119 119 132 161 115 184 114 101 112 106
CIP 384112.14 116 133 122 85 162 86 124 86 94 102 105
CIP 384724.13 178 163 148 154 182 125 168 154 140 175 126
CIP 390353.3 173 122 192 88 145 107 138 145 126 151 138
CIP676025 161 133 200 119 127 108 127 127 111 114 121
CIP 720090 128 158 165 96 133 117 133 123 93 128 99
CIP 800953 154 168 210 107 167 120 167 120 127 133 147
CIP 800983 153 115 85 133 194 133 164 133 133 139 140

Mean 149 140 152 110 160 114 144 124 112 129 121
' T1 = PXV alone: T2 = late blight alone: T3 = PXV+Late blight: T4 = Control (without inoculation);
DAP = Days after planting.

98 1997 Phil. Phvtopath. 33(2):87-102

Table 7. Tuber yield of different potato genotypes as influenced by the infection of potato
X potexVirus (PXV), late blight, and PXV+late blight in the field during dry
season, 1996.

Genotype Yield per plant'
PXV alone LB alone PXV+LB Control

CIP 375335.1 384.00cd 250.33d 300.33c 380.67c
CIP 384112.8 670.67a 562.00a 601.67a 701.33a
CIP 384112.14 369.67d 249.67d 280.33c 380.00c
CIP 384724.13 422.00b 350.33c 373.67b 422.67b
CIP 390353.3 240.00f 211.67e 224.33d 243.33e
CIP 676025 141.67g 116.00f 125.33e 149.67f
CIP 720090 87.33h 35.33g 57.00f 89.33g
CIP 800953 310.OOe 212.67e 239.00d 306.33d
CIP 800983 403.00bc 389.33b 400.67b 416.33b

Mean 336.48 264.15 289.15 343.52
CV (%) (main plot) 3.6
LSD (5%) (main plot) 7.49
LSD (1%) (main plot) 11.34
SIn column, means followed by a common letter are not significantly different at the 5% level by DMRT;
LB = Late blight

1997 Phil Phvtopath 33(2):87-102 99

obtained from CIP 384112.8 followed by infected plants showing mild mosaic
CIP 384724.13 (422.67 g/plant). symptoms were severely infected with late
blight. However, the identity of the viruses
was not mentioned. Nagaich and Prasad
DISCUSSION (1971) reported that the susceptibility of
potato varieties to Alternaria solani was
Under the conditions of our increased due to PYV and was decreased
experiments, PXV infection prior to late due to PXV infection.
blight inoculation delayed the appearance of
late blight symptoms in some potato In this study, it was not established
genotypes. The results support the findings that PXV infection prior to late blight
of Kalra and others (1989b). The increased inoculation increased the peroxidase
resistance to the penetration (infection activity of potato plants. High level of
efficiency) of P. infestans in PXV infected peroxidase activity was correlated with late
plants was similar to findings ofPietkiewicz blight resistance in potato plants (Fehrmann
(1974). Increase of resistance to and Dimond 1967). PXV infection did not
penetration of fungal zoospores after virus increase the cuticle thickness significantly.
infection might result from the destruction However, the plants that were more
of mycelium or even of the infection pegs. susceptible to PXV showed thicker cuticle
The reduction of sporulation period of P. and thickness increased with time after
infestans with prior PXV infection inoculation. In pepper fruits, the cuticle
observed in the present study might be due thickness was increased with maturity of
to change in the host nutritional status by the fruits which ultimately reduced the
virus infection. Reduced sporulation was infection of P. capsici (Biles and others
observed in plants infected with both late 1993) and such an increase of cuticle
blight and PXV compared with late blight thickness might have reduced the zoospore
alone. Similar results were reported by penetration of P. infestans.
Kalra and others (1989a). The mean lesion
diameter of PXV infected plants at 6 to 11 In the present experiment, it was
days after P. infestans inoculation was observed that late blight significantly
lower than those of late blight alone reduced the tuber yield. Potato genotypes
infected plants. PXV infection might with prior PXV infection reduced the
interfere with the development of P. disease severity of late blight. Thus, yield
infestans thereby increasing the resistance reduction was higher in plants inoculated
of the plant. with late blight alone than PXV+late blight
infection. Yield losses due to the diseases
Late blight infection ratings at have been shown to be positively correlated
different intervals were lower in PXV+late with the severity of the disease. The high
blight infected plants than those with late disease severity caused less potato tuber
blight alone. These results corroborated yield and it was also reported. that tuber
the findings of other workers (Muller and production stopped when 75% of the
Munro 1951; Nagaich and Prasad 1971; foliage was affected by late blight (Large
Pietkiewicz 1974 and 1975; and Kalra and 1952).
others 1989a). Contrary to the reports of The reduced susceptibility to late
Mukhopadhyay and Sengupta (1967), virus blight in potato plants pre-infected with

LOO 1997 Phil. Phytopath. 33(2):87-102

PXV might be due to the production or root rot disease incited by Gibberella zeae
depletion of certain metabolites (Blumer and Helminthosporium pedicillatum than
md others 1955), change in the nutritional were virus free ones. They assumed that
status of the host (Muller and Munro 1951) the increase root exudation from the root
hat inhibited zoospore penetration or by system of MDMV infected corm enhanced
nhibitory substances produced in or on the the fungal growth and provide higher
surface of the leaf thus reducing the inoculum potential of the root rot fungus in
chancess of infection. There were the rhizosphere. However, more research
suggestions that substances similar to should be undertaken to determine the
ahytoalexins could be responsible for physiological changes due to virus
increase resistance of virus infection to late inoculation in plants specially in host
alight (Heicht and Bateman 1964; Helton immune to the virus.
md Hubert 1968). It has also been
suggested that some physico-chemical
changess brought about by the virus might LITERATURE CITED
be responsible for the reduced susceptibility
to fungus (Mahmood and others 1974). ALEXOPOULOS CJ, MIMS CW. 1979.
Introductory Mycology. John Wiley and
Reduced susceptibility to Erysiphe Sons, Inc. 632p.
raminis was also recorded in barley dwarf
virus infected plants than virus-free plants ANONYMOUS. 1990. Potato late blight.

-eported that the development of and cuticle thickness of new Mexi<

3 by poor

section with bean yellow mosaic virus. BLUMER S, STALDER L, HARDER

U..U llIJui '-, V 11 1 II I UI I 1 v V,

t Alte,


md Ford (1971) reported that corn Benguet State University, La Trinidad,
eedlings infected with maize dwarf mosaic Benguet, Philippines, 92p.

ta J.r *, S l V I "T L f.J.t )11 V f"lI.J.fl
Preliminary results on breeding potato
through recurrent selection for
Phytophthora infestans resistance and
adaptation to highland tropics. Asian Pot J

CENTER). 1982. World potato facts. CIP,
Lima, Peru, 53p.

CENTER). 1994. Pathogen tested potato
cultivars for distribution. 3rd Ed., Lima,
Peru. 49p.

DARSOW U, WULFERT I. 1989. Studies
on the effect of secondary virus infection on
relative resistance to Phytophthora
infestans (Mont.) de Bary in some potato
cultivars. Archiv-fuer-Phytopathologie-
und-pflanzenschutz (German D.R.),

1 JIyluy tJiriulu nJyc )iuA. roL IX Z... Il1-

Nonspecific acquired resistance to
pathogens resulting from localized
infections by Thielaviopsis basicola or
viruses in tobacco\ leaves. Phytopathology

HELTON A, HUBERT JJ. 1968. Inducing
systemic resistance to Cytospora invasion
in Prunus domestic with localized Prunus
ringspot virus infection. Phytopathology

HENFLING JW. 1982. Field screening
procedures to evaluate resistance to late
blight. Technology Evaluation Series No.
1982-5, CIP, p.16.

HOSSAIN M. 1997. Purification and
antiserum production of potato X and Y

;-~;~=~Z~'-LZ~'~~L~?~ 1VI

SS--- _w / ['n71 rnuu. Yuviuun. j.iJ,,l;o/-iu

KHURANA SMP. 1990. Influence of Interaction between Alternaria solani at
extracts. Leachates and washing from potato viruses X and Y. Indian J Exp Bi
healthy'and potato virus Y-infected leaves 9:88-90.
on aggressiveness of Phytophthora
infestans. Zeitschrift fuer Pflanzen- PEREZ JC, DICCION TC, BALAKI E'
rankheiten-und-Pflanzenschutz, 97:551- 1992. Late blight resistance in potal
556. clones in the Philippines. Research resul
presented in a series of working paper
KAR RK, NANDA HP KABI T. 1985. CIP, Box 933, Manila, Philippines. 138p.
Activity of catalase and peroxidase during
natural and virus induced papaya leaf aging. PIETKIEWICZ J. 1974. Effect of virus
Indian J P1 Physiol 28:124-134. on the reaction of potato to Phytophthoi
infestans I. Characteristic of the reaction 1
KING LN, HAMPTON RE, DIACHUN S. P. infestans of plants infected with potal
1964. Resistance to Erysiphe polygoni of viruses X, Y, S, M and leaf roll. Phytopal
red clover infected with bean yellow mosaic Z 81:364-372.
virus. Science 146:1054-1055.
PIETKIEWICZ J. 1975. Effect of virus
LARGE FC. 1952. The interpretation of on the reaction of potato to Phytophthoi
progress curves for potato blight and other infestans I. Mechanism of changes in tl
plant diseases. PI Pathol 1:109-117. reaction to P. infestans in virus-infecte
plants. Phytopathol Z 82:49-55.
NAQVI AN, ALAM MM. 1974. Studies POTTER LR, JONES IT. 1981. Interactic

apnaeromeca jungena. nmaan rnytopatn4

1967. Susceptibility of some potat
varieties to the natural infection of lal
blight disease and the influence of mil
mosaic infection on its expression in tl
field. Indian Agriculturist 2:139-140.

reaction of virus infected potato plants t
Phytophthora infestans. Ann Appl Bi(

ri ratnol Ju: iJJ-iJy.

TU JC, FORD RE. 1971. Maize dwaw
mosaic virus predisposes corn to root rc
infection. Phytopathology 61:800-803.

UMAERUS V. 1959. The relationship
between peroxidase activity in potat.
leaves and resistance to Phytophthor
infestans. Am Pot J 36:124-131.

1997 Phil. Phvtopath. 33(2):103-109 103



Supported by the Japanese Society for Promotion of Science, Japan and IPB-
UPLB Basic Research Program.

Respectively, University Researcher, Institute of Plant Breeding (IPB), University
of the Philippines Los Baios (UPLB), College, Laguna, Philippines 4031, Associate
Professor, Department of Plant Pathology, UPLB, College, Laguna 4031 and Professor,
Laboratory of Plant Disease Science, Faculty of Agriculture, Gifu University, Gifu, Japan.

Key words: PCR-RFLP, rDNA, restriction map, Rhizoctonia solani

Restriction analysis of amplified rDNA internal transcribed spacer
region was done on six isolates of R. solani AGI-IA using three
endonucleases (EcoR1, Mbol and Hinfl). Restriction map was
constructed based on information from the restriction banding patterns and
double enzyme digestion. The 0.72 kb rDNA-ITS region including the
5.85 RNA gene exhibited six restriction sites using the three enzymes but
no differences on restriction site and number were found among the
isolates studied.


Rhizoctonia solani Kuihn is a soil-
borne fungus and a destructive pathogen of
corn, rice, sorghum, soybean, mungbean
and other important crops in the Philippines
(Pascual and Raymundo 1988; Tangonan
and Quebral 1992; Cu and others 1994). It
causes banded leaf and sheath blight
(BLSB) in corn, rice and sorghum, basal
sheath rot in sugarcane, blight and stem-rot
in soybean and mungbean and damping-off
in cotton. In corn, the disease starts from
the base, usually one month after planting,
forming typical banding pattern in the leaf
sheath. It could reach as high as the tassel

resulting to wilting of the leaves and
premature drying of the grains. It also
causes ear rot and was reported to be an
important component of the root rot
complex (Tangonan 1984). The disease is
more prevalent and severe in warm and
moist field condition. In the past years of
our surveys and collections of this
pathogen, the severity of the BLSB was
observed to vary from the different fields
even if planted with the same.cultivar.

Studies have shown the importance
of understanding fungal population.
Information on pathogen population
structure is needed to have a better

104 1997 Phil. Phvtopath. 33(2):103-109

knowledge of the dynamics of dispersal and
divergence of fungal pathogens which is the
basis for sound disease management.

Ribosomal RNA (rDNA) genes in
fungi are organized in clusters of tandem

freeze-dried at 80'C overnight. The
mycelium was ground in sterile mortar and
pestle. Genomic DNA was extracted
according to the procedure of Lee and
Taylor (1990) with some modifications.
Approximately 50mg of ground mycelium
of each isolate was suspended in 700ul

1997 Phil. Phytopath. 33(2):103-109 10

Table 1. List of isolates of Rhizoctonia so/ani AG 1-1A used in this study.

Isolate No. Isolate Name Host Origin Source

1 RS36 corn Luzon 1

2 RS3 corn Luzon 1

3 Sgr2 sugarcane Mindanao 1

4 RSR23 corn Mindanao 1

5 RSR3 rice Luzon 1

6 R-54 corn Japan 2

S1 = Cultures obtained from the Institute of Plant Breeding, University of the Philippines at Los Bailo
Philippines; 2 = Culture obtained from National Grassland Research Institute, Japan.

*m -- i --~n~- -i I I IM I

18S 5.8S


Figure 1. Location of the primers ITS1
(rDNA) internal transcribed spacers (I1


tnd ITS4 used for amplifying ribosomal DN
;), including the 5.8S ribosomal RNA gen

each), 1.25pl each of primers ITSI and
ITS4 (100pM each), 0.25 p1 Taq
polymerase (5U/pl) and 39.51l sterile
distilled water was prepared for each
sample and was added with one drop of
light mineral oil (Sigma). The mixtures
were amplified in a Perkin Elmer DNA
Thermal Cycler with a temperature profile
as follows: 25 cycles of 940C for 1min
(2min for the first cycle), 600C for Imin and
720C for Imin (2min for the final cycle).
After chloroform extraction, 3p1 amplified
rDNA together with 11ll bromcresol blue
and 21l sterile distilled water were mixed
and loaded into 1% agarose gel stained
with ethidium bromide. Electrophoresis
was done in 10x TBE at 50V for Ihr. The
amplified products were visualized under
UV light.

The amplified DNA samples were
precipitated with a mixture of 125pl
99.5% ETOH, 5pl 3M NaOAc and 11i
glycogen (20pg/pl), stored at -80c for
30mmin, dried in vacuum and the pellets
were dissolved in 25pl TE buffer.
Restriction analysis of amplified rDNA-ITS
region was done using three restriction
enzymes, viz. EcoR1, Mbol, and Hinfl
(Takara Shuzo Co. Ltd., Shiga, Japan). A
volume of 1211 reaction mixture containing
1.2pl 10x buffer, 11 restriction enzymes
(5U), 4pl amplified DNA and 5.8pl sterile
distilled water was prepared for each
sample and stored overnight under optimal
temperature (370C). Electrophoresis of the
digested samples was done using 2%
NuSieve (3:1) gel (low melting point
agarose gel), stained with ethidium
bromide, in 10x TBE buffer at 100V for
Ihr. Banding patterns were visualized
under UV transilluminator. In each gel,
pBr 322 was used as standard reference.

based on information from the restriction
banding patterns and double- enzyme


The rDNA-ITS region including
5.8S gene of each of the six isolates was
amplified using PCR and estimated as
0.72kb (Fig. 2). No differences in fragment
size were observed in all isolates. The
amplified products were digested by three
endonucleases, viz. EcoRl, Mbol, and
Hinfl. Six restriction sites existed in the
ITS-5.8S rDNA region (Fig. 4). The
restriction patterns for each specific enzyme
did not show restriction site and restriction
fragment length variation among the six
isolates (Fig. 3 and Table 2). Similar to the
findings of Liu and Sinclair (1993),
fragment size of 0.72kb in the rDNA-ITS
region and same restriction sites and map
(Fig. 4) using the three endonucleases in
the PCR-RFLP assay were observed.
However, unlike our results, they have
differentiated further AGI-1A into three
intraspecific groups (ISG) Our data
corresponded to ISG-A group of their
report. In our experiment, we used only

restriction anal)

With tl
simple PCR-RI
crops and rice
detect variatior
to clarify further
and the relation
R. solani AGI
analysis, high
observed amor
/, T- --- . J


use of this rapid an
? technique, more uplar
lates must be analyzed t
r genetic marker in ord(
he evolutionary dynamic
ip among populations <
A. Through restriction
evel of diversity ws
isolates within AGI-1.
... .

1 9W4 iLiMP IMI 2 1MI----AL v?~\ ~ ? r

1997 Phil. Phvtopath. 33(2):103-109 101

Sinclair 1993). DNA restriction mapping
showed that DNA length mutations,
S2 3 4 5 6 7 insertions or deletions were common
among genetically-distinct R. solani groups
720 bp (Liu and Sinclair 1992 and 1993). These
basic informations are crucial in developing
effective strategies for controlling BLSB in
corn and other cereal crops in the

Figure 2. PCR-amplified portions of nuclear
rDNA-ITS region using ITSI-ITS4 primers in six BRUNS TD, WHITE TJ, TAYLOR JW.
isolates of Rhizoctonia solani AG -A. Lane 1 = 1991. Fungal molecular systematics, Ann
RS36, 2 = RS3, 3 = Sgr2, 4 = RSR23, 5 = RSR3, 6
= R-54 and 7 = pBr322 (reference). Rev Ecol Syst 22: 525-564.

1 2 3 4 5 6 7 RC. 1988. Organization of the ribosomal
RNA genes of Schizophyllum commune.
Curr Genet 13: 417-424.
90 bp
PS. 1994. Sheath blight, a threat tc
intensive rice production systems. Phil
Phytopathol 30(2): 129.

b 370 bp 1987. Comparison of physical maps ol
350 bp ribosomal DNA repeating units in Pythium,
Phytophthora and Apodacha. J Gen
Microbiol 133: 2953-2959.

LEE SB, TAYLOR JW. 1990. Isolation ol
SDNA from fungal mycelia and single cells.
260 bp
2c6 bp JJ and WHITE TJ, ed. PCR Protocols, A
60 bp Guide to Methods and Applications.
40 bp Academic Press, San Diego. pp. 282-287.

LIU ZL, SINCLAIR JB. 1992. Genetic
Figure 3. DNA restriction digest patterns of six diversity ofRhizoctonia solani anastomosis
isolates of Rhizoctonia solani AGI-1A for rDNA- group 2. Phytopathology 82:778-787.
ITS region by endonucleases EcoRl (a), Hinfl (b).
.iArl Ah1,l (,I\ T inp AAcrrirntinn ic thp cimp ac in

108 1997 Phil. Phytopath. 33(2):103-10

330 330 330 330 330 330

Hinfl 370 370 370 370 370 370
350 350 350 350 350 350

Mbol 260 260 260 260 260 260
210 210 210 210 210 210
150 150 150 150 150 150
60 60 60 60 60 60
40 40 40 40 40 40

18S- 5.8 S rDNA __ 28S

I WY lL.f I U~L....I.. ti~\1 1~ .flf *nn

Differentiation of interspecific groups
within anastomosis group 1 of Rhizoctonia
solani using ribosomal DNA internal
transcribed spacer and isozyme
comparisons. Canadian PI Pathol 15:272-

Evaluation of resistance and yield loss in
sorghum due to Rhizoctonia sheath blight.
Phil J Crop Sci 13(1):37-42

1984. Ribosomal DNA spacer-length
polymorphism in barley: Mendelian
inheritance, chromosomal location and
population dynamics, Proc Nat'l Acad Sci
USA. 81: 8014-8018.

TANGONAN NG. 1984. Sorghum stalk
rot complex in Mindanao: Occurrence,

etiology and development as affected by
various cultural management practices and
weather effects. Ph.D. Dissertation,
University of the Philippines, Los Baflos,
College, Laguna.

Host index of Plant Diseases in the
Philippines. 2nd ed. 273p.

Ribosomal DNA restriction fragment length
polymorphism in Rhizoctonia solani.
Phytopathology 80: 151-158.

JW. 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes
for phytogenetics. In INNIS MA,
WHITE TJ. ed., PCR Protocols, A Guide
to Methods and Applications. Academic
Press, San Diego. pp. 315-322.



Respectively, Associate Professor, D4
Researcher, Department of Agronomy, Uni
Laguna, Philippines 4031.

Key words: corn, modeling, Perol
mildew, yield loss

General models of yield loss
Philippine downy mildew infection m
experiments under Philippine conditi
predictive models are: Y1 = 16.39 + C
11.556 log& xi (r2 = 0.7385") where'
yield per hectare, and X1 is percent
emergence. Initial validation, using (
disease were generated by differentia
showed good fit between theoretical a


For decades, Philippine downy
mildew (PDM), caused by Peronosclerospora
philippinensis (Weston) Shaw, a pathogen
indigenous to local habitats (Weston 1920
and 1923), has been a major limiting factor to
profitable corn production. It is considered as
the most destructive of the known downy
mildew-type diseases attacking corn
(Exconde 1970; Bonde 1982). Specific losses
in yield are highly correlated with percentages
of disease. For instance, Exconde and
Raymundo (1974) reported yield losses of 9,
26, 42, and 75% at 24, 40, 58, and 75%
infection, respectively in UPCA Var 3, a
susceptible variety. In sweet corn, percent



artment of Plant Pathology, and University
ersity of the Philippines Los Bafios, College,

'sclerospora philippinensis, Philippine downy

md grain yield in corn as affected by
re developed from data obtained from
is from 1969 to 1990. These general
109Xi (r2 = Q.7107"*) and Y2 = 76.99 -
i is percent grain yield loss, Y2 is grain
infection at one month after seedling
ta from an experiment where levels of
application of the fungicide metalaxyl,
I actual losses in yield of corn.

69.9, and 100 when mean percentages of
infection were 15.2, 38.9, 76.4, and 100,
respectively. Seedlings infected shortly after
emergence usually die. Those infected and
survived a month after emergence are stunted,
spindly, and formed deformed ears bearing
few kernels. Shortly thereafter, the corn plant
manifests a phenomenon called mature-plant
resistance (Sun and others 1966).

Among the measures recommended
for downy mildew control are planting of
resistant cultivars, cultural techniques such as
roguing, and synchronized planting to avoid
the build-up of inoculum, and the use of
fufgicides (Exconde 1970). The selection of
the best control measures requires a method

Raymundo 1974; Raymundo and Exconde
Several models have been proposed 1976), and from experiments on effects of
to quantify the relationship between disease fungicides (IPB 1970) were used as inputs in
severity and yield losses (Teng and Gaunt deriving simple models to relate yield losses
1981; Gaunt 1990). When a certain point in and grain yields to percent systemic infection.
the disease progress curve or during the crop All the studies from where the data were
growth stage appears important in the obtained had indicated that disease rating was
determination of yield then a critical-point done at one month after plant emergence.
model is used. When more than one growth Disease assessment at this stage of plant
stage or disease severity is involved then a growth, apparently, was in recognition of the
multiple-point model has been found onset of the phenomenon of mature-plant
applicable. The area-under-the-disease resistance to downy mildew (Sun and others
progress curve (AUDPC) model, which can 1966).
serve to indicate the cumulative amount of
disease, has also been utilized. A response- More than a hundred data pairs on
surface model, which accounts for losses in percent yield losses, percent infection, and
yield at specific levels of severity during grain yield representing wet and dry cropping
various stages of crop growth, is considered seasons, different years, and various corn
the most appropriate in many host-pathogen cultivars and genotypes were used in a
systems. These models, usually based on the regression modeling approach. In all cases,
regression form, Y (loss) = a + b (disease disease severity as the independent variable
severity), have been utilized extensively. Two was in the form of percent systemic infection.
simple linear models of this form were The dependent variables were percent yield
proposed by Tuleen and Frederiksen (1981) loss and grain yield. The statistical software
in simulating yield losses in grain sorghum MICROSTAT was utilized in developing the
due to sorghum downy mildew. These two models.
models were tested by Craig and others
(1989) using near-isogenic populations of Five criteria proposed by Teng and
sorghum. Gaunt (1981), namely, the coefficient of
determination, the F-statistic in the analysis of
This paper reports the development of variance, the standard error of the estimate of
general models that can account for corn yield dependent variable, the multiple correlation
losses due to Philippine downy mildew at coefficient, and the t-statistic were used in
different degrees of infection, selecting the most appropriate models.

Test of the validity of the models
MATERIALS AND METHODS involved comparison of theoretical downy
mildew disease progress curves with results of

I4 1 1U A r V *t I rI. __. __ _.*L 4 n1 .


The relationship between percent
downy mildew infection and grain yield loss
was best described by the model, Yi = 16.39
+ 08,09Xi where Yi is percent yield loss and
Xi is percent infection at one month after
emergence. The coefficient of determination,
r =.0.7107, indicative of the magnitude of
effect of the independent variable, percent
infection, on percent yield loss is highly
significant. Likewise, analysis showed that the
model, Y2 = 76.99 11.5561ogeXi where Y2
is grain yield per hectare, gave the most
acceptable explanation of the relationship
between downy mildew infection and grain
yield. A slightly larger coefficient of
determination, r2, with a value of 0.7385
which was significant, was obtained. The
model was arrived at after transformation of
the independent variable Xi.

The -grain yield loss model appears
acceptable as indicated by a close fit between
model estimates and data from the yield loss
trial utilizing differential application of the
fungicide metalaxyl (Fig. 1).

Models of yield losses due to downy
mildew of sorghum have been proposed and
used to simulate losses (Tuleen and
Frederiksen 1981). There are indications that
low incidence of systemic infection can thin
dense stands to optimnun plant densities that
would produce higher yields.

When infected seedlings die, the
competition for moisture and sunlight
encountered by adjoining seedlings is reduced
thereby providing a degree of compensatory
effect for the reduction in plant population.
This compensatory effect appears to be
greater in cases of uniform distribution of






0 28 40 54 68 98
Observed Percent Infection

Figure 1. Comparison between actual and predicted
percent yield loss in corn due to Philippine down

uniformly distributed (Schuh and others

It appears that the compensator)
effect is also dependent on the age of the
infected plant. Very likely, early death ol
downy mildew infected seedlings would allow
a higher degree of compensation than where
thinning due to disease occurs beyond the
early growth stages. Different amounts of loss
result when infection occurs at different
stages of crop growth as the corn plant ii
known to develop resistance as it matures
(Sun and others 1966).

Although the analysis utilized datu
obtained over a period of almost 20 years and
encompassing different growing conditions,
the models still have to be tested and
validated extensively. Testing should include a
i-Arsns n* nFnf n An-A +Ua n.aXwl ftl1.

I-'.' 1 1111. iA uVJ. jj11iViAt-iit Il__'i

w. Indian Phytopathol 23: 275-
RAYMUNDO AD. 1974. Efl
resistant varieties, fungicide ay
OR. ADVINCULA BA. 1970. roguinz for the control of corn c

L Il YIlU IUaJ UU.Lt t ly 1 lLtll V. L 1 "JId. J AAll.. A -
ersity of the Philippines, College Improvement Workshop, New
., Rice and Corn Project Annual Oct. 24-29, 1966.
72, College, Laguna.
DR, RAYMUNDO AD. 1974. systems of disease and yield k

114 1Yi mI ,rviOuamu. .7h l:.IV-I1.

,W- ayysICuls V. 1.1-1J't.

Simulating yield losses in grain sorghum di
to sorghum downy mildew. Agron J 73: 98:

TV JUVO I %.1' TV .A JVX. 17,&V. A "UpyUiJ VW %l
mildew of maize. JAgrRes 19: 97422.

WESTON WH JR. 1923. Production ar
dispersal of conidia of the Philippix
Sclerosporas of maize. J Agr Res 23:233!



Respectively, Senior Science Research Specialist and Laboratory Aide, Cotton
Research Development Institute Mindanao Area Research Center (CRDI-MARC),
'olomolok, South Cotabato, Philippines.

Key words: cotton, Fusarium oxysporum, Fusarium wilt, host range, race

The host range and pathogenic specialization of Pusarium
oxysporum f sp. vasinfectum (Fov) causing Fusarium wilt of cotton were
determined under screenhouse condition by using the root dipping
inoculation technique with different isolates of Fov obtained from cotton
farms of Polomolok, South Cotabato and General Santos City. Nine crops
namely: cotton, mungbean, cowpea, stringbean, peanut, corn, tossa jute,
sweet pepper, okra and three weed species namely goose grass, itch grass
and spiny amaranth were tested as host of Fov. Mungbean, cowpea,
stringbean, peanut, sweet pepper and corn were all infected, but the
infection was limited only at the hypocotyl. Tossa jute was severely
infected with all iknlatet from General Santn~ City Okra was found to he

Fov collected from Polomolok, South
were similar to race A of the path
differential cotton cultivars.


Wilt of cotton (Gossypium spp.) is
vascular disease caused by the soil borne
athogen, Fusarium oxysporum f sp.
asinfectum (ATK) W.C. Snyder & H.N.
plans. The disease is widespread and
auses substantial crop losses in most of the
lajor cotton-producing areas of the world.

In the Philippines, Fusarium wilt is
rst reported in cotton growing areas of
'nlnmnlnk Sniith Cntahatn and CGneral

Cotabato and General Santos City
gen based on their reactions to

antos City. Yield losses are estimated to
each 40% and can lower the farmers'
come from 10 to 100% (Cano and Catedral

The fungal pathogen has been
sported to have a wide range of host
including the family Leguminoseae,
lalvaceae and Solanaceae. The Fusariunm
tilt pathogen reported to occur in
southern Mindanao, Philippines has not
een characterized to what distinct race it

)ecies of cotton are included in the
:reening because of its importance as
-obable host range and source of genetic
ock for resistance, respectively. The
gnificance of establishing the host range
-s on giving recommendation on
usarium infested cotton production areas
j planting alternative crops.

The study was conducted at Cotton
research and Development Institute,
lindanao Area Research Center (CRDI-
IARC) Polomolok, South Cotabato from
ugust 1996 to March 1997 to determine
ie host range and pathogenic race of F.
tysporum f sp. vasinfectum (Fov).


ov Isolates

Different isolates of Fov were
:covered from wilted cotton plants from
atton production areas of Polomolok,
outh Cotabato and General Santos City.
ingle spore isolations were made from
iscolored vascular tissue in the upper
portion of the stem of the wilted plants.
ultures were grown and maintained in
otato dextrose agar and water agar,

athogenicity Test

Five isolates of Fov obtained from
tilted cotton plants were grown for 14 days
n PITA at ambient temperature. Cultured
lates were flooded with water and rubbed
rith sterile cotton buds to dislodge the
pores. Spore suspensions were adjusted
) 10' spores per ml using hemacytometer.
-a-A ,-F T ina Al wuhuih ia eii PptihlA tn

ays, seedlings were carefully removed and
ipped in the spore suspension for one
minute. Inoculated seedlings were
transplanted singly in plastic cups with
terilized soil. Plants were weekly assessed
until 6 weeks after inoculation for the
occurrence of symptom. Re-isolation of
ie pathogen from the discolored vascular
issue of the wilted plants was done.

)ifferential Reaction

A total of five isolates of Fov, two
rom Polomolok, South Cotabato (SC1 and
.C2) and three from General Santos City
GSC1, GSC2 and GSC3) were tested for
athogenic specialization. The differential
otton cultivars,- Acala 44 (Gossypium
irsutum), Ashmouni and Sakel (G.
arbadense) and Roseum (G. arboreum)
vere grown for 14 days before inoculation
vas done. Similar inoculation techniques
vere used as described above. Ten plants
vere used per cotton cultivar.

lost Range Test

Crop and weed species associated
vith cotton below were tested as host of

Common Name Scientific Name

'otton Gossypium hirsutum L
lungbean Phaseolus radiatus L.
peanut Arachis hypogea L.
:owpea Vigna ungiculata (L.)
itringbean Vigna sesquipedalis L.
)kra Abelmoschus esculentus L.
orn Zea mays L.
*weet pepper Capsicum annum L.
Ipiny amaranth Amaranthus spinosus

1997 Phil. PhytoDath. 33(2):115-121 117

Only four isolates of Fov (SC1, GSC1,
GSC2 and GSC3) were used in this
experiment. Seeds from weeds and tossa
jute were sown one month before
inoculation while other plants were sown 14
days before inoculation. Inoculation
technique previously described was used.

Disease Severity Rating

Disease severity rating (DSR) was
done by examining the vascular tissues of
different host plants using the rating scale
as follows:



1 No discoloration in the vascular

2 Discoloration confined to the

3 Discoloration confined to the midstem

4 Complete discoloration of the vascular

5 Plant dead.

The vascular browning index (VBI)
was taken from the first symptom
appearance until six weeks after inoculation
using the formula:

VBI DSR of individual plant x number of plants tested
total number of plants

Vascular Browning Index
1.0 1.9
2.0 2.9
3.0 3.9
4.0 5.0

Highly resistant
Highly Susceptible


Host Range of Fov

One isolate designated as SCI was
isolated from Polomolok, South Cotabato
and three isolates designated as GSCI
(Conel), GSC2 (Klinan 5), GSC3
(Mabuhay) were obtained from growing
areas of General Santos City.

Pathogenicity test using SCI isolate
showed that weeds associated with cotton
were resistant compared with leguminous
crops, corn and tossa jute. Okra and cotton
suffered severe infection of the vascular
system resulting to death of the plants
(Table 1).

Isolates obtained from General
Santos City showed almost a similar pattern
of infection. Goose grass was not infected
while the other weed species suffered mild
infection. Infection in corn, cowpea,
peanut, stringbean and mungbean was
limited to hypocotyl. Infection of tossa jute
was observed at the midstem section of the
plant. Okra and cotton were observed to
be highly susceptible to Fov (Table 2).

Cotton and okra were found to be
highly susceptible to all isolates of Fov.
Infected plants exhibited chlorotic leaves,
brownish discoloration of the stem and
stunted growth. The fungus was recovered
from the vascular and hypocotyl tissues of
the all affected plants.

Early reports on host range studies
of Fov in Tanzania, Egypt and USA
showed that plants belonging to genera
Glycine, Hibiscus, Physalis, Solanum and
Vigna were infected by Fov (Armstrong

ble 2. Reaction of different plant and weed species against GSC1, GSC2 and GSC3
isolate of Fusarium oxysporum f. sp. vasinfectum.

4eans followed by the same letters are not significantly different at 5% level DMRT.

997 Phil. Phvtooath. 33(2):115-121 119

mud Armstrong, 1960: Booth and Watson
964). Moreover, Grover and Singh (1970)
ias also shown that okra was naturally
infected with Fov. Isolates of Fov from
China were reported to infect okra and
;oybean (Chen and others 1985).

Smith and Snyder (1975) found that
,he pathogen may multiply rapidly in
symptomless hosts other than cotton. It
vas also noted that the pathogen appears to
survive indefinitely (perhaps
raprophytically) in field soils even in the
absence of cotton or okra and cannot be
eradicated economically from large areas.

Infected weeds, legumes, corn, okra
mnd tossa jute serve as sources of field
noculum thus enhancing the continuous
survival of Fov after the cotton growing
season. Fov cannot be_ economically
:radicated by chemical soil treatment, thus
:rop rotation using non-host crops and
breedingg of resistant varieties are the
Feasible ways of controlling the pathogen.

Pathogenicity Test on Different Cotton

The five isolates of Fov taken from
Polomolok, South Cotabatcf.iid, General
Santos City were pathogenic to G.
birsutum (Acala 44), G. barbadense
(Ashmouni and Sakel) except G. arboreum
rRnetuml (Table 3'

browning observed at the midstem section
of the plant during the final assessment.
'or cv. Roseum, the browning was limited
inly at the hypocotyl 6 wk after
inoculation. 'The fungus was, recovered
rom all affected plants. .

Differential set of cotton cultivars
vas used to determine Fov races
throughout the world. Currently, there are
ix distinct races restricted to defined
geographicc areas. Races 1 and 2 were
described in USA and Tanzania; race 3 in
igypt, Sudan and Israel; race 4 in India;
ace 5 in Sudan and race 6 in Brazil and
laraguay (Armstrong and Armstrong 1958,
960, 1978; Ibrahim 1966; Katan and Katan
988). Hillocks (1992) reported that there
vere 6 races, however, there are apparently
in additional 2 races (7 and 8) in China,
here a further two non-hosts (alfalfa and
)kra) were added to the differential set. In
conjunctionn with pathogenicity test,
kssigbetse and others (1994) made use of
andom amplified polymorphic DNA
RAPD) to assign races 1, 2 and 6 to race

The Fov isolates taken from
'olomolok, South Cotabato and General
Santos City showed reaction similar to
tace A. Fov isolates of Australia had also
a similar reaction (Roger Davis, personal
communication) .

12 I 100Q7 Plil "1hnna.h 122l.1.\ 11_C

Isolate/ Race" Acala 44 A

Race lb S
Race 2b S
Race 3 R
Race 4 R
Race5 R
Race 6b S


" Denotes pathogen-host interactions according to
b Race A as proposed by Assigbetse and others (191

:eptible) of a differential set of Gossypium t4
es of Fusarium oxysporum f. sp. vasinfectum.

,hmouni Sakel Roseum

S "S S


illocks (1992)

W97 Phil Phvtnnath 33(21. 11 r-1 1 121

)58. A race of the cotton wilt Fusarium
using wilt of yelredo soybean and flue-
ired tobacco. P1 Dis Rep 42:147-151.

960. American, Egyptian and Indian
cotton wilt Fusaria: their pathogenicity
id relationship to other wilt Fusaria.
*SDA Tech Bull 219, 19 p.

978. A new race (race 6) of the cotton
ilt Fusarium from Brazil. P1 Dis Rep 62:

usarium oxysporum f. sp. vasinfectum.
.M.I. Descriptions of pathogenic fungi
nd bacteria, No. 28.

assessment of Fusarium wilt infection in
elected cotton growing areas in Mindanao.
manual Accom. Report. CY 1991-1992.
RDI-MARC, Polomolok, South
'otabato, 2:160-168.

'HEN Q, JI X, SUN W. 1985.
identification of races of cotton wilt
'usarium in China. Agricultural Science
Uhina, No. 6.

K. 1996. Characterization of a population
f Fusarium oxysporum f. sp. vasinfectum
causing wilt of cotton in Australia. Aust J
Lgric Res 47:1143-56.

,ROVER RK, SINGH G. 1970. Pathology
vilt of okra (Abelmoschus esculentus (L)

Moench) caused by Fusarium oxysporum f
sp. vasinfectum, its host range and
histopathology. Indian J Agric Sci 40:989-

HILLOCKS RJ. 1992. Fusarium wilt. In
R.J. Hillocks ed., 'Cotton Disease' CAB
International, Wallinford, UK.pp. 127-160.

IBRAHIM FM. 1966. A new race of cotton
wilt in the Sudan Gezira. Cotton Grow Rev

KATAN T, KATAN J. 1988. Vegetative
compatibility grouping of Fusarium
oxysporum f. sp. vasinfectum from tissue
and the rhizosphere of cotton plants.
Phytopathology 78:852-855.

Persistence of Fusarium oxysporum f. sp.
vasinfectum in fields in the absence of
cotton. Phytopathology 65:190-192.

WOOD CM, EBBELS DL. (1972). Host
range and survival of Fusarium oxysporum
f sp. vasinfectum in Northern-Western
Tanzania. Cotton Grow Rev 49:79-82.


We acknowledge the kind
assistance of Mr. Roger D. Davis of the
Department of Primary Industries
Agricultural Research, Queensland,
Australia for giving the set of differential
cotton cultivars.

2 1997 Phil. Phvtonath. 33(21:122-129



Respectively, Research Associate Prc
*ofessor, Institute of Plant Breeding, Univer
Iguna, Philippines 4031.

Key words: avocado, Phytophthora ci

Phytophthora cinnamomi caus
for the first time in the Philippines.
wilting and drying of leaves, dieback
feeder roots and death of trees. The p
medium and purified using pancake rr
coralloid, hyaline, slender, becoming
Conidiophores are undifferentiated, sin
are borne terminally, ovoid to ellipsoi
flat, inconspicuous papilla on end oppo
conidia are produced in branches of co
fashion. Oospores are not observed.
globose to pyriform, terminal, or in
cluster. Zoospores are bean- or kidney
length attached to a concave side, hardly
largest mean colony diameter in modi
potato dextrose agar. Dense mycelia
modified carrot agar, oatmeal agar a
rosette pattern of colony morphology


Root rot in avocado is caused by
rytophthora cinnamomi Rands. The
thogen was first described by Rands in
22 as causing a stripe canker on
mamon in Sumatra (Rands 1922). It is
timated to be of Asian origin (Zentmyer
80). The disease is widespread and
curs in countries where avocados are



fessor, Former University Researcher and
sity of the Philippines, Los Bafos, College,

nnamomi, root rot

ing root rot in avocado is reported
Symptoms include color reduction,
of shoots, decay and blackening of
athogen is isolated using a selective
ethod. Young hypha are distinctly
thick-walled or tough and septate.
ple, sympodially branched. Conidia
d or elongate, hyaline, with broad,
site point of attachment. Secondary
nidiophores in successive sympodial
Chlamydospores are thick-walled,
short lateral branches, singly or in
-shaped with two flagella of unequal
y seen. P. cinnamomi produced the
led carrot agar and the shortest in
vere noted in potato dextrose agar,
nd V-8 juice agar. Camelloid or
vas very evident in potato dextrose

(Wager 1942; Coffey 1987), Australia
(Pegg and While 1987), Mexico (Zentmyer
1970), Israel (Pinkas 1989) and New
Zealand (Chee and Newhook 1965). In
California, USA alone, it was reported that
60 to 75% of the orchard was affected,
causing approximately an annual loss of
$30 million (Coffey 1987). In the state of
Quetaro, Mexico, the avocados were
eliminated due to this devastating disease

'resenuy, i is conslaerea as one oi me
nost troublesome soilborne pathogens not
,nly in avocado but in about 1000 crops
)ver 70 countries.

In the Philppines, avocados are one
)f the major and popular fruits due to its
iutritive value (Bergh and others 1976). It
ias been introduced as early as 1890 and
ince then avocado trees are found in
Almost all places in the country. It is eaten
Is fresh, processed into other foods, and as
mne of the ingredients in making cosmetics
and soap and ice cream. There has been no
report of root rot disease affecting
Avocados. Although many avocado trees
along peripheral roads, orchards and
)ackyards are dying, the causes of their
leath have not been ascertained.

This study was done to: (1) describe
he symptoms, (2) isolate the pathogen and
describe its morphological and cultural
characteristicss on various culture media.


Isolation of the Causal Organism

Feeder roots of a dead avocado tree
were collected from IPB fruit orchard.,
Tranca, Bay, Laguna. They were sealed in
-lean plastic bag and brought to the
laboratory. Roots were cut into 1 to 2cm
sections, dipped in 70% ethanol for 2 min
and rinsed with sterile distilled water. After
blotting dry in sterile filter paper, root
sections were plated onto corn meal agar
(CMA) containing pimaricin (10mg),
ampicillin (125mg), rifampicin (10mg) and
pentachloronitrobenzene (100mg) per liter
(PARP+CMA) (Solel and Pinkas 1984).
Purification from bacteria was done using
pancake method (Campbell and Sleeth

LYvt). iviyucii slp rIun rat-r-rlivi.
were picked and planted onto agar block
containingg dextrose (10g), ammonium
)hosphate (2g), potassium (2g), potassium
nitrate (1g), magnesium sulfate (1g) per
iter of distilled water. Blocks were
nverted in a sterile Petri dish an incubated
it 20-250C. Mycelia which grew up
throughh the agar were picked and
transferred to PDA slants. Pure cultures
were maintained in sterile mineral oil.
Symptoms of the disease were described.

Pathogenicity Test

Mycelial fragments of 5-day old
culturee of the fungus were placed on the
)ased of the roots of a 6-mo old avocado
plant. The fragments were covered with
noist cotton wad to prevent dehydration.
Inoculated seedlings were placed in the
screenhouse and observed for symptom

Morphological and Cultural

A 7-mm diameter agar disc of P.
cinnamomi was grown in nine agar media,
viz. Potato dextrose agar (PDA), corn meal
agar (CMA), yeast extract agar (YEA), V-
8 juice agar (V8JA), modified carrot agar
(MCA), potato carrot agar (PCA), soybean
seed decoction agar (SSDA), malt extract
agar (MEA) and oatmeal agar (OMA). The
seeded agar plates were incubated at 250C
for 5 days under continuous light. The
diameter of the colony and the type of the
mycelial growth were taken.

The morphology of hyphae,
conidiophores, chlamydospores, sporangia
and zoospores was described. For the
production of sporangia and zoospore
release, agar pieces from 7-day old culture

vv,/ rnn. rnytopatn. 33(z,:izz-izv 1,43

All F A"". A MTVWWW >M ^.^..-.

incubated for 10 days in complete darkness
at 24C. Zoospores release was induced by
stripping off the mycelia from the agar plate
and floated in 2ml sterile distilled water in
Petri dish, chilled to 50C for 30min
followed by a 7-hr incubation at 24C.



Leaves of infected trees are pale
green, wilt and fall readily. Shoots dieback
from the tips so that affected tree is
reduced to the base framework of dying
branches. Sudden death occurs on younger
trees while the effect is gradual for older
trees. Feeder roots are black, decayed and
few in number. Rapid decline of trees may
occur under severe waterlogging.

Isolation of the Causal Organism

The pathogen was isolated with
difficulty due to bacterial contaminations.
PARP cocktail developed by Solel and
Pinkas (1984) caused partial purification of
the cultures since in most cases, some
bacterial species cannot be totally
eliminated with this cocktail. However, the
use of pancake method proved to be an
efficient purification procedure. Growth of
the fungus was observed 3 to 4 days after

Pathogenicity Test

The inoculated seedlings started
showing symptoms of wilting and dieback
one month after inoculation and was
completely dead after 2 mo. The plant
failed to produce feeder roots. The

affected roots.

Cultural Characteristics

Of the nine media tested, MCA
gave the largest mean colony growth
diameter (59.7mm) while PDA, the shortest
(32.2mm) (Table 1 and Fig. 3). The
mycelial growth was slightly dense in PCA,
MCA, OMA and V8JA and scarce on other
media. Although very dense, the mycelia
were hardly seen on OMA. In PDA, the
colony had camelloid or rosette pattern, a
unique characteristic. The camelloid
pattern was observed at the center of the
colony in PCA. This is due to the presence
of potato in PCA. Chen and Zentmyer
(1970) and Savage and others '(1968)
recommended V8J broth for growth and
sporangium production for most species of

Under the microscope, distinct
coralloid (hyphal swelling or vesicles) type
of hypha was observed on all media tested.
However, the density and size of vesicles
varied (Table 2). According to Ribeiro
(1978), the type of hyphal swellings and
rosette pattern of colony growth on PDA
were the distinctive characters that
differentiate P. cinnamomi from other
Phytophthora species.

Morphology of the Fungus

Young hypha are distinctly
coralloid, hyaline, slender, becoming thick-
walled or tough and septate with age (Fig.
la and 2b). Conidiophores are
undifferentiated, simple, hyaline, thin-
walled, with broad, flat, inconspicuous
papilla on end opposite point of attachment
(Fig. 2a). Secondary conidia are produced

1997 Phil. Phytopath. 33(2):122-129 125

Table 1. Mycelial growth ofPhytophthora cinnamomi on different agar media.

Agar Medium Colony Diameter (mm) per hr
24 48 72 96 120 Mean

modified carrot 23.5 43.2 62.3 81.8 88.0 59.7
malt extract 19.9 42.6 54.0 80.5 88.0 57.0
potato carrot 19.7 38.0 56.8 73.0 88.0 55.1
oatmeal 15.4 28.5 39.0 49.0 88.0 44.0
V-8 juice 18.2 30.5 42.0 55.8 70.5 43.3
yeast extract 18.1 30.6 42.9 50.0 62.4 40.8
corn meal 16.3 23.8 37.5 47.0 62.2 37.4
soybean seed decoction 16.6 28.5 36.4 43.9 54.8 36.0
potato dextrose 13.1 23.5 33.7 35.2 55.6 32.2

Table 2. Morphological characteristics of colony and hyphae ofPhytophthora cinnamomi
grown in different agar media, 5 days after incubation at 28-300C under
continuous light.

Agar Medium Colony Growth Hyphal Growth

modified carrot slightly dense very few but big hyphal swelling
malt extract very scarce few but small hyphal swelling
potato carrot slightly dense, slightly camelloid at few hyphal swelling
the center
V-8 juice slightly dense, fluffy profuse and big hyphal swelling
yeast extract scarce few hyphal swelling
corm meal very scarce few hyphal swelling
soybean seed decoction very scarce at periphery, dense at profuse and big hyphal swelling
potato dextrose camelloid or rosette pattern profuse and big hyphal swelling

120 Z17 / rui. riyupuau. JI. a ,1; -


Figure 1. Young (a) and mature (b) hypha
coralloid (rounded vesicles or hyphal swi
successive sympodial fashion stained with la

a b

f Phytophthora cinnamomi. Note the distin<
Ilings) type of hypha, secondary conidia i
,tophenol cotton blue (c), chlamydospores (c

771 T rnlu. rIVLyUauI*I. JJ11 .Ih-ilY IA1

sympoaiai rasmon (rig. ic). are tnicKc
walled, globose to pyriform, singly or ir
cluster, terminal or in short lateral branches
(Fig., Id). Oospores are not observed.

Sporangia or zoosporangia (Fig. 2a)
are produced following the method
described above. P. cinnamomi starts tc
release zoospores after incubating the
mycelia at 50C for 30min followed by
incubation at 240C for 7hr. Zoosporangie
arise from unbranched hyphae (Fig. 2b).
Zoospores start to be released in distilled
water after 30min to one hour at 240C.
Spore case of a zoosporangium partially
collapse after zoospore release (Fig. 2b).
Zoospores are bean- or kidney-shaped with
two flagella of unequal length attached to a
concave side. Flagella are hardly seen.
Zoospores remain motile for few hours
after which they encyst. Exposure tc
continuous darkness inhibits the mycelial
growth of the fungus. At 21-240C, the
fungus is capable of producing good yield
of zoospores (Byrt and Grant 1979). The
release of zoospores is induced upon
exposing the fungus at very low
temperature preferably 50C (Chen and
Zentmyer 1970) and returning to 240C.
Temperature below 180C delayed
synchronous zoospores release (Byrt and
Grant 1979). According to Palzer (1976),
the small drop in temperature could induce
zoospores release in P. cinnamomi.


WB. 1976. Avocado rootstock breeding
especially in relation to Phytophthora. Acta
Hortic 57:237-240.

BYKI P, (RANT BR. 1979. Som
conditions governing zoospore production
in axenic cultures of Phytophthorn
cinnamomi Rands. Aust J Bot 27:103-115.

root rot of guayule caused by Pythiun
ultimum. Phytopathology 35:636-639.

Improved methods for use in studies o
Phytophthora cinnamomi Rands and othe
Phytophthora species. New Zealand
Agric Res 8:88.

Production of sporangia by Phytophthorn
cinnamomi in axenic culture. Mycologii

COFFEY MD. 1987. Phytophthora roo
rot of avocado: An integrated approach t<
control in California. P1 Dis 71:1046.

PALZER C. 1976. Zoospore inoculun
potential of Phytophthora cinnamomi
Ph.D Thesis, University of Wester

Phytophthora control in Australia. Soutl
African Avocado Growers Assoc Yearbool

PINKAS Y. 1989. The arrest of avocado
root rot in Israel. Conference oi
Phytophthora disease of Citrus and other
crops in the Mediterranean area, EPPO
Palerma, Italy.

RANDS RD. 1922. Streepkamker vai
kaneel, veroorzaakt dor Phytophthorn
cinnamomi n. sp. Mededeleingern van he
Instuut Plantenziekten ten. 54:41.


1997 Phil. Phvtonath. 33(2):122-129





-a. J Cramer, Caduz, cinnamomi and

GALLEGLY ME. 1968. Homothalism, Phytophthora cinnamomi: Evidence tha
heterothalism and interspecific is not an indigenous fungus in
hybridization in the genus Phytophthora. Americas. Phytopathology 67:1373.
Phytopathology 58:1004-1021.
ZENTMYER GA. 1980. Phytophthe
SOLEL Z, PINKAS Y. 1984. A modified cinnamomi and the disease it cause
selective medium for detecting Phytophthora Monograph No. 10, Ax
Phytophthora cinnamomi on avocado Phytopath Soc, St. Paul, MN 96p.
roots. Phytopathology 74:506-508.

130 1997 Phil. Phytopath. 33(2):130-133




Respectively, Professor, Department of Plant Pathology and Science Research
Assistant, Plant Pathology Research Laboratory, Crops Research Division, USM
Agricultural Research Center (USMARC), University of Southern Mindanao, Kabacan,
Cotabato, Philippines.

Key words: durian, Durio zibethinus, leaf blight, Rhizoctonia solam

Rhizoctonia leaf blight disease of durian is heretofore reported for
the first time. Symptom is characterized by the browning of the leaf along
the tip and margin and progressing until the whole leaf becomes blighted.
Rhizoctonia solani was identified as its causal fungal pathogen. In culture,
the fungus produces the typical brown sclerotia (1-2mm) in 3 wk time;
microscopically, the characteristic right-angled mycelia measure 38.83 x
8.23m. Inoculated healthy durian seedlings developed the characteristic
Symptom in 3 to 4 wk.

INTRODUCTION durian trees at Serquifia's farm, Kidapawan,
Cotabato. They noted in their findings a
Prior to 1984, there were only three 100% incidence of Phytophthora disease in
diseases of durian reported in the fruit bearing durian trees. Severity of
Philippines (Tangonan and Quebral 1992). infection showed an average of 65%. They
These were fruit rot caused by Diplodia described the symptoms as follows:
duriones, leafspot caused by Placosphaeria brownish red gum in the collar region,
duriones, and bark disease caused by roots, and extending to the trunk; some
Trametespersonii. In 1984, Alincomot and revealed the pathogen developed the
others (1984) from USM, Kabacan, characteristic symptom 14 days after
Cotabato reported that Corticium inoculation. Pamplona and Garcia (1993)
salmonicolor causing pink disease of of USMARC-CEMARRDEC published a
rubber could also infect durian seedlings; technoguide on the commercial production
and in 1990, Baliton reported algal spot of durian and they reported Phytophthora
due to Cephaleuros virescens attacking stem canker disease attacking durian in
durian leaves. Two years later, Aquino and nurseries as well as in newly and already
others (1992) studied the occurrence and established plantations. They clainted that
symptoms of Phytophthora disease of such Phytophthora disease of durian is one

1997 Phil. Phvtopath. 33(2):130-133 131

of the most serious constraints to its Isolation and Identification of the
production and management. In 1994, Pathogen
Vilbar collected stem and root rot infected
durian plant parts from 3-year-old trees at Infected plant parts were collected,
Polomaling, Polomolok, South Cotabato. placed in plastic bags, and brought to the
She noted an 82.50% infection of durian laboratory for diagnosis (Fig. 1). Diseased
seedlings in her unheated plants but with plant parts were washed in tap water.
application oflkamantigue extract there was Direct microscopic examination of the
34.44 to 89.10% disease control when diseased plant parts was done.
applied as eradicative measure. Similarly in
1994, Laquihon and Jover (1994) observed Using standard laboratory
high incidence of. Phytophthora disease procedures, tissues were sectioned (0.5 to
(39.21 to 86.36%) in durian orchards at 2mm) disinfected with 10% chlorox for 2
four municipalities in Cotabato, Manungol, min, washed.with three changes of sterile
Saguing, Mateo, and Balindog. A year distilled water, blotted dry and planted onto
later, Tagacay and Dionio (1995) identified previously plated potato sucrose agar
a new foliar disease of durian leaf blight. (PSA). Pure cultures were maintained in
caused by Fusarium sp. The disease was PSA flat bottles.
first noted at USMARC and Paco,
Kidapawan. Pathogenicity Test

Tangonan and Cuambot (1996) in Two-week-old Rhizoctonia solani
their study of durian field disease observed Kihn was inoculated to healthy durian
another leaf blight disease in orchards of seedlings (about 3ft tall and 2cm stem
Cotabato particularly at Saguing, Makilala diameter) using swabbing technique. The
and Kidapawan areas. The occurrence of inoculated seedlings were covered with
this disease and its causal organism is clean transparent plastic bags to maintain
hereby described and reported in this paper. humid condition.

This study aimed at determining the
etiology of a leaf blight disease affecting RESULTS AND DISCUSSION
durian seedlings and fruit bearing trees.

MATERIALS AND METHODS Rhizoctonia leaf blight disease of
durian starts on the leaf tip and margins
Disease Survey until the entire leaf turns brown resulting to
defoliation. At first look, it appears like a
Various durian, orchards and sun-scalding symptom but upon direct
nurseries in. Cotabato province were examination, the structures ofR. solani are
surveyed for disease incidence. In the present in the blighted tissue.
course of the survey (1994 to 1996), durian
leaf blight was noted for the first time.




1. Durian leaves naturally and artificial'
g the characteristic blighting symptom.

tom) infected w

Figure 2. Rhizoctonia solani in culture showing typical dark sclerotial bodies (left) and


filings developed the Identification and cont]
ig symptom in 3 to 4 durian. Abstr Bibl Ur
Ition revealed the same from 1990-95 p. 140.
R solani found in

134 1997 Phil. PhvtoDath. 33(2):134-140

Abstracts of Paper Presented and
Submitted during the 28* Anniversary
of the Pest Management Council of the
Philippines, Inc. and Annual Scientific
Meeting of the Philippine Phytopatho-
logical Society, Inc. in loilo City, May


Development and comparison of
methods to screen abaca varieties
against Fusarium oxysporum f. sp.
cubense. L.M. Borines, G.N. Bastasa and
R.B. Cardines, Department of Plant
Protection, ViSCA, Baybay, Leyte.

Techniques for screening abaca
varieties for resistance to the Fusarium wilt
fungus Fusarium oxysporum f sp. cubense
were evaluated in the laboratory and pot
experiments. Pseudostem cross sections of
eight recommended varieties were
inoculated with suspension (103 spores/ml)
of the fungus and vascular discoloration
was measured. Tiny sections of corn
eyebuds were dipped in the spore
suspension and place on a depression slide
and the sporulation of the fungus was
assessed. In a experiment, sterilized soil
was infested with the fungus and planted
with the different abaca varieties. In a
separate pot experiment, the soil was
infested when the plants were grown for 2
months. Internal vascular discoloration,
external wilting and' basal splitting were
used as parameters in measuring the
reaction of the varieties. Regardless of
inoculation methods, Linawaan variety was
consistently resistant. Results varied
slightly between the laboratory and pot
experiments. Linawaan, Linino, and Putian
showed resistance to the disease while

Inosa, Laylay and Minenonga were
susceptible in terms of vascular
discoloration. Inosa and Laylay also
showed basal splitting and external wilt
symptom. Pot screening was more reliable
than laboratory screening and internal
vascular discoloration was more reliable
parameter in measuring the reaction of

Identification of suitable parameter for
resistance screening at the blast nursery
using molecular markers. R.E. Tabien,
S.R.M. Pinson, M.A. Marchetti, Plant
Breeding and Biotechnology Division,
PhilRice, Maligaya, Muiioz, Nueva Ecija,
USDA-ARS, Route 7, Box 999, Imes
Road, Beaumont, TX77713, USA.

Importance of parameter in
screening for specific trait depends on
reliability and this is generally associated
with heritability of the trait or the
parameter being measured. Association of
molecular markers with the trait, therefore,
will shown their genetic basis. Three
parameters: area under disease progress
curve (AUDPC), percent disease leaf area
(%DLA), Standard Evaluation System for
blast (SES rating), were used to evaluate
resistance of 245 recombinant inbred lines
from 'Lemont' and 'Teqing' cross at the
blast nursery using 167 molecular markers
evenly distributed in 12 linkage group of
rice, nine quantitative trait loci (QTLs)
were localized for AUDPC, six for %DLA
and eight for SES rating, indicating
heritable form of blast resistance, although
the three parameters were highly correlated
(r = 0.95), only three QTLs were associated
to all parameters. Analyses of weekly
scores using SES rating and %DLA
revealed temporal expression of these
QTLs. Both AUDPC and SES rating can

1 y rnn. rnvopamn. M33it:m1q-1iu 13

identify more QTLs than %DLA and these has only three genes. These were Pi-ql,
loci control large portion of resistance Pi-qS, and Pi-q6 for AUDPC and %DLA
variation at the blast nursery. However, and Pi-q5, Pi-q6 and Pi-b2 for SES rating.
SES rating was preferred due to its These results suggest that gene selection
simplicity and early detection of heritable and their interaction have to be considered
variation at the blast nursery. before gene pyramiding.

Gene selection, interaction and Disease carry-over of Rhizoctonia solani
pyramiding for blast resistance in rice. in rice-onion cropping system. L.M.
R.E. Tabien, Z. Li, A.H. Paterson, M.A. Sanches, S.A. Miller and S.R. Obien.
Marchetti, S.R.M. Pinson, Plant Breeding PhilRice, Maligaya, Mufioz, Nueva Ecija;
and Biotechnology Division, PhilRice, Ohio State University, OARDC, Dept. of
Maligaya, Muiioz, Nueva Ecija, Soil and Plant Pathology, 1680 Madison Ave.,
Crop Science Department, Texas A & M Wooster, OH 44691.
University, College Station. TX77840,
USA; USDA-ARS, Route 7, Box 999, This study was conducted to
Imes Road, Beaumont, TX77713, USA. determine a) the disease carry-over of
Rhizoctonia solani from rice and weeds to
With the breakdown of several onions and b) the cellular fatty acids
major genes. Combination of genes known composition of both isolates by gas
as gene pyramiding was recommended. chromatography procedures. R. solani
Recent reports, however, indicated from rice and weeds inoculated to onions
interaction of the genes, thus increased caused seedling rot or root rot. Both of
number of genes is not necessarily equated these isolates grew rapidly and produced
to better resistance. Four major genes for large spherical sclerotial bodies on potato
blast resistance (Pi-ql, Pi-q5, and Pi-b2) dextrose agar. These multinucleate isolates
were tagged from a population of had in common eight fatty acids such as
recombinant inbred lines. Using the myristic (14:0), pentadecanoic (15:0),
molecular markers as tag for each gene, palmitic (16:0), 7-palmitic *(16:1 cis 7),
lines with 0, 1, 2, 3 and 4 genes were heptadecanoic *(17:0), 9-heptadecanoic
evaluated at the blast nursery using three (17:1 cis 9), oleic (18:1 cis 9) and steric
parameters: area under disease progress (18:0) Fatty acids present in great amounts
curve (AUDPC), percent diseased leaf area were palmitic and oleic. These preliminary
(%DLA) and Standard Evaluation system results suggest that R. solani from rice and
for blast (SES rating). Results showed weeds are similar and onion is also another
both direct gene effect and gene X host plant.
interaction. Pi-q6 significantly affected
AUDPC and SES rating while Pi-ql
contributed significant variation for Evaluation of advanced breeding lines
AUDPC and %DLA. Pi-q5 affected all for resistance to rice tungro disease. R.C.
parameters but not Pi-b2. In most cases, Cabunagan, E. Angeles, E.R. Tiongco, S.
Pi-q6 interacted with other major genes but Villareal, P.S. Teng and G.S. Khush, IRRI;
not Pi-b2 gene. Regression analyses T.C.B. Chancellor, NRI; X.H. Troung,
rive.aled that the best fitting combinations PhilRice; I.G. Astika, BPTP, Bali and A.

V" 1007 Phil Phvtnnath AAMA.1A4AA14

Muis, MRIMCC, South Sulawes
Indonesia; A.K. Chowdhury, BCKV, We!
Bengal and T. Gahapthy, TNAU, Tam
Nadu, India.

Eighty-one advanced breeding line
developed at IRRI from crosses of I
longistaminata (20 lines), Utri Rajapan (3
lines), Utri Merah (20 lines), ARC 11554 (
lines) and Habiganj DW8 (6 lines) wit
susceptible line IR 1561 228 3 3 wer
evaluated by test tube inoculation metho
for their resistance to or tolerance of ric
tungro viruses. Four of the most promising
lines with different sources of resistance
were further tested, together with
selection of varieties in replicated 8 x 8 r
plots at two sites in the Philippine!
Indonesia and India. A line with resistance
derived from Utri Merah had low infection
with rice tungro spherical and bacillifon
viruses in three-countries, suggesting the
this source of resistance is likely to b
effective at a wide range of location. Line
derived from ARC 11554 and Balima
Putih also showed promising results.

Detection of Pseudomonas solanacearut
in potato tubers by polymerase chai
reaction. M.P. Natural and J.E. Villi
Department of Plant Pathology, UPLE
College, Laguna and Institute of Plar
Breeding, UPLB, College, Laguna.

Seven pairs of primer developed fc
specific biovars of Pseudomona
solanacearum in Australia were tested o
Philippine isolates. The thermal cycle!
DNA, MgCls and primer concentration
were optimized until good amplification c
DNA was obtained. Primer pair 759F an
760R was the best primers. The therms
cycles of 1 cycle 95 C for 2 min, 60 C for
min and 72 C for 1.5 min; then followed b

50 cycles of 94 C for 18 sec, 60 C for 18
sec. And 72 C for 18 sec; and a final cycle
of 72 C for 5 min and 4 C rest resulted in
consistent amplification of a 281 base pair
DNA. Using whole cells the detection limit
was estimated as 26 cells the detection limit
was estimated as 26 cells per 25 ul PCR
mix. The same PCR conditions were able
to detect P. solanacearum in naturally
infected tubers.

Evaluation of rice germplasm to rice
tungro viruses under different disease
pressures. L.S. Sebastian, G.V.
Maramara, D.A. Tabanao, E.B. Tabelin and
E.R. Tiongco, PhilRice, Maligaya
Experiment Station, Mufioz, Nueva Ecija
and Midsayap Experiment Station, Bual
Norte, Midsayap, North Cotabato.

Field screening of 56 rice
germplasm (IR and PSB recommended
varieties, IR 22 mutant lines and TN 1 X
ARC 11554 lines) to the tungro viruses was
conducted at PhilRice stations in Nueva
Ecija and North Cotabato with respectively
where low and high tungro disease
pressures. In Nueva Ecija, 22 entries were
visually scored to have a low disease
incidence (<21%) at 64 DAT. ELISA of
leaf samples of these plants revealed low
RTBV and RTSV and RTBV alone
infections (<21%) but with high RTSV -
alone infection (>60%). In addition, 4
entries (IR 74, PSB Rc 18, PSB Rc 34 and
TI-11-8, a TN 1 x ARC 11554 line) have
low disease incidence (<5%) and very low
infection of both RTBV and RTSV (0-4%).
IR 66 and another TN 1 x ARC 11554 line
have high disease incidence (>60%) but
ELISA showed that they were infected with
RTBV only. However, in North Cotabato,
only 5 entries (IR56, IR62, 3 TNI> 11554 lines and mutant line of IR 22)

1997 Phil. Phytopath. 33(2):134-140 137

retained their low infection to both RTBV
and RTSV and RTBV alone (<26%) but
have high infection of RTSV alone. Only
TI-11-8 recorded a very low infection to
both or either RTBV and RTSV indicating
high level of resistance to these viruses.
The results show the current reaction of the
present set of varieties to tungro infection.


Molecular mapping of resistance to
tungro in Utri Rajapan (Oryza sativa L.)
M.G.V. Maramara, L.S. Sebastian, H.C.
Martin and E.R. Tiongco, Plant Breeding
and Biotechnology Division, PhilRice,
Mufioz, Nueva Ecija, Philippines.

Tungro remains as the most
damaging virus disease in rice and the
development of varieties tolerant or
resistant to this disease is a priority
objective of the Philippine rice breeding
program. Utri Rajapan has resistance to
rice tungro spherical virus (RTSV). To
determine the chromosomal location of the
resistance gene at RTSV in Utri Rajapan,
138 F2 plants derived from TN1 x Ytri
Rajapan were evaluated using restriction
fragment length polymorphism (RFLP) and
tungro resistance screening in the
greenhouse. DNA from the parental lines
and the F2 plants were digested with eight
restriction enzymes and probed with DNA
markers using non-radioactive labeling
method. Parental survey from RFLP
polymorphism using 175 DNA markers
showed that 49 markers were polymorphic.
The polymorphic markers are now being
hybridized with the mapping population to
determine their segregation. Twenty F3
seedlings/F23 family were inoculated at the
rate of 4 GLH Der seedling to determine the

ELISA. Visual scoring showed 11 lines
having high resistance and moderate
resistance to tungro. Only one line had low
virus titer to RTBV (15%) and RTSV
(15%). Twelve lines have low virus titers
to RTSV but all lines have low ELISA
readings and visual scores indicating
resistance reactions to tungro. The results
of the phenotypic data and segregation of
the polymorphic markers will be analyzed
for co-segregation to determine the map
location of the RTSV resistance. gene.

Molecular characterization of two rice
tungro spherical virus variants. MA.
L.M. Yambao, P.Q. Cabauatan and Ossmat
Azzam, IRRI, Los Bafios, Laguna.

Rice tungro disease is associated
with two viruses, rice tungro spherical virus
(RTSV), a single stranded RNA virus, and
rice tungro bacilliform virus (RTBV), a
double stranded DNA virus. These two
viruses and several of their variants are
found throughout Southeast Asia.
Cabauatan and others (IRRN 19:2, June
1994) have reported the presence of two
RTSV A, the IRRI maintained strain, and
Vt6 strain, the resistance-breaking strain to
TKM6 source of resistance, which was
identified in Midsayap, North Cotabato,
Philippines. A reverse transcriptase-
polymerase chain reaction (RT-PCR) assay
was developed to amplify cDNA from coat
protein coding regions of RTSV genome
and enzyme-based restriction fragment
length polymorphisms (RFLPs) were
detected between the two RNA from
RTSV, strain A and Vt6-infected plants and
a primer, RSCPsC-3607 that is
complementary to the 3' end of coat
protein 2 region of RTSV. The cDNA was
then amplified by regular PCR using
RSCP1V-2453 primer, which anneals at the

13R 1007 Phil Phwtnntoh 11/'7\.11.1AAn

5' end of coat protein 1 region, and the
same RSCP2V-3607 primer used in the
cDNA synthesis step. PCR products of
1,154 kbp, covering the two presence of
RTSV, HindII and BstYi restriction
digestion of amplified PCR products from
both RTSV strains showed different
patterns while PstI and XhoI restriction
digestion did not. Using these two
enzymes patterns, we were able to
differentiate the two strains. This technique
is applied to examine inter- and intrafield
genetic diversity of natural RTSV
populations in the Philippines.

Clonal Analysis of Rhizoctonia solani
AG1-1A isolates. C.B. Pascual and M.
Hyakumachi, IPB, UPLB, College, Laguna
and Gifu Univ., Japan.

Eighty-two isolates of Rhizoctonia
solani collected from different host species
and geographic areas in the Philippines
were found to belong to anastomosis group
1-1A. Clonal analysis of these isolates was
done thru hyphal fusion and lysis evaluation
between isolates. Results were discussed.

Comparison of two mass screening
methodologies for tungro resistance. E.
Coloqiuo, E.R. Tiongco, R.C. Cabanugan
and Ossmat Azzam. IRRI, Los Bafios,

Using 16 rice accessions with
known reactions to rice tungro viruses and
their vector, we compared the inoculation
efficiency, consistency and accuracy of two
mass screening methodologies for tungro
resistance: the water tray and the potted
plant methods. After 4-day continuous
insect acquisition on an inoculum source,

inoculation of the 16 rice accessions was
done with insect reacquisition after each
inoculation while for the water tray
method, three serial inoculation were done
prior to insect reacquisition. Symptom
severity and percent of infection were
measured visually 3-4 weeks post
inoculation and only in the third trial,
selected lines were also scored using the
enzyme-linked immunosorbent assay
(ELISA) for both tungro viruses to
measure the accuracy of visual scoring.
Results from three trials show that both
methods are consistent but the water tray
method has higher inoculation efficiency
and is more accurate than the potted
method. In addition, large number of
samples could be handled effectively using
the water tray method. Our future goal is
to decentralize this methodology to national
programs who are involved in mass
screening for tungro disease resistance.

In vivo, screening of promising
antagonists against postharvest
pathogen of sweetpotato and yam. M.K.
Palomar, E. Salamat, V. Palermo and G.
Edurise, Department of Plant Protection,
ViSCA, Baybay, Leyte.

Eleven (11) promising isolates from
in vitro bioassay were tested in vivo using
sweetpotato and yam tubers. Two (2)
isolates F145 (healthy cassava roots) and
Trichoderma (corn) showed consistent
antagonistic effect against Botryodiplodia
theobromae, M. phaseolina and Sclerotium
rolfsii. On the other hand, F145 (healthy
cassava roots), Fl (sweetpotato tissue
culture), F150 (coconut. trunk),
Trichoderma from yam, F128 (yam tuber)
were found as the most effective'against B.

lurvey of Fusarium species in soils from
mall upland mixed farms of Cavite. A.
langalang, R.G. Vidon, Biological Science
department Don Severino Agricultural
college Indang, Cavite, Philippines.

Composite soil samples were
collected from selected mixed farm in each
pland towns namely: Alfonso, General
Lguinaldo, Indang, Magallanes, Silang,
4aragondon, Amadeo, Mendez and
'agaytay. Nine Fusarium species were
violated using the soil dilution plate
technique and peptone PCNB agar, namely,
. merismoides, F. lateritium, F dimerum,
. solani, F. oxysporum, F. longipes, F.
ompactum, F. equiseti and F. semitectum.
. oxysporum and F. solani were the two
nost abundant and frequently isolated
presenting 87% of the isolates. The other
even species were isolated less frequently.

oymptomatology and identification of
pathogens associated with stem disease
complex in periwinkle. T.O. Dizon, S.V.
iar and L.A. Reyes, IPB, UPLB, College,

A stem disease complex of
periwinkle [Catahranthus roseus (L.) Don.]
locally known as chicirica is reported.
item rot was first reported to be caused by
Uizoctonia solani. However, this study
reports that a number of pathogens has
ieen causing stem rot, a serious and major
disease. The disease was found to be
caused by Sclerotium rolfsii, Fusarium
oniliforme, F. solani and Phomopsis sp.
)ne or more pathogens can be infecting a
ingle periwinkle plant. The symptom of
he disease may start at any part of the stem
_X .. ,. ._. 1-_-. "I-- 'r1_ .iar--

becoming brown to dark brown. The
otting may cover the whole or part of the
tem. The rotting may progress upward,
causing wilting and later death of the plant.
'ungal structures such as -pycnidia,
clerotial bodies or mycelial growth were
observed on the surface of affected tissue.

isterase phenotype determination for
identification of Meloidogyne species in
ice-based cropping areas. I.R. Soriano
nd J. Claude Prot, IRRI, Los Bafios,

Electrophoretic characterization of
sterases in 140 Meloidogyne populations
collected from 10 rice-based cropping areas
i Luzon and Sarangani Province showed
bree different phenotypes. The phenotype
fSI ofM. graminicola was observed in all
populations from rice planted during the
vet and dry seasons. The same phenotype
vas noted from Allium cepa, A. sativum,
irassica chinesis, Brassica olerecea,
'yperus iria and Echinocloa colona
obtained during the dry season. Eleven
plants grown after rice during the dry
eason were found to harbor M. incognita
ihenotype II. M javanica phenotype J3
vas observed in four crops planted after
ice during the dry season. Mixed
populations were noted from root samples
if L. ,esculentum (M. incognita and M
avanica) A. sativum (M. graminicola and
. javanica) and B. oleracea (M.
raminicola and M javanica) Two or
nore Meloidogyne populations may be
)resent in a ricefield and each may infect
oots of suitable hosts. Otherwise, a
populationn may wait until a preferred host
s planted and in some cases when the host
s favorable for both populations, a mixed
~~lj'u n4-^ hara j-lpanar l Tcaev' mP

14U 1997 Phil. Prhtooth. 33(2):134-

Scnnr snonmn ne Tvnen on one c

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