• TABLE OF CONTENTS
HIDE
 Front Cover
 Table of Contents
 Front Matter
 Detection of maize stripe tenuvirus...
 Cloning and sequence analysis of...
 Cultural and molecular characterization...
 Integrated management of onion...
 Molecular techniques for routine...
 Transmission of banana bract mosiac...
 Isolation of a tobamovirus from...
 Sclerotium rolfsii causing fruit...
 Abstracts of paper presented during...
 Back Cover














Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00090520/00046
 Material Information
Title: Journal of tropical plant pathology
Series Title: Journal of tropical plant pathology.
Alternate Title: Journal of Philippine phytopathology
Philippine phytopathology
Physical Description: v. : ill. (some col.) ; 26 cm.
Language: English
Creator: Philippine Phytopathological Society
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
College Laguna
Publication Date: January-December 2003
Frequency: semiannual
regular
 Subjects
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
 Notes
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00046
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
    Table of Contents
        Table of Contents
    Front Matter
        Front Matter
    Detection of maize stripe tenuvirus in rottboellia cochinchinensis and its transmission to corn, Zea Mays
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
    Cloning and sequence analysis of the coat protien gene of zucchini yellow mosiac potyvirus
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
    Cultural and molecular characterization of the stenocarpella macrospora (Earle) Sutton causing leaf blight, ear rot and stalk rot in maize
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
    Integrated management of onion antracnose [colletotrichum gloeosporioides (Penzig) Penzig & Sacc.]
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
    Molecular techniques for routine disease indexing of sugarcane
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
    Transmission of banana bract mosiac virus by three aphid species in the Philippines
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
    Isolation of a tobamovirus from bottlegourd, Lagenaria siceraria Standl.
        Page 66
        Page 67
        Page 68
        Page 69
    Sclerotium rolfsii causing fruit rot of jackfruit (atrocarpus heterophyllus L.)
        Page 70
        Page 71
        Page 72
    Abstracts of paper presented during the 34th pest management council of the Philippines conference held at Cebu City on May 6-9, 2003
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
    Back Cover
        Page 85
        Page 86
Full Text


ICCO fl4 4 r- AfoA


JOURNAL OF

PLANT PAT
Formerly Philippine I


TROPICAL
S I r" 'Mr'\


I
1; 7~6y..
:i~3` i.-_ ?'~s

~PFYY,
F:~b









PWcrl

4w


JOURNAL OF TROPICAL PLANT PATHOLOGY
Published by The Philippine Phytopathological Society, Inc.


OFFICERS OF THE PHILIPPINE PHYTOPATHOLOGICAL SOCIETY, INC.


2002-2003


2003-2004


President
Vice President
Secretary
Treasurer
Auditor
Business Manager
PRO
Board Members



Ex-Officio


E.B. Gergon
R.A. Zorilla
N.V. Desamero
F.M. dela Cueva
T.O. Dizon
A.D. Raymundo
O.S. Opina
C.M. Vera Cruz
M.P. Natural
E.G. Sison
R.E. Tabien
P.M. Magdalita


President
Vice President
Secretary
Treasurer
Auditor
Business Manager
PRO
Board Members



Ex-Officio


R.A. Zorilla
G.A. Peratta
P.G. Gonzales
F.M. dela Cueva
O.S. Opina
R.V. Abgona
LA D. Lando
T.O. Dizon
M.P. Natural
C.M. Vera Cruz
MaA.G. Maghuyop
E.B. Gergon


EDITORIAL BOARD


Editor-In-Chief
Associate Editors

Circulation Manager


Teodora.O. Dizon
Rizaldo G. Bayot
Oscar S. Opina
Teodora O. Dizon


SUSTAINING ASSOCIATES

AgChem Manufacturing Corporation
BASF Phil., Inc.
Bayer Crop Science
Pioneer Hi-Bred
Syngenta Phil, Inc.
CropKing Chemicals, Inc.
DOW AGRO SCIENCE


Cover photo


Jackfruit infected with frit rot causdl by a&5GOtinw [rf" Sacc.













ME 39 NUMBER 1 & 2 JANUARY TO DECEMBER 2003


i of maize stripe
inensis and its tr
rro and N.B. Baj

and sequence an
zucchini yellow m
illamor, E.Y. ArdM

and molecular ch
pella macrospor
it, ear rot and sta
:cual and C.A. F

d management c
Wioides (Penzig)
?rto, M. V. Duca a

ir techniques for
:ane
a Cueva, M.P. de
Luzaran

;sion of banana t
ecies in the Philii


CONTENTS

tenuivirus in Rottboellia
transmission to corn, Zea mays
et 1-15

alysis of the coat protein
osaic potyvirus
ales and N.B. Bajet 16-35

aracterization of the
a (Earle) Sutton causing
lk rot in maize
?elevante 36-42

>f onion anthracnose [Colletotrichum
Penzig & Sacc.)
nd S.A. Miller 43-48

routine disease indexing

. Ocampo, J.B. Ferrater
49-57

bract mosaic virus by three
pines


Tadura, L.V. Magnaye, M.A. Alforque,
ores and L. Kenvon 58-65










Journal of Tropical Plant Pathology 39:1-15


DETECTION OF MAIZE STRIPE TENUIVIRUS IN ROTTBOELLIA
COCHINCHINENSIS AND ITS TRANSMISSION
TO CORN, ZEA MAYS'


J. T. NAVARRO and N. B. BAJET2

'Portion of the thesis adjudged as Best Undergraduate Thesis in Plant Pathology
during the Annual Convention of the Pest Management Council of the Philippines
(PMCP), 06-08 May 2002, Davao City.

2Respectively, former student and Professor, Department of Plant Pathology,
College of Agriculture, University of the Philippines Los Banos, College, Laguna.


Two types of foliar symptoms were observed on Rottboellia
cochinchinensis in plots around the UPLB Biological Sciences
building: Type A comprised of very small dots and thin pale yellow
short lines along the veins and were more numerous and prominent
on newly emerged leaf. The more commonly observed was Type B
symptoms which consist of long and narrow chlorotic stripes wider
at the base, taper and end toward the middle of the leaf and the
developing younger leaves showed complete chlorosis. Results of
enzyme-linked immunosorbent assay (ELISA) showed that maize
stripe tenuivirus (MStpV), rice grassy stunt tenuivirus (RGSV),
sugarcanelmaize dwarf mosaic potyvirus (SCIMDMV), and maize
mosaic rhabdovirus (MMV) were present in the symptomatic plants
with MStpV the most commonly occurring virus with 77.0% of the
samples infected by it. This virus was either alone (26.7%) or in
combination (50.3%) with SCIMDMV, MMV or RGSV in the
symptomatic R. cochinchinensis but the viruses were not detected
in nonsymptomatic plants. A 15.8 kDa protein was observed with the
MStpV-ELISA positive but not with MStpV-ELISA negative samples
in SDS-PAGE gels. A signal corresponding to this protein appeared
on the membrane probed with the MStpV antiserum in western blot
indicating that R. cochinchinensis was infected with MStpV. The
MStpV was transmitted from R. cochinchinensis to corn by the
insect vector Peregrinus maidis that was infesting that plant but
neither with the non-MStpV exposed vector nor from MStpV
diseased R. cochinchinenssis alone. Both R. exaltate and the P.
maidis serve as reservoir for MStpV. MStpV as a threat to corn
production in the country is discussed.

Key Words: Itch grass, virus, serology, planthopper, epidemiology


INTRODUCTION "Aguingay", is a grass (Graminae or
Poacea), and is one of the three most
Rottboellia cochinchinensis [syn.=R. important weeds in sugarcane, corn and
exaltata L.f.] (Itch grass) or locally called upland rice in the Philippines (Madrid et







avaro and Baet


al., 1972; Pancho and Obien, 1995). It is
erect and may reach 4 m tall and a
vigorous competitor of 18 crops in 28
countries. It has fiberglass-like needles on
its leaf sheath that cause painful
infections when these break off after
penetrating the flesh of the hand (Holm et
al., 1977).
In a study on the prevalence of
maize stripe tenuivirus (MStpV) in corn
plants around the University of the
Philippines Los Batios campus (UPLB)
(Navarro and Bajet, 2001), a number of
R. cochinchinensis plants showed
symptoms typical of virus infection. These
foliar symptoms are very similar to those
exhibited by corn or maize (Zea mays L.)
plants found to be associated mostly with
MStpV and to a lesser extent,
sugarcane/maize dwarf mosaic potyvirus
(SC/MDMV) (De Luna, 1990; Navarro and
Bajet, 2001). This weed species is
reported to be one of the hosts of MStpV
(Brunt et al., 1996) and the delphacid,
Peregrinus maidis (Ashmead) (Catindig et
al., 1996; Tsai, 1996), an insect vector of
a number of plant viruses that infect corn
(Brunt et al., 1996; White, 1999).
In the Philippines, a number of local
researchers showed that grass weeds,
including R. cochinchinensis are hosts of
viruses that infect abaca (Celino and
Martinez, 1956; Eloja and Magnaye,
1996; Escober, 1956; Legaspi, 1955;
Protacio, 1953) or rice (Khan et al., 1991).
Tangonan and Quebral (1992) listed
mosaic as a disease of R.
cochinchinensis and also mentioned
sugarcane mosaic virus (SCMV) and
potato virus Y under this plant. However,
the relationship between R
cochinchinensis with mosaic and these
specific viruses was not determined.
MStpV infection of R cochinchiensis has
not been reported in the country and that
serological analysis like enzyme linked
immunosorbent assay (EUSA) and
Western blot have not been applied to
assay this weed -species for virus
infection. MStpV is a member of the
genus Tenuivirus (Falk and Tsai, 1998).
The other members are rice grassy stunt


virus (RGSV), rice stripe virus (RSV), and
rice hoja blanca virus (RHBV) (Brunt at
al., 1996). The tenuiviruses produce'large
amount of noncepsid protein (NCP) in the
leaves of infected plants that can be used
in assaying the virus in the infected plants
(Falk et al., 1987; Falk and Tsai, 1983;
Gingery, et al., 1981; Miranda and
Koganezawa, 1995).
The use of sensitive and highly
discriminating serological techniques, like
ELISA and western blot, to confirm the
observations of mosaic on R.
cochinchinensis would increase our
understanding of the nature of those
symptoms on this weed species in the
country. ELISA has been one of the very
useful and commonly used tools in
detecting the presence of viruses in
plants (Bajet, 2001) and was used locally
for the assay of MStpV in com (De Luna,
1990; Navarro and Bajet, 2001). Likewise,
western blot was applied in the studies of
various properties of the coat protein of
the Tenuivirus, including the
demonstration that a large amount of
MStpV-specific NCP is produced in corn
plants infected with MStpV (Gingery et al.,
1981) and in rice infected with RGSV
(Miranda and Kaganezawa, 1995). A
great advantage of this technique is that it
identifies the virus by two independent
properties of its coat prein-molecular
weight and serological specicity
(Matthews, 1992).
The objective of this study was to
determine if the mosaic observed on R.
cochinchinensis, herein referred to as
"Itch grass stripe', around UPLB is
infected with the maie stripe tenuivirus
(MStpV) and other viruses using ELISA
and western blot and to evaluate if the
MStpV is transmissible to corn plants.

MATERIALS AND METHODS

Area of vinrs disease coalaton and
sources of antdsum Leaves of R.
cochkchminesis showing mosaic were
collected from plots around the
greenhouses in the Biological Science




















ns, Sugarcane
rus (SC/MDMv
art, University
MN), Maize
; courtesy of F
University, V\
tenuivirus
*sy of Prof. B
mia, Davis, C
tenuivirus NC
1 protein (RG
3.J. Miranda,


homogenized with tme carbonate DuTrer, and slightly moaitlea (Navarro, zu
centrifuged briefly and the supernatant Sambrook et al., 1989).
was collected and used in the assay. The
ELISA microtiter plates used was Falcon Transmission of the MStpV in
3912 (Becton Dickinson Labware, cochinchinensis ("ltchgrass stripe"]
Oxnard, CA) supplied locally by Medical corn. Two experiments were conduct
Test Systems (Medtest). A sample with to determine if the MStpV-infec
absorbance at 405 nm (A405 nm) greater "Aguingay", herein referred to
than the threshold value which is the "Itchgrass stripe" is a source of MStpV
mean (m) of healthy control plus 3 corn. Experiment 1 consisted of 1
standard deviations (m+3 SD) was groups of R. cochinchinensis plants. C


r"-+--+i-^ ^f niv= +rirM






















containing three-four pots of 7-10 day old same symptomatic plant showed longer,
sweet corn seedlings, the bag opened to wider stripes and in some leaves, the
retrieve the insect infested weed and the whole lamina waas chlorotic or
weed carefully laid at the base of the corn completely whitish. Most of the samples
seedlings. After 5-7 days, the corn obtained were of Type B but each
seedlings were sprayed with an symptom type looked very similar to those
insecticide and the dried "Aguingay" observed on corn that were found
removed. The inoculated corn seedlings infected with SC/MDMV and MStpV
were observed for 18-30 days. (Navarro and Bajet, 2001).
In experiment 2, essentially the same
set up was followed but the P. maidis and Serological Analysis of "ltchgrass
the weed were separated prior to stripe" by ELISA. Table 1 shows the
inoculation as follows. The bags of weed mean absorbance values at 405 nm of 30
infested by the insect were placed inside field-collected symptomatic samples of R.
a 4-6 C chamber for about 20-30 min then exaltata as assayed by ELISA using
immediately the bags were retrieved, antiserum of SC/MDMV, MMV, MStpV
shaken to dislodge the insects from the and RGSV. Eleven samples (sample 1, 5,
whorl and leaf axils and then the weed 6, 8, 9, 10, 13, 19, 23, 24 and 26) showed
was placed inside separate cages of corn absorbance values higher than threshold
seedlings as above. The P. maidis that value of 0.45 for SC/MDMV. Absorbance
were dislodged and collected inside the values of these samples ranged from 0.47
bags were identified and picked out and to 2.26, hence were positive for the virus.
transferred to another set of corn The absorbance value of the healthy anc
seedlings and the inoculated plants buffer controls was 0.43 and 0.0C
maintained in insect cages for 6 wk. respectively.
With regard to the reaction of the
RESULTS AND DISCUSSION samples to MMV antiserum, 15 samples
(samples 4, 6, 8, 9, 10, 11, 13, 18, 19, 23,
Symptoms observed on R. 24, 26, 27, 28, and 29) showed
cochinchinensis. R. cochinchinensis is a absorbance values ranging from 0.49 tc
common weed in UPLB. The samples 0.88. The mean absorbance of the
collected showed two types of symptoms. healthy and buffer control was 0.42 anc
One type (type a, Fig. 1A) was a fine 0.00, respectively. These samples were
mosaic consisting of small dots and very considered positive for the antigen oi


--A 12-;-Ar~ nia








Detection of maize strine


and 24 had lower absorbance values than with MStpV. No sample was four
the threshold value or negative for reacted only to the RGSV-CP anti,
MStpV. The mean absorbance of healthy Samples with double infections i
and buffer controls was 0.41 and 0.00, samples 4, 11 and 28 (with MM
respectively. MStpV), sample 3 (with SC/MDM
Twelve of the 30 samples reacted MMV), and 4 samples (sample 17,
positively to RGSV-CP antiserum and 27) with MStpV and RGSV
(samples 9, 10, 13, 17, 18, 19, 20, 23, 25, samples were triply infected, sar
26, 27, and 29). The samples gave (SC/MDMV, MMV, MStpV), saml
absorbance values ranging from 0.99- (SC/MDMV, MMV, RGSV) and si
1.96 that were higher than its threshold 18 and 29 (MMV, MStpV, RGSV
value of 0.89. The mean absorbance of four viruses simultaneously infect
healthy and buffer controls was 0.88 and samples (sample 9, 13, 19, 23 and
0.00, respectively. Eleven of the 12 (92%) SC/MDMV had the lowest F
RGSV-positive samples also showed infection (33.3%) followed by
positive reaction with the MStpV-NCP (40.0%) and MMV (50.0%). MStp
antiserum. These results indicate that the the most commonly detected
RGSV-CP antibodies recognize MStpV (77.0%) indicative of an associa
and the MStpV-NCP antibodies recognize MStpV with the stripping symptom
RGSV. It also indicates, but yet to be 2 Type B) similar to those express
proven, that these two viruses have corn suggesting that such sympton
common epitopes recognized by their be a consistent biological phenot
respective antibodies. the hosts infected with the virus
MStpV and RGSV are members of samples collected showed mosi
genus Tenuivirus and that RGSV has Type B symptom (Fig. 1) as it was
been recognized as having biological and noticeable than the other type in th
molecular properties similar to MStpV In most cases, the MStpV-ir


M~tpv ana Kijv inciuae
transovarial transmission by de
planthoppers, (2) NCP cai
concentrated by the differer
precipitation procedure, (3)
stranded RNA, (4) viruses are th
stranded particles, (5) NCP
needle-shaped crystals and large i
of NCP found in infected leav
failure of detection of NC
planthoppers with ELISA evei
Western blot analysis. Likewis
reaction of the R. cochinch
extracts with both MStpV and
antiserum could be a consequence
indirect ELISA method used in the
It allows detection of a broader n
related viruses with a single ani
(Koenig, 1981, Koenig and Paul, 1
The samples infected by or
virus were sample 1 with SC/I
sample 24 with MMV, and 8 sample


(1) viruses, had quite high abso
icid values. Similar results were
be observed with those infected
pH SC/MDMV. Since absorbance is i
gle- proportional to antigen or
ine- concentration, the MStpV or SCI
ned could still have multiplied and a
unt high titers in cells/tissues even
(6) presence of the other viruses (Cla
in Adams, 1977; Matthews, 1992).
with The above results show
the symptomatic R. cochinchi
Isis especially those with the Type B ,
iSV symptoms were infected with MStp
the other symptom type were also it
idy. with the same virus and to a
of extent, with SC/MDMV or MMV
rum and in combination, and perhaps
!). RGSV. This result confirms the re
one Lugod and Dalmacio (1988) and ac
MV, virus to the list of diseases of "Agi
or Itch grass reported in the Philipp








Navarro and Bajet


by Tangonan and Quebral (1992) and the
world (Brunt et al., 1996). Our findings
were similar to and complements results
obtained previously that MStpV was the
most common virus detected in corn
plants growing in the same or nearby
areas where the R. cochinchinensis
samples were obtained (De Luna, 1990;
Navarro and Bajet, 2001).
Two other viruses were identified that
were also infecting R. cochinchinensis
and these were SCIMDMV and MMV.
This result confirms the report that R.
cochinchinensis is a host of these two
viruses (Brunt et al., 1996; Lugod and
Dalmacio, 1998; Rozenkranz, 1980;
White, 1999). SC/MDMV is transmitted
non-persistently by several aphid species
and this is probably the same or a closely
serologically related strain of the virus
causing mosaic of "Aguingay" (Tangonan
and Quebral, 1992) and by others who
claimed to be a source of viruses for
abaca (Celino and Martinez, 1956; Eloja
and Magnaye, 1966; Escober, 1956;
Legaspi, 1955; Protacio, 1953). MMV is
transmitted persistently by the same
MStpV insect vector (Brunt et al., 1996;
White, 1999). Similar to MStpV, this is the
first report as far as it is known, of
infection of R cochinchinensis by these
two above-mentioned viruses in the
Philippines.
On the other hand, 3 samples
(samples 3, 7 and 12) among the 30
symptomatic samples showed negative
reaction to the four antisera used in the
assay. The symptoms could have been
caused by another pathogen or different
virus that is not recognized by any of the
antiserum used. On the other hand, the
samples could have been infected but the
processed tissues had virus titers below
the lowest limit of sensitivity of detection
by the ELISA procedure (Clark and
Adams, 1977). The absorbance values of
these samples are quite dose to the
threshold values for al the viruses (Table
1).
To further confirm the previous
findings on MStpV (Table 1), another
batch of R. cochinchinensis samples


showing only the Type B symptoms (Fig.
1B) were obtained and assayed in ELISA
with the MStpV antiserum. The sap of
infected corn showed high absorbance
values of 0.18 at sap dilution of 0.000025
and 1.95 at sap dilution 0.25. The sap of
healthy corn showed absorbance value of
0.03 to 0.22 at the corresponding sap
dilutions. With the infected R.
cochinchinensis at sap dilution of
0.000025, absorbance values ranging
from 0.00 to 0.76 (mean = 0.37) were
obtained while values ranging from 0.49
to 2.11 (mean = 1.75) were obtained at
sap dilution of 0.25. The sap of healthy R.
cochinchinensis showed a corresponding
absorbance value of 0.00 (mean = 0.00)
and 0.00 to 0.30 (mean = 0.15) at sap
dilution of 0.000025 and 0.25,
respectively (Table 2).
The consistent positive reaction of
the MStpV-NCP antiserum with the
extracts of symptomatic cor and R.
cochinchinensis but negative with the
healthy control counterparts at various
dilutions tested indicates a highly
probable and close serological
relationship, if not identity, between the
MStpV that infects the weed and com
from around UPLB with the MStpV-
infecting corn in USA as the antiserum
used in assays was from the USA. Brunt
et al. (1996) mentioned that serological or
nucleic acid hybridization tests can be
specifically used to check and establish
the identity to an unknown virus after
identifying its natural hosts and establish
which viruses have been isolated. If
reagents are available however,
reciprocal tests may have to be done with
their homologous and heterologous
antiserum diluted 2-fold to provide a
better idea of the degree of antigenic
relatedness between the MStpV of corn in
the USA and that of the MStpV of iR
cochinchinensis and corn in the
Philippines (Jaegle and Van
Regenmortel, 1985, Matthews, 1992).

Western Blot Analysis of "ltchgrass
Stripe"







ustetaon or maize 5


sus-PAGE analysis or "ltcngrass standard curve where calculations wer
stripe" proteins In general, the protein extrapolated from, among others (Hame
samples extracted following the and Rickwood, 1990; Navarro, 200'
procedure of Carillo (1995), Saldajeno Sambrook et al., 1989). SC/MDMV capsi
and Bajet (1998) and Miranda and protein is bout 35 kDa and MM
Koganezawa (1995) show slight preparations show protein species (
differences in the gross protein profiles about 75 kDa, 50 kDa, 45 kDa, and 3
(Navarro, 2001). A distinct major protein kDa (Brunt et al, 1996). A similar stuck
band was consistently resolved, relatively using antibodies specific to the capsi
abundant but of apparently uneven protein of the MStpV, MMV an
intensities, migrated similar to the 18.4 SC/MDMV could help resolve its identity
kDa protein marker in all the six samples
assayed. Also, a less distinct protein Western blot analysis of "Itchgrau
species was resolved intermediate of the stripe" The major protein band of aboa
43 kDa and 29 kDa protein markers and a 15.8 kDa found in infected I
similar but broad somewhat smearing cochinchinensis reacted to the MStp\
bands with the other extraction protocols. NCP antiserum as shown by the sign
The distinct major protein band that that developed, although of differed
migrated similar to the 18.4 kDa marker intensities, on the same position occupied
had an approximate molecular weight of by this 15.8 kDa protein on tl
15.8 kDa (Navarro, 2001) which is similar membrane. No such signal developed
and close to the report of 16.3 kDa for the along the whole lane filled with extracts
MStpV-NCP (Gingery et al., 1981). These MStpV-NCP ELISA negative I
results demonstrate that infected R. cochinchinensis. Similarly, the sarr
cochinchinensis, like corn, the NCP is protein in MStpV-infected corn, as shom
also produced abundantly and thus, by ELISA, showed the signal but not wi
confirm and extend the report that within healthy corn (data not shown). The.
the cells of tenuivirus-infected plants, the results indicate that the 15.8 kDa prote
NCP is the most abundant protein (Falk produced in infected R. cochinchinensis
and Tsai, 1983, 1998). The differences in also the NCP and is closely related to,
the intensities of the protein bands in the not the same as the NCP produced
gel could have been due to the uneven MStpV-infected corn. The differences
amounts of protein loaded which, in turn, the intensities of the signals among tt
could have been affected by stage of samples could have been due to tt
infection of the sample (Navarro, 2001). amounts of protein resolved in SD!
The less distinct protein species that PAGE and blotted onto the membrane, <
was resolved intermediate of the 43 kDa mentioned above. All these results
and 29 kDa protein markers had an serological studies indicate that tt
approximate molecular weight of 39.8 disease agents in East Africa, tt
kDa. This was probably the capsid protein Americas, Australia, and now in tt
of MStpV or either of the other co- Philippines are related (Kulkarni, 197
infecting viruses. The capsid protein of Gingery et al, 1979; De Luna, 199
the MStpV was reported to be about 32 Navarro, 2001; Navarro and Bajet, 2001











IStpV-infected "Aguingay" with the P. inoculated to sweet corn s
is colonies developed initial (NBBajet, unpublished results:
toms of fine chlorotic somewhat inoculation tests from the infecl
lated dots between its minor veins of seedlings to "Aguingay" shall
!s that developed about 3-4 wk from confirm the role of the planthoppe
sure with the inoculum (Table 3). As The results demonstrate 1
particular leaf further developed and MStpV was transmitted, in gros
ended, the "brush stroke" chlorotic by the planthopper, P. maidis i
ig of about 0.3-0.5 cm wide and 2-3 infected "Aguingay" to corn. Th
mng was evident at its base starting was not specifically design
e 5th week. The leaf that developed demonstrate and confirm the p
after showed much more prominent type of relationship between the \
osis or the whole leaf was chlorotic. the planthopper vector as this is
e was no com seedling that known (Gingery et al., 1979; I
loped the symptoms with those 1973; Tsai, 1975). Hence, thi
sed to the inoculum consisting of the colonized plants were cut and
I colonies and MStpV ELISA- inoculum or virus sources after:
tive "Aguingay" plants (healthy) of daily monitoring for the present,
e 3). Other researchers found that colonies of the particular insec


Later forrr
mpletely <
ulkami, 1i
Experimc
;s expo.
alone or 1
"Aguinga
i seedling
is colonie!


ad to the insect from healthy Philippines (Exconde, 1977). Lik
gay" or from those healthy and this study extends on to the Phili
'-infected "Aguingay" plants alone and supports the results of Ginger
oarl Mf thm inet-t4 ITabhla '\ Thoa 14 /IQO41\ 4hat a.|* #f hk )A irnFIla


179: Grebel


k. ---.. -_--A r^ftilj


.. l..f Th-a imilp m--I
, Idamfln "Theiam, C-;Vnlft- rvffm ,.,








LJWL%-l.IIJI I V I I ICI41U QLI I1JJ


(L.) Monch.], barley (Hordeum vulgare L.) with MStpV alone. R. cochinchinensih
and R. cochinchinensis (Kulkarni, 1973; samples showing only the striping
Lastra, 1977). Navarro (2001) cited symptoms tested in ELISA show the
reports of others that the disease is consistent detection of MStpV suggesting
widespread throughout tropical and that such a symptom could be E
subtropical areas of the world and consistent biological phenotype of the
probably follows the distribution of P. hosts infected with the virus.
maidis. This includes Australia, East Infection of this weed by the virus
Africa, Amani and Tanzania, North to was further confirmed using SDS-PAGE
South America, Trinidad, Hawaii, Cuba, and western blot. In SDS-PAGE gels, z
Mauritius, and now the Philippines. 15.8 kDa protein was consistently
Thus, R. cochinchinensis serves as observed with the symptomatic, ELISW
reservoir for MStpV, as shown by our positive R. cochinchinensis and corn bu
study. The extent or distribution of MStpV not with the ELISA negative
infection in R. cochinchinensis and corn symptomless or healthy controls. Ir
around the country has to be determined, western blot, the 15.8 kDa protein signa
however. The weed is the favored host of appeared strongly when probed with th(
P. maidis outside of corn and therefore same MStpV-NCP antiserum. No suct
also a reservoir of P. maidis, as shown reaction signal developed with extracts o
locally by Catindig et al., (1996). The the healthy, ELISA negative R
insect is also the most significant insect cochinchinensis and corn samples. Thes(
vector of MStpV as this virus persists in it results indicated that the 15.9kDa proteir
and is also transmitted to its offspring. It was the MStpV-NCP and the virus in R
has also the strong potential of spreading cochinchinensis was MStpV or a virus
the virus even at long distances because closely related to the MStpV that infect,
it is very mobile. Adults were rarely corn in the USA.
observed in the weed between 10 am-4 Another but less distinct band o
pm on sunny and windy days unlike the about 39.8 kDa was also observed ir
nymphs that are quite sedentary in the SDS-PAGE gels and this was probably!


organisms are common in the Philippines
and are quite hard to control. These
conditions provide mechanisms for the
persistence and survival of the MStpV ir
the environment every year. Thus, this
virus disease, as it causes significant
yield reductions especially in sweet corn
presents a significant threat to corr
production in the country. The control o
R. cochinchinensis should be integrated
in in whatever scheme devised anc
implemented for the virus disease.

SUMMARY AND CONCLUSION

MStpV was the most common virus
detected by ELISA in symptomatic R
cochinchinensis samples with percent
infection of about 77.0% in combination


by ELISA or SDS-PAGE. More studies
have to be done for a better
understanding of the identity of this 39.E
kDa protein. Taken all the results of the
serologically analyses together, MStp\
infects and is common in R
cochinchinensis, especially with those
showing the stripe symptoms.
R. cochinchinensis serves as a
reservoir of MStpV and the virus spreads
within the R. cochinchinensis population
through its vector, P. maidis. The plant is
also a host and a reservoir of the insec
vector as colonies, in various stages o'
development, in its whorls and leaf axils
Since MStpV has been reported to cause
substantial yield losses in corn, the virus







I av =IIV Ca lu )IOjL


CuCilIlUfffIurI~wS brnuuIu oa Gcunsa reIu in
the development and implementation of
MStpV management.

ACKNOWLEDGEMENT

We would like to thank the Bureau of
Agricultural Research, Department of
Agriculture (BAR-DA) for the generous
financial support, Prof. B. Lockhart,
University of Minnesota, St., Paul. MN;
Prof. D. Gordon, Ohio State University,
Wooster, OH; Prof. B. Falk, University of
California, Davis CA; and Dr. G.J.
Miranda, International Rice Research
Institute (IRRI), Los Bafios, Laguna, the
Philippines for the gift of antiserum and to
Dr. Alberto T. Barrion, IRRI for confirming
the identity and the nice photographs of
Peregrinus maidis.

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TSAI, JH and TA ZITTER. 1982.
Transmission characteristics of








Detection of maize stripe


Table 1. Absorbance at 405 nm of symptomatic Rottboellia cochinchinensis using antiserum of
sugarcane.maize dwarf mosaic potyvirus (SC/MDMV), maize mosaic rhabdovirus
(MMV), maize stripe tenuivirus non-capsid protein (MStpV-NCP) and rice grassy stunt
tenuivirus capsid protein (RGSV-CP) in ELISA

Antiserum
Samplea SC/ MMV MStpV- RGSV- Remarksb
MDMV NCP CP
1 2.21 0.47 0.19 0.71 SC/MDMV
2 0.16 0.29 1.04 0.68 MStpV
3 0.18 0.38 0.14 0.42 No infection
4 0.42 0.63 2.02 0.88 MMV + MStpV
5 0.58 0.45 0.51 0.68 SC/MDMV + MStpV
6 0.84 0.57 0.62 0.88 SC/MDMV + MMV + MStpV
7 0.22 0.37 0.13 0.48 No infection
8 2.17 0.60 0.24 0.76 SC/MDMV + MMV
9 2.26 0.86 0.73 1.61 All four
10 2.18 0.54 0.15 1.46 SC/MDMV + MMV + RGSV
11 0.26 0.60 1.04 0.71 MMV + MStpV
12 0.24 0.47 0.22 0.81 No infection
13 1.56 0.82 0.87 1.61 All four
14 0.24 0.45 2.21 0.59 MStpV
15 0.22 0.32 2.26 0.80 MStpV
16 0.23 0.28 2.26 0.89 MStpV
17 0.20 0.45 2.11 0.99 MStpV + RGSV
18 0.39 0.76 1.57 1.96 MMV + MStpV+ RGSV
19 2.21 0.88 2.57 2.01 All four
20 0.18 0.45 2.16 1.31 MStpV + RGSV
21 0.16 0.35 2.25 0.57 MStpV
22 0.26 0.35 1.62 0.45 MStpV
23 1.86 0.64 0.47 1.59 All four
24 0.47 0.49 0.32 0.74 SC/MDMV + MMV
25 0.24 0.44 1.52 1.10 MStpV + RGSV
26 2.14 0.67 0.53 1.65 All four
27 0.42 0.61 1.79 1.18 MMV+MStpV+RGSV
28 0.38 0.57 2.15 0.85 MMV + MStpV
29 0.24 0.55 2.22 1.26 MMV + MStpV + RGSV
30 0.14 0.43 2.23 0.87 MStpV
Buffer 0.00 0.00 0.00 0.00 -
A healthy 0.43 0.42 0.41 0.88 -
M + 3SDO 0.45 0.47 0.44 0.89 -
% Infection 33.3 50.0 76.7 40.00 -
Sample was homogenized at 1 part: 3 parts buffer. From the preparation, aliquots were
obtained and assayed in ELISA using the indicated antiserum at a dilution of 1:1000. Samples
1 and 24 showed Type A symptom; all others had type B symptoms.
b Virus/virUses: SC/MDMV = sugarcane/maize dwarf mosaic potyvirus; MMV = maize mosaic
rhabdovirus; MStpV=maize stripe tenuivirus;RGSV = rice grassy stunt tenuivirus.
0 Absorbance value higher than threshold value which is mean (m) of healthy plus 3 standard
deviation (m+3SD) is considered positive. Values are means of two wells.








Navarro and Bajet


Table 2. Absorbance values (405 nm) of sap dilutions of symptomatic and healthy "Aguingay"
(Rottboellia cochinchinensis.) and cor (Zea mays L.) using antiserum to the maize
stripe tenuivirus non-capsid protein (MStpV-NCP)

Sample Sap Dilution"
2.5 2.5 2.5 2. 2.
A. M.StpV-NCP antiserum__
Symptomatic corn 1.95 1.96. 1.84 0.56 0.18
Symptomatic R. cochinchinensis 1 1.96 1.98 1.90 1.21 0.30
Symptomatic R cochinchinensis 2 2.11 2.12 2.05 2.05 0.62
Symptomatic R. cochinchinensis 3 2.11 2.12 2.04 1.97 0.19
Symptomatic R. cochinchinensis 4 0.49 0.50 0.41 0.00 0.00
Symptomatic R. cochinchinensis 5 2.09 2.10 2.14 1.94 0.76
Healthy com 0.22 0.31 0.18 0.00 0.03
Healthy R. cochinchinensis 1 0.00 0.26 0.01 0.00 0.00
Healthy R. cochinchinensis 2 0.30 0.43 0.20 0.13 0.00
Means:
Symptomatic R. cochinchinensis 1.75 1,76 1.71 1.43 0.37
Healthy R. cochinchinensis 0.15 0.34 0.11 0.06 0.00
A symptomatic sample typically showing the Type B symptom was homogenized at a ratio of 1g in 3 ml
carbonate buffer. From the preparation, aliquots were obtained and assayed in ELISA using the
antiserum at a dilution of 1:1000 as specified in the Materials and Methods. Values are means of two
wells.



Table 3. Transmission of the maize stripe tenuivirus (MStpV) in "Aguingay" (Rottboellia
cochinchinensis.) ("Itchgrass stripe") to corn (Zea mays L.) seedlings using the
indicated inoculum.

Inoculum Trial Number Total Infected
1 2
Experiment 1':
R. cochinchinensis, MStpV-ELISA positive
with Peregrinus maidis colonies 2/12a 2/158 4/27
R. cochinchinensis, MStpV-ELISA negative
with P. maidis colonies 0/15 0/15 0130
Experiment 2:
P. maidis alone (from R. cochinchinensis,
MStpV-ELISA positive) 3/15 2/12 5/27
P. maidis alone (from R. cochinchinensis,
MStpV-ELISA negative) 0/15 0/12 0/24
R. cochinchinensis alone, MStpV-ELISA
positive 0/12 0/12
R. cochinchinensis alone, MStpV-ELISA
negativeI 1 0/12 0/12
" Numerator is the number of corn seedlings that developed the stripe symptoms and were MStpV positive in
ELISA; denominator is the total number of corn seedlings exposed to the inoculum.
Source of inoculum was covered with plastic bag, carefully cut and placed at the base of the corn
seedlings inside a cage.
The P. maidis inoculum was dislodged from the R. cochinchinensis by bagging the infested plant and
storing it with the insect in a 4-6 C chamber for 20-30 min then shaken. The P. maidis was liberated on
and the R. cochinchinensis placed at the base of the corn seedlings inside separate insect cages.









Detection of maize stripe


Figure 1. Leaves of RdttboelHa cochinchinensis. showing two types of symptoms
of virus infection. Type A = fine mosaic consisting of small dots and
very thin or fine pale yellow lines of various lengths or chlorotic
areas in between and parallel to the minor veins, and Type B =
small chlorotic fine stripes wider at the base, taper and end toward
the middle of the leaf. Note the newly emerging leaves show longer,
wider stripes and the whole lamina chlorotic or whitish. C = healthy.
control.








I1 of Tropical Plant Patholoq'


ONING AND SEQUENCE ANALYSIS OF THE COAT PROTEIN
GENE OF ZUCCHINI YELLOW MOSAIC POTYVIRUS


D.E.V. VILLAMOR, E.Y. ARDALES and N.B. BAJET

University Research Associate, Assistant Professor and Professor, respectively,
rtment of Plant Pathology, College of Agriculture, University of the Philippines Los
s, College, Laguna


Reverse-transcriptase polymerase chain reaction was used to
amplify the full length zucchini yellow mosaic potyvirus coat protein
(ZYMV CP) gene from a squash infected with papaya ringspot
potyvirus (PRSV) and ZYMV. We designed ZYMV-CP gene specific
primers based on the multiple sequence alignments of several ZYMV
and PRSV isolates downloaded from the genbank. A PCR product of
about 837 base-pairs was obtained which corresponded to the
length of the region flanked by the primers. The amplified DNA was
cloned and the sequence of two representative clones was
determined. Nucleotide sequence analysis revealed that the cloned
PCR product also consisted of 837 nucleotides as reported for the
coat protein gene of ZYMV isolates from other countries. The two
clones differed from each other by only one nucleotide with clone 1
containing guanine (G) and clone 2 adenine (A) at the 12th position.
The amino acid sequence of the putative protein, consisting of 279
amino acids with an approximate molecular weight of 31 kDa, was
conserved for both clones. However. SDS-PAGE analysis of the











- .uiu.IJ, uIn I I umILI I I IL, uVILt I ,auoIuO y in tnis paper, we aescriDe Tne
devastating epidemics (Provvidenti, cloning of the coat protein gene of one
2000). Philippine isolate of ZYMV. Our strategy
In the Philippines, ZYMV is a was PCR based cloning using primers
relatively new virus affecting cucurbits. It specific for the full length ZYMV coat
was first reported in squash by Valencia protein gene.
et al. (1996). Since that time, however,
the virus has not received much MATERIALS AND METHODS
importance compared to other
pathogens of cucurbits as only a few Virus isolate propagation and
scientists or none at all have been purification. Several symptomatic
working on this field. A quantitative squash leaf samples were collected in
estimate of losses due to ZYMV has yet Bukidnon. The samples were assayed by
to be accounted. Recently, a number of ELISA for the presence of the following
symptomatic squash from different viruses: ZYMV, PRSV, CMV and WMV.
provinces in the country were identified by One sample (designated as SqZYMV)
ELISA as ZYMV-infected (NBBajet, showed a high absorbance reading for
manuscript in preparation). With this ZYMV and low absorbance reading for
scenario, therefore, the current status of the other viruses (NBBajet, manuscript in
ZYMV in cucurbits in the country is preparation). Sap from this sample was
expected to be much more significant. used for the propagation of ZYMV by
The strategies to control ZYMV are mechanical inoculation of 15-20 two-
are almost similar to all other aphid week-old healthy squash seedlings. The
transmitted viruses. The control of aphid inoculated seedlings were kept inside the
vectors using insecticides and cultural greenhouse for 21-24 days. Infected
practices such as intercropping and leaves were then harvested and the
reflective mulches have been employed putative virus was purified as described
(Shukla, 1994) but these strategies are by Suzuku et al. (1989). The
less effective compared to the use of concentration of the purified virus
rpciqtnrnt wariftic Vin rorfn Wi .cictant r.... -. ..-^- ,4 t .. \ /IL


engineering (recombinant DNA
technology). The latter approach gives
rise to what is known as pathogen-
derived resistance (PDR) that has gained
much popularity over the years. This
strategy involves the insertion of a
pathogen/virus gene into the plant's
genome and the resulting plant, called
transgenic, is resistant to the same
pathogen/virus. Coat protein mediated
resistance is the most common PDR
strategy employed against plant viruses
(Beachy et al., 1990; Fitchen and Beachy,
1993) and has proven successful in
conferring resistance to crops against
many virus groups, including ZYMV
(Namba et al., 1992; Fang and Grumet,
1993). A specific requirement of this
strategy is the isolation of the viral coat


Uc I I III II. UI Z..1t Il1111i ll r-UIl Ull,
1966).

Serological analysis by ELISA and
western blot. The indirect form of ELISA
(Koenig, 1981) was used to assay for the
presence of ZYMV and PRSV in the
SqZYMV and the purified virus
preparation. Antiserum of ZYMV came
from Dr. Y. Amemiya, Chiba University,
Japan while PRSV antiserum was kindly
provided by Dr. C. Malpica, formerly of
the International Laboratory for Tropical
Agricultural Biotechnology (ILTAB), The
Scripps Research Institute, La Jolla, CA
USA.
Western blot analysis was done to
detect the presence of the ZYMV coat
protein in the purified virus preparation.


VllldlllUI, /lUdalts dilU DdJUL







Cloning and sequence analysis


An equal volume of Laemmli sample
buffer (Laemmli, 1970) was added to the
purified virus and the mixture was heated
for 3 min in boiling water. The proteins
were separated by SDS-PAGE in a
12.5% acrylamide-bisacrylamide gel and
visualized by staining with Coomassie
blue. Estimation of the distance traveled
by the proteins was made by comparison
with protein molecular weight markers
(Gibco BRL, Life Technologies).
The separated proteins were
electrophoretically transferred to a 0.45-
pm nitrocellulose membrane (MSI,
Westboro, MA, USA) using a Bio-Rad
mini trans-blot cell (Bio-Rad Laboratories)
according to the manufacturers
instructions. After transfer, the membrane
was rinsed with Tris buffer-saline
(TBS:4.84 g Tris, 58.48 g NaCI, 1 I
distilled water and adjusted to pH 7.5) for
10 min. The membrane was incubated in
blocking solution (5% non fat dry milk in
TBS) for 30 min at room temperature,
washed twice (15 min each) with Tris
buffer-saline containing Tween 20 (TBS:
0.5 ml Tween 20 in 1 I TBS), and probed
with either ZYMV or PRSV antiserum
diluted 1:1000 in antibody buffer (5% non
fat dry milk in TBS). The membrane was
washed as described previously prior to
incubation for 1 hr at room temperature in
goat-anti rabbit IgG-alkaline phosphate
(GARAP) diluted to 1:5000 in antibody
buffer. The membrane was washed with
TBS as above and finally with TBS for 30
min. Bound GARAP was detected with
t',e substrates 5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium
(BCIP/NBT) in alkaline phosphate (AP)
color development buffer. When the
desired band appeared, the membrane
was washed with diluted water to stop the
reaction.

Total RNA extraction and first strand
cDNA synthesis. Total RNA from the
purified virus preparation was extracted
following the procedure of Miranda
(1999). Total RNAs were also extracted
from leaves of SqZYMV, healthy squash,
PRSV infected papaya and healthy


papaya using the Plant RNA Reagent kit
(Invitrogen cat no. 12322-012) following
the manufacturer's instructions.
First strand cDNAs were
synthesized from the purified viral RNA
and from total RNAs extracted from
healthy and infected plants using the first
strand cDNA synthesis kit (Invitrogen cat.
no. 11904-018) according to the
manufacturer's instructions. Two primers
were used: (1) the oligo (dT)12-18 primer
was used to all total RNAs and (2) a
specific primer for the ZYMV coat protein
gene (GSP: ZYMV-CPR-Ncol) was used
for RNAs from SqZYMV and the purified
virus.

Primer design and polymerase chain
reaction (PCR). The nucleotide
sequences of the coat protein (CP) genes
from several isolates of ZYMV and PRSV
were downloaded from the genbank
(http://www.ncbi.nlm.nih.gov) and were
aligned using the program ClustalX
(Thompson et al., 1997). Specific primers
for the coat protein gene of each virus
were designed from regions where there
were considerable nucleotide differences
between ZYMV and PRSV in the
alignment but with high degrees of
identity among isolates of the same virus
(Fig. 2). A total of 30 sequences of
ZYMZV-CP genes were downloaded. For
PRSV-CP, a total of 19 sequences were
obtained representing only the Southeast
Asian isolates.
The primer pair for the full length
ZYMV coat protein gene (837
nucleotides) was designated as ZYMV-
CPF-Ncol and ZYMV-CPR-Ncol. The
forward primer, ZYMV-CPF-Ncol,
consisted of 24 bases (18 nucleotides
specific to the ZYMV-CP gene plus the 6
nucleotides comprising the recognition
sequence of Ncol) and had a degenerate
base R at position 18. On the other hand,
the reverse primer (ZYMV-CPR-Ncol)
consisted of 31 bases (25 nucleotides
specific to the ZYMV-CP gene plus the 6
nucleotides comprising the recognition
sequence of Ncol) and had two
degenerated bases at positions 16 and 28








Villamor, Ardales and Bajet


with R and Y, respectively. Primers for the
PRSV-CP gene were designed to amplify
the 3' portion of the gene of
approximately 480 nucleotides. The
forward primer, PRSV-CPF, was 25
nucleotides long and had degenerate
bases at positions 8 and 11 with Y and R,
respectively. There were no degenerate
bases for the reverse primer, PRSV-CPR
(Table 1).

PCR cloning and sequence analysis of
the ZYMV-CP gene. Two ml of the
previously synthesized first strand cDNA
was used as a template for PCR
amplification (Biometra Uno). The PCR
reaction mix (20ul final volume) consisted
of 0.5 pM of each primer pair (either the
primer pair for ZYMV-CP or PRSV-CP),
20 mM Tris-HCL (pH 8.0), 50 mM KCI,
0.2 mM dNTP mix, 1.5 mM MgCI2, and
0.5 U Taq polymerase. The amplification
conditions consisted of heating at 94 C (2
min); followed by 25 cycles of
denaturation at 94 C (1 min), primer
annealing at 55 C (1 min), and extension
at 72 C (1 min); and a final extension at
72 C for 7 min. Ten ml of the PCR
products were analyzed by
electrophoresis through a 2% agarose gel
prepared in 1x TAE buffer.
When the 837 bp band was
observed after PCR, two to four ml of the
amplified products were cloned into the
topoisomerase (TOPO) activated vector
and the recombinant plasmid was
transformed into the TOP 10 E. coli
competent cells (Invitrogen cat. no.
K4600-01) as described by the
manufacturer. Five transformants were
selected and confirmed for the presence
of an insert by colony PCR and restriction
enzyme digestion. In the case of colony
PCR, transformant were resuspended in
the PCR cocktail as described previously.
The same set of conditions was adopted
as described above except that the initial
incubation time was increased from 2 to
10 min. For restriction enzyme digestion,
plasmid DNA was extracted from the
selected transformants using the alkaline
lysis method for small scale plasmid


isolation as described by Sambrook et al.
(1989). The plasmid DNA was digested
with Hind III to linearize the TOPO vector
and followed by EcoRI to drop the insert
(the putative ZYMV CP gene) from the
TOPO vector.
DNA from two representative clones
were sent to the Waikato DNA
Sequencing Facility, University of
Waikato, Hamilton, New Zealand for
sequencing using the external T7 and
SP6 primers. The nucleotide and the
predicted amino acid sequences of the
clones were assembled using the BioEdit
Software (Hall, 1999), a biological
sequence editor and analysis program.
The pairwise alignment menu of the
software was used to obtain and compare
the percent nucleotide and amino acid
sequence identities of the clones with the
corresponding sequences of the coat
protein gene of ZYMV isolates from other
countries.

RESULTS

Virus isolation, purification and
serological assay. Initial mosaic
symptom was observed on squash leaves
7-9 days after mechanical inoculation with
sap from SqZYMV. After 21 days, the
youngest fully expanded leaf showed
more severe symptoms that fit those
described and claimed as typical of ZYMV
infection (Fig. 1A). When a portion of this
leaf was assayed in ELISA, to gave a
very high absorbance value of 0.98 with
the antiserum of ZYMV and also weakly
reacted with antiserum of PRSV with an
absorbance value of 0.13. The isolate
was further propagated onto another
batch of squash seedlings and
subsequently, purified the putative virus
following the procedure of Suzuki et al.
(1989). Along the 20-50% sucrose density
gradient, a diffuse light scattering band
was observed occupying a portion of the
gradient between the 30-40% sucrose
concentrations (Fig. 1B). This band was
collected manually and the sucrose was
removed by diluting it in buffer and
pelleted by centrifugation at 60000 rpm in










3eckman Ti80 rotor for 1 hr. An aliquot of
he pellet from the band showed a UV
absorption profile typical of a
lucleoprotein (data not shown). Virus
rield was in the range of 2.5-5 mg virus
)er 100 g of infected tissue.
When the purified virus was
analyzed by SDS-PAGE, a protein of
approximately 36 kDa was observed (Fig.
IC, lane 3). In western blot, this protein
)ands reacted strongly to ZYMV
intiserum (Fig. 1D, lane 3) and weakly to
PRSV (Fig. 1E, lane 3) antiserum.
Moreover, an additional protein band of
approximately 29 kDa, which was not
observedd in SDS-PAGE, reacted to
EYMV but not to PRSV As.

:irst strand cDNA synthesis and PCR.
Specific PCR primers for the coat protein
genes of ZYMV and PRSV are
)resented in Table 1. The primers were
:ested for RT-PCR amplification of the
)urified virus and the crude extracts of
3qZYMV (Figg. 3A and B). With the
ZYMV primers, an 837 bp band was
amplified from the purified virus (Fig. 3A,
anes 2 and 3) and from crude extracts
:Fig. 3B, lanes 2 and 3). This was
observed for both the oligo (dT)12-18 and
3SP primed templates. On the other
land, a 480 bp band was amplified using
:he PRSV primers but was observed only
.or the oligo (dT) 12-18 primed cDNAs (Fig.
3A, lane 7 and 8; Fig. 3B, lane 7 and 8).
:or both primers, no amplification
products were obtained from healthy
controls (Fig. 3A, lanes 5, 6, 10 and 11;
cig. 3B, lanes 5 and 9). A 480 bp band
was amplified from PRSV infected
papaya using the PRSV primers (Fig. 3A,
ane 9) but there was no amplification
products when the ZYMV primers were
ised (Fig. 3A, lane 4).

Cloning and sequence analysis. Thee
337 band that was amplified from the
purified virus using the ZYMV primers
was successfully cloned into the TOO
vector. When analyzed by colony PCR,
all the putative cloned selected


)resumably harboring the full length
!YMV-CP gene produced the expected
137 bp band (Fig. 4A). Moreover,
estriction enzyme digestion of the
,lasmid DNA from the five clones also
revealed the 837 bp band (Fig. 4B).
Two of the five clones were
sequenced and the sequence obtained
revealed that the length of the cloned
ragment is indeed 837 nucleotide (Fig. 5)
is reported for the coat protein gene of
!YMV isolates from other countries. The
wo clones differed from each other by
)nly one nucleotide with clone 1
containingg guanine (G) and clone 1
adenine (A) at the 12th position. However,
he predicted amino acid sequence of the
wo clones was conserved as their
difference occurred only at the 3rd base
positionn of the 4th codon (Glutamine, Q)
Fig. 5). The predicted amino acid
sequence of the clones consists of 279
*esidues with an approximate molecular
weight of 31 kDa.
Comparisons of the nucleotide and
amino acid sequences of the two clones
with other 30 ZYMV isolates from other
countriess showed that the two clones
were 83-90% identical at the nucleotide
evel. At the amino acid level, the two
clones shared sequence identities of 89-
95% with the lower range observed only
n South Korea (SKI) and Reunion Island
solates (Table 2).

DISCUSSION

Dne of the requirements of gene cloning
s the isolation of a nucleic acid from an
organism of choice. This template must
De "pure" and free of nucleic acids from
theirr sources so that the resulting clones
are not contaminated. In the case of
3lant viruses, this is normally achieved by
extracting the viral genomic DNA or RNA
Trom purified preparations. However, with
:he advent of PCR based cloning, the
-equirement of "pure" nucleic acid
preparation is not necessary because a
specific primer pair could be used to
amplify a desired gene. In this paper, we










reporrea me '-Ul oasea cloning or tl
ZYMV-CP gene from squash infected w
ZYMV and PRSV.
PRSV and ZYMV belong to t
Potyviridae family of plant viruses. One
the major problems encountered
serology of potyviruses has been t
observation that antisera raised agair
individual members to this group cro
react with one another (Shukla et
1994). This phenomenon is due to t
presence of cross reacting epitopes in tl
highly homologous core region of tl
potyvirus coat proteins (Shukla et E
1988; Shukla et al., 1989a, b, c, i
SqZYMV was one from of the samples
symptomatic squash collected
Bukidnon. Our initial assays of tt
sample and the virus purified from it see
to follow the phenomenon stated abo
because: 1) By enzyme-linki
immunosorbent assay (ELIS
experiment, this isolate reacted stron
to the ZYMV antiserum with
absorbance value of 0.98 but weal
reacted to the PRSV antisenr
absorbancee value of 0.13); 2) when tl
purified putative virus was assayed
ELISA, the same pattern of results w
observed as above (i.e. with strong at
weak reactions for ZYMV and PRS
antisera, respectively; data not know
3) in western blot analysis of the purifii
virus, a protein of about 36 kDa had
strong and weak reaction to ZYMV ai
PRSV As, respectively (Fig. 1D, Fig.1E
Based on these results, we hypothesize
that our SqZYMV is purely ZYMV infect
and that its reaction to PRSV As is jusi
cross reaction brought about by tl
phenomenon described above.
In order to prove our hypothes
RT-PCR was done using primers speci
for the ZYMV-CP and PRSV-CP gene
Two types of first strand cDNA we
synthesized from the total RNA extract
from our purified virus preparation ai
from crude SqZYMV: 1) the first type w;
synthesized using an oligo (dT)12-18 prim
and 2) the second one was primed with
ZYMV coat protein gene specific prim<


synthesized from appropriate contrc
coming from total RNA extracts of PRE
infected papaya and healthy papaya ai
squash. Oligo (dT)12-18 primer primes tl
synthesis of cDNAs from organism
whose RNAs contain a poly A tail regic
at their 3' ends. Such RNAs include tl
mRNAs of eukaryotes (e.g. plants) ai
bacteria and the genomes of some-Rrl
viruses like the potyviruses. The potyvir
genome consists of a single stranded
positive sense linear RNA moleci
containing a poly A tail at their 3' ei
(Brunt et al., 1990; Matthews, 199
Shukla et al., 1994). GSPs, on the oth
hand, only primed the synthesis of tl
target gene. In this study, we used
reverse PCR primer (ZYMV-CPR-Nci
for the first strand cDNA synthesis of tl
full length ZYMV-CP gene.
The results of our RT-PCR reveal
two pattems: 1) An 837 bp product w;
amplified from the purified vin
preparation and SqZYMV using tl
primers for the full length ZYMV-CP gen
This product corresponded to the leng
of the region flanked by the primers ai
was obtained for both the oligo (dT)12
and GSP primed cDNAs (Fig. 3A, lani
2-3; Fig. 3B, lanes 2-4), 2). However
when the PRSV-CP gene primers we
used, the expected amplification prodi
of 480 bp was only observed from tl
oligo (dT)12-18 primed cDNAs (Fig. 3
lane 7; Fig. 3B, lane 7 and 8). This resi
indicated that mixture of PRSV and ZYIN
cDNAs were synthesized from the G.
(ZYMV-CPR-Ncol). These results cleat
show that our SqZYMV isolate and tl
purified virus preparation derived from
contains both PRSV and ZYMV. Henc
the positive reaction of SqZYMV and tl
purified virus preparation to both ZYN
and PRSV antiserum in ELISA was n
due to cross reaction but is an indicatic
of the presence of the two viruses in ti
extract.
The presence of two or mo
pathogens in an infected plant is comm(
for viral diseases and has been w
recognized as a rule rather th,
exception in the field. As for ZYMV, it


V111CA111VI, mlualub3








I and sequence analysis


frequently occurs together with other
common cucurbit viruses (Davis and
Mizuki, 19871; Brunt et al., 1990; Ullman
et al., 1991; Luis-Artega et al., 1998). It is
therefore not surprising to note that
SqZYMV from squash was doubly
infected with PRSV and ZYMV. These
two viruses are relatively difficult to
separate from each other because they
share common physical and biochemical
properties such as the presence of same
aphid vector species, non-persistent
pattern of transmission, common hosts
and same buoyant density in cesium
chloride gradients (Brunt et al., 1990).
Our attempts to biologically separate
these two viruses were not successful;
thus, we did not pursue this aspect further
and so we proceeded to clone the full-
length coat protein gene of ZYMV via the
PCR strategy. Through PCR, we have
demonstrated that these pairs of primers
were specific for each virus (Fig. 3A and
3B).
The results presented in Fig. 4A and
Fig.4B indicated that we have cloned the
full length ZYMV-CP gene. The sequence
of the two representative clones
comprised of 837 nucleotides (Fig. 5) as
reported for the coat protein gene of
ZYMV isolate, from other countries.
Although, the two clones differed by one
nucleotide, their predicted amino acid
sequence was conserved consisting of
279 residues with an estimated molecular
weight of 31 kDa. However, our SDS-
PAGE estimates revealed a higher value
of approximately 36 kDa (Fig. 1C). SDS-
PAGE estimates of the coat protein of
these two viruses have been reported by
other workers to be 36 kDa for both
PRSV (Gonsalves and Ishii, 1981; Yeh
and Gonsalves, 1985) and ZYMV (Lisa et
al., 1981). The 29 kDa protein band that
reacted to ZYMV As in western blot was a
result of partial degradation as was
observed by Suzuki et al. (1989) for


these methods, protein and gene
sequencing provide the most accurate
estimates. Therefore, the molecular
weight of Z-YMV-CP is around 31 kDa as
estimated from the predicted amino acid
sequence and the discrepancy obtained
from our SDS PAGE value was due to the
variation inherent of the method (Hames
and Rickwood, 1990).
The nucleotide and the predicted
amino acid sequences of our clone were
compared with those of the coat protein
gene of ZYMV isolates from other
countries. Overall, our clone exhibited
sequence identities of 83-90% and 89-
95% at the nucleotide and amino acid
level, respectively (Table 2). Comparison
of coat protein sequence data among
potyvirus has been widely accepted as a
molecular basis to differentiate distinct
viruses from its strains. Generally, distinct
potyvirus species possess amino acid
sequence identities ranging from 38-71%
whereas strains of the same virus
exhibited a 90-99% range and have N-
terminal sequences that are very similar
(Shukla and Ward, 1989; Shukla et al.,
1994) Our clone exhibited a lower range
value of 89% amino acid identity to the
South Korea (SKI) and Reunion Island
(RI) isolates, two out of thirty sequence of
ZYMV-CP gene examined. The same
value of 89% amino acid sequence
identity was also demonstrated between a
ZYMV isolate from US (Florida strain) and
from Reunion Island (Lin et al., 2000) and
yet they are considered strains of the
same virus. Therefore our clone is a
strain of ZYMV.
Examination of the predicted amino
acid sequence of our clone has revealed
the presence of the DAG motif at the N
terminal portion of the gene (Fig. 2). This
motif was essential for aphid transmission
(Atreya et al., 1990. Atreya et al., 1991;
Gal-on et al., 1992) and its alteration has
been shown to abolish its aphid
transmissibility as in the case of a
Singapore and an Israel isolate with GAG
and DTG, respectively, replacing DAG
(Lee et al., 1992). An exception to this is








villamor, Araales ar


from Taiwan with a DAT motif instead I
DAG (Lin et al., 2000). In the case of oi
isolate SqZYMV, we have yet to condu
insect transmission studies to determine
it is transmissible by aphids.
A study conducted by Lin et a
(2000) revealed four distinct genotypes
ZYMV based on their phylogenet
analysis of the Taiwanese ZYMV co
protein genes including isolates fro
other countries. Thomson et al. (199
distinguished WMV-2 from Australia
ZYMV by RT-PCR and examined tt
phylogenetic relationships among isolatE
by comparison of several publish(
potyvirus. In plant virology, phylogenel
studies determine relationships amor
similar genes from different strains which
may reveal a picture of variability up
the species level. An analysis
sequence variability is an essent
component in disease manageme
because it reveals the genetic structure
a pathogen/virus population. Mo
importantly, sequence variability has
implications in the use of CP genes f
transgenic resistance. Transger
resistance may only be effective agair
closely related viruses, strains or isolate
This specificity probably depends on tl
degree of relatedness of the transgene
the CP of the challenging virus (Nelson
al., 1988; Quemada et al., 1991, Sande
et al., 1992; Lornonosff 1995). In view
this, it is important to note that when c
clone was compared to ZYMV-(
isolates from other geographic origins, \
observed that no isolate had a greal
than 95% sequence identity (Table 2)
is possible that our clone represents
distinct strain or genotype to ZYMV bu
needs further investigation. We are E
trying to examine the phylogene
relationship of our clone to other ZYIV
CP isolates from other countries. T
biological properties of SqZYMV are al
still being determined. Moreover, we E
on the process of collecting ZYF
isolates from different regions of t
country in order to determine if differ


are prevalent ana nopeTully, aerermrn
the degree of variability among thes
strains. Nevertheless, the result
presented in this study demonstrate fi
the first time the molecular cloning ar
sequencing of the coat protein gene of a
isolate of ZYMV in the Philippines. Th
isolate can be used as a reference fi
comparison with other unknown strains i
the country.

LITERATURE CITED

ATREYA, CD B. RACCAH and T
PIRONE. 1990. A point mutation
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ATREYA, PL, CD ATREYA and
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BEACHY, RN, S LOESCH-FRIES ar
NE TURNER. 1990. Coat protei
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BRUNT, A, K CRABTREE and
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FITCHEN, JH and RN BEACHY. 19,
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ROSNER and B RACCAH. 1992. A
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LIN, SS, RF HOU and SD YEH. 2000. BEACHY. 1988. Virus tolerance, plant
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SHUKLA, DD, M TOSIC, J JILKA, R
QUEMADA, HD, D GONSALVES and JL FORD, RW TOLER and MA,
SLIGNTOM. 1991. Expression of coat LANGHAM. 1989c. Taxonomy (
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virus strain C in tobacco: Protection and sugarcane in Australia and th
against infections by CMV strains United States as determined b
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SAMBROOK, J, EF FRITSCH and T 79:223-229.
MANIATIS. 1989. Molecular cloning:
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Harbor, N.Y. WARD. 1989d. Localization of virui
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mosiac potyvirus and
r mosaic virus. Proceedings
" Annual Conference PMCP.
ty. May 1996.






ners used for cloning and reverse-transcriptase-polymerase chain reaction (R
Z) amplification.


ner Designation Sequence*


gene primers

PF-Ncol (forward) 5"-CCATGGTCAGGCACTCARCCAACT-3'
PR-Ncol (reverse) 5'-CCATGGCTGCATTGTRTTCACACCTAGYAAG-3'

'gene primers

PF (forward) 5'-CTCAATTYGARAAGTGGTATGAGGG-3'
PR (reverse) 5'-TCTCCGTGTTTTCTTCCTTGTTACTG-3'

e code: R = A+G and Y = C+T











Table 2. Percentage nucleotide and amino acid sequence identities of the coat protein gi
the Philippine isolate of ZYMV and isolates from other countries.

ZYMV Isolates Genbank Accession Percent Identity
Number Nucleotide Amino acic
Taiwan-NT1 AF127933 90 94
Taiwan-NLM1 AF127932 90 94
Taiwan-CY2 AF127930 89 94
Taiwan-TN3 AF127929 90 94
Taiwan-TC1 AF127931 90 94
Singapore X62662 86 91
Malaysia N/A* 86 92
Japan AB063251 90 95
South Korea 1 AF062518 86 89
South Korea 2 AJ429071 90 94
China-Hangzhou AF435425 90 94
China-Dongyang AF486822 89 94
China-Ningbo AY074810 90 94
China-Shanxi AY074808 83 90
China-Beijing AY074809 90 94
China-Hainan AF486823 88 93
China-Shandong AF513552 83 91
Italy ZYE420020 90 95
Germany-Berlin AJ420019 90 95
Slovenia AJ420018 90 93
Austria AJ420016 90 93


Hunagary-S1 AJ45995
Hungary-S2 AJ45995
Israel M35095
Reunion Island L29569
USA-Connecticut D00692
USA-California L31350
USA-Florida D13914
*Sequence information was not available
at the following IP address http://w~


89 93
90 93
90 94
84 89
90 94
90 95
89 91
rom the genbank and was obtained from the inti
igri.upm.edu.my/~szakaria/zymv.html


\/ill-mnr ArrinllaC anri Paida








Cloning and sequence analysis


Figure 1. A) Symptoms of zucchini yellow mosaic potyvirus in squash 21 days after mechanical
inoculation with sap frofn SqZYMV, B) Light scattering band (arrow) containing the
putative virus from SqZYMV in 20-50% sucrose gradient. C) SDS-PAGE of the
dissociated proteins of the purified virus. Lane 1, protein molecular markers; lane 2,
protein preparation from healthy squash; lane 3, protein preparation from the purified
virus, D&E) Western blots of the same gel in C probed with ZYMV (D) and PRSV (E)
antisera.










Villamor, Ardales and Bajet


YMV -.Korea -Ihx'
:Z Y W -t rT a T* SKnI







ZYXV Tn- TW-CY
S YV Tn TWT Tl
ZYXV-Tnw-WT'
ZfIl(v-TJS-TLI
prTMV -uSA- YL
ZTMV- USA-CW
ZYM1V-Ua- 3


YTMV- chana. 3bn
T.YMV Anat1A.rit
ZYV-Rungalry-s~
YW.VRunqary-KR
zYMV luraa ry K
YwMV-r -ungary Z4
SYMV- S I Nra


zyNV-OTsajnJapan
ZTY4V-S R ewa-ny
YWs-V ChIa-Hn
ptaw H T at ti-y



PRSV Tavirwn-Pg


PERSV-Ta-w Tn

PWSgV-Thaa. -Bsinga
PM.SV-Thax Cait
PR V-Tha. -CR l

PPRSV- Th a 1 --ChU2
PRSV- Tha i!. cKlt
PRSV-ThaX -akbM
PRSV-Tb.al MiA
'PRSv-Thathay-i
PRBV-halaysa.a
PRSV KrPS
PRBV-ZIndant -2
PRV In 1
;p.-im,i Rl


#20;
- r ;- -- I -'.- '-* cj :. | ,:*|'-^? -''"'


;*Xs :-;A" *^~-;"'M:W~




** ';: : ; t *,< ; y r -~ a i -a ~ 3 s t ?;$




: ; *:,*; *c; R .i '





4E4
*j1
** *~- yr- .* rriec ^y yte t;:: gfI" -:fii;;:~ ~ o%







-; : : -. *- -.!. W T & -,
*V - ^ -T












: -E : 3

:At 4 : -4 u- ,T *,4*^f^'s s y


Figure 2. Positions of the annealing sites of the PRSV-CP and ZYMV-CP gene primers derived
from the multiple sequence alignment of a coat protein genes from several isolates of
each virus. The alignment was made using the ClustalX program (Thompson et al.,
1997). Primers for full length ZYMV-CP and a portion of the PRSV-CP genes were
selected from regions enclosed in black and blue boxes, respectively.










Cloning and sequence analysis


--'lu-lw-
iitrf Cha -ulir"




mle-n.RII~
Wittr-Magapge









-ranr-a~u
*WM tv4

Win*simm5G
ra~dt-',tt



nw-c'
rn-mv-txu












rn-Thatue1
Wlslilldlll**











rnu-Cal-at




13-auf
aMgwgagy-r
Wlg-'lru-1lg
nIB
, ..g l-lo
,ppa gliAtta

ggperg-aggag**S
agg.ggaggg.Zags
AMP*MaiaB.
#e~t~ag
AIgpn~l
NWF. 4W-T


.. .... . I. I. ... .... .. -.-l *. .-I


' 45 "J"eU, G-\* c c-~ A 1 AGI-T.Z -O




% - I -
,'45 .1 -1....1 -,r. v ,S 4
745 r. .a 7 mc +.7



'4. .tfV :" .[ w1T ,.sr It..k". .4
':95 p, J+r9 t..J., .. -t4. Ynt-3 f. il ,,, u 6.4
"4% ..+ r I t *- ri t : 1'-6. ",
' -W; -W. r -







'46 -. "; ,, '.; r.- -,, 04

745a ,'" "" .r. ." "4

'.6 "c ; 'i" .'-*.'*"W k.l
1 "i f f 't. % !u
7-p i ,*T .. ac


o 16N-



-. ,- 'T ':" -' q. 04
.., : -- r- G %94

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.~0 g4
*~,. 9


--. r-

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+-


-I


-I

-I.


033


86 S






'I,-. 5" Y
.;-&' 6,,
t AP-,-*'

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rr-


Figure 2 (continued)

















*;T" W' 390 4& 4l9 4 *2
r.I
i i n.P ,h(
;.',v ^;n -inA-Sh l * '` '""-

V'i^' arngaputv

r iv pr;i-^l ind '1
Vp~ Pea .r nd;
-Yj~y i., ..,. 1-. *".-- - : ^ ..
3'-yw OIt'aa-sorU *""*'
MY Vn T-7N-TN: ^ '.
.'..IM -S **f-as-*-'' ^ "
Y:'MV-ln orea-2
;:?W-Tn S-T.C "
. ."W Tn ",


IYWV iruany
V- aral n :

:f M P -pan -. .
STWV-S -- -
ZyW-Chav*-Inan .


I SV-T a I *i py
PrIsV-Tar-T -



PtSV-ThU-im2 '











Cloning and sequence analysis


I . i ,:l ., li i. i. ;. t . .. .

*.**W -ChU'tri a etr-g : I '^ !. --* ^ -
I I -.1 9 rs

1)' If p


*rW '.I: tCT'-I'!] iR-i ,* *: /. ,
ZfiNV-Cu i 'Th '.': : *"









",V C*.hin~a Bi~ng
V;:M'- A --r'a









YMV liunAy FL
,',tL' n **n- .






-uyMY IfunAry 8
:,v IsA c : l
:;VMV Jt tAll
: : Y ?f yt s K o* 1
?.'!M~rlRa-lvst i ''. *'

.:um"tnhgary-3;' 52
YHV- bf'igary-K :*






2tNV Chiagmr Dy
.v1V 5TaVanIP
WV-Bu lary-y -


siNV tay-B


:;?MV-siov st *''*
*;fy-% a S K oi *
=I;V -JI p i<' '.
".XtfV.s s re a- l 1'* "Y* 1 *1 1.;1;1 :


xRV ? ~-P q
PRSqV l-TaX -cy :
PRSv-Taj.v-C

PR1V-ThaiT .


0.V Th. R~1
?BRSV-T;'i*-7nJ r- ; :'. **




lb S' Thei Cah
rrL TbMial k





RSV KPS
PRSV- -*' ".
VB 91 ~3r r
PkfSV- 1 Rat :


Figure 2. Continued








Villamor, Ardales and Bajet


1,1118 h Pff*m



(II 3 h



~I WI hri,


Figure 3.Agarose gel electrophoresis of reverse transcriptase polymerase chain reaction
products using ZYMV and PRSV CP gene primers. A) RT-PCR products derived from
the purified virus. Lane 1, 1 kb DNA ladder; lane 2 and 7, purified ZYMV (cDNA
synthesized using oligo (dT)12-18); lanes 3 and 8, purified ZYMV (cDNA synthesized
using GSP: ZYMV-CPR-Ncol); lanes 4 and 9, preparation from PRSV infected papaya;
lanes 5 and 10, preparation from healthy papaya; lanes 6 and 11, water. Note:
Amplification at lanes 2-6 and 7-11 used ZYMV and PRSV primers, respectively. B)
RT-PCR products derived from the crude extracts of SqZYMV. Lane 1, 1kb DNA
ladder; lanes 2 and 6, SqZYMV (cDNA synthesized using GSP: ZYMV-CPR-Ncol);
lanes 3 and 7, SqZYMV (cDNA synthesized using oligo (dT)12018; lanes 4 and 8,
purified ZYMV (cDNA synthesized using oligo (dT)12-18); lanes 5 and 9 healthy squash.
Note: Amplification at lanes 2-5 and 6-9 used ZYMV and PRSV primers, respectively.







Cloning and sequence analysis


.4f1 hp







B


4.361 bp..1..1

2.322 b
,027 hp











Figure 4.Confirmation of positive clones by PCR and restriction enzyme digestion. A) Agarose
gel electrophoresis of polymerase chain reaction products derived from the five
bacterial transformants containing the putative ZYMV CP gene. Lane 1, 1 kb DNA
ladder; lanes 2-6, five transformants; lane 7 non-transformed cells; lane 8, water; lanes
9 and 010, purified ZYMV. B) Agarose gel electrophoresis of the, putative plasmid ZYMV
CP gene clones after digestion with EcoRI and Hindlll. Lape. 1 and 12, DNA/Hind Ill
fragmentss; lanes 2-6 and 7-11 are EcoRI, and HindIll cut ONANHind III fragments;,aris
2-6 and 7-ft' are EcoRI and Hindill cut TOPO plasmid vector containing the putative
ZYMV C genhe, respecively.












Villamor, Ardales and Bajet


I' -- V A. A


3 1s _L 3


D r o Y


3 s A
*"" V f1 A C. S KE .. ***


A- y X


V 6 c a


-~c ); -a.


i ,I* ,





.4 ~


~ & z I Q


Philippine isolate of ZYMV. The 12h nucleotide is represented by N (itlicized and
colored blue) and is G and A for clones 1 and 2, respectively. The DAG motif is

enclosed in a box and highlighted in red.


- i
n a. o T


5 5


"A t 1.o










of Tropical Plant Patholoav 39:36-,


. otui.J~p;ll;ac IId.usivjpud, uipluuid ulsedde, GUilnlUK suDSiraie, t-'uK








Pascual and Relevante


range. S. macrospora appears to be more
generally widespread in warm and humid
climates than S. maydis. S. macrospora
was first reported in the Philippines in
1931 by Stevens and Celino as the
causal organism of leaf spot of corn.
The use of molecular techniques is a
more specific and sensitive approach of
identifying and detecting microorganisms
although conventional methods are often
used as a routine (Xia and Achar, 2000).
A variety of molecular tools have been
successfully established for various
organisms. In many fungi, analysis of the
internal transcribed spacer (ITS) region
has been used for differentiation of
species (Brisbane et al., 1995; Lovic et
al., 1995; Shi et al., 1996; Hyun and
Clark, 1998; Guzman et al., 1999).
Ribosomal RNA genes in fungi are
conserved and contain sequence
components reflecting different
evolutionary rates, which are
phylogenetically and taxonomically
informative (Bruns et al., 1991; Liu and
Sinclair, 1993). Up to now, there have
been no extensive studies done on S.
macrospora. The objective of this study is
to obtain information on the characteristic
of S. macrospora using conventional and
PCR techniques.

MATERIALS AND METHODS

Colony morphology of isolates. The
pathogen was isolated from the margins
of diseased leaf tissues and corn seeds
of infected ears. Leaf sections (5x5
mm2) and corn seeds were soaked in
10% sodium hypochlorite for 4 min,
washed 3x with sterile distilled water, blot-
dried on sterile paper towel, placed on
plated potato dextrose agar (PDA) and
incubated at 250C under continuous light.
The isolates were maintained at 250C on
PDA. S. macrospora isolates from
Mindanao and Northern Luzon were
grown on different media such as PDA,
oatmeal agar (OMA), V8 juice agar, corn
plant decoction agar and different


substrates which include corn plant parts
such as leaves, stalks, grains and corn
husk cut about 1 x 2 cm2 and toothpick
soaked in potato-V8 broth. The substrates
were placed in bottles or flasks and were
sterilized. About 5 mm2 mycelial disk was
cut from the margin of expanding young
cultures on PDA, placed at the center of
plated media and in the sterile substrates
and incubated at 250C and at room
temperature (300C) under continuous
light. The colony characteristics of the
isolates were evaluated visually everyday
for 4 weeks after incubation and further
observation for pycnidia formation was
done for the next one month. The mycelial
growth rate was also determined daily at
15, 20, 25, 28, 30, 32 and 350C on PDA
for 5 days. Each treatment was replicated
three times.

DNA isolation and quantification. The
genomic DNA of S. macrospora was
isolated following the procedure of Lee
and Taylor (1990) with some
modifications. Pure fungal culture was
grown on oatmeal broth at room
temperature for 10-15 days. Harvested
mycelia were powdered in liquid nitrogen
with a sterile mortar and pestle,
transferred into 1.5 ml microcentrifuge
tubes, suspended in 500 ul lysis buffer
(50 mM Tris-HCL, pH 7.4, 50 mM EDTA,
3% sodium dodecyl sulfate and 1%
mercaptoethanol), vortexed and
incubated at 650C for 1 hr in a waterbath.
The samples were extracted twice with
500 pl phenol/chloroform/isoamyl alcohol
25:24:1 v/v/v). After centrifugation, DNA
was precipitated by adding 10 pl 3M
sodium acetate and 500 ul isopropanol
into the aqueous phase and centrifuged
for 5 min. The pellets were rinsed with
70% ethanol, air-dried for 1 hr and
resuspended in 100 pl TE buffer (10 mM
Tris-HCL, pH 7.5 and 0.1 mM EDTA).
DNA was pooled and 100ul TE buffer was
added. DNA was treated with 5 pl RNase,
incubated at 370C for 1 hr and extracted
*Sequentially with 500 pl equilibrated











phenol/chloroform/isoamyl alcohol into light brown once pycnidia start to
25:24:1). After RNase digestion and form. Black pycnidia containing
)henol-chloroform/isoamyl alcohol- pycnidiospores were formed in OMA, V-8
extraction, the DNA concentration was and corn husk substrate three weeks after
estimated by running the samples incubation and 6 weeks after incubation in
together with a standard DNA marker of PDA. Pycnidia were not formed in other
mnown concentration on 1% agarose gel substrates. OMA and corn husk produced
it 50 V/cm for 45 min. The gel was the most abundant pycnidia based on
stained in 0.5 pg/ml ethidium bromide visual observation. Most of the pycnidia
solution for 15 min and photographed, were submerged in the agar although
Fhe DNA samples were stored at 40C some were formed as aggregates on the
intil use. surface of OMA (Table 2, Fig. 1).
Formation of pycnidia was very rare in
>CR amplification of rDNA-ITS region. PDA and once formed, they were all
The rDNA-ITS region including 5.8s gene submerged in the agar, hence no
vas amplified through polymerase chain pycnidiospore was recovered. Similar
action (PCR) using the universal results was reported by Alovera (2001) on
>rimers, ITS1: 5' OMA. Corn husk can be used as an
CCGTAGGTGAACCTGCGG 3' and inexpensive substrate for inoculum
TS4: 5' TCCTCCGCTTATTGATATGC 3'. production of S. macrospora especially
amplification reactions were performed in for large-scale screening for resistance to
5 ul reaction volumes containing 1x Taq this pathogen, which was proven in the
icubation buffer, 40 uM each of dATP, previous findings (Pascual et al., 2002).
ICTP, dGTP and dTTP, 0.3 uM primer, Growth curves of 10 isolates were
iO ng of genomic DNA, 1 U Taq DNA generated from mycelia growth over time.
)olymerase (Gibco, BRI) and sterile PCR The optimum temperature for the isolates
vater (Gibco, BRI). Amplification was tested was within 25-320C based from
lone on PTC-Cooled Thermal Cycler these growth curves. Other workers had a
Rising the following reaction conditions notion that S. macrospora thrive best at
iitial denaturation at 930C for 1 min colder places because the disease was
allowed bv nhc r k, h L.. -


>r 2 min at 720C with a final extension for
min at 720C. Five (5) pi of each reaction
as analyzed on 1.5% agarose gel run at
O V/cm for 45 min in 1x TAE: buffer.

RESULTS AND DISCUSSION

characteristics of S. macrospora
olates. The ten isolates used in this
:udy are listed in Table 1. The isolates
produced a vigorous and cottony white
iycelia on OMA and relatively thin grey-
hite mycelia on PDA after 1-week
cubation. No differences on mycelial
)lor and colony characteristics were
suallv observed amnnn the inlntAc nn


--i 1 %In l ',iJULiaLO iLU all U Im>dU Id
during our survey and collection activity
nd through communication with other
ssearchers (Alovera, 2001; Dalmacio,
001). But since the optimum
temperature for growth of S. macrospora
ras within 25-320C range, this suggests
iat colder temperature is not the critical
actor for severity of the disease in corn
reas in the highlands of Bukidnon and
;otabato. Moisture played an important
)le in disease development as observed
I our previous screening activities at IPB
disease nursery. The set of samples for


130







Pascual and Relevante


and ear infection than those sets that
were watered only once a day (data not
shown). However, production of pycnidia
was affected by temperature. Culture of
S. macrospora on OMA and corn husk
substrate produced more pycnidia at
colder temperature (250C) than at room
temperature (300C) (Table 2). This
implied a great possibility of high
inoculum density leading to high
incidence of corn leaf and ear infection at
colder temperature.

Amplification of rDNA-ITS region. In
this study, the ITS region rDNA, including
the 5.8S rDNA from isolates of S.
macrospora, was amplified. All isolates
revealed the same size of PCR product,
approximately 575 bp, after amplification
with ITS1 and 1TS4 primers (Fig. 3).
Similar to the result of Xia and Achar
(2000) on their work on PCR markers for
the differentiation and detection of S.
maydis in maize seeds, 575 bp fragment
size was obtained also from S. maydis
and S. macrospora ITS region. They were
able to design species-specific primer for
S. maydis from their amplified product.
Our results indicate that the 575 bp
amplified ITS region of S. macrospora
could be useful for rapid detection and
identification of this pathogen.
To detect any variation among
isolates, restriction enzyme digestion of
the amplified ITS region will be conducted
as was done by other workers (Liu and
Sinclair, 1993; Pascual et al., 2000;
Priyatmojo et al., 2001 and Xia and
Achar, 2000). This technique relies on
only one specific area of differentiation
and does not take into account variation
of the entire genome (Lovic et al., 1995;
Hyun and Clark, 1998). The use of other
PCR-based molecular markers like rep-
PCR and RAPD may provide also the
extent of variation among different
isolates (Toda et al., 1999).


This study aims to propose a
simple process of producing abundant
inoculum and methods of identifying
isolates of S. macrospora using
conventional and molecular procedures.
The basic information obtained from
these techniques set the groundwork for
inoculum production for Stenocarpella
leaf blight and ear rot resistance
evaluation for large-scale screening and
for studying the population structures of
S. macrospora.

LITERATURE CITED

Alovera, RB. 2001. Effect of
environmental factors on
Stenocarpella macrospora (Earle)
Sutton and disease yield loss
relationship as affected by site
inoculation and inoculum
concentration in corn (Zea mays L.).
PhD Dissertation, UPLB, College,
Laguna, Philippines. 122 p.

Brisbane, PG, SM Neat, CE Pankhurst,
NS Scott and MR Thomas. 1995:
Sequence-tagged site markers to
identify Rhizoctonia solani AG4 or 8
infecting wheat in South Australia.
Phytopathology 85:1423-1427.

Bruns, TD, TJ White and JW Taylor.
1991. Fungal Molecular Systematics.
Ann. Rev. Ecol. Syst. 22:525-564.

Dorrance, AE, OL Miller and HL
Warren. 1999. Comparison of
Stenocarpella maydis isolates for
isozyme and cultural characteristics.
Plant Dis. 83:675-680.

Guzman, P, P Gepts, S Temple, ABC
Mkandawire and RL Gilbertson.
1999. Detection and differentiation of
Phaeoisariopsis griseola isolates with
the polymerase chain reaction and
group-specific primers. Plant Dis.
83:37-42.










IV danU IM % ,Idi. IUO0. /- ldlySi ai
usarium lateritium using RAPD p
rDNA RFLP techniques. Mycol.
102:1259-1264. Pasc
S
,B. 1942. Natural mode of ei
nce of fungi into corn ears and m
Symptoms that indicate el
tion. J. Agric. Res. 64:421-442. S
Ti
,FM and AE Rossi. 1983.
ocarpella macrospora (syn = Priya
idia macrospora) and S. maydis Ti
maydis) compares as pathogens K
rn. Plant Dis. 67: 725-729. C
01
and JW Taylor. 1990. Isolation G
JA from fungal mycelia and single ni
as. In: Innis. M.A., D.H. Gelfand, p
Sninsky, T.J. White (eds), PCR
)cols. A guide to Methods and Shi,
cations. Academic Press, San C
3. 282-287 pp. ol
w
IR, RD Martyn and ME Miller. ril
. Sequence analysis of the ITS p
ns of rDNA in Monosporascus
to evaluate its potential for PCR- Steve
ated detection. Phytopathology di
55-661. A

.and JB Sinclair. 1993. Toda
rentiation of intraspecific groups 1!
i AG-1 of Rhizoctonia solani w
Sribosomal DNA internal ar
cribed spacer and isozyme R
arisons. Can. J. Pathol. 15:272- M

Xia,
RF. 1989. Field Crops Diseases ar
Book. 2nd ed. AVI Book. Van pc
*and Reinhold, New York. 108- th
)p. Si
J.
, CB, T Toda, AD Raymundo
M Hyakumachi. 2000.
acterization by conventional
liques and PCR of Rhizoctonia
1i isolates causing banded leaf InstitL
and E


baiadLII UIIyIIL I1 illlitie. ridaii
ology 49(1):108-118.

, CB, PS Guzman and AM
ear. 2002. Reliable and
omical inoculum production
od and disease resistance
nation techniques to
ocarpella macrospora in maize. J.
Plant Pathol. 38:1-8.

lojo, A, VE Escopalao, NG
onan, CB Pascual, H Suga, K
!yama and M Hyakumachi. 2001.
acterization of a new sub-group
Thizoctonia solani anastomosis
p 1 (AG1-ID), causal agent of a
)tic leaf spot in coffee.
)pathology 91:1054-1061.

, P Loomis, D Christian, LM
s and H Leung. 1996. Analysis
e genetic relationship among the
it bunt fungi using RAPD and
omal DNA markers.
)pathology 86:311-318.

, FL and MS Celino. 1931. Two
ises caused by Diplodia. Phil.
.20:370-374.

M Hyakumachi and DK Arora.
. Genetic relatedness among and
1 different Rhizoctonia solani
tomosis groups as assessed by
), ERIC and REP-PCR.
biol. Res. 154:247-258.

ind PN Achar. 2001. Random
fied polymorphic DNA and
erase chain reaction markers for
differentiation and detection of
)carpella maydis in maize seeds.
ytopathol. 149: 35-44.

CKNOWLEDGEMENT

lis work was supported by the
of Plant Breeding, UP Los Bahos
3AR.











Table 1. Geographic origin and colony c
from infected corn leaf and eai

Isolate Geographic Origin

DillPB IPB, UP Los Bafios
Di2TSCot Tupi, South Cotabato
Di3Aur Aurora
Di4Delsa Dammang East
Echague, Isabela
Di5BSIsa Benito Soliva, Isabela
Di6MBuk Malaybalay, Bukidnon
Di7MFBuk Manolo Fortich,
Bukidnon
Di8Lbuk Libona, Bukidnon
Di9DMBuk Dalwangan, Malaybala
Bukidnon
Di10QBuk Quezon, Bukidnon


Table 2. Pycnidia formation of Stenocc
media and substrates1 and tw

Medium/ Pycnidia
Substrate Formation2
25uC 30uC
Potato Dextrose + None
Agar
Oatmeal Agar +++ ++

V-8 juice Agar +++ +
Corn leaves None None
decoction agar
Corn leaf None None
substrate
Corn stalk None None
substrate
Corn grain None None
substrate
Corn husk +++ +++
substrate
Toothpick None None
substrate
The same result for ten S. macrospora
2Based on visual observation; + = few a
+++ = abundant amount of pycnidia;
3 WAI weeks after incubation


acrensucs or srenocarpea ni~auuspuira Isuida


Colony Characteristics
Potato Dextrose Agar Oatmeal Ag.
Off-white, thin Cottony white, thi
Off-white, thin Cottony white, thi
Off-white, thin, flat Cottony white, thi
Off-white, thin, flat Cottony white, thi

Off-white, thin Cottony white, thi
Off-white, slightly thick Cottony white, thi
Off-white, thin Cottony white, thi

Off-white, thin, flat Cottony white, th
Off-white, slightly thick Cottony white, th

Off-white, slightly thick Cottony white, th


4la marcospora isolates grown in different culture
*mperature conditions

Onset of Pycnidia Formation Other Observ
(WAI)3 on Pycnidi
25uC 30UC
6 All submerged
agar
3 5 submerged; so
form aggregate
3 6 mostly submer
-







3 5 On hush surface



lates.
unt, ++ = moderate amount,





"I I-








Journal of Tropical Plant Pathology 39:43-48


INTEGRATED MANAGEMENT OF ONION ANTHRACNOSE
[COLLETOTRICHUM GLOEOSPORIOIDES (PENZIG)
PENZIG & SACC.]


R.T. ALBERTO1, M.V. DUCA2 and S.A. MILLER3

'Professor 1, Department of Pest Management, College of Agriculture, Central
Luzon State University, Science City of Muioz, Philippines; 2Science Research
Specialist, Crop Protection Division, Philippine Rice Research Institute, Maligaya,
Science City of Muioz, Nueva Ecija, Philippines; 3Associate Professor, Department of
Plant Pathology, College of Agriculture and Environmental Science, OARDC, Ohio State
University, Wooster, OHIO, USA.


A field study was conducted at PhilRice Central Experiment
Station in Maligaya, Science City of Muiioz, Nueva Ecija to develop
integrated management strategies against anthracnose of onion.
Evaluation ;of different combinations of disease management
practices showed that wider spacing (18 x 20 cm) of onion seedlings
with low nitrogen (60 kglha) application and mancozeb application at
7 days interval significantly reduced the incidence, severity and area
under the disease progress curve (AUDPC) of anthracnose of onion.
However, the yield did not differ significantly with the other
treatments except with the treatment with no mancozeb, standard
spacing and low nitrogen.

Key Words: integrated management, anthracnose, onion


INTRODUCTION

Anthracnose is currently the most
destructive disease of onion in the
Philippines (Alberto et al., 2001). Its
occurrence in onion-growing areas in
Luzon was observed only in the past
three years. It is caused by Colletotrichum
gloeosporioides and its most prominent
symptom is twisting of leaves and stems,
thus popularly called "twister" by the
farmers (Baltazar, 2001). In the 2001
cropping season, anthracnose destroyed
several onion farms in Luzon when
unusually heavy rainfall occurred in
February of the said year.
At present, the use of chemical
fungicides is the only effective means of
control. In using chemical control,
however, it is important to know the


frequency of application with less
negative impact to non-target organisms.
The fungicides should be used effectively
in combination with other control
measures with less environmental and
health hazards and to prevent the
development of resistance of the
pathogen to the fungicides. Moreover,
attempts were also made to develop an
alternative control strategies other than
the use of fungicides.
The study was conducted to compare
cultural and chemical control and their
combinations to manage anthracnose of
onion and to determine the effective
frequency of application of commercial
fungicide against anthracnose of onion.










-w ,,.., WmlxeU w 5ln siciRer nloes5ICK, on
water) and applied at the rate of
s conducted at PhilRice ai/ha 5 days after symptom deve
ment Station, Maligaya, Only the amount of nitrogen (N]
M iAnh 7 Mi llvna Friin frnm ..-, : ..':. k ^-, I.- ... I


houses. Thirtv Dlots


accommodate six treatments with five
replications each and arranged in RCBD.
Each plot was prepared into a raised bed.
The treatments were:

* T1 mancozeb (at the rate of 800g
ai/ha applied at 7 days interval,
wide spacing (18 cm x 20 cm),
low N (60 kg/ha)
* T2 No mancozeb, wide spacing,
standard N (120 kg/ha)
* T3 mancozeb applied at 14 days
interval, wide spacing low N
* T4 mancozeb applied at 7 DI,
standard spacing (10 cm x 15
cm), standard N
* T5 No mancozeb, standard
spacing, low N
* T6 Control (No mancozeb,
standard spacing, standard N)

Forty-five days-old seedlings were
transplanted in raised beds with eight
rows per bed at the plant spacing
required in the treatments. Three weeks
after transplanting, the plants were
inoculated with 14-day-old spore
suspension of C. gloeosporioides (spore
density of 2.5 x106 with 10 drops of
Tween 80/liter of the suspension). The
inoculated plants were covered with
plastic sheaths overnight and removed
the following morning. This was done for
3 consecutive days to induce infection.
During the day, overhead irrigation (Alha
Rainbird, 1.5-2.0 bars water pressure with
rotating nozzles) was used to simulate
rainfall and to maintain near 100%
relative humidity. Relative humidity was
maintained by running the overhead
irrigation from 9.0 am to 3:00 pm when it
was very hot and from 10:00 am to 2:00
pm when it was cloudy. Mancozeb was


handweeding.
Plants in all plots were evaluated for
ease incidence and disease severity at
ekly intervals using the following
mula:

disease Incidence = No. of Infected Plants x 100
Total no. of plants

disease severity=n(1)+n(3)+n(5)+n(7)+n(9) x 100
Nx9

lere: n= number of infected plants
classified by grade according
to scale
N = total number of samples

Scale Percent Infected
Plant Part

0 none
1 1-5
3 6-12
5 13-25
7 26-50
9 >50

Based on the disease severity,
JDPC was computed using the
Lowing formula by Shaner and Finney
977) as follows:
n
JDPC= Z [Yi+nl+Yi/2][Xi+1-Xi]
1=1

'here: Yi = anthracnose severity (per
unit) at ith observation
Xi = time (days) at the ith
observation
N = total no. of observation

ie percent control was computed using
a formula:

% Control = CIV -TIV x 100
.lv













Where: CIV = Control Incidence Value
TIV = Treatment Incidence
Value

Onion bulbs from 1m2 area at th
center of each plot were harvested
counted, cleaned and weighed. Dal
gathered were summarized, analyze
using SAS and means compared among
the treatments by Duncan Multiple Rang
Test (DMRT)

RESULTS AND DISCUSSION

Combination of different cultural an
disease management practice
significantly reduced the incidence an
severity of anthracnose in onion. Amon
the different combinations, wider spacin
(18 x 20 cm) of planting with low nitrogen
(60kg/ha) application and application (
mancozeb at 7 days interval (T1) had th
lowest incidence (Fig.1), severity an
AUDPC (Table 2) of anthracnose which
resulted in higher percent control of 71'
42 days after inoculation (Table 1). Wid4
distance of planting actually reduces th
high relative humidity in the plant
microenvironment which is unfavorable
for anthracnose development. Lo
nitrogen application, on the other hanm
makes the onion leaf tissues harder an
less succulent to attack of the pathogen
resu,4 whiettls-etmi to what has bee
described bytaWamer and co-worke
,Cerkaukas anl Zhang (2003) in soft rot (
chinese cabbage, Webster (1997) in steel
rot of rice and Anonymous (2002)
anthracnose of avocado. These factors
combination with weekly application (
mancozeb provided adequate protectic
of onion plants against anthracnose und<
field conditions.
Lower percentage reduction
disease incidence (67.50%) was achieve
in plants not soraved with mancozeb. an


spacing and low N (T5) resulted in
66.50% and 63.75% control, respectively
which is better than the combination of
mancozeb application at 7 days interval,
standard spacing and standard N (T4)
with 61.75% control. The highest
incidence (40.0%), severity (19.4%) and
AUDPC (29.4) of anthracnose was
observed in onion plants planted at
standard spacing (10 x 15 cm) applied
with standard rate of nitrogen and not
sprayed with mancozeb (T6).
The yields did not vary significatnly
among the different treatments except
between T2 (No Mancozeb, wide spacing
and standard N) and T5 (No Mancozeb,
standard spacing and low N) where the
lowest and highest yields were obtained,
respectively (Fig. 2). The difference
between these two treatments were the
plant spacing and rate of nitrogen (N)
application indicating that the application
of mancozeb did not have any significant
effect on yield. Though the disease
incidence, severity and AUDPC in these
two treatments (T2 and T5) were
significantly different from each other, it
appeared that plant spacing and nitrogen
(N) rate are more critical in affecting yield
irrespective of the incidence of the
disease.
Results further showed that wider
spacing (18 x 20 cm) might have reduced
disease incidence and severity but had
resulted in -1wer yields compared to
those with the standard spacing of 10 x
15 cm. In the standard spacing, plant
density was higher than in the wide
spacing, thus, higher yields were obtained
despite the higher incidence and severity
of the disease.

CONCLUSION










application might help in the control
especially if the application is made at
weekly interval. Plant spacing of 10 cm x
15 cm gave higher yields compared with
the wide spacing of 18 cm x 20 cm y
although disease incidence and severity
were lower at wider spacing. Low nitrogen
(N) application is a very practical means
of managing the disease. It is easy to
implement and the effect in controlling the
disease is significant.

LITERATURE CITED

ALBERTO R.T, MS DUCA, AND S.A.
MILLER. 2001. First report of
anthracnose of onion (Allium cepa L.),
causes by Colletotrichum
gloeosporioides (Penzig) Penzig &
Sacc. in the Philippines. J. Tropical
Plant Pathol. 37(1):46-51. 1
Ii
ANONYMOUS, 2002. Responses in C
tropical fruit to Colletotrichum. (I
Cooperative Research Center for C
Tropical Plant Protection. 45p. \
L
BALTAZAR, AM. 2001. Integrated pest F
management in rice-vegetable ir
systems: Mature and promising F
technologies. Paper presentation. C
PhilRice external review. Philippine C
Rice Research Institute. Maligaya, L
Mufoz, Nueya Ecija. a
c
SHANER, G, ABE FfilEY. 1977. The
effect of nitrbgen fertilization on the


expression of slow-mildewing
resistance in knox wheat.
Phytopathology 67:1051-1056.

RNER, JR, CERKAUKAS, T ZHANG.
2003. Nitrogen management for
controlling petiole spotting, bacterial
soft rot of chinese cabbage.
Agriculture and Agri-food Canada,
Greenhouse and Processing Crops
Research Center, harrow. 55p.

BSTER. RK. 1997. Update on pest
management crop development.
Department of Plant Pathology,
University of California, Davis. 6(1): 1-
9.

ACKNOWLEDGEMENT

s research was supported by the
grated Pest Management
aborative Research Support Program
I CRSP) which was funded by USAID
nt No. LAG-G-00-93-00053-00 with
linia Polytechnic Institute and State
/ersity as management entity,
ippine Rice Research Institute as lead
itution and University of the
ippines Los Baios, Central Luzon
:e University, Leyte State University,
o State University, Pennsylvania State
/ersity and Asian Vegetable Research
Development Center as
aborating institutions.


46











Table 1. Percent incidence and control of


Management Practices


T1 Mancozeb 7DI, Wide spacing, Low I
T2 No Mancozeb, Wide spacing, Std. F
T3 Mancozeb 14 DI, Wide spacing, Lov
T4 Mancozeb 7DI, Std. Spacing, Std. h
T5 No mancozeb, Std. Spacing, Low N
T6 Control (No Mancozeb, Std. Spacini

Means followed by the same letters are r
significance.


Table 2. Percent incidence and area unde
anthracnose forty-two days after


Management Practices


T1 Mancozeb 7DI, Wide spacing, Low I
T2 No Mancozeb, Wide spacing, Std. tF
T3 Mancozeb 14 DI, Wide spacing, Lo\
T4 Mancozeb 7DI, Std. Spacing, Std. Ih
T5 No mancozeb, Std. Spacing, Low N
T6 Control (No Mancozeb, Std. Spacin!

Means followed by the same letters are r
significance.


:hraCnose forty-two days after inoculation.


% Incidence* % Control


11.6d 71.00
13.0c 67.50
1 13.4c 66.50
15.3b 61.75
14.5b 63.75
Std. N) 40.0a 0.00

significantly different at 5% DMRT level of



he disease progress curve (AUDPC) of
3culation.


% Incidence* AUDPC


13.9d 23.9d
13.9d 23.9d
J 13.9d 23.9d
15.0c 25.0c
16.1b 26.1b
Std. N) 19.4a 29.4a

significantly different at 5% DMRT level of








Alberto, Duca and Miller


45 0





20.0

10,0
5C"
0:.


14 28 35 42


-- tManconrt. 'a Vkle
spacing -ow N
-- W&o*Mancoueb. VWce
spacing Sa N
gpecing L N
+ No Manc rt-t S ld


spacing Low N
Si- M"rDt : Std
spaecMg cSd N

spacing LIM* N
--* -Cntrol. tC1 itCang
Std N


Days after inocustion



Figure 1. Disease progress curves of the incidence of anthracnose in onion at different
combinations of disease management practices (CES, PhilRice, Munoz, Nueva Ecija,
2003 Dry Season).


17.;
.t ?


14 t!
>p
0


ir1 ;
s


Treatments


Figure 2. Yield of onion (Takii's) at different combination of disease management practices (CES,
PhilRice, Mufloz, Nueva Ecija, 2003 Dry Sbason).


)C 'f
'I'
;JJ


r.


'J











MOLECULAR TECHNIQI
INDEXING (


F.M. DELA CUEVA, M. P. I
and R.

'University Researcher, Graduate F
College of Agriculture, University of the I
Pathologist, PHILSURIN Research Static


Disease indexing scheme
and efficient diagnostic assay
presence of important disease
materials. Diagnostic tools that
of pathogens include polymer;
and bacterial diseases and rev
reaction (RT-PCR) for diseases
used to detect a range of sugar
scald, ratoon stunting disease
syndrome (YLS) in introduce
showed that these nucleic a
identify materials that are ii
symptoms are not visible, the
new diseases or new strains of

Key Words: Indexing, sugarcane, polym(


INTRODUCTION

Germplasm exchange has always bee
an important component of any cro
varietal improvement program. Th
movement of germplasm involves a ris
of accidentally introducing new disease
or new strains of pathogen along with th
host plant material. In order to minimize
the risk, effective indexing procedures ar
required to ensure that distribute
material is free of pests that are c
quarantine concern (Frison and Putte
1993). Disease detection is done while
4*-- nl-n1 -r --A flllna 4-l r '. r,.*, ;n -, ;- l


ES FOR ROUTINE DISEASE
: SUGARCANE


OCAMPO1, J. B. FERRATER1
LUZARAN2

search Assistant, Institute of Plant Breeding,
lippines Los Baios, College, Laguna, 2Plant
VMC Compound, Victorias City.


evolving the use of sensitive, rapid
was developed to determine the
in sugarcane plants and planting
ive been adapted for the detection
a chain reaction (PCR) for fungal
se transcription-polymerase chain
viral origin. These tools had been
ne pathogens associated with leaf
(RSD), mosaic and yellow leaf
varieties of sugarcane. Results
I-based techniques could easily
*cted with pathogens even the
)y minimizing the introduction of
a pathogen.

se chain reaction, diseases


Diseases are major constraints in
sugarcane production. Their outbreak is
often the principal factor that inflicts
sizeable damage which alters the
production level, yet, only a few recognize
the importance of this problem and know
how to control these natural hazards (The
Philippine Recommends for Sugarcane,
2001). The problem becomes more
complicated when several pests occur in
the field at any one time. Downy mildew,
smut, leaf scald, RSD, mosaic, YLS and
Fiji disease virus (FDV) are among the
major diseases of sugarcane that are
responsible for heavy losses in
sugarcane. The ability to recognize these
rHicoQnac their cwmntnmc Q 1A10ll !I thQ










the pests to be able to initiate effective
control measures.
Incidence of leaf scald in the field
causes 20% reduction in the number of
stalks as well as a decrease in sucrose
content (Hoy and Grisham, 1994).
Because of the severity and variability of
leaf scald, Xanthomonas albilineans is
considered to pose a threat to all
sugarcane growing countries. It is
considered to be a phytosanitary threat
worthy of quarantine regulation (Frison
and Putter, 1993). In the Philippines, leaf
scald is not considered as a major threat
to sugarcane production probably
because it has not been observed to
occur in epidemic level or only the chronic
phase of the disease is present in the
country which could have been mistaken
as symptom of other common diseases.
Yield loss caused by RSD can
exceed 30% for a three-year crop cycle in
some cultivars (Grisham, 1991). A great
concern with RSD is that the bacterial
pathogen can exist for extended periods
of time in apparently healthy plants. Since
sugarcane is a vegetatively propagated
crop, the pathogen can be readily
disseminated between fields regions and
even countries.
Mosaic has caused serious
economic losses and has been involved
in the decline and failure of important
commercial clones in several countries. In
the Philippines, sugarcane mosaic (SCM)
and sorghum mosaic have been reported
but strains of these two viruses are not
yet known.
In Brazil, losses of sugarcane as
high as 50% have occurred from YLS
(Vega et al., 1997). Virion particles from
YLS-diseased plants may be transmitted
to disease-free plants with Melanaphis
sachari and Rhopalosiphum maidis as
vectors, to produce YLS (Scagliusi and
Lockhart, 2000).
There are diseases that are not yet
endemic in the country. Diagnosis of


symptom expressions are affected which
make diagnosis difficult and unreliable.
This also leads to spread of diseases
from one locality/country to another
particularly when the disease has a latent
phase. To address these potential
problems, disease indexing scheme
involving the use of nucleic .acid-based
protocols (Polymerase chain Reaction
and Reverse Transcription-Polymerase
Chain Reaction) was developed at the
Institute of Plant Breeding, UPLB to
determine the presence of important
diseases in introduced varieties of
sugarcane. This study is an integral
component of the PHILSURIN-CFC
Funded Project on "Sugarcane Variety
Improvement in South East Asia and the
Pacific for Enhanced and Sustainable
Productivity Disease Indexing
Component".

MATERIALS AND METHODS

Disease Indexing Scheme

Introduced varieties from sugarcane
growing countries like Bangladesh,
Indonesia, Malaysia, Thailand and
Australia were planted in the IPB Post
Entry Quarantine Greenhouse. Introduced
materials from France were planted in the
Philsurin Research Station, VMC
Compound, Victorias, City. After
emergence, the plants were indexed
visually for downy mildew, smut, leaf
scald, mosaic and other viral diseases.
Four months after planting, the plants
were indexed for the presence of mosaic
(RT-PCR), leaf scald (PCR), RSD (PCR),
and FDV (visually and PCR). Ten mo
after planting, the plants were indexed for
the presence of mosaic (RT-PCR), RSD
(PCR), leaf scald (isolation/PCR), YLS
(RT-PCR). Twelve months after planting,
sugarcane plants were ratooned,
replanted and the scheme was repeated.
After 18 to 24 mo in the greenhouse, the











that showed latency inside the
greenhouse.

Nucleic Acid-Based Detection of
Diseases

Polymerase Chain Reaction (PCR)

Leaf Scald (Xanthomonas albilineans).
A leaf diffusate sample was prepared by
soaking slices of leaf tissues in 1 ml
sterile water in a sterile microfuge tube
overnight at room temperature. The
diffusate was transferred by pipetting to a
fresh tube and centrifuged at 10,000 rpm
for 10 min to precipitate any bacteria. The
pellet was resuspended in an equal
volume of water and a 1 ul aliquot was
used in a PCR assay (Pan et al., 1997).
Two sets of primers were used in
the detection. One set which was
developed by Pan and co-workers (1997)
involved the use of forward primer PGBL1
and the reverse primer PGBL2 which
yielded a 288 bp product. The other set
was developed by Wang (1999) using
XAF and XAR primers with a PCR
product size of 600 bp. These two primers
were designed on the basis of a multiple
sequence alignment among sequences of
X. albilineans and were more effective for
quarantine as well as for routine
diagnosis (Pan et al., 1999).
Preparation of PCR reaction mixture
as well as thermal cycling program was
done following their respective protocol.

Ratoon Stunting Disease (Leifsonia
xyli subsp. xyli). Vascular sap samples
were used most often in the diagnosis of
RSD. About 100 ul of sap was extracted
using sterilized pliers in the open end of a
freshly sectioned internode and collecting
the sap directly into a microfuge tube. The
sap samples were centrifuged for 5 min
(13,000 rpm) to precipitate bacteria. The
pellets were washed once with 250 ul


)CR was done using the primers, L1 and
31, which were developed by Pan et al.
1998). The reaction mixture and PCR
)rofile were done following the protocol of
'an et al (1998).

Reverse Transcription-Polymerase
Phain Reaction (RT-PCR) .

llosaic. Primers developed by Smith and
le Velde (1994) using S400 551 and
3400 910 which yielded 360 base pairs
productt were used in the detection of
nosaic.
The reaction mixture and RT-PCR
profile were done following the protocol of
Smith and de Velde (1994).

fellow Leaf Syndrome. RT-PCR was
jone using the primers, YLS 111 and
fLS 462, which were developed by Mike
rey (personal communication). The
actionn mixture as well as thermal
:ycling program was done following the
protocol of Irey.

RESULTS AND DISCUSSION

Disease Indexing Scheme

afterr emergence, no disease was
observedd in all introduced varieties of
sugarcane from Bangladesh, Indonesia,
Malaysia, Thailand, France and Australia.
Four and ten mo after planting, the
presence of leaf scald, mosaic, RSD and
YLS were observed from introduced
varieties of sugarcane.
The presence of leaf scald could
occur in two forms, the chronic phase and
the acute phase (Ricaud and Ryan,
1989). The chronic phase was
characterized by several external
symptoms. The most typical symptom of
the disease was the presence on the leaf
lamina of a white pencil-line streak about
^ _i 1 -- A -' -


II I_








,=J rA= CU-pMrW%


previous symptom expression. The onset
of this condition generally follows a period
of stress, especially prolonged dry
weather or dry weather following a rainy
period.
Mosaic was identified primarily by its
leaf symptoms, which vary in intensity
with the cane clone, growing conditions,
temperature, and strain of the virus
involved. The general symptom was a
pattern on the leaf lamina of contrasting
shades of green, often of island of normal
green on a background of paler green or
yellowish chlorotic areas. The chlorotic
areas are most evident in young, rapidly
growing leaves and are particularly
distinct in the basal portion of the leaves;
the older leaves appear more normal as
chlorotic areas tend to develop the normal
green color with age (Koike and Gillaspie,
1989).
RSD does not show any specific
external symptoms usually found in the
majority of plant diseases. Internally, the
discoloration of the vascular bundles of
stalk was apparent. If a diseased stalk
was cut longitudinally with a sharp knife,
the discoloration found in the node of a
mature cane is in the shape of dots or
commas and a general pink color in the
nodes of very young canes (The
Philippine Recommends for Sugarcane,
2001). YLS was characterized by
yellowing of the leaves and central veins,
yellowing being more intense near the
leaf tips. In severe cases, veins became
reddish, and necrosis developed along
the leaf edges, beginning at the leaf tip
and extending to the base of the leaf
(Avila et al., 2001).
Under open quarantine, varieties
from Bangladesh and CIRAD were
planted and being observed for the
presence of diseases.

Nucleic Acid-Based Detection
of Diseases


Leaf Scald. Results showed that 4 mo
after planting, seven out of forty-seven
Varieties from first batch from
CIRAD (France) yielded positive reaction
to leaf scald (Table 1). Most of the plants
that were positive to leaf scald through
PCR were asymptomatic under
greenhouse condition but yielded strong
bands when assayed using the two sets
of PCR primers (Fig. 1).
Ten months after planting, one out
of six varieties from Malaysia, six out of
seven varieties from Thailand and twenty-
six out of sixty eight varieties from CIRAD
(France) yielded positive reaction to leaf
scald (Table 1). These plants were
negative to the disease when assayed 4
mo after planting. It was noted, however,
that typical chronic symptoms of the
disease such as white pencil-line streaks
started to appear 6 mo after planting.

Ratoon Stunting Disease. Four mo after
planting, twenty-four out of sixty eight
varieties from CIRAD (France) yielded
positive reaction to RSD. Ten months
after planting, 5 out of 9 varieties from
Malaysia and 1 out of 7 varieties from
Thailand yielded positive reaction to RSD
(Table 1, Fig. 2).

Mosaic. Four mo after planting, all
varieties from Bangladesh and Indonesia,
3 out of 6 varieties from Malaysia, 5 out of
7 varieties from Thailand and 67 out of 68
varieties from CIRAD (France) showed
positive reaction to SCMV (Table 1).
Ten mo after planting, 4 out of 9
varieties from Bangladesh, all varieties
from Thailand and 28 out of 68 varieties
from CIRAD (France) showed positive
reaction to SCMV. No mosaic was
observed in varietied obtained from
Indonesia and Malaysia (Table 1).

Yellnw I maf Cvnmmrnma A smrrih, frnm










bUMMAKY ANU UUNULUSlUN

Routine indexing of sugarcane for tl
presence of important pathogens shoL
be done to minimize the risk
introducing pathogens or new variants
pathogens in sugarcane producil
regions through efficient, sensitive ai
reliable means of identifying pathoge
present in the plant. Inter-district ai
inter-country movement of plantii
materials should be properly monitored
prevent the spread of pathogens tt
could pose a threat to sugar industi
Diseases that are of minor important
should also be identified and observed
prevent them from reaching epiden
level.
The molecular-based protocols c;
now be used for practical screening
plants and planting materials for tl
presence of important pathogens
sugarcane particularly those that a
difficult to assess visually.

LITERATURE CITED

AVILA, R, MC ARRIETA,
VILLALOBOS and L MOIREIR
2001. First report of sugarcane yellc
leaf virus (ScYLV) in Costa Ric
Plant Dis. 85:919.

PRISON, A and CAJ PUTTER. 199
FAO/IBPGR Technical Guidelines f
the Safe Movement of Sugarcar
Germplasm. Rome, Italy: Food ar
Agriculture Organization of the UnitE
Nations. Rome/Intemational Board f
Plant Genetic Resources. 44 pp.

GRISHAM, MP.. 1991. Effect of ratoc
stunting disease on yield of sugarcar
grown in multiple three-year planting
Phytopathology 81:337-340.

HOY, JW and MP GRISHAM. 199
Sugarcane leaf scald distribution


KOIKE, H. and AG GILLASPIE, JR.
1989. Diseases of Sugarcane: Major
Diseases as cited by Ricaud, C.B.T.
Egan, A.G. Gillaspie, Jr. and C.G.
Hughes. Elsevier, Amsterdam. pp.
301-322.

PAN, YB, MP GRISHAM and DM
BURNER. 1997. A polymerase chain
reaction protocol of the detection of
Xanthomonas albilineans, the causal
agent of sugarcane leaf scald
disease. Plant Dis. 81:189-194.

PAN, YB, MP GRISHAM, DM BURNER,
KE DAMANN, JR and Q WEI. 1998.
A polymerace chain reaction protocol
for the detection of Clavibacter xyli
subsp. xyli, the causal bacterium of
sugarcane ratoon disease. Plant Dis.
82:258-290.

PAN, YB, MP GRISHAM, DM BURNER,
BL LEGENDRE and Q WEI. 1999.
Development of polymerase chain
reaction primers highly specific for
Xanthomonas albilineans, the causal
bacterium of sugarcane leaf scald
disease. Plant Dis. 83:218-222.

RICAUD, C. and CC RYAN. 1989.
Diseases of Sugarcane: Major
Diseases as cited by Ricaud, C.B.T.
Egan, A.G. Gillaspie, Jr. and C.G.
Hudhes. Elsevier, Armsterdam. pp.
301-322.

SCAGLIUSI, S. and BE LOCKHART.
2000. Transmission, characterization
and serology of a luteovirus
associated with yellow leaf syndrome
of sugarcane. Phytopathology 90:120-
124.

SMITH, GR and R VAN DE VELDE.
1994. Detection of sugarcane mosaic
virus and Fiji disease virus in
diseased sugarcane using the


0_








dela Cueva, de Ocampo


THE PHILIPPINE RECOMMENDS FOR
SUGARCANE. 2001. PCARRD, Los
Baros, Laguna. 269 pp.

VEGA, JS, SNM SCALAGUSI and EU
WAIN. 1997. Sugarcane yellow leaf
disease in Brazil: Evidence of
association with a luteovirus. Plant
Dis. 81:21-26.


WANG ET AL. 1999. Comparison of
PCR, Bio-PCR, DIA, ELISA and
isolation on semi-selective medium for
detection of Xanthomonas albilineans,
the causal agent of leaf scald of
sugarcane. Plant Pathology 48:245-
252.


Table 1. Results of disease indexing of Bangladesh, Indonesia, Malaysia, Thailand and
CIRD (France) varieties for leaf scald, sugarcane mosaic virus (SCMV), ratoon
stunting disease (RSD) and yellow leaf syndrome (YLS) four and ten months
(mo) after planting.

Reaction
Varieties Leaf scald SCMV RSD YLS
4 mo 10 mo 4 mo 10 mo 4 mo 10 mo 4 mo 10 mo
Bangladesh
1 s batch
ISD 20 ND + + ND + +
ISD 28 ND + + ND + -
ISD 24 ND + + ND + -
ISD 16 ND + ND +

2Nu batch
ISD 25 -
ISD 21 +
ISD 29__+ -
ISD 30 -
ISD 31 _

Indonesia
1 s batch
PS81-362 ND + ND -
PS10029 ND + ND -
PSGM5052 ND + ND ND ND -
PSBM9044 ND + ND ND ND -
PSCO90-2411 ND + ND ND ND

2" batch
PS90-3092 ND-
PS862 ND +
PS893 ND
PS851 ND -
PS82-1007 ND +

Malaysia


7 6r-3t4Z


ND


ND


--------------* ------- ------------- 1 -- ND-- _______ ___ __ I








82-1089 ND + + ND ND -
GPB-5 ND + ND ND +
80-13-90 ND ND ND -
80-A-1867 ND ND -
GP76-321 ND -. ND -

Thailand
Uthong-1 ND + + ND -
K8865 ND + + + ND + -
K8892 ND + + + ND -
K8469 ND + + ND -
K8887 ND + + + ND +
K84-200 ND + + + ND -
K76-4 ND + + ND -

CIRAD (France)
1st batch
FR95-183 + + ND -
FR95-195 + + + + ND -
FR95-223 + ND -
FR95-224 + ND -
FR95-240 + + + ND -
FR95-244 + + + ND -
FR95-245 + + ND -
FR95-259 + + ND -
FR95-263 + ND -
FR95-328 + + ND -
FR95-392 + + + ND -
FR95-355 + + + ND -
FR95-364 + ND -
FR95-382 + + ND -
FR95-406 + + + ND -
FR95-417 + + + ND -
FR95-418 + + + + ND -
FR95-437 + + + ND -
FR95-506 + + + ND -
FR95-556 + + + ND -
FR95-559 + ND -
FR95-563 + + + ND -
FR95-575 + + ND -
FR95-579 + + + ND -
FR95-618 + + ND -
FR95-638 + ND -
FR95-643 + + + ND -
FR95-653 + + + ND -
FR95-706 + + ND -
FR95-2112 + + + ND -
FR95-2117 + + ND -
FR95-2155 + + + ND -


FR95-2159


+









FR95-2179 +
FR95-2194 +
FR95-2189 +
FR95-2199 +
FR95-2200 + +
FR95-2222 +
FR95-2230 + +
FR95-2233 +
FR95-2247 -
FR95-2248 + +
FR95-2258 +
FR95-2260 +
FR95-2345 +

2N batch ND -
FR96-65 ND +
FR96-423 ND +
FR96-416 ND +
FR96-418 ND +
FR96-22 ND +
FR96-428 ND +
FR96-238 ND + +
FR96-405 ND + +
FR96-53 ND +
FR96-31 ND + +
FR96-29 ND +
FR96-626 ND + +
FR96-229 ND +
FR96-62 ND + +
FR96-74 ND +
FR96-628 ND +
FR96-33 ND +
FR96-620 ND +
FR96-95 ND +
FR96-512 ND +
FR96-333 ND + +
ND Not Detected, negative, + positive


ND
+ ND
ND
ND
+ ND
+ ND
+ ND
+ ND
ND
ND
+ ND
+ + ND
+ ND

ND ND -
+ ND ND
+ ND ND
+ ND ND
+ ND ND -
- ND ND
+ ND ND -
S ND ND -
I ND ND -
+_ ND ND
S ND ND
+ ND ND -
ND ND
I ND ND
I ND ND
I ND ND -
ND ND
I ND ND
ND ND
ND ND
ND ND
+- ND ND




Molecular techniques 5

HiBiXjP---












APHID SPECIES IN THE PHILIPPINES


L.E. HERRADURA1, L.V. MAGNAYE1, M.A. ALFORQUE1,
MA. A. LOBRES1 and L. KENYON2

1Supervising Agriculturist, retired Supervising Agriculturist, Research Assistant and
Agriculturist1, respectively, 'Bureau of Plant Industry, Davao National Crop Research
and Development Center, Bago-Oshiro, Davao City and 2Virologist, Natural Resources
Institute, Chatham, UK


Banana bract mosaic virus (BBrMV) was transmitted in a non-
persistent manner by the three aphid species, Pentalonia
nigronervosa, Aphis gossypii and Rhopalosip)hum maidis on
different Musa spp. Further study of thot virus-vector relationship of
BBrMV using P. nigronervosa was conducted. Results of the study
showed that the optimum acquisition feeding time of 30 min and
infection feeding time of 24 hr could produce symptom expression
on the plants. Five aphids can transmit the virus at 15% efficiency.

Key Words: Banana bract mosaic virus, banana cultivars, aphid species


INTRODUCTION BBrMV is becoming more common in
these three countries and could be
Banana bract mosaic virus (BBrMV) is a causing significant losses in production
potyvirus (Bateson and Dale, 1995; and fruit quality in some areas. Yield
Thomas et al., Muiez, 1992) that was reduction of about 40% has been
observed as early as 1979 affecting observed in cv Lakatan in the Philippines
banana plants in Mindanao (Magnaye (Roperos and Magnaye, 1991; Kenyon et
and Espino, 1990). It is commonly found al., 1996).
infecting the cooking banana varieties BBrMV has been reported to be
Saba and Cardaba but can also infect transmitted by at least three aphid
dessert varieties like Cavendish species. Magnaye and Espino (1990), in
(Magnaye and Herradura, 1997; Magnaye their preliminary transmission tests,
and Espino, 1990; Muiez, 1992). showed that Aphis gossypii and
BBrMV-infected plants 'show Rhopalosiphum maidis could transmit the
continuous pale green to pale brown virus. Muiez (1992) and Diekmann and
spindle-shaped streaks or long Putter (1996) reported that Pentalonia
continuous and discontinuous stripes nigronervosa can also transmit the virus
irregularly scattered along the petiole and in a non-persistent manner. Since the
leaf lamina. Leaf symptoms may or may knowledge of virus-vector relationship of
not be observed (Magnaye and Espino, the efficient vector of the virus is limited,
1990). the study aims to optimize and
Observations from India, Sri Lanka standardize the transmission of the
and the Philippines have indicated that potyvirus causing banana bract mosaic
from and between Musa spp.








Herradura, Magnaye, Alforque 59

MATERIALS AND METHODS infection feeding time (IFT) of 24 hr.
Transmission was done separately for the
BBrMV. Naturally field-infected suckers different aphid species using six test plant
from three banana cultivars showing per trial.
symptoms of Banana bract mosaic virus
(BBrMV) were collected and planted in Acquisition feeding time (AFT). AFT
the screenhouses at Davao National Crop was determined by allowing the non-
Research and Development Center, viruliferous aphids to feed on BBrMV-
Bureau of Plant Industry, Davao City for infected leaf pieces for 1, 5, 15 and 30
transmission studies. These were reared min. Twenty viruliferous aphids were then
and maintained as three isolates of transferred to healthy banana plants for
BBrMV for the transmission work. Isolate 24 hr infection feeding time (IFT) before
1 was from cv Cardaba (BBB) from Bago spraying with insecticide (Sevin XLR) to
Oshiro, Davao City, Mindanao, Isolate 2 kill the aphids.
was from cv Latundan (AAB) collected
from Nueva Vizcaya and Isolate 3 was Inoculation feeding time (IFT). After 30
from cv Pisang Surung (AA), a Malaysian min AFT, twenty viruliferous aphids were
accession planted at the Southeast Asia allowed to feed on healthy banana tests
Banana Genebank. plants for 6, 12, 24 and 48 hr before
spraying with Sevin XLR to kill the aphids.
Mass Rearing of Aphids. Three aphids
species, P. nigronervosa, A. gossypii and Number of aphids. To determine the
R. maidis were field-collected originally minimum number of aphids required to
from banana, Euphorbia hirta (Asthma transmit BBrMV, after 30 min AFT on
plant) and Echinocloa coloma (Jungle infected leaves, 1, 5, 10, 15 and 20
rice), respectively. The aphids were aphids previously starved for 3 hr were
transferred to their respective host plants transferred to cv Lakatan plantlets (3-4
grown from seeds and kept in separate leaf stage) and allowed to feed for 24 hr.
insect cages. For banana, tissue cultured Ten test plants were used for each
cv Lakatan plantlets indexed against treatment and replicated four times. After
BBrMV were used. The nymphs were infection feeding, Sevin XLR was sprayed
transferred to another batch of new on the plants to kill the aphids. The plants
healthy host plants for each generation of were kept inside the screenhouse for
aphid for at least 4 generations before symptom observation. Days to first
they were cultured and mass reared for symptom expression were also recorded.
the transmission experiments.
RESULTS
Vector Transmission
Isolates. All three isolates of BBrMV
Efficiency of transmission. Parallel exhibited the characteristics spindle
tests were done to determine the most streak symptoms on the lamina and
efficient vector of the virus among the pseudostem (Fig.1A). Magnaye and
reported aphid species. The most efficient Espino (1990) and Muiez (1992) reported
vector was used in the succeeding tests. similar symptoms on cv Cavendish
Twenty non-viruliferous aphids previously clones. However, on the field-infected cv
reared on healthy host plants were Latundan suckers collected from Bonfal,
starved for 3 hr in Petri plates prior to Nueva Vizcaya, Luzon (Isolate 2), light
acquisition feeding of 30 min on BBrMV- green bands were observed along the
infected leaf pieces from the three veins (Fig. 1B). This is the first time that
isolates. The aphids were then such symptoms have been associated
transferred to healthy test plants, i.e. with BBrMV. Samples exhibiting these
banana cv Lakatan and allowed an symptoms have been subjected to







w.I I I ;1 1Q1 I i i| l OI I I V1 | JlJ Id l Id

enzyme linked immunosorbent assay observed. The minimum number of
(ELISA) at Queensland Department of aphids that can effect transmission was
Primary Industries (QDPI), Australia and five with an efficiency of 15%.
reacted positively to antisera against
BBrMV (Herradura and Magnaye, 1998). Symptoms on Musa spp. Using P.
nigronervosa. R. maidis and A. gossypii,
Vector Transmission BBrMV was transmitted to members of
the Musaceae. Characteristic initial
Efficiency of transmission. All three symptoms observed on the lamina of
isolates of BBrMV were successfully inoculated plants of cv Lakatan were
transmitted to cv Lakatan, cv Butuhan, cv sDindle streaks, which would sometimes


d R. maidis on the three other inoci
)wed that P. nigronervosa symptom,
;t efficient in transmittina the hbndc nn I


nfirmed that
in the non-
it three aphid
potyvirus. Of
niaronervosa


us H. nigronervosa aphids. Using 40%. In the experir
js, 50% transmission efficiency transmission efficiel
gained 15 DAI (Table 2). This was cv Lakayan pl
]hest transmission efficiency nigronervosa (Table
in the experiment. Chlorotic All three isolat
streaks and vein enlargement on able to infect and ca
la were the typical symptoms










symptoms ir
scuminata ar
P. nign
-ninni7inn ar


,o Musa genotypes, M. gossypii and R. maidis in a non-persistent
1. balbisiana. manner.
vosa is specialized in


aphid species tested in this study are BATESON, M.F. and J.L. DALE. 1995.
nyv'l' r ,u"<- N/,lil..=, 'i,/" d'k.i n n"r"i' tff".,#eM nl^J' r',i-lln f -A A r't I,, uk * j;,k ,,; j ,MJ M. :* -* A.


o other plant sl


denenerate


readily transmit
than by other apl
R. maidis
commonly founc
within banana ai
Their capability ir
their prevalence
these areas r
important in the
field. While the
colonize on Mu
grasses and ban
~nrmrn t.,rh;~ ,Ie


II LJLJIIIVIV LU U0I IIIU
by P. nigronervo.
species
d A. gossypii a
I weeds around ar
Ibaca growing area
nsmitting BBrMV ar
their natural hosts
e them potential
sad of BBrMV in tt
were not found
they can migrate
particularly the ala


KMANN, D.R. and 1
1996. Musa, 2nd edi-
Technical Guideline.
Movement of Gerrr
Food and Agriculture
the United
Rome/International
Resources Institute, F

RRADURA, L. E
MAGNAYE. 1998.
banana bract mos,


U.J. PUTTER.
i. FAO/IPGRI
for the Safe
ism, No. 15
organization of
Nations,
ant Genetic
ne, 28 pp.

and L.V.
electionn of
(BBrMV) in


l-hI oIL I Mlny I IIUIVI
SIcc V\/ ntrir- DOk--^


ed hosts of the aphi'
minimize their rapi
reduce the chance t
.s to banana plants.


KENYON, L.,
WARBURTOI
CHANCELLC
C. FOOT. 1


...- -.. .. ...... ...... v .. n .vvu. II i I I iL4l1IV I il 1. V *I.JW I I 1 1 If 1VI*
bands along the veins of the lamina. Protection Programme Project
Plants exhibiting these symptoms should A0127/X0258 Final Technical Report.
not be over looked in eradicating infected National Resources Institute,
plants or in selecting plant materials as Chatham Maritime, UK.
explants for tissue culture.
The three aphid species tested were MAGNAYE, L.V. and R.R.C. ESPINO.
capable of acquiring and transmitting the 1990. Note: Banana bract mosaic, a
three isolates of BBrMV but they differ in new disease of banana. I.
their efficiency of transmission. P. Symptomatology. Philipp. Agric.
nigronervosa was the most efficient with 73:55-59.
an optimum acquisition feeding period of
30 min and infection feeding period of 24 MAGNAYE, L.V. and L.E.


virus trans:


Mufez (1992) support the findings Technical Repo







62 Transmission of banana

MUNEZ, A.R. 1992. Symptomatology, THOMAS, J.E., A.D.W. GEERING, C.F.
transmission and purification of GAMBLEY, A.F. KESSLING and M.
banana bract mosaic virus (BBMV) in WHITE. 1997. Purification, properties
'Giant Cavendish' banana. MS Thesis, and diagnosis of banana bract mosaic
University of the Philippines, Los potyvirus and its distinction from
Baios, Laguna, Philippines. abaca mosaic potyvirus.
Phytopathology 87:698-705.
ROPEROS, N.I. and L.V. MAGNAYE.
1991. Status of Banana Diseases in ACKNOWLEDGEMENT
the Phil. In: Valmayor, R.V. Umali, B,
and Bejosano, C.P. (eds) Banana Natural Resources Institute, United
Diseases in Asia and the Pacific. Kingdom and Queensland Department of
Proc. of a Technical Meeting on Primary Industries, Australia as
Diseases Affecting Banana and collaborators of the project.
Plantain in Asia and the Pacific,
Brisbane, Australia 15-18 April, 1991.









Herradura, Magnaye, Alforque


Table 1. Efficiency of three aphid species in transmitting banana bract mosaic virus to three
different Musa genotypes.


Transmission Efficiency (%)
Test Pentalonia nigronervosa Rhopalosiphum maidis Aphis gossypii
Plants Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate
1 2 3 1 2 3 1 2 3
Lakatan 50 50 50 0 16 0 0 33 33
(Musa
acuminata
AAA)
Butuhan 50 16 16 0 0 0 0 0 0
(Musa
balbisiana
BB)
BBrMV isolates:


* Isolate 1 cv
* Isolate 2 cv
* Isolate 3 cv


Cardaba (BBB); Bago Oshiro, Davao City, Mindanao
Latundan (AAB); Bonfal, Nueva Vizcaya, Puzon
Pisang surung (AA); Malaysia


Table 2. Transmission efficiency of (%) banana bract mosaic virus using Pentalonia nigronervosa.

No. of aphids Transmission Symptom Expression Symptom
per plant Efficiency (%) (No. of days)
1 0
5 15 37 Faint chlorotic streaks on the
youngest leaf, enlargement of
veins
10 20 36 Chlorotic streaks on the
youngest leaf, enlargement of
veins
15 30 16 Chlorotic streaks on the
youngest leaf, enlargement of
veins
20 50 15 Chlorotic streaks on the
youngest leaf, enlargement of
veins
* Average of 4 replicates with 10 plants/treatment








Transmission of banana


Table 3. Effect of acquisition feeding time (AFT) on banana bract mosaic virus transmission
efficiency using Pentalonia nigronervosa


AFT Transmission Efficiency (%) Symptom Expression
(minutes) (No. of days after inoculation)

1 30 29
5 30 29
15 40 23
30 50 15
* Average of 4 replicates. Ten plants/treatment.


Table 4. Effect of inoculation feeding time (IFT) on banana bract mosaic virus transmission
efficiency using Pentalonia nigronervosa


IFT Transmission Efficiency (%) Symptom Expression
(hours) (No. of days after inoculation)

48 50 15
24 50 16
12 20 22
6 20 23
Average of 4 replicates. Ten plants/treatment.








Herradura -Magnayp, Alforque


Figure 1. Symptoms of banana bract mosaic on infected plant. A. spindle streaks on
lamina, B. chlrotic band on midrib and veins of lamina, and C. twisting
of petiole on inoculated plants.







Journal of Tropical Plant Pathology 39:66-69


ISOLATION OF A TOBAMOVIRUS FROM BOTTLEGOURD,
LAGENARIA SICERARIA STANDL.


L.M. DOLORES and A.R. ALCACHUPAS

Respectively, University Researcher and Laboratory Technician, Institute of Plant
Breeding, College of Agriculture, University of the Philippines Los Baiios, College,
Laguna.


A rigid rod shaped, sap transmissible virus was isolated and
identified as cucumber green mottle mosaic virus (CGMMV) from a
severely infected "upo" (bottlegourd) collected from a farm in
Pangasinan. The pure virus isolate was obtained by single lesion
isolation and through a series of mechanical inoculations from local
lesion host to systemic host. The virus caused local lesions to
Chenopodium amaranticolor and C. quinoa while mild to severe
systemic symptoms were exhibited by cucumber and bottlegourd.
The negative stain of partially purified extracts of the putative virus
showed rigid rod particles under the electron microscope. Indirect
ELISA yielded positive reactions to the tobacco mosaic virus (TMV)
antiserum only but none against PRSV, WMV-2, ZYMV, and CMV.

Based on the results of bioassay, electron microscopy and
ELISA tests, the virus was determined to be a strain of CGMMV, a
tobamovirus.

Key Words: tobamovirus, bottlegourd, cucumber green mottle virus, enzyme
linked immunosorbent assay


INTRODUCTION

Bottlegourd (Lagenaria siceraria Standl.)
popularly known as "upo" is an important
cash crop in the Philippines. Like any
other crop, upo is susceptible to viruses.
Several viruses including PRSV, WMV-2,
ZYMV and CMV have been detected from
naturally infected cucurbitaceous crops by
ELISA (IPB Annual Report, 1998).
Recently, a rigid rod shaped transmissible
virus was isolated from a severely
infected upo collected from a farm in
Pangasinan. The virus was almost similar
to Cucumber green mottle mosaic virus
(CGMMV) identified in Taiwan (Chen and


Wang, 1986) CGMMV belongs to
tobamovirus group with rigid rod particles
and occurs in several strains most of
which are hosted in cucurbita (Hollings et
al., 1975; Raychaudure and Varma,
1978). The watermelon strain of Japan
causes slight leaf mottling and dwarfing
on watermelon (Komuro et al., 1971).
Other strains have been recognized
according to serological and differential
hosts (Brcak et al., 1962; Holling et al.,
1975; Nozu et al., 1971).
This study aims to identify a
tobamovirus isolated from upo.










wir-lu |. |-Um-- 'liu I-WnL I I % %wJ WILII UIUU IIlly UIUIUIIl IUI I III dL IUU
temperature, 100 pl of each prima
Isolation of the virus. The virus isolate antibody was loaded into each well ar
was obtained by single lesion isolation incubated for 2 hr. The plates were the
from Chenopodium quinoa and then back washed and the second antibody (Go
inoculation to bottlegourd. The virus was antirabbit conjugate, Sigma A 802
purified in the host through a series of diluted 1000 times in blocking solutic
mechanical inoculations from local lesions were loaded into each well and incubate
host (C. quinoa) to systemic host for 2 hr at 370C. The plates were washed
(bottlegourd), 4-5 times and then again with PBST as above. Finally, tt
propagated in bottlegourd for use as substrate (p-nitrophenyl phosphate at
source of virus in subsequent assays. mg/ml in diethanolamine buffer) wi
added at 100 pl and incubated for 1 hr
Host range and symptomatology. The room temperature. Wells were observe
host range of the virus was determined by for yellow color as positive reaction f
mechanical inoculation of the pure virus each specific antigen/antibody binding.
isolate onto different host plant species
belonging to Chenopodiaceae, RESULTS AND DISCUSSION
Solanaceae, and Cucurbitaceae.
Inoculated plants were kept inside the Cucumber green mottle mosaic vir
greenhouse and observed for symptoms. (CGMMV) was isolated from a field grove
upo by single lesion isolation usir
Electron microscopy. The virus isolate Chenopodium quinoa. The virus induce
was partially purified following the necrotic lesion on C. amaranticolor (Fi
procedure described for tobamovirus 1). Inoculated bottlegourd showed slig
(Gooding and Hebert, 1967) which mosaic symptoms at the early stage ar
involved virus clarification using 8% later developed a well defined ve
butanol and virus precipitation by PEG banding with green blisters, le
and sodium chloride. The partially purified outgrowths and deformed leaf (Fig. 2).
virus suspension was negatively stained cucumber, a very prominent vein clearir
with 2% phosphotungstic acid (pH 6.5) symptoms appeared 2 wk aft
and examined under a Hitachi model inoculation (Fig. 3). The virus was n
electron microscope of the National able to infect the following tests plan
Institute of Molecular and Biotechnology, namely, Nicotiana glutinos
UP Los Baros. Chenopodium murale, Datura meth
Citrullus vulgaris and C. moschat
Indirect Enzyme Linked Datura stramonium exhibited a mi
Immunosorbent Assay (ELISA). Indirect mosaic symptoms only (Table 1).
ELISA was performed as previously The negative stain of partial
described (Crowther, 1995) using the purified extracts of the putative vin
different reference antisera namely, showed rigid rod particles of tt
Cucumber mosaic virus (CMV), tobamovirus group under an electric
Watermelon mosaic virus 2 (WMV-2), microscope (Fig. 4).
Papaya ringspot virus (PRSV), Zucchini Indirect ELISA gave positi)
yellow mosaic virus (ZYMV) and tobacco reactions only to the TMV antiserum b
mosaic virus (TMV). Briefly, samples not to CMV, PRSV, WMV-2, and ZYIV
were extracted in coating buffer (50 mM suggesting its relatedness to TMV,
sodium carbonate, pH 9.6) and loaded member of the tobamovirus group.
directly in microtiter plates (100 pl/well). Based on the results of bioassa
The plates were incubated overnight at electron microscopy and ELISA tests, tt
40C and then washed with PBST 3 times virus was determined to be a strain
3 min interval. After blocking the plates C~MMV Reverat strains of CGMMV


r\_l____ ___I LI___I_.~_







! ME


ve been recorded so far based on die
,ults of serology and indicator hosts
allings et al., 1975; Nozu et al., 1971).
e type strain induced local lesions on
amaranticolor but not to D. stramonium
d Petunia hybrida. Cucumber virus 4
luces striking symptoms on cucumber
its, local lesions to C. amaranticolor
d systemic mottling on C. mural. The
st European strain produced local
ions on tobacco cvs. Samsun and
nthi (Brcak et al, 1962, Hollings et al.,
75). Komuro (1971) reported a
panese strain of CGMMV which caused
iere fruit distortion in cucumber fruit
d local lesions on D. stramonium but
t on C. amaranticolor. The Indian strain
causes lesion on C. amaranticolor but
infection on tobacco while muskmelon
ain neither infects C. amaranticolor nor
stramonium. In 1986, Chen and Wang
Taiwan found a new strain of CGMMV
m bottlegourd which caused chlorotic
ions on C. amaranticolor, symptomless
action on D. stramonium and P. hybrida
d no infection on watermelon and C.
irale. According to these results, our
is isolate was almost similar to the
iwan isolate causing chlorotic lesions to
amaranticolor and C. quinoa, moderate
severe mosaic on cucumber, no
action on C. vulgaris, C. murale and C.
isehata. Our local isolate however,
Feared in its relation to D. stramonium
duringg mild mosaic symptoms against
Ssymptomless reaction of the Taiwan
late.
This is the first report of the
:urrence of CGMMV, a tobamovirus,
bottlegourd in the Philippines.
bamovirus is extremely stable and has
vide host range. Control measures are
aded to prevent the spread of CGMMV
;ucurbit fields.

LITERATURE CITED

iCAK, J, M VERYCHOVA and M
CECI. 1962. Infection of tobacco and
some Chenopodium species by


cucumber virus 4 (and 3) and by its
nucleic add. Virology 16:105-114.

IEN, Mand S WANG. 1986. A strain
of cucumber green mottle mosaic virus
on bottlegourd in Taiwan.

tOWTHER, JR. 1995. Methods in
Molecular Biology Vol. 42-ELISA
Theory and Practice. Humana Press,
Totowa, NJ.

ODING, JB and TT HERBERT. 1967.
A simple technique of purification of
tobacco mosaic virus in large
quantities. Phytopathology 57:1285.

)UJNGS, M, Y KOMURO and H
TOCICHARA. 1975. Cucumber
green mottle mosaic virus. CMI/AAB.
Description of Plant Viruses. No. 154.

STITUTE OF PLANT BREEDING.
1998. Annual Report UP Los Baflos,
College, Laguna.

)MURO, Y, H TOCHICHARA, R
FUKATSU, Y NAGAI and S
YONEYAMA. 1971. Cucumber green
mottle mosaic virus (watermelon strain)
in watermelon and its bearing in
deterioration of watermelon fruit known
as "konnyaku disease". Annals of
Phytopathological Society of Japan.
37:34-42.

)ZU, Y, H TOCHICHARA, Y KOMURO
and V OKADA. 1971. Chemical and
immunological charaterization of
cucumber green mottle virus
(watermelon strain) protein. Virology
45:577.

iYCHAUDHURI M and A VARMA
1978. Mosaic disease of muskmelon
caused by a minor variant of cucumber
green mottle mosaic virus.
Phytopathologische Zeis Chrift 93:
120-125.








Dolores and Alcachupas


Figure 1. Chenopodium quinoa showing chlorotic local lesions.


Figure 2. Bottlegourd showing typical CGMMV symptoms of (a) green vein
banding and (b) severe blistering mosaic with leaf outgrowths.


Figure 3. Cucumber exhibiting dark green Figure 4. Negative stain of CGMMV showing
and vein clearing symptoms. rigid rod particles in 2% phospho-
tungstic acid (PTA).








of Tropical Plant Pathology


"LEROTIUM ROLFSII CAUSING F
(ARTOCARPUS HETER


N.G. TANGONAN and %

respectively University Prc
lonan(~hotmail.com, Tel/Fax 63-64-248-2E
Pathology Research Laboratory, Crops I
Irch Center (USMARC). University of Soul
Sin part from the USM-funded study on "IntE
ses of Durian, Jackfruit, Lanzones, and Mar


The incidence of Sclerotium rolfii S
is therefore reported for the first time as a
Sclerotium fruit rot of jackfruit (Artocarp
noted at Kisante, Makilala, Cotabato laE
mycelial growth and abundant sclerotial I
were noted on the surface and caused
oatmeal agar, the fungus exhibited a fan-s
narrow aerial mycelial strands. The hypha
with clamp connections at both sides
sclerotia first appeared white, then tumr
mature measure 1-2 mm and develop on
after incubation. Pathogenicity test on he;
reaction one week after inoculation.

rords: Jackfruit, fruit rot, Sclerotium rolfsii


INTRODUCTION ripe
var
*uit (Artocarpus heterophyllus L.), apr
F known as "langka or nangka", is a frui
ar fruit in the Philippines. Its
sing demand locally has disi
Iraged farmers to plant more the
uit because it can be sold directly to lea
,tailers or wholesalers. It is planted glo
' in home gardens and mixed yoL
rds as vegetable and fruit crop. die
ruit is the largest tree-borne fruit in the
orid, reaching 80 pounds in weight gre
p to 36 inches long and 20 inches in the
ster. The young fruit is cooked as the
able, pickled or canned while the cat


UIT ROT OF JACKFRUIT
PHYLLUS L.)


". SOLILAP

ssor/Corresponding author:
) and Science Research Assistant,
search Division, USM Agricultural
rn Mindanao, Kabacan, Cotabato.
rated Disease Management of Field
g".


c. causing fruit rot of jackfruit
w disease in the Philippines.
heterophyllus L.) was first
March 2003. White cottony
dies of the fungal pathogen
ting of the affected fruit. In
aped white colony with many
measured 7.90 x 177.80 pm
some septations. Spherial
tan to dark brown as they
e colony surface 5 to 7 days
hy jackfruit revealed positive





ruit is eaten fresh and made into
is local delicacies or used as
:izers and flavorings to ice cream,
lends and beverages.
Pathak (1980) reported that
ses could become limiting factors in
ultivation of jackfruit. These include
spot caused by Colletotrichum
sporioides, which infects leaves,
) tender shoots and leaf petioles,
ack, caused by Botryodiplodia
iromae, which infects the young
I twigs extending along the veins of
.af edges resulting to the death of
vigs and branches, and pink disease
)d by Corticium salmonicolor,









*- ---------.*-- -. I- -. ----.,
reas, affects stem and branches causing
eath of the trees when severely infected.
in the other hand, Rhizopus fruit rot of
ickfruit caused by R. stolonifer had been
.ported to cause 30% yield loss in the
hilippines, India, Okinawa and Bonin
Lands (AICAF, 1995). Several diseases
ave been reported to attack jackfruit in
ie Philippines such as leaf spot
,ercospora artocarpi), fruitlet rot
Choanephora cucurbitarum and C.
ifundibulifera), pink disease (Corticium
almonicolor), leaf disease (Dichotomella
reolata and Diplodia artocarpina), fruit
ecay (Diplodia sp.), fruit
>t/inflorescence rot (Rhizopus
rtocarpus), soft rot (R. nigricans), stem
>t (Sclerotium rolfsii) and sooty mold
ripospoerium sp.) (Tangonan, 1999).
Sclerotium rolfsii is known to cause
diseases on a wide range of agricultural
nd horticultural crops affecting all parts
nd stages of crops. It infects more than
00 species of monocotyledonous and
icotyledonous plants (Hall, 1991;
angonan,1999).
The study aimed to characterize fruit
)t of jackfruit based on symptoms, to
lentify its causal pathogen, and describe
s cultural and morphological
characteristics.

MATERIALS AND METHODS

collectionn of specimens and isolation
f the suspected causal fungus.
disease specimens were collected from
ickfruit orchards at Kisante, Makilala,
;otabato. Direct microscopic examination
Ias done to observe for fungal structures.
he suspected causal fungus was first
,olated and grown in potato sucrose agar
200 g potato, 10 g sugar, 18 g agar in
000 ml distilled water). Subsequent
ansfers were done on oatmeal agar (60
oatmeal, 20 g agar, in 1000 ml water)
ntil pure culture was obtained.


nature jackfruit still attached on trees
rere inoculated with the fungal isolate.
wo-week-old pure culture of the
suspected fungus in a suspension of its
lycelial growth with 5 sclerotial bodies
vas inoculated to a healthy fruit by patch
method then covered with transparent
plastic for 48 hr to maintain moisture and
humidity favorable for its initial growth.
diseasee reaction was' monitored and
recorded.

Jentification of the fungal isolate
iased on its cultural and
morphological characteristics. The
ungal isolate was identified through its
characteristic symptom expression in
ackfruit; its cultural growth. was also
loted and described; and the hyphae and
clerotial bodies were measured.

RESULTS AND DISCUSSION

descriptionn of the disease. Sclerotium
ruit rot was identified as a new disease of
ackfruit. A white, thick cottony mycelial
growth ramified over the surface of the
ifected fruit and rapidly spreads during
favorable conditions. Affected tissues
exhibited rotting arid abundant sclerotial
bodies were likewise observed (Fig. 1).

culturall and morphological
characterization of the suspected
ungal pathogen. The pathogen
*xhibited a fan-shaped white colonies
with many narrow mycelial strands on
oatmeal agar (Fig. 2). The pathogen
apidly covered the whole area of a Petri
late in 3 days at room temperature.
"he hyphae measured 7.90 x 177.80 pm
vith two septations. Sclerotia developed
in the colony surface 5 to 7 days after
icubation and at first it appeared as
white, then turned tan to dark brown.
developedd sclerotia were small and
;pherical, 1-2 mm in size (Fig. 2). The










romsii Sacc.

Pathogecinity test. Pathogenicity tesi
revealed positive reaction, 7 days after
inoculation. Inoculation portion of the fruil
rotted and exhibited white mycelia growth
and produced sclerotial bodies. Typical
symptom observed was the same with the
naturally infected fruit. Based on such
findings, fruit rot of jackfruit is thus
confirmed as caused by Sclerotium rolfsi
Sacc.

LITERATURE CITED

AICAF. 1995. Disease of Tropical Fruil
Trees. Association for International
Cooperation by Agriculture and
Forestry (AICAF). Japan. 142 p.

















Figure 1. Jackfruit infected w











Figure 2. Two-week old cultui
White cottony myce
bodies.


Diseases. The Americar
Phytopathological Society. St. Paul
MN, USA:AP Press.

PATHAK, VN. 1980. Diseases oi
Tropical Crops. Oxford and IBN
Publishing Co., 309pp,

TANGONAN, NG. 1999. Host Index ol
Plant Diseases in the Philippines. 3"
ed. Department of Agriculture-
Philippine Rice Research Institute
(DA-PhilRice), Murioz, Nueva Ecija
University of Southern Mindanao
Kabacan, Cotabato, Philippines, 40E
PP.

TANGONAN, NG and SE SOLILAP
2003. Sclerotium fruit rot: A new
disease of jackfruit. USM RDEF
Monitor 23(2):9.















Sclertium rofsii Sacc.











of Scerotium rofsi on oatmeal agar.
with dark brown spherical sclerotial











ABSTR/
Presented During the 34
Philippine Con
on


Oral Presentation

CADANG-CADANG DISEA
CONTAINMENT PROGRAM. M,
Rodriguez, EP Pacumbaba, JC Orense
Raul, R Alfiler and GG Manalo. PCA-AI
Guinobatan, Albay

The Cadang-cadang disease, caused by
coconut cadang-cadang viroid is ende
only in the Bicol provinces and in isola
locations in Quezon, Samar and Bili
provinces. Since it was first recognized in
1930s, cadang-cadang was estimated
have killed about 30 million palms. During
last five years, an import ban has bi
imposed on young fresh coconuts (bul
desiccated coconut, nata de cc
makapuno, coconut in powder and sli
form and fiber by-products by sevi
countries due to the disease and its allele
danger to human health. But the threat
cadang-cadang disease can be county
because of availability of rapid, relia
detection methods, technical manpo,
expertise and facilities in the country.
A comprehensive and sustains
disease containment program is presented
establish the required conditions that
remove the present biases and doubts rai
by importing countries on Philippine cocc
products. The program has three compone
geared towards the resumption of cocc
exports. The identification and accredita
of disease-free areas will assure impor
that coconuts will be taken only from diseE
free palms that have been subjected to hi(
sensitive CCCVd detection techniqL
Establishment of buffer zones in Luzon
Visayas will prevent the further spread of
disease and confine it to certain areas of
country. Eradication of the disease will
done by removal of diseased palms and ol
possible sources of inoculum in the field
replanting with certified disease-free, I


T OF PAPERS
'est Management Council of the
since held at Cebu City
y 6-9, 2003


yielding planting materials.
implementation of the program relies
on the successful collaboration of the
SAlbay Research Center with the othi
units of the Philippine Coconut Authc
well as other concerned agencies such
Department of Agriculture, the BPI
Quarantine Service, the Local Gove
Units and DTI Bureau of Product Stani

I MARKER-AIDED TRANSFER
S BACTERIAL BLIGHT AND RICE TI
DISEASE RESISTANCE GENES
TRANSGENIC LINES TO RECOMME
VARIETIES OF RICE. S Khondk
Pastor, AP Anglecer, DA Tabanao <
Sebastian, Plant Breeding and Biotect
Division, PHILRICE, Science City of I
I Nueva Ecija

I Three cross combinations were ger
From the initial intercrossing of tt
I transgenic parents (IR72-82-3-4-;
S 147.8, IR72-82-13-2-2/T309-147.4 anm
82-3-13-2-2/T309-147.8). All F1
generated from the initial intercrossin
S genotyped using PCR-based Sei
S Tagged Sites analysis and confirm
I successful transfer of both BLB an
I resistance genes. All the selected
t containing both BB and tungro (
resistant transgenes were backcrossi
t the four recurrent parents (IR64, PSE
PSB Rc82 and IR72976-AC1). At IE
S plants of the 12 cross combination
planted and genotyped for the press
S cloned resistance genes. Plants hav
desired genes based on genotype!
I selected, emasculated and backcross
their respective recurrent parents. f
BC1F1 cross combinations generate
plants and these plants were plant
r surveyed by molecular assay using thi
I PCR assay. A total of 28 plants cor
I both resistance transgenes were select










ssed with their respective recurrent cc
BC2F1 seeds from a total of seven re
)mbinations are planted with at least nc
s per cross to allow the materials for th
ping and genotyping and afterwards
i of the resistant plants. Cl
TI
kTION OF MULTILINES FOR YI
TRIAL LEAF BLIGHT IN RICE. RR Vi
MC Abalos2, MP Fernando2, H
CM Vera Cruz3 and M Bustamam4, RI
A & M University, Agricultural re
h and Extension Center, U.S.A., ZL
reeding and Biotechnology Division, (Z
;E, Science City of Muroz. Nueva wi
RRI, Research Institute for Food ZI
biotechnology, Bogor, Indonesia. pr
all
genes for bacterial leaf blight (BLB) is(
Available and being pyramided in P(
genetic backgrounds like IR64 and in cc
:ess of the development, lines with fla
ene and gene combinations can be ini
id. These lines are typical isolines se
1 be tapped in the development of re'
s. Through Asian Rice Biotechnology sh
(ARBN), elite lines with different thi
*re developed and exchange of lines otl
ated. Two lines from Indonesia, Bio 1 on
) 2, containing Xa-5 and Xa-7, gu
rely in IR64 background and 3 lines 2
I IR 64 background from PhilRice but th(
ig xa-5 and Xa-21 were evaluated ac
with mixtures of these lines for these 31
He
32-19-3-4 had the highest average ZN
3.60t/ha across wet season of 2001 ap
)2, followed by AR32-19-3-3 with the
both significantly higher than the cIc
:ks, PSB Rc28 and IR64. Due to the fro
of disease pressures, however, dry ex
data showed comparative yields. an
Ind of IR64 and PSB Rc28 were le\
affected by BLB for 2 wet seasons
)ining these susceptible varieties with
stant lines increases the yield when


jVOIU LU I iQLUI 0 II II ItIcLIUI I. r\ Jj.Ju
bination of resistant and susceptible line
Ited to moderate bacterial infection and
ly similar maturity and grain qualities with
of the pure stand.

NING AND SEQUENCE ANALYSIS OF
COAT PROTEIN GENE OF ZUCCHINI
LOW MOSAIC POTYVIRUS. DE
nor, EY Ardales and NB Bajet, UPLB-CA

trse-transcriptase polymerase chain
tion was used to amplify the full length of
hini yellow mosaic potyvirus coat protein
1V-CP) gene from a squash infection
papaya ringspot potyvirus (PRSV) and
V. We designed ZYMV-CP gene specific
ars based on the multiple sequence
ments of several ZYMV and PRSV
tes downloaded from the genbank. A
product of about 837 base-pairs
*sponded to the length of the region
ed by the primers. This DNA was cloned
i topoisomerase activated vector and the
ence of two representative clones
aled that their nucleotide sequence also
red the 837 nucleotides as reported for
'YMV isolates from other countries. The
Stwo clones differed from each other by
one nucleotide at the 12th position with
ine (G) and adenine (A) for clones 1 and
;pectively. The amino acid sequence of
putative protein, consisting of 279 amino
; with approximate molecular weight of
:Da, was conserved for both clones.
ever, SDS-PAGE analysis of the purified
revealed a higher value of
)ximately 36 kDa. The nucleotides and
predictedd amino acid sequence of our
were compared with ZYMV isolates
other countries. Overall, our clone
ited high sequence identities of 83-90%
19-95% at the nucleotide and amino acid
respectively, indicating that it is ZYMV.










INTERACTION OF Xa GENES
DIFFERENT MAINTAINER L
BACKGROUND TO HISTORICAL )
CONTEMPORARY RACES
XANTHOMONAS ORAZAE PV. ORYZAJ
LM Briones1, ED Redoha2, MP Natural3,
Vera Cruz4, and H Leung4. 1Leyte S
University, 2PHILRICE, Science City
Munoz,.Nueva Ecija, 3 UPLB-CA and 41RF

Single major genes usually confer resists
of certain rice varieties against bact
blight. Combining major resistance genes
variety (gene pyramiding) is said to pro
higher level of resistance and delays
breakdown of resistance. Four maintain
lines used for hybrid rice breeding
introgressed single and combination of
and Xa7 genes were inoculated with
historical (race 1 PX061 and race
PX086) and three contemporary races (
3 PX0340, race 9 PX0339 and race
PX0341) of Xanthomonas oryzar pv ory
to: 1) determine the reaction of individu;
combination of these genes to the diffE
pathogen races, 2) determine the effect
line background to the expression of
genes, and 3) detect possible (
interactions in conferring resistance 1
particular race.
The Xa4 and Xa7 genes in IRB
(control) and in the four maint,
background (IR58025, IR62829B, Lian B,
913 B, either singly or in combine
provided the expected reaction to
diagnostic races (i.e. Xa4-resistant to ra
and Xa7 resistant to race 2). All the
with both Xa4 and Xa7 showed sh
lesions and provided resistance to r
races than those containing single ge
Xa4 and Xa7 genes together in diff
maintainer background, however, impart
complementary gene action by conferri
higher level of resistance to race 3-(PX0O
and to race 10 (PX0341) in Lial
background. By examining the reaction
these genes in the four B lines to ra,
(PX0339), a segregation of an unknown


I resistance gene in IR58025B, IR6282!
: 913 B was observed which was not dE
S previously using the diagnostic rac
* differentiate Xa4 and Xa7.

1 REVERSE TRANSCRI
i POLYMERACE CHAIN REACTION
f PCR) OF CITRUS TRI
CLOSTEROVIRUS COAT PROTEIh
CP) GENE. GA Atienza1 DE Villarr
e Herradura2, and NB Bajet', 4UPLB-C
iI DNCRDC, Bago-Oshiro, Davao City.

3 The full-length putative coat prote
e gene of citrus tristeza closteroviru,
) was amplified by reverse trans
1 polymerase chain reaction (R"
4 Primers for full length CTV-CP was d
o based on the multiple sequence alii
of several CTV isolates downloaded 1
e genebank (http:/www/ncbi.nlm.nih.goi
nucleic acid extracts from partially
CTV served as template for first strain
r synthesis. PCR was conducted us
it following parameters: initial denatur
if 940C for 2 min followed by 30 cycles
3 each denaturation (940C), annealing
a and extension (720C) and a final exte
a 720C for 7 min. Agarose gel electron
of the RT-PCR products reveal
7 presence of an approximately 670 I
*r corresponding to and expected of
d length CTV-CP gene. The same be
I, absent on water control template. Th
a shows the application of RT-PCF
1 Philippine isolate of CTV. Clonii
s expression of the CTV-CP gene are c
'r to further characterize this CTV P
a isolate.

it ASSOCIATION OF CANDIDATE DE
a GENES FROM RICE, BARLEY ANC
a WITH QUALITATIVE AND QUANTI
) RESISTANCE IN RICE. CM Vera (
3 Ramalingam2, K Kukreja2, JM Chitt
)f Wul, SW Lee2, M Baraoidan1, ML (
9 MB Cohen', SH Hulbert2, JE Leach2,










Leung', 'lRRI and 2Kansas State University,
USA

Candidate genes involved in recognition
(resistance gene analogs, RDAs) and general
plant defense (putative defense response,
DR genes) were used as molecular markers
to test for association with resistance in rice
to blast, bacterial blight (BB), sheath blight
and brown planthopper (DPH). The 118
marker loci were either PCR-based RGA
markers or RFLP markers that included
RGAs or putative DR genes from rice, barley
and maize. The markers were placed on an
existing RFLP map generated from mapping
population of 116 double-haploid (DH) lines
derived from a cross between an improved
indica rice variety, IR64, and a traditional
japonica variety, Azucena.
Most of the RGAs and DR genes
detected single loci with variable copy
number and mapped on different
chromosomes. Clusters of RGAs were
observed, most notably on chromosome 11
where man known blast and BB resistance
genes and quantitative trait loci (QTL) for
blast, BB, sheath blight and BPH were
located. Major resistance genes and QTL for
blast and BB resistance located on different
chromosomes were associated with several
candidate genes. Six putative QTL for BB
were located to chromosomes 2, 3, 5,7 and 8
and nine QTL for BPH resistances were
located to chromosomes 3, 4, 6, 11 and 12.
The alleles of QTL for BPH resistance were
mostly from IR64 and each explained
between 11.4 and 20.6 percent of the
phenotypic variance. Several candidate RGA
and DR gene markers were associated with
QTL from the pathogens and pest. One
candidate DR gene, oxalate oxidase and
several RGAs were mapped to BB QTL.
Dihydrofolate reductase thymidylate
synthase co-localized with two BPH QTL
associated with plant response to feeding
and also to blast QTL. Blast QTL were also
associated with aldose reductase, oxalate
oxidase, JA-Myb and peroxidase markers.


Abstracts of papers
The frame map provides reference points to
select candidate genes for co-segregation
analysis using other mapping population,
isogenic lines and mutants.

PETCHAY MILD BLISTER: A PETCHAY
DISEASE LIKELY TO BE A VIRUS. NB
Bajet, PM Barcial and L Galvez, UPLB-CA.

A petchay plant showing mosaic and mild
blistering on its leaves was observed from a
lot in Malvar, Batangas, brought to and
maintained at the Plant Pathology, UPLB for
preliminary studies. Leaf extract of this plant
was urated in 10 mM sodium phosphate
buffer, pH 7.2 and mechanically inoculated on
2-4 wk old healthy petchay seedlings. Initial
symptoms were mild vein clearing and slight
downward curling of the newly emerging leaf.
More advanced symptoms were stunting and
leaves developing above the inoculated leaf
showing mosaic, mild blistering, and severely
reduced size. Mustard and radish seedlings
mechanically inoculated with extract of these
symptomatic petchay plants showed
conspicuous mosaic, blistering and mild
mottling, respectively. These symptoms
persisted on the plants but without or very
minor stunting. Extract of healthy petchay did
not indicate any of the above symptoms on
these three plant species. The putative virus
was assayed by purification through
differential centrifugation followed by rate
zonal ultra-centrifugation in a cesium chloride
gradient. Two light scattering bands were
observed about halfway down the SW55
tube. Band 1 was more distinct and the less
intense second band was below band 1.
These bands were collected separately and
UV 260 nm and 280 nm analysis show that
both bands are nucleoproteins. These results
partly indicate that the disease is viral.
"Petchay mild blister" and petchay mild blister
virus or PMBV are proposing names. The
isolate is being maintained and further
characterized for a more definitive
identification and taxonomic grouping.



































Metoiaogyne spp. was rouna. cost of large scale fermentation at optil
root galling was less on the pH 6.0-7.0 and temperature of 28-30 C.
n, Tay, Ngu Thoc and chosen PGPR/BNF inoculants from Ce
Luzon and Los Bahos successfully with
plantt reaction to Meloidoygne the lyophilization treatment, thus effect
tnamese Musa genotypes, as prolonging the shelf life of the preparation
)types FHIA-01, FHIA-02 and Vital NT can cut fertilizer costs by
rL %*a? I/*i ia+Cri I inr4r Fiilr4 -gmr r. n ;,..j, -.ill K- ^ IC A ; O / in








78


n the host, there is a specmc gene to BB, resistance to PX0339. I wenty-4
conditioningg virulence or avirulence in the varieties were also resistant to PX0339
pathogen. Of 26 Xa genes for resistance to Phenotypic and molecular analy
,acterial blight (BB) caused by Xoo, the Xa4 84 NPT lines and 17- NPT donor vc
3ene confers specific resistance to race detected the presence of Xa4, xa5, X<
>X061. Due to the diversity of germplasm xa13 although some lines with Xa4, Xi
use in breeding for various traits, the or xa5/Xa7 showed susceptible reaction
resistance of 12 strains of Xoo of 322 races except PX079 and PX0339. IR
classical elite lines, 44 IR and PSBRc 20-3-2-3 and IR 72976-AC 1
varieties and 84 new plant types (NPTs) of Xa4/xa5/Xa7/xa13 and have very
rice was analyzed using phenotypic lesions (0.9-4.5 cm) against all Xoo
approaches aided by molecular markers that Seven NPT donor varieties exhibitec
tagged five known Xa genes. The resistance resistance to BB indicative of the Xa:
Df NPT donor cultivars was also determined suggesting that Xa3 may also be pre:
and compared to the NPT lines. PCR some of the NPTs.
markers linked to Xa4, xa5, xa13 and Xa21 The gene-for-gene relationship (
were used to detect the presence of these Xoo- pathosystem was demonstrated f<
genes. Pedigree analyses using the with single Xa genes. For lines with
International Crop Information System more Xa genes, it was difficult to disti
database were performed on all lines to trace the effect of one gene on the host pher
the possible sources of resistance gene Thus, the use of molecular markers a
detected. the detection of two or more Xa genes
Two hundred and eight classical elite rice lines used in the study.
lines were resistant to PX061 indicating the
presence of Xa4. PCR primers for Xa4 USE OF RESISTANT VARIETIES II
confirm these results. Xa7 and xa5 were also MANAGEMENT OF RICE TU
detected in 2 and 10 lines, respectively, as DISEASE IN ILOILO. RC Cabunag
indicated by their reaction to Xoo and Sandig2, A Pamplona1 and II Ryong
confirmed by PCR markers. Twenty-seven 'IRRI and 2Provincial Agriculture
elite lines were resistant to most of the Xoo Department of Agriculture, Iloilo.
races indicating the presence of several Xa
genes. PCR markers detected gene Tungro disease is considered as the
combinations Xa4/xa5, Xa4/Xa21, and important virus disease of rice in Sou
xa5/Xa21 in these lines. Pedigree analyses Southeast Asia. Although several
show that these genes have been methods against the disease had beer
introgressed from donor, cultivars TKM6, effective, epidemics still occur perio
IR1545-33a, IRBB7 and Oryza barthii (now About 2,000 ha of rice crops were da
0. longitaminata) for Xa4, Xa5, Xa7 and by tungro disease in Iloilo pn


MIIUWIII Illy l I l CV I u1 rI U i Ldla l LU IIIUr l Anl
aces has Xa4/xa55.
Previous studies on BB resistant
utilized 6 races of Xoo. Using 12 strains
(oo, an unidentified gene conferrii
resistance to PX0339 was detected in sevel
slite lines singly or in combinations with Xa,


susceptlDie vanety IKt4.
Deployment of varieties with tt
resistance from different sources appear
be very effective for management of tt
disease in the province of Iloilo. F
seasons (WS 2001 and DS 2002) we
evaluated the performance of three tung







tracts of naDers


istant lines developed by IRRI at farmer's
is in the three sites of Iloilo province
irotac Nuevo, Ajuy and Lemery). The
gro resistant lines were 1) IR69726-29-1-
(Matatag 2, released as a tungro stop gap
iety in 2001, 2) IR 69726-116-1-3-2-2
itatag 1), 3) IR 73885-1-4-2-3-1-6
itatag 9, released as another tungro stop
) variety in 2001) and 4) 50:50 mixture of
34 and Matatag 9. IR 62 and IR 64 served
check varieties. About 1 kg seeds of each
iety were provided for farmer cooperators
1 in each site, three plantings (early,
dium and late) were made to an area of
)ut 0.25 ha.
Results revealed significantly low tungro
actionss on all of the three tungro resistant
,s tested, even when planted later than the
rounding areas where the infection
luency of IR64 still remained high. Results
o showed greater than 50% reduction of
gro infection in the mixture planting plots
npared to IR64 monoculture plots.
iough the infection in the mixture plots
re not as low as that of the pure tungro
istant lines, the mixing of Matatag 9 with
64 (phenotypically similar) could give
ners the chance to cultivate IR 64, which
highly preferred in the area because of its
h yield and good eating quality ratios.
ice WS 2002, we have been conducting a
lilar expanded trial using the variety
(ture of different ratios. Planting of different
gro resistant lines is being promoted
oughout the province of Iloilo as part of the
PITSAKA Project activities of IRRI,
ilRice and the Provincial Agriculture Office.

TECTION OF DISEASES IN
PRODUCED VARIETIES OF
GARCANE. FM dela Cueva, M P de
ampo, JB Ferrater, RT Luzaran, LM
lores and MB Palacpac, IPB, UPLB-CA
d PEQS, Philsurin.

the Philippines, the quarantine and
exing of foreign varieties of sugarcane is
trusted by the Philippine Sugar Research


Institute Foundation, Inc. (PHILSURIN) to 1
Post Entry Quarantine of the Institute of PI
Breeding, UPLB. These varieties are be
observed for the presence of pests E
pathogens potentially present in the plants
plant parts. Diagnostic tools used
pathogen detection include nucleic-a
based technique like polymerase ch
reaction (PCR) for fungal and .bacte
diseases and reverse transcript
polymerase chain reaction (RT-PCR)
disease of viral origin. Antibody bas
techniques like evaporative binding enzyr
linked immunosorbent assay (EB-ELISA) c
dot blot immunoassay (DBIA) are also be
employed to detect diseases caused
viruses and bacteria. Results of index
showed that leaf scald caused
Xanthomonas albilineans was present in5
of 10 varieties of sugarcane from Thailanc
out Of 9 varieties from Malaysia, 1 out of
varieties from Bangladesh, and 14 out of
varieties from France. Likewise, sugarc,
mosaic and sorghum mosaic viruses w
also detected from introduced varieties
sugarcane. This study showed that index
of introduced varieties should be routir
done to minimize introduction of new dise;
or new strains of the pathogens.

BANANA STREAK BADNAVIRUS IN T
PHILIPPINES. LE Herradura, MA Alforc
and Ma. A Lobres, BPI-DNCRDC, Davao

Banana streak disease caused by
badnavirus is considered a serious disease
Africa (Lassoudiere, 1974). In the Philippir
the disease has been reported to occui
some banana cultivars since 1994.
presence and spread of the disease w
determined through a survey in major bans
growing provinces of the country.
The paper presents the occurrence
BSV and the recent observations of
diseases in 2003. The most comn
symptom observed is chlorotic streaks on
lamina, which eventually turns necrotic. Sli







Abstracts of pap


triability in symptom expression among,
jitivars was observed.

NTAGONISTIC POTENTIAL OI
BACTERIA TO KEY PATHOGENS OI
ICE. JS Nazar, HX Truong, RG Corales, AE
Ilanueva, SA Miller and B Tadeo
HILRICE, Science City of Munoz, Nuev,
cija.

sneficial microorganisms, generally, have
e potential to cure rice diseases. This stud,
med to explore all the potential
icroorganisms with antagonistic ability,
against some common rice diseases
different microorganisms were isolated fron
healthy rice plant including soil around th<
ot.
Fifteen isolates of Bacillus pumilus, B
acerans, Actinomyces and Pseudomona.
utzeri were antagonistic to sheath bligh
pathogen (Rhizoctonia solani), nine t(
bacterial leaf blight pathogen (Xanthomona,
yzae pv oryzae), ten against rice bias
pathogen (Pyricularia grisea), one against
akanae pathogen (Fusarium moniliforme
id seven against both sheath blight an(
icterial leaf blight pathogen.
Other species such as Arthrobacte
stidinolovoran, B. pumilis, Corynebacteriun
luaticum and Paenibacillus polymyxa were
)le to enhance the development of the rice
ot system and seedling height comparable
nitrogen fixing Azotobacter. Development
microbial inoculant composed of these
omising isolates is going-on.

BIOLOGICAL CONTROL OF SOIL-BORNE
ITHOGENS USING BACILLUS SP. (LEP
18) IN ONION. MSV Duca1, RT Alberto2, LE
adua3 and SA Miller4, 'PHILRICE, Science
ty of Munoz, Nueva Ecija, 2CLSU, Science
ty of Muroz, 3UPLB-CA and 4Ohio State
diversity, USA

eld studies were conducted at the PhilRice
DGROCOMA/IPM CRSP Demo Farm ir
ongabon, Nueva Ecija to determine effective


delivery system of Bacillus sp. (LEP 11
biocontrol agent against soil-bome patho(
in onion cultivar Red Pinoy from Januai
April 2002. The incidence of bulb rot and
root was lower in plants drenched with
118 at 3 and 60 days after transplanting
in plants with roots dipped into the bact
suspension before transplanting
drenched with the bacterial suspension
days after transplanting.

MAPPING SOILBORNE PL
PATHOGENS IN MT. PROVINCE, BSU,
CHARMP AND LGU IN COLLABORAI
FOR RESPONSIVE DISE
MANAGEMENT. LA Lando1, AK Nagpala1
Bacbac (BSU) and CPicpican2. 1Departr
of Plant Pathology, College of Agricull
BSU, La Trinidad, Benguet and 2 GIS U
CHARMP, DA-CAR, Baguio Dairy F
Baguio City

Ralstonia solancearum, the causal orgai
of the bacterial wilt disease of solanaceE
crops was isolated from twelve soil sam
out of the 47 samples taken from
vegetable-producing sites in the CHAR
covered areas in Mt. Province. Of the 12
with Ralstonia, seven sites had crops
disease severity rating higher than 20%.
Seven economically important
three minor soilborne pathogens v
isolated. Plasmodiophora brassicae was
noted in the sites surveyed. Five pote
biocontrol species were isolated. T[
included Bacillus, Penicillium, Pseudomo
Trichoderma and Paecilomyces. The c
microorganisms isolated rema
unidentified.

GRAFTING AS AN EFFECT
MANAGEMENT STRATEGY AGAII
BACTERIAL WILT OF EGGPLANT.
Opina', RT Alberto2, SE Miller3, 1IPB, UF
CA, 2CLSU, Science City of Munoz, NL
Ecija, 3Ohio State University, USA










erial wilt, caused by Ralstonia
7acearum, is a major limiting factor in the
i scale production of eggplant in the
cs and subtropics. The study was
lucted to determine the effectiveness of
ing in reducing bacterial wilt of popular
arial wilt-susceptible eggplant varieties.
)lant cultivars resistant to Ralstonia
nacearum were grafted for use as
;tocks to high yielding bacterial wilt
eptible farmer's and commercial varieties
ggplant. The grafted and non-grafted
Ilings were planted in naturally bacterial
nfested farmer's field in Pangasinan and
ia Ecija and in the disease nursery at
B. The incidence of bacterial wilt was
itored in each plot at weekly intervals.
ificant difference in the incidence of
arial wilt were observed between grafted
non-grafted eggplant in both farmer's
Bacterial wilt incidence in the
eptible green variety was reduced in
ed plants by 93% in Nueva Ecija and
in Pangasinan compares to non-grafted
ts. Disease incidence in grafted Abar was
significantly reduced by 86% in Nueva
i. Bacterial wilt disease was significantly
ced in Casino and Abar scions on Eg
rootstocks compared to the non-grafted
rols.

ARDING BULB ROT INCIDENCE IN
)N WITH SELECTED CURING AND
RAGE METHODS. SE Santiago1, DT
32, RT Alberto2 and SA Miller3,
LRICE, Science City of Muroz, 2CLSU,
nce City of Mu~oz, 3Ohio State
ersity, USA.

ium-size Red Creole onion were cured
igh sun-drying and site-drying, placed in
>ags and wooden crates with net capacity
) kg and stored at 270C and in cold room
'C. Bulbs stored in 270C lasted for 13
were prematurely terminated on the 17
< of storage with high percentage of


ound bulbs. Bulbs in 270C incurred a 10'
weekly rate of rotting compared to 1% rotting
t 0C. Bulbs in net bags incurred 8% week
ate of rotting while 4% of bulbs in woode
rates rotted. Sun-dried bulbs had relative
>wer bulb rot incidence while site-dried bult
ad the highest rot incidence regardless <
torage temperature and packaging materiE
aspergillus niger, A. flavus, Fusarium sp. an
'. cepacia were detected. 'A. niger was th
predominant bulb rot microorganisms at 270
whilee Fusarium sp. was prevalent at 0C.

IEMATICIDAL ACTIVITY OF SELECTED
'LANT SPECIES AGAINST THE ROO"
[NOT NEMATODE (MELOIDOGYN
;RAMINICOLA) AFFECTING BUL
)NION. RM Gapasin1, DKM Donayre', C
;anchez and SA Miler2, 'LSU, Baybay, Leyl
Ind 2 Ohio State University, USA

leem, kuyot, papaya and Euphorb
regardless of form such as fresh extrac
hopped leaves/tuber and powdi
significantly reduced the number of galls ar
lematode population in roots and in so
plantt height and fresh weights of onion wei
highest with the application of kuyot E
powder. Except for kuyot, however, neer
papaya and Euphorbia applied as powdi
significantly reduced the nematoc
population in the soil. Their effects wei
comparable to the nematicide, Mocap.

EFFECTT OF VAM APPLIED SINGLY OR I
COMBINATIONN WITH SELECTED
ORGANICC MATERIALS ON THE RIC
tOOT-KNOT NEMATODE MELOIDOGYN
YRAMINICOLA, AFFECTING ONION. R
3apasin', DKM Donayre1 and SA Miler2
LSU, Baybay, Leyte and 2Ohio Stal
University, USA

rAM treated-plants and those combined wil
organic materials have higher fresh weigh
compared to nematode-treated contr
treatment. The highest fresh weight was








ADstracts OT paP


)Dservea in VAM-COW manure comDnanio
68g/plant). Reduction in gall counts in onio
oots due to VAM-organic materi
combinationn ranged from 42 to 69% over th,
if nematode alone. VAM spore counts in sc
ind VAM infection in roots were high in VAI
reated-plants. In most cases, the addition (
organic material improved spore production
comparedd to nematode plus VAM treatment

WUSA GERMPLASM INFORMATION
SYSTEM (MGIS): A TOOL FOR GENETIC'
RESOURCES MANAGEMENT. V Ro;
NIBAP AP, c/o IRRI.

The International Network for th
improvement of Banana and Plantai
INIBAP) has established the electron
changee of germplasm data through th
Alusa Germplasm Information Systel
MGIS). The MGIS is aimed to collei
detailed and standardized information on th
varieties stored in the different collection
throughout the world and to facilitate access
or the international scientific community. Th
database contains records of accessions hel
including taxonomic description;
norphological characterization, agronom
evaluations, evaluations to stores
susceptibility to diseases and photos. MGI
s intended for three types of users: (
:urators of collections who has a primary ro
since they enter the information in MGI
which intends to be a tool for the efficiei
management of their collections; (2) INIBA
which centralizes, verifies, diffuses an
nanages the information provided by th
collections ; and (3) researchers interested
he information collected in MGIS. At present
here are 15 countries and institutions around
he world contributing information in th
database. Steps are now being done for thl
AGIS to be accessed through the INIBA
vebsite.

THE INTERNATIONAL MUSA TESTING
PROGRAMME: A GLOBAL PROGRAM
rO EVALUATION ELITE MUSA VARIETIES


1-UK tti-CItN I UIStASt MANAULMI
AB Molina, Jr.1 and Ma AG Maghu
'INIBAP-AP, c/o IRRI.

In the last 15 years, Musa breE
programme within the INIBAP network
made significant accomplishments rest
to an increasing number of new, high yie
and disease resistant varieties of ban
and plantain. These varieties include
dessert and cooking types, a numbt
which are considered to hold good poti
for smallholder production in Asia. T
improved varieties are made avai
through the INIBAP coordinated program
The IMTP through an appropriate Ma
Transfer Agreement. IMTP is a collabor
effort to evaluate elite Musa varieties
suitable sites worldwide and was
developed to carry out detailed evaluation
new materials, to obtain information on
resistance/tolerance to the major pests
diseases of banana, the black and y
sigatoka caused by Mycosphaerella fiji
and M. musicola, respectively, anm
Fusarium wilt caused by Fus&
oxysporum f.sp. cubense.
IMTP phase II started in 1996 whe
different countries initiated their field
using 9 different elite varieties
Honduras, Taiwan, Brazil and Cuba. Re
showed that several of these var
particularly the FHIA lines are resista
Sigatoka diseases and Fusarium wilt.
Phase III is now in progress, where n
varieties are included. In Asia, the follc
countries are participating: India, Philipp
Indonesia, Malaysia, Sri Lanka, Bangla(
Australia, China and Vietnam. IMTP var
that are adaptable for commercial us
farmers are available for multiplication
distribution by identified National Respoi
Centers (NRCs) in Asian countries. Ir
Philippines, IPB-UPLB and BPI-DNC
were designated as the NRCs.

RISK ANALYSIS OF ABACA V
DISEASE EPIDEMICS AND DECISION-










AGEMENT STRATEGIES IN THE
IPPINES. AD Raymundo, CS dela Cruz
WM Basio. UPLB-CA

risk of epidemics of bunchy-top and
iic virus diseases in abaca was analyzed
analytical modeling, computer simulation
Geographic Information System (GIS)
biology.
Based on rates of infection computed
I analytical models, destructive levels of
amics can occur in the next few years in
Visayas region. GIS modeling has
rmed this prediction. Computer
ation, utilizing detailed cycles of disease
the insect vector, likewise, provided a
ir forecast.
3ased on the calculated risks, sustained
ise eradication drives aided by
biologicall information are being
nmended to prevent this prediction from
ig into reality.

TIVARIATE STATISTICAL ANALYSIS
THE VIRULENCE OF
VOCARPELLA MACROSPORA AND
r RESISTANCE TO
JOCARPELLA LEAF BLIGHT IN
N. Sutoyo and AD Raymundo, UPLB-


variability of 119 isolates of
)carpella macrospora, collected from
ent corn-growing areas in the Philippines
studied in culture and in host plants while
expression of host resistance to
)carpella leaf blight in corn was
mined in 37 corn populations from the
pines and Indonesia. Various
onents of virulence and resistance were
ured and analyzed using the
'ariate statistical method Principal
)onent Analysis.
Variability of S. macrospora indeed
td among the isolates studied. In host
;, variability of the isolates in incubation
j, latent period, pycnidial production,


!xisted. The most virulent isolates produce
earlierr incubation period and shorter later
period, had more pycnidia, elicited bigger
esion size and higher percent disease
everity as well as faster rate of increase i
esion size and apparent infection rate
principal Component analysis showed the
ie first component, which accounted fc
lore than half of the total variance, identified
ie isolate Sm-14 as the most viruler
allowed by Sm-13.
Host resistance in the population
tested varied in terms of incubation period
tent period, pycnidial production, lesion siz
nd percent disease severity. Population
resistant to Stenocarpella leaf bligl
expressed relatively longer incubatio
eriod, longer latent period, less number c
ycnidial production, smaller lesion size an
)wer percent disease severity. Principc
omponent analysis of resistant variable
showed that the first component, which
accounted for more than half of the tote
ariance, was consecutively dominated b
percent disease severity, pycnidiE
reduction, latent period, incubation period
nd lesion size.

TRANSMISSION, SYMPTOMATOLOG'
,ND SEROLOGICAL IDENTIFICATION 01
EATHERY MOTTLE VIRUS COMPLEX II
WEET POTATO. LM Dolores, GN Yebrol
nd VM Aquino, IPB, UPLB-CA

t least three viruses have been detected
causing the leaf curl syndrome in swee
otato. These include Sweet potato feather
bottle virus (SPFMV), Sweet potato chlorotil
3cks virus (SPCFV) and Sweet potato
ilorotic stunt virus (SPCSV). Among these
ruses, SPFMV was found to be the mos
widespread and predominating virus in the
disease complex. SPFMV caused mild tc
moderate virus symptoms only if it occurred(
single infection. However, plants exhibited
lore severe symptoms when exposed t(
hiteflies. Based on differential reactions to


____ _________











nuII uOLI, I IUOLO IlIi Ipr uIIIutca aIusa Callu I.
some SPFMV isolates were isolated and
iracterized and confirmed positive for
gle infection with SPFMV. Results of NCM
SA varied among isolates.


ri-u cxo WI pral












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