Front Cover
 Breeding for resistance to papaya...
 Isolation, purification and partial...
 Population dynamics of nephottetix...
 First report of antracnose of onion...
 Occurrence of papaya ringspot potyvirus...
 Analysis of factors affecting the...
 Back Cover

Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00090520/00044
 Material Information
Title: Journal of tropical plant pathology
Series Title: Journal of tropical plant pathology.
Alternate Title: Journal of Philippine phytopathology
Philippine phytopathology
Physical Description: v. : ill. (some col.) ; 26 cm.
Language: English
Creator: Philippine Phytopathological Society
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
College Laguna
Publication Date: January-June 2001
Frequency: semiannual
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00044
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
    Breeding for resistance to papaya ringspot virus
        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
    Isolation, purification and partial characterization of potato X potexvirus (PVX) in the Philippines
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
    Population dynamics of nephottetix virescens in relation to tungro disease progression in pure and mixed stands of rice cultivars with different types of resistance
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
    First report of antracnose of onion (allium cepa L.), caused by colletotrichum gloeosporioides (penzig) Penzig & Sacc., in the Philippines
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
    Occurrence of papaya ringspot potyvirus in mindanao
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    Analysis of factors affecting the occurrence of rice tungro epidemics in the Philippines and implications to disease management
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
    Back Cover
        Page 85
        Page 86
Full Text



it -L 3 1 ar-un,,


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46prp lil" 7m L a1' 9

(Formerly Phili
Official Publication of

President C. A. B
Vice President R. A. Zi
Secretary T. 0. D
Treasurer L. C. LI
Auditor N. L. 0
Buss. Manager P. M. N
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Buss. Manager P. M. N
Board Members N. B. B
T. U. D
M. P. N
E. R. T
Ex-Officio A. M. 5

Journal of Tr

A. D. Raymundo
R.G. Bayot
N.B. Bajet



Subscriptions: Communications should be addressed to th
Laguna 4031. Philippine Phytopathology, published semi-annual
members in good standing and Sustaining Associates. For others
free and payable in advance. Membership in the Philippine Phyto1
by the Secretary upon request Page Charge: The Editorial Board
-nn-a -nmmn lratP +innn thp navmrnt r.nnnhilitv of their reFearcl

ne Phytopathology)
' Tropical Plant Pathology

01 2001-2002
qued P. M. Magdalita
a R. E. Tambien
I T. 0. Dizon
:an N. L. Opina
a E. B. Gergon
dalita A. D. Raymundo
a E. B. Gergon
dalita A. D. Raymundo
nundo N. B. Bajet
t L. C. Lapitan
ay R. A. Zorilla
ral N. V. Desamero
gco C. M. Veracruz
ihin C. A. Baniqued

:al Plant Pathology
ard 2000-2001

Associate Editor
Associate Editor


.EASURER, P.P.S. c/o Departmer t of Plant Pathology, UPLB, Collq
; the official publication of Tropi cal Plant Pathology. It is sent free
s P100.00 per copy (domestic) an S25.00 per copy elsewhere, post
logical Society, Inc. Information regarding membership will be suppli
rves the right to charge some authi rs a modest amount of each publish
'- -- - -9--. ;-,*.t..t, A A ,.. f t. m1? .t rrt- -

Phkrtnnsthnln>rtnal Rn.iptv Tnr.

" I



A portion of the PhD thesis of the senior author submitted to the Postgraduate Studies, The University of
Queensland. Brisbane. Australia. Given the Best Paper Award by the Philippine Phytopathological Society during the
3 1" Annual Scientific Convention of the Pest Management Council of the Philippines, Inc. held at the Hotel Supreme,
Baguio City on May 3-6. 2000.

'University Researcher, Institute of Plant Breeding. College of Agriculture, University of the Philippines Los
Bafios, College, Laguna, Philippines: 2Senior Lecturer, School of Biomolecular and Biomedical Science, Griffith
University-Nathan Campus, Brisbane, Australia 4111: and 3Professor, School of Land and Food Sciences, The University
of Queensland. Brisbane. Australia 4072.

Interspecific hybridization between Carica papaya and the resistant wild species (Carica
cauliflora Jacq.) was done to create hybrids containing resistance to papaya ringspot virus
(PRSV). Two thousand one hundred hybrid embryos (4.8%) from 43,736 seeds were isolated
90 to 120 days after cross-pollination. The hybrid embryos were germinated for five days
onto a medium containing 0.5 strength De Fossard nutrients plus; 6-benzylaminopurine
(BAP 0.25 mM), a-naphthalene acetic acid (NAA; 0.25 mM), gibberelic acid (GA3; 10
mM), sucrose (58 mM) and agar (8 g 1). One thousand nine hundred eighty and one
(1,981) C. papaya x C cauliflora hybrid embryos were germinated and 485 hybrid plant
grown successfully. Because the numbers of main leaf veins as well as the leaf margins
were found to be intermediate between C. papaya and C. cauliflora, they were used as
morphological markers of hybridity. An analysis of the putative hybrid plants by random
amplified polymorphic DNA (RAPD) using five Operon primers showed that all 120 putative
hybrids analyzed were genetic hybrids of C papaya x C. cauliflora. The virus did not infect
the 34 hybrids and C cauliflora plants after manual inoculation and field tests. Similarly,
the back-inoculation test indicated that all the 34 hybrids and C cauliflora plants did not
carry the virus, while the C. papaya plants did. The plate trapped antigen-enzyme linked
immunosorbent assay (PTA-ELISA) confirmed that the virus was absent in the 34 hybrids
and C cauliflora plants but occurred in the C papaya plants. Taken together, these three
tests strongly suggest that all the C papaya x C cauliflora hybrid plants are resistant to
PRSV, and that this resistance was inherited from C. cauliflora.

Key words: papaya ringspot virus, resistance, embryo rescue, random amplified polymorphic DNA markers,
enzyme-linked immunosorbent assay


Papaya (Carica papaya L.) is a popular high value
crop in many countries. The Philippines ranks eight in
world papaya production. Papaya production in Luzon
used to be a profitable business before the outbreak of
the devastating papaya ringspot virus (PRSV) that
practically wiped out the industry in Cavite. In 1994,
production declined by about 80% in Region IV and yield
losses amounted to ca. P56.6 M (Quebral et al.. 1994).
Since 1982. the disease has been spreading like wildfire
over long distances (Magdalita et al., 1989) and is now
widespread all over the Luzon mainland and other islands

including Palawan. Mindoro. Marinduque, the Visayan
group including Negros, Leyte. Aklan and Panay. and
recently South Cotabato in Mindanao (Herradura et al..

The most reliable solution to PRSV is the
development of truly resistant papaya cultivars (Mekako
and Nakasone. 1975; Manshardt, 1992). A good source
of resistance within the species has yet to be found (Cook
and Zettler, 1970), but C. cauliflora Jacq., a wild relative
of papaya, does show resistance (Alvizo and Rojkind,
1987; Magdalita et al., 1988). Introduction of the
resistance from this species to the cultivated papaya has

been attempted (Jim6nez and Horovitz, 1958; Mekako
and Nakasone, 1975), but these efforts failed mainly
because the hybrid embryos died in the developing fruit,
and no attempt was made to use a tissue culture step to
rescue them. In succeeding studies, an embryo rescue
technique was used (Manshardt and Wenslaff, 1989; Chen
et al., 1991), but hybrid survival was very low.

Interspecific hybridization of papaya and
C. cauliflora has been limited to the use of cultivars
(e.g. Sunrise Solo) adapted to a specific environment
(e.g. Hawaii) and are accepted only by a certain group
of consumers. If these studies were to be successful,
the resistant cultivar developed may not perform well
under a wide range of environmental conditions, and/or
may not be acceptable to local consumers. Thus,
interspecific hybridization work needs to be undertaken
on papaya x C. cauliflora crosses, which specifically
target papaya cultivars suitable for local consumption.

Hybridity confirmation of putative interspecific
hybrid plants in the past has been via morphological
(Khuspe et al. 1980) or biochemical (i.e. isozyme)
analysis (Manshardt and Wenslaff, 1989; Chen et al.,
1991). However, these techniques are highly prone to
environmental influences and internal factors (e.g. genetic
segregation and epistasis) and therefore their use as
techniques for hybridity confirmation is limited (Beckman
and Soller. 1986; Tanksley et al., 1989). Despite such
limitations, morphological markers are still useful for the
preliminary identification of hybrid plants. An improved
method for the confirmation of hybridity utilizes DNA
markers (Williams et al., 1990; Welsh and McClelland;

In the past, screening methods for PRSV resistance
in hybrid plants were limited to the use of manual
inoculation and/or field tests (Khuspe et al., 1980;
Chen et al., 1991). An additional test, an ELISA (Clark
and Adams. 1977) which can detect the actual
presence of a virus, needs to be included as another
component for a more reliable screening method. This
study was conducted to: a) investigate the use of
interspecific hybridization and embryo rescue for creating
papaya x C. cauliflora hybrids. b) explore the use of
morphological and RAPD markers to confirm the
hybridity status of the putative hybrids and, c) confirm
the resistance status of the hybrids to PRSV using
different screening methods. In this study, an ELISA
was used for virus indexing in the manually inoculated
or field-planted hybrids so as to confirm their resistance
to PRSV.


Plant Materials

The flowers and pollen used for interspecific
hybridization were produced in field-grown trees. Fifteen
papaya cultivars (Cariflora, Honey Gold, Improved 4165,
Kamerunga, Known You No. 1, Majestic, Red Luck,
Shailer, Washington, 0003, 0004, 0051, 0052, 2001, 4002)
susceptible to PRSV and a C. cauliflora line resistant
to PRSV were used. All trees were planted two meters
apart in field plots (72 m long, 1 m wide) aligned in a
north-south direction.

Interspecific Hybridization

Pistillate flowers of papaya at full-ballon stage were
bagged using white paper bags (12 cm in length, 6 cm in
width) and cross-pollinated three days later with viable
C. cauliflora pollen. Reciprocal cross-pollinations were
also undertaken. At the same time, papaya pollen was
transferred to pistillate papaya flowers which were
bagged immediately. Unpollinated pistillate papaya
flowers were also bagged for use as controls.

An experiment was conducted to identify a papaya
cultivar that produces a high number of putative hybrid
embryos when hybridized with C. cauliflora. Thirty
pistillate flowers from each of 15 papaya cultivars were
cross-pollinated with C. caulflora pollen. In reciprocal
crosses, 10 C. cauliflora pistillate flowers were cross-
pollinated with pollen from each of the 15 papaya
cultivars. The number of successfully formed fruit was
counted 14 days after pollination and converted to a
percentage of those flowers cross-pollinated. From the
90 day-old fruit, hybrid seed was removed and counted.
The putative hybrid embryo was isolated from the seed
using a pair of sterile forceps with the aid of a binocular

Embryo Rescue

Embryos used for all experiments were from sib-
pollinated C. papaya or from interspecific hybridizations
between papaya x C. cauliflora. The fruits were
washed using a detergent solution (5%, v/v), rinsed with
distilled water and surface-sterilized with 70% ethanol
before being bisected in a laminar air flow cabinet. The
seeds were aseptically removed from the central fruit
cavity, and the embryos aseptically isolated with the aid
of forceps and a binocular stereo-microscope. All media
used in all experiments contained sucrose (58 mM) and

2 Volume 37 (1) January.June 2001 Journal of Tropical Plant Pathology

2 Volume 37 (1) January-June 2001

Journal of Tropical Plant Pathology

Bacto-agar (8 g L-'). unless otherwise stated. The media
used for embryo rescue were dispensed (20 mL) into
plastic Petri dishes or polycarbonate tubes. The isolated
embryos were then cultured on one of several
germination media. All cultures were incubated at 25"C
under a constant photosynthetic photon flux density (ca.
120 mmol m2's-') which provided a 16 h photoperiod.

Ninety-day-old embryos were incubated for seven
days on a 0.5-strength De Fossard (DF) to which was
added gibberelic acid (GA,: 0 or 10 mM). 6-
benzylaminopurine (BAP: 0 or 0.25 mM). and a-
naphthalene acetic acid (NAA: 0 or 0.25 mM). The
medium of Manshardt and Wenslaff (1989) containing
0.5 strength MS nutrients and BAP (0.89 mM) and NAA
(2.67 mM) was used as a standard control to which these
treatments were compared. After a pre-incubation
period of seven days. the percent germination on each
medium was assessed. After 60 days of further
incubation on a plant growth regulator-free (PGR-free)
plantlet growth medium containing full-strength DF
nutrients, sucrose (58 mM) and agar (8 g L-'). the
seedlings were removed from the culture vessels and
assessed for dry weight. height. leaf number and root
length. The statistical method used in the experiment
was a completely randomized design with 10 replicate
plates, each plate with 20 embryos for each of the various
treatments. Twenty replicate seedlings were used in
the analysis of growth characters. Differences between
treatments were tested by protected least significant

A large number of putative interspecific hybrid plants
were produced. Putative hybrid embryos (2.100)
produced from fruit crosses were pre-incubated on a
liquid germination medium for five days before being
incubated on a PGR-free plantlet growth medium for a
further 60 days. Subsequently. the putative hybrid
seedlings were removed from the culture vessels. Their
roots were washed with tap water to remove the agar
and grown in trays (70 x 19 x 8 cm, length/width/depth)
containing a steam-pasteurized peat: perlite: polystyrene
mix (1:1:1). The seedlings were acclimatized following
the procedures of Drew (1988).

Morphological Analysis

Plant height, stem diameter, petiole length, leaf length
and laminar length were measured 120 days after the
putative hybrid plants were established in soil. For this
study. 20 hybrid and five 120-day-old parent plants
selected at random were used. The presence or absence

of leaf wax, petiole hairs, plant vigor, leaf serration.
venation, general leaf appearance, stem, petiole or flower
color, flowers per node and sex form were also assessed.

Random Amplified Polymorphic DNA (RAPD)

The RAPD banding pattern of 120 putative hybrids
and three parent plants each of papaya and C. cauliflora
was determined 120 days after the plants were established
in soil!. Young leaves near the apex were cut, individually
washed in distilled water and dried, after which the
midribs were removed. The remaining leaf tissues (1 g)
were then placed in a sterile polycarbonate tube and
stored in a biofreezer at -70oC until ready for use. The
modified CTAB (hexadecyltrimethylammlonium bromide,
Sigma Chemical Co., St Louis, Missouri. USA) method
of Graham et al. (1994) was used for all DNA
extractions. The isolated DNA was dissolved in 100
mL Tris-EDTA (TE) buffer (10 mM Tris-HC1, pH 8.0. 1
mM EDTA, pH 8.0 and treated with 2 mL RNAse A
(10 mg mL-'. Sigma Chemical Co., St Louis, Missouri.
USA). The DNA concentration was determined using
a fluorometer (TKO 100 Mini Fluorometer, Hoefer
Scientific Inst.. San Francisco, California, USA) following
the procedures provided by the manufacturer.

Seventy-two 10-mer primers (Operon primers.
Operon Technologies Inc.. Alameda. California, USA)
were tested for their ability to amplify portions of the
DNA obtained from a single putative hybrid, a papaya
and a C. cauliflora plant. Of these, five primers that
produced the highest degree of polymorphism were used
for analyzing the hybridity of the 120 putative hybrid
plants. The polymerase chain reaction (PCR) was carried
out in 25 mL reaction volumes containing 20 ng of isolated
genomic DNA. 2 mM of each primer, 0.01 mM
deoxyribonucleotide triphosphates (dNTPs; Promega Co..
Madison, USA). 3 mM MgCI,, 50 mM KC1, 10 mM Tris-
HCI and 1.0 unit Taq DNA polymerase (Boehringer
Mannheim. Mannheim. Germnnany). The mixture was
overlaid with 30 mL of light mineral oil. The thennrmal
cycling program used was: one initial cycle of
denaturation at 94"C for five minutes followed by 40
cycles of denaturation at 94"C for one minute, annealing
at 35"C for one minute and extension at 72"C for two
minutes (Perkin-Elmer 480 DNA Thermal Cycler.
Perkin-Elmer Cetus Co., Norwalk. Connecticut, USA).
The PCR-amplified products were analyzed by
electrophoresis on 1.2% agarose gel in Ix Tris-borate
(45 mM)-EDTA (TBE: ImM) buffer. pH 8.0 with across
field of 100 v for 2.5 h. The gel was then stained with

P.M. Magdalita et al. ~IoIume 37 (1) January.June 2001 3

\lolume 37 (1) January-June 2001 3

P.M. Magdalita et al.

ethidium bromide for 20 minutes and then photographed
under UV light using a transilluminator.

Screening for PRSV Resistance

Greenhouse and Laboratory Screening. This
study was conducted at the Plant Pathology Unit,
Queensland Department of Primary Industries,
Indooroopilly, Queensland, Australia. The interspecific
hybrid and parent plants were screened for PRSV
resistance using a manual inoculation test. The inoculum
was obtained from PRSV-P-infected zucchini squash
plants grown in the greenhouse (Thomas and Dodman,
1993). All 22 interspecific hybrids, three papaya and
three C. caulflora plants were inoculated with PRSV.
The PRSV isolate was multiplied in zucchini squash (cv.
Green Ruffles), prepared by homogenizing infected leaf
tissue in 0.1 M potassium phosphate buffer (pH 7.1)
containing sodium sulphite (1%, w/v) and applied by
rubbing onto carborundum-dusted leaves gently using the
forefinger. All plants were inoculated twice, seven days
apart. The hybrid, papaya and C. caulflora plants were
observed for symptom development 30 days after
inoculation. Three plants each of the interspecific hybrids,
papaya and C. cauliflora were used as non-inoculated

PRSV infection in all test plants screened for
resistance was detected by back-inoculation into zucchini
squash plants (five squash plants/entry). With the non-
inoculated controls, the sap extracted from each of the
hybrids, papaya and C. cauliflora was then inoculated
into five healthy squash plants for use as a control. The
presence or absence of PRSV in all inoculated squash
plants was assessed 30 days after inoculation. The
presence or absence of PRSV in all manually inoculated
test plants and non-inoculated control plants was tested
30 days after inoculation using a plate-trapped antigen-
enzyme linked immunosorbent assay (PTA-ELISA) as
described by Thomas and Dodman (1993) using
antiserum to PRSV. The reaction solution absorbance
(at 405 nm) was read using an ELISA plate reader.
Samples were considered positive for PRSV infection
when the mean absorbance exceeded twice the mean
absorbance of the non-inoculated controls (Thomas and
Dodman, 1993).

Field and laboratory screening. Another batch
of 12 hybrid, three papaya and three C. caulibfra plants
was screened for PRSV resistance in a field trial with
naturally high PRSV incidence. The field was located

in Bridgemandowns in Southeast Queensland, Australia.
All plants were initially inoculated with PRSV inoculum
in the glasshouse and planted in the field three days after
inoculation. They were assessed for PRSV infection
through the presence of symptoms 30 days after
inoculation. The presence or absence of PRSV was
confirmed by a PTA-ELISA test as described above.
Leaf samples from one plant each of the non-inoculated
hybrid, papaya and C. cauliflora that were maintained
in the glasshouse were also collected for use as control.

All experiments involving manual inoculation tests
undertaken in the glasshouse and immunological tests
undertaken in the laboratory were conducted using the
completely randomized design. In the field screening
experiment, the plants were planted 1.5 m apart in a
field plot (37.5 m long, 1 m wide) aligned along a north-
south direction. This field plot was adjacent to other
plots planted to PRSV-infected papaya plants.


Interspecific Hybridization

The percent successful crosses obtained from the
15 papaya cultivars hybridized with C. cauliJlora ranged
from 17 to 53% (Table 1). Similarly, the number of
putative hybrid embryos and seeds ranged from 0 to 10
and from 221 to 3.846, respectively. Out of the 15
cultivars, only 4 (cvs. 2001, 0003, 4002 and Improved
4165) produced fruit and seed with viable putative hybrid
embryos. The ability of these papaya cultivars to produce
putative hybrid embryos with C. cauliflora suggests that
they are partially compatible and more tolerant ofgenomic
disturbances created by a foreign Carica species than
other cultivars (eg. Cariflora, Honey Gold, Known You
No. 1). Cultivar 2001 produced the highest number (10)
of putative viable embryos. Two types of hybrid embryos.
viz. single (Fig. 1A) or multiple (Fig. IB) were observed
following interspecific crosses.

It can be speculated that cultivar 2001 may contain
a gene for higher crossability than the other three
cultivars. For example, in wheat x maize intergeneric
crosses, it has been found that a specific wheat variety
contains kr 1 and 2 recessive alleles that promote
crossability between wheat and maize (Laurie and
Bennett, 1987). An earlier study had demonstrated that
most cultivars were more incompatible with C.
cauliflora than cv. Washington or Sunrise Solo

4 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

Journal of Tropical Plant Pathology

4 Volume 37 (1) January-June 2001

[anshardt and Wenslaff, 1989). All reciprocal crosses
solvingg 150 C. cauliflora pistillate flowers cross-
llinated with pollen from the 15 papaya cultivars were
successful in forming fruit (Table 1), suggesting strong
;ompatibility of the C. cauliflora genome to papaya.

Embryo Rescue

A 0.5 strength DF nutrients supplemented with BAP
25 mM). NAA (0.25 mM). and GA, (10.0 mM)
luced 100% germination of 90-day-old embryos (Table
This medium was subsequently used for germinating
papaya x C. cauliflora hybrid embryos. The
cellent germination of embryos could be attributed to
: use of GA3. This plant growth regulator has been
mdto be a promoter of embryo germination in avocado
kene and Barlass, 1969) and in papaya (Bertocci et
1994). The resulting seedlings had significantly
<0.05) higher dry weight, height, leaf number and root
igth than those produced in the other treatments, while
aducing only a low amount of callus. However, the
mnts were etiolated, which could be due to the side
'ect ofGA3 (Wareing and Phillips, 1986). This plant
awth regulator could cause excessive cell division and
nationn due to stimulated mitotic activity in the sub-
ical region of the stem (Sachs, 1965), a condition
-ectly related to etiolation ofthe plants produced. Good
rmination (94%) occurred in a 0.5 strength MS
trients supplemented with BAP (0.89 mM) and NAA
.67 mM) but the regenerated seedlings produced large
entities of callus and resulted in poor quality seedlings
short stature, with less leaves and poor root systems.
ie results are consistent with the findings of Manshardt
d Wenslaff (1989). Poor germination (71%) and
owth were obtained on a 0.5 strength DF nutrients
thout GA,, BAP or NAA.

A liquid or a solid form of the best germination
medium previously mentioned promoted 100%
rmination of putative hybrid embryos, but the seedlings
oduced on a liquid germination medium had
gnificantly (P<0.05) better growth characters than
ose produced on a solid germination medium
'able 3). This is possible because the absorption of
itrients by the putative hybrid embryos was faster
hen cultured in a liquid than on a solid medium. In oil
Lim (Rabechault et al., 1968) and cocoa (Pence et al.,
)80), a liquid medium induced rapid growth of
>ung embryos compared with those on solid
medium. The papaya embryos germinated on a liquid
:rmination medium produced seedlings that were
Iler and had greener and healthier cotyledonay leaves

in the seedlings produced on a solid germination
:dium. The result suggests that the liquid formulation
led as a catalyst of growth as it promoted the growth
weak embryos by more than 50% compared with
)wth on a solid medium.

From 338 fruit crosses, 43,736 seeds were formed
d a batch of 2,100 (5%) putative hybrid embryos was
)duced. This suggests that successful pollination and
utilization had taken place, but some embryos failed to
velop due to abortion of the zygote. The liquid
rmination medium previously developed consistently
amoted the germination of single embryos and their
velopment into whole plants. In contrast, the multiple
ibryos underwent direct somatic embryogenesis and
aduced multiple plants. There was efficient production
putative hybrid plants, which developed into
)rphologically normal plants, of which 485 were
oduced. In previous studies, a limited number of
erspecific hybrid plants were produced, many of which
d abnormal morphoany of which had abnormal morpho,
if fasciation and distortion, and roots covered with
Ilus (Manshardt and Wenslaff, 1989, Chen et al., 1991).

Morphological Analysis

The putative hybrid plants had a morphology that
is not seen before in either parent, identical to one or
both parents, and intermediate between those of the
,o parents (Table 4). Out of 18 characters of papaya
C. cauliflora, only two were intermediate of the two
.rents in all putative hybrid plants examined. These
arphological characters may be useful for the screening
the potential hybrid plants prior to a more detailed
:netic analysis. Intermediate morphological
characteristics have been used previously for the
notification of papaya x C. cauliflora hybrid plants
lhuspe et al., 1980, Chen et al., 1991). One of the
termediate characters was the number of main leaf
ins, which was consistently seven in all of the 20 hybrid
ants studied, in contrast to the maternal (papaya) and
Lteral (C. cauliflora) plants, which had nine and five,
spectively. The second character was the leaf margin,
which has two types: type 1 is an elongated leaf with
:ep serrations and has narrow and pointed teeth (Fig. 2
>ttom-left), while type 2 is a broad leaf with shallow
rrations, and has broad and slightly convex teeth (Fig.
bottom-right). Out of the 20 putative hybrid plants
:amined, 16 (80%) had the type 1 leaf morphology.
his suggests the strong genetic influence of the C.
ruliflora male parent. Five characters such as a solitary
power per node and hermaphrodite flower type, cupped

I. Maudalita et al. me M Ii) January-June ZUU1 ~

1. Miadalita et al.

mew3 (-i) janary-june zuui 0

leaf appearance, short stature, and low vigor were seen
in all putative hybrid plants, indicating the uniqueness of
the hybrids.
RAPD Analysis

The 72 10-mer primers screened for the amplification
of segments of the genome of papaya, C. cauliflora
and putative hybrid generated a total of 238, 263 and
182 RAPD bands, respectively. The lower number of
RAPD bands present in the hybrid plants compared to
that of the parents may suggest competition for primer
binding sites of similar homology. A similar primer
binding competition has been seen in interspecific
somatic hybrids of potato, Solanum tuberosum x S.
brevidens (Xu et al., 1983). A high level of
polymorphism (68%) in RAPD banding patterns existed
between papaya and C. cauliflora, which indicates that
these two species are distant relatives in the genus
Carica as also reported by Jobin-Decor et al. (1995).
Five of the 72 10-mer primers (OPA-07, OPA-09, OPA-
19, OPB-12 and OPC-06) consistently amplified
papaya and C. cauliflora DNA sequences in all of the
120 hybrid plants. Using these five primers, a total of 10
polymorphic bands (2 bands from each primer, 5 bands
from each parent) were amplified in the hybrid plants.
For example, primers OPA-09 (Fig. 3A), OPA-07, OPA-
19 and OPB-12 (Fig. 3B) amplified an 800, 1,600, 1,400
and 940 bp fragments respectively, in the hybrid plants
which were from the paternal parent. Likewise, the same
primers amplified an 850, 1,200, 900 and 830 bp fragment
respectively; in the hybrid plants that were from the
maternal parent (Fig. 3A&B). Since a DNA fragment
from each parent was consistently amplified in the hybrid,
this implies the presence of two chromosome sets or
parts of two chromosome sets in the hybrid (one from
each parent). For example, using OPA-09, an 850 bp
and an 800 bp DNA fragment from papaya and C.
cauliflora, respectively, were identified in some of the
putative hybrids (Fig. 3A). These RAPD markers
generated indicate that the putative hybrids produced are
true genetic hybrids of papaya and C. cauliflora. The
same type of markers was used for confirming the
hybridity of Solanum (Waugh et al., 1992) and Oryza
(Farooq et al., 1994) interspecific hybrids.

Screening for PRSV Resistance

Glasshouse and Laboratory Screening. All 22
papaya x C. cauliflora hybrids did not show any PRSV
symptoms when assessed 30 days after inoculation (Table
5 and Fig. 4A-center). Likewise, the three C. caulilflora
plants did not show any PRSV symptoms. The result

proves that the hybrid and C. cauliflora plants are
resistant to PRSV. This suggests that the hybrids
have inherited the PRSV resistance from C. cauliflora.
This is similar to the observation reported by Khuspe
et al. (1980) with their papaya (cv. Washington)
x C. cauliflora hybrid plants. However, the three papaya
plants exhibited typical PRSV symptoms such as leaf
mottle or chlorosis, and water-soaked lesions on the
petioles (Fig. 4A-right). All non-inoculated control plants
did not show any PRSV symptoms (Table 5).

In a back inoculation test, the 22 hybrid and three C.
cauliflora plants tested negative for PRSV while the
three papaya plants tested positive (Table 5). The
infection in papaya was indicated by the typical leaf
chlorosis or mottle symptoms produced in back-
inoculated squash plants. In addition, all non-inoculated
hybrid, papaya and C. cauliflora plants were negative
for PRSV infection, as symptoms were not produced in
the squash plants (Table 5). Furthermore, in PTA-ELISA
test, all 22 hybrid and three C. cauliflora plants were
negative for PRSV infection, while the three papaya
plants were positive (Table 5). The mean absorbance at
405 nm of all hybrid and C. cauliflora plant extracts did
not exceed twice the mean absorbance of their non-
inoculated controls, indicating the absence of PRSV
infection (Fig. 5). This indicates that the hybrids and
their C. cauliflora parents are genetically resistant to
PRSV However, the mean absorbance of the papaya
plant extracts was several times higher than their non-
inoculated controls, indicating the presence of PRSV
infection. All non-inoculated hybrids, papaya and C.
cauliflora plants tested negative for PRSV infection.
(Table 5).

Field and Laboratory Screening. Twelve hybrids
did not show PRSV symptoms when observed 33 days
after inoculation or when they were planted 30 days after
in the field with PRSV present (Fig. 4B-center).
Similarly, the three C. cauliflora (Fig. 4B-right) plants
planted in the field showed resistance to PRSV, while
the three papaya (Fig. 4B-left) plants showed typical
symptoms. The non-inoculated hybrid, papaya and C.
cauliflora plants had no PRSV symptoms.

All hybrid and C. cauliflora plants were negatively
indexed for PRSV infection while papaya plants were
positively indexed as shown by a PTA-ELISA test. This
is shown by the mean absorbance of the hybrid and C.
cauliflora plants, which did not exceed twice the mean
absorbance of their non-inoculated control (Fig. 6). This
finding confirms that the inoculated and field-planted

6 Volmune 37 (1) Januavy-June 2001 Journal of Tropical Plant Pathology

6 Volufne 37 (1) January-June 2001

Journal of Tropical Plant Pathology

hybrid and C. cauliflora plants contain genetic resistance GRAHAM GC, MYERS P, HENRY RJ. 1994. A simplified
to PRSV and constitutes conclusive evidence for method for the preparation of fungal genomic DNA for
resistance. However, in the positively-indexed papaya PCR and RAPD analysis. BioTechniques 16:48-50.
plants, the mean absorbance was several times higher
than that of the non-inoculated control. The non-
Detection of papaya ringspot virus (PRSV) in Southern
inoculated hybrid, papaya and ('. cauliflora plants were Mindanao. Proc. 3 V Annual Scentific Convention of
negatively indexed for PRSV infection. In this study. the Pest Management Council of the Philippines. Baguio
the manual inoculation test. further complemented bv a City. MaN 3-6, 2)000
PTA-ELISA test and a field screening test. proved that
all hybrids are resistant to PRSV JIMENEZH,HOROVITZS. 1958. Cnizabilidadentreespecies
de Canca. Agron. Trop. 7:207-215.

1996. RAPD and isozyme analysis of genetic
ADAMS PA, KAUFMAN PB, IKIVMA H. 1973. EflectsofGA relationships between ('aricapapava and wild relatives
.nd sucrose oi the growth of oat (.A-era .watra L.) stems Genetic Resources. and Crop Evol. 44:1-7
segments Plant Phvsiol. 51:1102-1108.
mancha anular del papaya en ('arica caulillora. Rev. Utilization of tissue culture to isolate interspecific
Mex. Dc Fitopal. 5:6 1-02 hybrids in Carica L.. pp. 1 98-205. In: Rao PS. Heble MR.
Chadha. MS. (Eds.). Plant Tissue Culture. Genetic
BECKMAN JS. SOLLER M. 1986. Restriction fragment Manipulation and Somatic Hybridization of Plant Cells.
length polymorphism and genetic improvement of Bhamba Atomic Research Centre, India.
agriculltral species Euphltica 35:111-124.
LAURIE DA, BENNETT MD. 1987. The effect of the
BERTOCCI FD, VOGLERJN, DREW RA. 1994. Influenceof crossabilito loci Krl and Kr2 on fertilization frequency in
culture media on the germination of papaya ('arica hexaploid wheat x maize crosses. Theor. Appl. Genet.
papaya L.) whole seeds. mature and immature embryos. 73:403-409.
In: Abstracts VIII"' International Congress of Plant
Tissue and Cell Culture. 12-17 June 1994. Florence. MAGDALITAPM.VILLEGASVN,PIMENTELRBBAYOT
Italh p 46 RG. 1988. Reaction of papaya (('arica papava L.) and
related ('arica species to ringspot vius. Philipp J. Crop
CHEN MH. CHEN CC. WANG DN.CHEN FC.(1991). Somatic Sc. 13:129-132.
eiiibriogenesis and plant rcgencration from immature
embryos of ('arica papaya x (. cauliflora cultured in MAGDALITA PM, OPINA OS, ESPINO RRC. VILLEGAS
vitro Can. J. Bot 69:1913-1918 VN. 1989. Epidemiology of papaya ringspol in the
Philippines. Philipp. Phytopathol. 25:1-1 1.
CLARK MF. ADAMS AN. 1977. Characteristics of the
microplate method of enzyme-linked imniunosorbent MANSHARDT RM. 1992. Papaya, pp. 489-511. In:
assay for the detection of plant viruses. J. Gen. Virol. Hammnerschlag F.A. and Litz. R.E. (Eds.). Biotechnology
34:475483 in Agriculture No. 8 Biotechnology of Perennial Fruil
Crops. Alden Press Ltd.. Oxford
COOK AA, ZETTLER FW. 1970. Susceptibility of papaya
cultivars to papaya ringspot and papaya mosaic virus. MANSHARDT RM, WENSLAFF TF. 1989. Zygotic
Plant Dis Reptr. 54-893-895. polycmbrvony in interspecific hybrids oft 'arica papavy
and C. cauliflora..I. Amer. Soc. Hort. Sci. 114:684-689.
DREW RA. 1988. Rapid clonal propagation of papaya in
vitro from mature field-grown Irces. HortSci 23:609-611. MEKAKO HU, NAKASONE HY. 1975. Interspecilic
hybridization among 6 ('arica species. J. Amer. Soc. Hort.
FAROOQ S, SHAH TM. IQBAL N, ARIF M. 1994. The use Sci. 100:237-242.
of RAPD markers for identification of wild and cultivated
rice species and their F, hybrids. In: Abstracts 4"' PENCE VC,HASEGAWAPM,JANICKJ. 1980. Initiation
International Congress of Plant Molecular Biology. 19- and development of asexual embryos of Theohbrioa
24 June 1994. Amsterdam. The Netherlands. p. 1872. cacao L. in vitro. Z. Pflanzenphysiol. 98:1-14.

P.M. Magdalita et al. Volume 37 (1) January-June 2001 7

State of the art of papaya ringspot virus in the Philippines. markers for the detection of gene introgression in potato
In: Report of the Ad Hoc Committee to Formulate PlantCellReptr. 11:466-469.
Comprehensive R and D Action Plan for the Control of
the Papaya Ringspot. University of the Philippines at WELSH J, Mc CLELLAND M. 1990. Fingerprinting genome
Los Bafios, College. Laguna. Philippines. 14pp. using PCR with PCR with arbitrary primers. Nucl. Acid
:: Res. 16:7213-7218.
RABECHAULT H, AHEE J, GUENIN G. 1968. Recherches
sur la culture in vitrodes embryons de palmier a huile WILLIAMS JK, KUBELIKAR, LIVAK KJ, RAFALSKI JA
(Elaeis guineensis Jacq.) IV. Effets d 1l teneur en eau des TINGEY SV. DNA polymorplusms amplifiedby arbitrary
noix et de la duree de leur staockage. Oleagineus 23:233- primers are useful as genetic markers. Nucleic Acids Re!
237 18:6531-6535.

SACHS RM. 1965. Stem elongation. Annu. Rev. Plant Physiol. XU YS, CLARK MS, PEHU E. 1993. Use ofRAPD markers 1
16:73-96 screen somatic hybrids between Solanum tuberosum an
S brevidens. Plant Cell Reptr. 12:107-109
SKENE KGM. BARLASS M. 1969. Stimulation of germination

Differentiation in Plants. Pergamon Press Ltd.. Oxford. and Jeorge Piperides) are highly appreciated and we
343 pp. remembered.

An..-~nl -f T,--i,.l PI-f~tnln

8 Volume 37


23 0 2317

Nutrients (pM) (pM) (/M) n weight t (cm) (no.) Length n Index
s (%) (mg) (cm) Index

DF 0.00 0.00 ().00 71.0 a 58.0 a 2.4a 5.0 a 5.2a 0.0 a 1.0b
DF 0.25 0.25 00.00 74.0 a 60.3b 2.9b 6.2 b 6.5b 0.0 a 3.0 c
DF 0.00 0.00 10.00 96.0 b 63.1c 5.5d 8.4d 7.7 d 4.0 c 0.0 a
DF 0.25 0.25 10.00 100.0b 65.0d 9.1e 10.0 e 8.5 e 3.0b 1.0b
MS 0.89 2.67 00.00 94.0b 62.0b 3.4c 7.4c 7.3c 0.0 a 4.0 d

ata followed by the same letter are not significantly different at P<0.05.

tble 3. Germination on 0.5 strength De Fossard nutrients supplemented with BAP (0.25 pM) and GA, (10 pM) and growth ofthe
seedlings produced from 90 to 120 day-old Carica papaya x C. cauliflora putative hybrid embryos.

K Maqdalita et al. ie 37 III January-June 2001 A

I. Macdalita et al.

ie 37 111 Januarvy-June 2001

lesions on

I .....
A | ,')-F 14!

Journal of Tronical Plant Patholoov

Table 6. The number of plants inoculated and field-planted, the number infected and, the niumber with symptoms of papaya
ringspot virus. The reaction of the test plants to a plate trapped antigen-enzyme-linked immunosorbent assay is also

Treatment Plants Number of Number of Symptoms that Reaction to
plants plants developed 33 days after PTA-ELISA
survived infected field-planting

Inoculated Hybrid 12 0 nil
C. papaya 3 3 leaf chlorosis and +
severe distortion; water
soak lesions on petioles

C. cauliflora 3 0 nil

Non-inoculated Hybrid 3 0 nil
C. papaya 3 0 nil
C. cauliflora 3 0 nil

Fig 1. A) A single putative hybrid embryo ofCarica papaya x C. cauliflora (center), C. papaya (left) and C. cauliflora (right)
and: B) putative multiple hybrid embryos produced at 90 days post-pollination. Scale bar represents 1 cm for A and 0.2
cmfor B-

C: papaya C cauliflora

C papaya x C caulflora

Fig. 2 Leaf margin profiles of 120-day-old Carica papaya and C. cauliflora and the two distinct leaf margin types oftheputative
hybrid plants. Type 1: elongated leaf with deep serrations. and narrow and pointed teeth (bottom-left) and; Type 2: broad
leaf with shallow serrations. and broad and slightly convex teeth (bottom-right). Scale bar represents 5 cm.

Voltffm 36 June~eceniber 2000 11

P.M. Maqdalita et al.

10 11 12 13 14

' **

1 2 3 4 5 6 7 8 9 101 1 12 13 14

Fig 3. RAPD band profiled generated by primers OPA-09 and OPB-12, resolved in 1.2% agarose and stained with ethidium
bromide. A) OPA-09; Lanes 1-11: hybrids, 12: Carica cauliflora, 13: C. papaya, 14: molecular weight marker. Note thatthe
850 bp fragment of C. papaya (*) and the 800 bp fragment of C. Cauliflora (**) are present in some of the hybrids (lanes
2,3,5-7 and 9-11) but the 800 bp fragment is absent in the other hybrids (lanes 1,4 and 8); B) OPB-12: Lane 1: C. papaya,
2: C. cauliflora, 3-13: hybrids, 14: molecular weight marker. Note that the 830 bp fragment of C. papaya (*) and 940 bp
fragment of C. cauliflora (**) are present in some of the hybrids (lanes 4-13) but the 940 bp fragment is absent in 1 hybrid
(lane 3)

12 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

12 Volume 37 (1) January-June 2001

Journal of Tropical Plant Pathology

Fig. 4. Plants undergoing screening for resistance to papaya ringspot virus (PRSV) in the glasshouse (A) and in the field (B).
A): The hybrid (center) and C. cauliflora (left) exhibit no viral symptoms. C. papaya (right) exhibits typical symptoms
such as leaf mottling and chlorosis (arrows). B): The hybrid (center) and C. cauliflora (right) exhibit no viral symptoms.
C. papaya (left) exhibits typical symptoms such as leaf mottling, chlorosis and distortion (arrows). Scale bar represents
10 cm for A and 5 cm for B.

P.M. Magdalita et al.

Volume 37 (1) January-June 2001 13


0.6 -

0.4 -

0.2 -

S0 2 4 6 8 10 12 14 16 18 20 22 24

SCarica papaya Carica caulflra
< I

0 2 4 6

I t-T-
0 2 4 6

Test Plant Number

Fig. 5. Absorbance values (at 405nm) obtained using a plate trapped antigen-enzyme linked immunosorbent assay from 22
hybrid. C. papaya and C. cauliflora plants being screened for PRSV resistance in the greenhouse. Absorbance at 405 nm
ofthe non-inoculated hybrids and parents are shown by the asterisk. The bars represent the mean and standard error from
two replicate measurements taken on each plant.


0.8 -


14 Volume 37 (1) January-June 2001

Journal of Tropical Plant Pathology

0.2 -


A' A-

I I 6
2 4 6

8 10 12

Test Plant Number

Fig. 6. Absorbance values (at 405nnm) obtained using a plate trapped antigen-enzyme linked immunosorbent assay from 12
hybrids, C. papaya and C. cauliflora plants being screened for PRSV resistance in the field. Absorbance at 405 nm of the
non-inoculated hybrid and parent plants are shown by the asterisk. The bars represent the mean and standard error from
two replicate measurements taken on each plant.


I lllllllli

0.2 -

0.1 -


Carica papaya

_I i

I 2
S 2 4

Carica cauliflora


P.M. Magdalita et al.

Volume 36 June-December 2000 15







Portion of the Ph. D. dissertation of the senior author submitted to the Graduate School, University of the
Philippines Los Bafios (UPLB), College, Laguna, Philippines

SRespectively. Senior Officer. Tuber Crops Research Center, Bangladesh Agricultural Research Institute (BARI),
Gazipur. Bangladesh and 2Professor, Department of Plant Pathology, UPLB. College, Laguna, Philippines.

Potato X potexvirus (PVX), isolated from symptomatic potato samples collected from
Benguet and Laguna, was identified through their serological reactions in ELISA and host
range studies. The dilution end point was 10 7; the thermal inactivation point was 750 C;
and the longevity in vitro was 17 days. The molecular weight of the viral coat protein was
about 30,000 daltons. The virus particles were flexuous rods of about 520 nm x 13 nm.
Among the isolates, ringspot and normal mild mosaic strains were identified with the
former much more prevalent.

The virus was purified through sucrose gradient centrifugation and the yield of
purified PVX was higher in infected samples maintained in Benguet (49.84-55.55 mg
virus/kg leaf samples) than those from Laguna (12.88 mg virus/kg leaf samples). PVX
antiserum produced in rabbits with a titer between 106 to 10-7 in ELISA was able to detect
the presence of PVX in some field samples of potato, tomato, and pepper.

Keywords: Potato. potexvirus, purification, serology


Potato (Solanum tuherosum L.) is an important
vegetable as well as food crop that grows well in
many countries including the Philippines. Potato
production in the Philippines is concentrated in the
high and mid-elevated areas of Benguet and Mountain
Provinces in Northern Luzon and in some areas of
Mindanao In the Benguet and Mountain Provinces,
potato is considered as the most important economic
crop and occupies an area of about 4000-4500 ha with
a yield ranging from 18-22 tons/ha (Potts el al.. 1982).

Potatoes are affected by about 25 viruses and one
viroid (Hooker. 1987). Among them. potato X
potexvirus (PVX) is the third most important after
potato leaf roll luteovirus (PLRV) and potato Y
potvvirus (PVY). A total of seven viruses of potato
namely. PLRV. PVY. PVX. potato S carlavirus
(PSV). potato M carlavirus (PMV). potato A potyviris
(PVA). and tobacco mosaic tobamov\irus (TMV). have
so far been reported in the Philippines (Talens. 1979:
Balaoing et al.. 1979: Baloing and Verzola. 1980: and
Luis. 1981).

Gupta et al. (1985) reported that the common
viruses like PVY and PLRV singly reduced yield by
up to 60-75% while the mild k nes like PVX. PSV, and
PMV reduced yield by 1( -30%. Salazar (1989)
reported a potential yield loss up to 90% with severe
virus infections. In the Philipuine. single infection by
PVX and PSV reduced yield up to 35% at 20 x 30 cm
distance of planting. Triple infections with PVX,
PVY. and PLRV reduced yield up to 40% at 40 x 30
cm spacing (Luis, 1981)

There has been no concerted and sustained effort
to increase productivity of potato in the country
through virus disease management. One draw back is
the unavailability of more reliable and sensitive
techniques to detect and identify viruses in the field or
to ascertain the presence/absence of these viruses in
vegetatively-propagated potato planting materials.
Most viruses that are in vegetatively-propagated
planting materials cause much more severe losses if
not total crop failure. Thus. this study was under
taken to determine the occurrence of different strains
of PVX on potato in Benguet, and to purify and
produce antiserum to the virus


The experiments were conducted at the
Department of Plant Pathology. University of the
Philippines Los Baflos (UPLB). Laguna: and at the
Northern Philippines Root Crops Research and
Training Center (NPRCRTC). La Trinidad. Benguel
from February. 1995 to Ma\. 1997.

Collection and Assay of the Symptomatic
Potato Samples

Symptomatic samples were collected from several
locations as follows: three fiom the Institute of Plant
Breeding. University of the Philippines Los Bafios
(IPB-UPLB). College. Laguna: two from the Baguio
National Crop Research and Development Center
(BNCRDC). Bureau of Plant Industry (BPI). Baguio
City: ten from the experimental farms of NPRCRTC
and Benguet State University (BSU). La Trinidad.
Benguet: one from the experimental farms of IPB. La
Trinidad. Benguet and five from farmers' field in
Natubay. Benguet. The collected samples were
brought to the greenhouse and each was separately
inoculated after maceration using sterilized mortar and
pestle in 1.0 M phosphate buffer. pH 7.0 and abrasive
carborundumm powder) to a set of indicator plants
namely, Nicotiana hittinosa. N. tabacum var. White
Burlev and Samsun. N. hcnthamiana. 'Phvsalis
floritdan. (;1omphrena gIohosat. ( henopodilm
amaranticulor. C. qjlinoa. Datura metel. D.
stramonium and Lycopersicon esculentum. These
indicator plants were assayed for the common potato
viruses PVX. PVY and PVS in enzyme linked
immunosorbent assay (ELISA) using kits from the
International Potato Center (CIP)

Isolation and Propagation of PVX

Isolation was done follow ing standard potato virus
isolation nrocedures thrnnch mechanical innolnVitinn

transmission manner using the aphids Myzus persicae.
Rhopalosiphum maidis and Aphis gossypil Witi
samples doubly infected with PVX and PVY. thesc
viruses were separated through inoculation intc
immune hosts. It has been reported that D.
stramonium is immune to PVY but susceptible tc
PVX and some potato genotypes are also immune tc
either PVY or PVX (Hossain. 1997).

Biological Characterization of PVX Isolates

The virus isolates were characterized through
symptomatology and host range, and physical
properties in vitro like dilution end point (DEP).
thermal inactivation point (TIP), and longevity in vitro
(LIV). The strains of the virus were identified though
host range and symptomatology followed by cross
protection tests in La Trinidad using C. amaranticolor
and N tabacum var. White Burley.

SDS-PAGE Analysis

About 0.2g of PVX-infected N. glutinosa leaf
sample collected from the greenhouse was
homogenized on ice in a mortar and pestle with
extraction buffer containing 0.1 M Tris HCI, pH 6.8
containing 0.01 M EDTA. The extract was
transferred into an Eppcndorf tube and placed on ice
for 0.5 hr. The tube was centrifugated at 14.000 rpm
for 5 minutes in an Eppendorf microfuge. With 100
-il of supernatant. 400.l of cold acetone was added
and placed on ice for 15 min. With the pellet. 50 -tl of
loading buffer (0.125 M Tris, pH 6.8. 4% SDS. 20%
glycerol. 10% 2-mercaptoethanol and 0.001%
bromphenol blue) was added and boiled for 5 min
The controls were health N. glutinosa leaf sample
processed similarly as above and purified PVX. The
purified virus sample was prepared as follows: 40 pl
nure vinrses (1 mir/mnl added with 10 nd1 7% qn<

M. Hossain. N.B. Baiel

Vnitimp 17 Ill Aaniin-v --hiina qnni 17

0.05 M phosphate buffer, pH 7.2, stirred and again
Electron Microscopy of Samples clarified low speed centrifugation at 2,500xg for 5
min. The supernatant was collected and centrifugated
The isolates were examined with an electron through two cycles on performed 20-50% linear
microscope as follows. A leaf tissue from each isolate sucrose density gradient in 0.01 M phosphate buffer.
was homogenized in 0.05 M phosphate buffer. pH 7.0 pH 7.2 containing 0.01 M EDTA at 70,000xg using a
at 1:5 ratio. A collodion coated copper grids (400 Beckman SW 28 rotor for 90 min. The virus band
mesh) were allowed to float on a drop of the PVX was collected manually and diluted with 0.01 M
antiserum diluted 1:10 for 1 hr at room temperature. phosphate buffer, pH 7.2 and the virus was
The grid were washed with 0.06 M PBS containing sedimented through centrifugation at 125,000xg
1% BSA (PBS+BSA). A 50 utl drop of homogenized (Beckman Ti45 rotor) for 90 minutes. The virus pellet
zap was added and incubated for 30 minutes at room was then resuspended with 0.01 M phosphate buffer.
temperature. The grids were washed with the pH 7.2. The concentration of the virus was
PBS-PSA twice followed bN washing in 20 drops of determined in a UV spectrophotometer using PVX
distilled water. After blotting the excess wash extinction coefficient of 2.97 (Noordam. 1973) and
solution the grids were then added with 5 drops of the yield per kg plant sample was calculated. The
20% uranvl acetate and blotted dry immediately. The purified vinrs was kept at -20"C until use.
grids were observed under the transmission electron
microscope. Philips TEM 410 at the International Rice Antiserum Production
Research Institute (IRRI.).
Rabbit was immunized intramuscularly at 7- and
Purification and Antiserum Production 14-day intervals. For each dose of injection 0.2 mg
purified virus, was resuspended in 500 ml, 0.01 M
Virus purification was done following the phosphate buffer, pH 7.2, mixed emulsified white 500
protocol provided by Dr. U. Jayasinghe, CIP. Briefly. pl adjuvant. After the 4th injection, the rabbit was
N. glutinosu or N tabacum var. White Burley with killed and the blood collected and allowed to'cloth.
typical symptoms were harvested 20-22 days after The serum was collected and then clarified by low
inoculation. The leaf samples were homogenized in a speed centrifugation (8,000 x g for 10 min) and kept at
pre-cooled blender in 2 volumes of cold 0.1 M -20 "C. The antiserum titer was determined by
phosphate buffer. pH 8.0 containing 0.2% 2- comparing reaction with the pre-immune serum with
mercatoethanol and 10% ethanol. The sap was PVX infected leaf samples and purified PVX in
extracted through 3 layers of gauze cloth and ELISA.
centrifugated at about 8.000xg in a Sorvall RC-2B
Centrifuge for 20 min at 4'C. Its applicability to detect PVX in field samples of
potato. tomato, and pepper collected from La
The supernatant was collected and to it 1% Triton Trinidad. Central Experimental Station (CES),
X-100 was added then stirred for lhr. at 4' C. The UPLB. and Batangas (Lipa City), respectively, was
supernatant was again clarified by centrifugation at compared with an antiserum of PVX from CIP, Peru.
6.000xg for 20 min at 4"C. The supernatant was The positive controls were the vimrs isolates
collected and to it while stirring, 0.2 M NaCI and 4% maintained in the green house and the purified virus.
polyethylene glycol (PEG. MW 6000-8000) were The negative controls were buffer alone, healthy N.
added slowly and the mixture stirred for 1 hr. at 4" C glutinosa and N. tabacum.
and then allowed to stand for additional 1 hr at room
temperature. The virus was recovered through centri-
fuigation at 8.000xg for 30 min and the PEG pallet was RESULTS
collected and resuspended in 8 ml (for every 50 g
tissue sample) of 0.05 M phosphate buffer. pH 8.0 Assay of PXV
containing 1% Triton X-100 and then centrifugated at
8,000xg for 10 min. The supernatant was collected The potato samples collected from various sources
and underlaid with 30% sucrose (in 0.05 M phosphate had symptoms typical of PVX. Results of the ELISA
buffer. pH 8) and centrifuged at 75,000xg for 150 indicate that of these 123 samples tested 103 were
min. in a Beckman Optima XL-80 Ultracentrifuge. positive for PVX infection. 3 samples were mixed
The pallet was collected and resuspended in 0.5 ml of infected with PVX and PSV and 1 sample was

18 Volume 37 (1) JanuarV-June 2001

Journal of Tropical Plant Pathology

combined infected with PVY and PVX (Table 1).

Host Range Symptomatology of PVX

The results of the mechanical inoculation of PVX
on a series of test plants maintained at La Trinidad
and Los Bailos conditions show that vein clearing and
vein banding followed by systemic mottling
developed in N. glutinosa in 8-10 days at La Trinidad
conditions (Table 2). Under Los Bafios conditions,
these symptoms appeared 10-19 days after inoculation
and the vein clearing symptom was not so prominent.
Most of the plants produced very severe mottling and
vein banding only and only three of them had mild
symptoms at La Trinidad conditions. N. tabacum var.
White Burley and Samsun produced chlorotic
ringspots (Fig. 1) followed by vein clearing and
systemic mosaic symptoms at both locations within 6-
10 days after inoculation Severe systemic mosaic
symptoms were produced on N. bethamiana in 10-12
days after inoculation at both locations. On P.
floridana, severe systemic mosaic and stunting of the
plants were noted 8-10 days after inoculation. On G.
globosa, local lesions with chlorotic center and
conspicuous red margins appeared within 7 days after
inoculation (Fig. 1). Chlorotic local lesions were
observed on C. amaranticolor and C. quinoa 5-8 days
after inoculation in both locations. The virus
produced systemic mosaic, vein banding and mottling
symptoms on both D. metel and D. stramonium 9-10
days after inoculation at both locations. On L.
esculentum. the virus produced vein clearing followed
by systemic mosaic symptoms 10-12 days after
inoculation in both locations.

Characterization of PVX Isolates

The physical properties in vitro of PVX on C.
amaranticolor were as follows: DEP and TIP was 10-7
and 75 C. respectively and the LIV was 17 days at
Los Bailos conditions. The estimated molecular
weight of PVX coat protein was around 30.000
daltons (Fig. 2). Numerous particles consisting of
elongated flexuous rods (Fig. 3) with modal length of
about 520 nm long and 13 nm wide were observed
under the transmission electron microscope.

Identification of Virus Strains

There were two strains of PVX, the ringspot and
the normal mild mosaic, identified with the PVX
samples. The ringspots strain was more prevalent as it
was isolated from 18 samples (85.71%), while 3

samples (14.29%) were infected with the normal mild
mosaic strain (Table 3).

The ringspot strain produced chlorotic ringspot,
vein clearing, systemic mosaic and mottling symp-
toms on N. tabacum var. White Burley and Samsun
while the mild mosaic strain of the virus did not
produce any chlorotic on this host plant. On N.
tabacum var. White Burley, the mild mosaic strain
cross-protected the ringspot strain of the virus.

Purification and Production of Antiserum of PVX

After sucrose density (20-50%) gradient centrifu-
gation, a virus containing zone was observed at about
the middle of the tube (Fig. 4). The zone was broad
and thick and approximately 2 cm wide. This band,
when pelleted and examined sprectrophotometncally.
produced typical nucleoprotein spectrum with a
maximum absorption at 260 nm and minimum at 240
nm. The yield of virus with fresh leaf samples from
La Trinidad was 49.8 mg/kg. The samples from the
same location but stored at -200C for 35 days showed
that the virus yield was about 12% higher (Hossain,
1997). The samples from Los Bafios had virus yields
of 12.9 mg/kg fresh samples. The mean ratio of
absorbance at 260/280 nm was 1.68 (Hossain, 1997).
Positive reaction with infected leaf samples in ELISA
was observed with the antiserum even when diluted at

Reaction of Antiserum with Field Samples

The locally produced PVX antiserum with a
dilution of 5x10-6 performed as well as the antiserum
obtained from CIP with dilution of 2x103 (Table 4).
For symptomatic tomato samples collected from the
Central Experimental Station (CES), UPLB, 3 showed
positive reaction to PVX (Table 4). Out of 38 pepper
samples collected from Batangas, 7 showed positive
reaction to PVX antiserum in ELISA (Table 5)


Mostly single infection by PVX was detected by
ELISA with the symptomatic samples. PVY and PVS
were also found associated with a few symptomatic
samples. The present findings of high incidence of
single infection of PVX with most of the symptomatic
samples do not confirm the report of Luis (1981)
where she reported only 3.33% of single infection of
PVX. On the other hand, Talens (1979) reported
14.8% single infection of PVX and about 4.1% had

M. Hossain, N.B. Bajet Volume 37 (1) January -June 2001 19

M. Hossain, N.B. Bajet

Volume 37 (1) January -June 2001 19

PVX + PVY In the present study 57.14% showed
single infection with PVX symptomatic samples and
the remaining samples were mixed infected with two
PVX symptomatic samples produced vein
clearing and vein banding followed by systemic
mosaic on N. tabacum var. White Burley and Samsun.
necrotic local lesion on G. globosa and clorotic local
lesion on ( amaranticolor. The results were similar
to the findings of other researchers in other countries
(Thomson. 1956. Sampson and Taylor. 1968) and
confirmed the findings of Talens (1979). The result of
studies on physical properties in vitro of PVX. the
molecular weight of PVX coat protein and the
dimension of the virus particles were also as reported
by Brunt et al. (1990). These results support the view
that it is really PVX that was isolated. Additionally.
the tests for contamination by PVX were negative.

Two strains of PVX namely. ringspot and normal
mild mosaic, were detected. Earlier. Talens (1979)
reported the presence of a ringspot strain of PVX in
the Philippines. The present study confirms the
findings of Talens (1979). In addition, it reports the
existence of the normal mild mosaic strain of PVX.
Similar to the findings of Talens (1979). the ringspot
strain was more prevalent than the normal mosaic
strain. This is the first report of isolation and
identification of the latter in the Philippines.

There were some variations in the expression of
symptoms of the host plant between Los Bailos and La
Trinidad conditions for PVX. Most of the indicator
host plants produced prominent and typical symptoms
at La Trinidad condition but showed mild or no
symptoms at Los Bailos condition on some host
plants. In general. at Los Bailos. it took longer for
symptoms to be expressed. The possible reason lies in
the varying weather conditions especially temperature.
where the maximum ranges from 29-38C in Los
Bafos condition while that in La Trinidad was 20-
26"C. Similar observations were also noted with our
studies on PVY (data not shown). It was reported that
most of the potato viruses produce typical
characteristic symptoms on known indicator host at
20-25"C (Delgado, Sanchez and Grogan. 1966). Brunt
et al. (1990) also reported that some strains of PVX
cause no symptoms at high glasshouse temperatures.

The ringspot strain of PVX was successfully
multiplied in N. glutinosa and N. tabacum var White
Burley at La Trinidad. Benguet and Los Bailos.
Laguna and was purified following the protocol

described above. The purified virus had a spectrum
typical of a nucleoprotein and a mean A260/280 nm
ratio of 1.68. Hyung et al. (1977) found a A260/280
nm ratio of 1.4. The preservation of infected samples
after harvest at -20C for a short period of 35 days did
not reduce the yield of purified virus, hence the virus
was stable. Although no comparative analysis was
done, it was noted that higher virus yield was obtained
from samples propagated and multiplied at La
Trinidad than at Los Bafios conditions. These
observations further support the theory that this virus
prefers to multiplies in hosts much more efficiently at
lower temperatures, such as those common in La
Trinidad. in addition to the production of much more
distinct/severe symptoms.

PVX antiserum produced in rabbits was of high
titer and reacted to homologous antigen (purified as
well as PVX-infected leaves) but not to PVY and to
healthy leaf extracts indicating specificity. The
antiserum was able to detect or establish association
of PVX with some symptomatic potato, tomato and
pepper samples collected from fields in La Trinidad,
Los Bafios and Lipa City. respectively. These results
demonstrate the presence of this virus in these crops
and locations.

This is the first report in the country of the
occurrence of another PVX strain, successful
propagation, purification and production of antiserum
to this virus. Similar studies on isolation.
transmission, propagation. and purification of PVX.
among other major potato viruses was reported bx
Aquino et al. (1996). The availability of the antiserum
to this vinrs in the country opens up better prospects
for a more efficient and reliable detection of this
particular virus that may lead to more effective
management, including effective certification and
production of clean planting materials by micropro-
pagation. and epidemiological studies among others.


FLORES. 1996. Isolation, transmission, propagation
and purification of major potato viruses: potato virus X
(PVX), potato virus Y (PVY) and potato leaf roll virus
(PLRV). Proc. 27t Anniv. and Annu. Conv. Pest
Management Council of the Philippines, May 7-10,
Davao City, p121.

The Philippines Recommends for Potatoes. PCARRD,

20 Volume 37 (1) January.June 2001 Journal of Tropical Plant Pathology

20 Volume 37 (1) January-June 2001

Journal of Tropical Plant Pathology

Los Bafios, Laguna, Philippines. 67pp.

POTTS MJ, LM PACUZ, EO SANO. 1982. White potato
yield survey: Benguet province (Philippines). Philipp.
Agric. 65:385-393.

PROMEGA. 1991. Protocols and Application Guide. 2n'd
Ed.. Promega Corp., 2800 Wood Hollow Road,
Madison, WI, USA.

RICKWOOD D, BD HAMES. 1990 Gel electroporesis of
protein. A practical approach. 2n"d Ed. IRL Press.
Oxford University N.Y. 383pp.

SALAZAR LF. 1989. A worldwide problem: Potato virus
disease control. CIP Circular 17.

SAMPSON PJ, RH TAYLOR. 1968 A comparison of
the electron microscope, microprecipitin tests and
indicator plants for the detections of potato viruses S.
X, and Y. Phytopathology 58:489-493.

TALENS LT. 1979. Potato viruses in the Philippines:
Detection and identification of potato viruses X, S, and
Y. Philipp. Agric. 62:144-148.

TALENS LT. 1979. Potato viruses in the Philippines:
Identification of a ringspot strain of PVX. Philipp.
Agric. 62:183-190.

THOMSON AD. 1956. Definition of potato virus X strains
present in New Zealand potato vaneties. Austr. J.
Agric Res.. 7:527-537.


The authors wish to thank Dr. Upali Jayasinghe. CIP,
Baguio for providing antisera of potato viruses and other
various information pertaining to potato virology and of the
laboratory facilities at La Trinidad, Benguet, to Dr. Ossmat
Azzam., IRRI and Dr. F.C. Sta Cruz. IRRI for the help with
the electron microscopy. and to Dr. Asuncion K.
Raymundo. Institute of Biological Sciences, UPLB for the
use of the Beckman Ultracentrifuge.

BALAOING VJ, EA VERSOLA 1980. Pest/disease
aspects of potato m the Philippines, pp. 48-54. In Proc.
11" Anniuuv. and Annu. Comn. Pest Control Council
Philippines. College. Laguna. Philippines

BRUNT A, K CRABTREE, A GIBBS. 1990. Viruses of
Tropical Plants. C.A.B. Intl. & A.C.I. A.R. 707 pp.

CLARK JM Jr., RL SWITZER 1977. Experimental
Blochemistrl. 2"' ed W.H Freeman and Comp.. San
Francisco. 335pp

Chenopodium quinoa. a local lesion assay host for
potato virus Y. Phytopathology 56:1394-1396

SRIVASTAVA. 1985, Perspectives in Plant Virology.
Printers House. India. Vol. 1. 322pp.

HOOKER WJ. 1987. Les viroses de terre. In: Bulletin
d'information Technique 19:129-136.

HOSSAIN M. 1997. Purification and antiserun production
of potato X and Y viruses and the effects of these
viruses on the development of late blight in selected
potato (Solanum tuheroxum L) genotypes. Unpublished
Ph.D. Thesis. University of the Philippines Los Bafios.
College. Laguna 4031. Philippines. 198pp.

1977. Studies on purification and serology of potato
virus X. Korean J. Plant Pathol. 16: 101-104.

LUIS JS. 1981. Virus incidence and yield loss in certified
red pontiac and uncertified conchita white potato
cultivars. Unpublished M.S. Thesis, University of the
Philippines Los Bafios. College. Laguna. 4031.
Philippines. 123pp.

NOORDAM D. 1973. Identification of Plant Viruses:
Methods and Experiments. Cent. Agric. Publ. Doc..
Wageningen, 207pp.

M. Hossain. N.B. Bajet

Volume 37 (1) January -June 2001 21

Table 1. Reaction of different indicator host inoculated with potato X potexvirus (PVX) maintained in the greenhouse an
tested in enzyme linked immunosorbent assay.

Indicator Host Number Number that reacted + to
Inoculated Antiserum

Nicotiana glutinosa 60 60 1 1
N. tabacum (Sumsun) 2 2 -
N. tabacum (White Burley) 9 9 -
N. bethamiana 2 2 -
P. floridana 6 6 -
Chenopodium amaranticolor 8 6 -
Datura metel 5 4 1
D. stramonium 7 7 -
L. esculentum 5 5 -
S. tuberosum 3 2 1
N glutinosa (healthy) 3 -
N. tahacum (Sumsun) (healthy) 1
N. tabacum (White Burley) (healthy) -
P. floridana (healthy) 1- -
C. amaranticolor (healthy) -
D. stramonium (healthy) 1- -
S. tuberosum (healthy) -
Buffer (7 wells) 7 --
Total 123 103 1 3
Legend: "-- means no reaction observed

22 Volume 37 (1 Journal 01 Tropical Plant Patholoc

22 Volume 37 11

Journal of Tropical Plant Patholo!:

iult J. rULdLU A J ULXAVIIU (r VA) CILU 1.3 Laliih 1UVILLLuOIUIIUadLjU 11Uil 3auli /vL IVUIaIt aL Ll.a JiL ILuAIIu, U- Engl t

SI. Isolate Place of collection and variety Virus and strain
No. Number
1 PVX1 IPB greenhouse, Los Bafios (Liza) RS
2 PVX2 IPB greenhouse, Los Bafios (Liza) RS
3 PVX3 IPB greenhouse. Los Bafios (Liza) RS
4 PVX Potato field, Natubay (BSU-P03) RS
5 PVX Potato field, Natubay (HPS-11/67) RS & SV
6 PVX Potato field. Natubay (Granola) RS
7 PVX card. BSU field, La Trinidad (Granola) Mild str.
8 PVX BSU field, La Trinidad (Granola) RS
9 PVX Potato field, Natubay (BSU-P03) RS
10 PVX Potato field, Natubay (BSU-P03) RS & PSV
11 PVX NPRCRTC, La Trinidad (Granola) RS
12 PVX NPRCRTC. La Trinidad (Granola) Mild str.
13 PVX NPRCRTC, La Trinidad (BSU-P03) Mild str.
14 PVX NPRCRTC. La Trinidad (Granola) RS
15 PVX IPB garden. La Trinidad (Gennplasm) RS
16 PVX BPI. Baguio (Granola) RS
17 PVX BPI. Baguio (Faunosa) RS
18 PVX BSU field, La Trinidad (Granola) RS & PSV
19 PVX BSU field, La Trinidad (Granola) RS
20 PVX BSU field, La Trinidad (BSU-P03) RS
21 PVX NPRCRTC. La Trinidad (LB differential) RS
RS Ring spot strain of PXV and
Mild str. Normal mild mosaic strain of PVX.

.Hossain, N.B. Bajet Volume 37 (1) January -June 2001 23

Table 4. Comparison of the reaction of potato and tomato samples infected with Potato X potexvirus (PVX) to antiserum
produced locally and from CIP in ELISA.

Antiserum to PVX
CIP AbsorbanceAbsorbance
(A405 nm) (A405 nm)

Granola (healthy) 0.03 0.02
HPS 11/67 0.03 0.07 +
HPS 11/67 0.01 0.21 +
HPS 11/67 0.01 0.21 +
BSU-P03 0.33 + 0.29 +
BSU-P04 0.03 0.24 +
Atlantic 0.03 0.22 +
PVX isolate >3.00 + 0.90 +
Purified PVX 1.44 + 0.35 +
Purified PVY 0.00 0.00
Buffer 0.00 0.00
M+4SD 0.07 0.03

17 (3 x 7) 0.06 + -0.01
123 (4 x3) 0.01 -0.02
Hofit 0.00 0.00 +
16 (9 x2) 0.01 0.02 +
Imp. pope 0.03 0.04 +
16 (9 x 2) 0.03 0.04 +
PVX isolate >3.00 + 0.80 +
Purified PVX 2.12 + 0.39 +
Healthy tomato 0.01 0.01
Buffer 0.00 0.00
M+4SD 0.03 0.01
Total infected 6 14
Antiserum dilution 5 x 10 fobr locally produced
2 x 10" for CIP
Antigen dilution 1: 5
Mean absorbance of 2 wells/sample

24 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

Table 5. Association o potato X potexvinrs (PVY) and potato Y potyvirus (PVY) in pepper samples from Batangas tested

Antiserum to PVX Antiserum to PVY
Absorbance Remarks Absorbance Remarks
(A405 nm) (A405 nm)

1171048-1 0.109 0.142
171060-1 0.108 0.236 +
I171046-2 0.324 0.371 +
171018 0.132 0.427 +
171063 0.175 0.468 +
171048 0.320 + 0.183
171056 0.010 0.230
171044 0.010 0.101
176001 0.027 0.149
171020-2 0.140 0.356 +
1171022-1 0.091 0.260 +
II71064 0.169 0.366 +
II71020 0.216 0.377 +
1171036-1 0.807 0.235 +
1171062 0.336 + 0.171 +
1171048-2 0.139 0.187
1171060-3 0.012 0.112
1171036-2 0.371 + 0.212
171060-2 0.044 0.178
1171046-1 0.099 0.212
1171057 0.093 0.211
171023 0.080 0.314 +
171057 0.098 0.207
1171015 0.083 0.214
1171048-3 0.079 0.276 +
171048-3 0.061 0.208
171066 0.051 0.024
1171066 0.053 -0.185
171020 0.080 0.222
171034 0.093 0.216
ICHKA 0.111 0.169
171013 0.197 0.232
176006 0.031 + 0.092
1171048-2 0.054 0.009
1171070 0.045 0.197
1171022-2 0.070 0.168
1171048-1 0.007 0.261 +
U71048-3 0.078 0.152
PVX GH N. glutinosa >3.000 + 0.185
PVY N. glutinosa 0.139 -1.332 +
N. glutinosa (healthy) 0.136 0.252 +
Buffer -0.020 --0.026
M+4SD 0.191 0.232
To!al infected 8 14

Vohane 37 (1) January-June 2001 25

M. Hossain and N.B. Bajet

Volume 37 (1) January-June 2001 25

Fig. 1. Reaction of different tests plants to potato X potexvirus (PVX) by sap inoculation:
chlorotic ring spot on Nicotiana tabacum var. White Burley (left) and necrotic local
lesions on Gomphrena globosa (right)

26 Volmune 37(1) January-June 2001 Journal of Tropical Plant Pathology

26 Volume 37 (1) January-June 2001

Journal of Tropical Plant PathmIM

(x 000)





18.4- a

Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel
(12%) electrophoresis of potato X potexvirus
(PVX). Lane 1, protein marker; lane 2,
purified PVX; lane 3, PVX2 infected
Nicotiana glutinosa; and lane 4,
healthy N. glutinosa

Fig. 3. Electron micrograph of potato X
potexvirus (PVX) particles.
Magnification x 29, 000.


Broad PXV


Fig. 4. Sucrose gradient (20%-50%) centrifugation of partially purified
extracts of potato X potexvirus (PVX) infected tobacco (Nicotiana
tabacum) showing a relatively broad light scattering bahd.

27 Volume 37 (1) January-June 2001 Journal or Tropical Plant Pathology

Journal of Tropical Plant Pathology

27 Volume 37 (1) January-June 2001



Supported by the Philippine Rice Research Institute. Mufioz, Nueva Ecija and the Department of Science and
Technology. Bicutan. MetroManila. Portion of the senior author's Ph.D. dissertation submitted to the Graduate School
of the University of the Philippines Los Baiios (UPLB), College, Laguna, Philippines

'Associate Professor, College of Agriculture, Western Mindanao State University, Zamboanga City, 2Professor,
Department of Plant Pathology. UPLB. and 'Professor. Department of Entomology, UPLB, College, Laguna

Populations of the green leafhopper Nephottetix virescens varied among pure and mixed
stands of rice cultivars with different types of resistance in three sites during the dry and
wet seasons of 1999. In most cases, the mixtures of two (IR-64 and PSBRc 18), three (IR-
64, PSBRc 18 and IR-72), and four (IR-64, PSBRc 18, IR-72 and PSBRc34) in close (15 x
20 cm) and wider spacing (25 x 25 cm) had higher GLH populations. For instance, in
Barangay Talisayan with high disease incidence in some of these mixtures, diverse geno-
types constituting these mixtures had no effect in suppressing disease development and
reducing GLH populations. Population build-up was rapid in the susceptible cultivar IR-
64. Pure stands of resistant cultivars also had high GLH as in IR-64.

There was significant correlation between incidence and vector population in IR-64,
PSBRc 34, IR-72, mixtures of two cultivars (IR-64 and PSBRc 18) and mixtures of four
cultivars (IR-64, PSBRc 18, IR-72 and PSBRc 34) planted within rows. However, disease
incidence in the mixtures of three, four cultivars in rows, those planted in closed (15 x 20
cm) and wide spacing (25 x 25 cm) and mixtures of four cultivars (IR-64, PSBRc 18, IR-72
and PSBRc 34) in alternate rows had no correlation with GLH population.

N. virescens rapidly increased at 15 DAT and peaked at 36-43 DAT. The most critical
stage was at 15 DAT when population build-up was rapid as plants were young and suscep-
tible to infection.

Keywords: Population dynamics, rice tungro disease, Nephotettix virescens, mixed stands, resistance


Insect vectors perform an important role in the
development of tungro disease. a serious threat to rice
production in the tropics (Chancellor et al.. 1996). The
increase oftungro in rice fields is often preceded by the
increase in the population of green leafloppers (GLH).
Nephotettix virescens. (Distant) (Suzuki el al.. 1992).
The availability of the host and favorable environmental
conditions may favor the increase of viruliferous insects
For instance, the introduction of high yielding cultivars
and intensive use of fertilizers are mainly responsible
for the population build-up of vector populations and
tungro outbreaks (Suzuki et al., 1992). Vector
populations, however. often fluctuate due to differences

in cropping practices adopted by farmers.

Multiple rice cropping systems over large areas
provide a continuous source of plant hosts and allow a
year-round development of insect pests particularly green
leafhoppers, plant hoppers, stemborers and leaf folders
(Litsinger, 1989). Further, asynchronous planting dates
and overlapping rice crops enable leafhoppers and viruses
to survive (Chancellor et al., 1999).

Viruses and their vectors have been able to adapt
easily to homogenous plantings of cultivars over large
areas. Consequently, the use of heterogeneous crop
populations was proposed and eventually practiced.
Diversity in such heterogeneous environment has been

2B Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

28 Volurne 37 (1) January-June 2001

Journal of Tropical Plant Pathology

considered to be an essential characteristic promoting
balanced polymorphism in natural ecosystems (Browning,

Vector populations vary with the kind of variety grown
in the field as well as with environmental fluctuations.
Rainfall, temperature and sunshine are known to affect
vector abundance (Pathak, 1972). The use of varieties
resistant to the vector has a profound effect in the
development of the disease. However, these varieties
tend to succumb to infection due to the emergence of
new vector biotypes. Several strains of the virus ranging
from mild to severe have been reported based on
different symptoms and reactions to rice cultivars (Hull.

Fanrers' practices are contributory to the build-up
of vector and vimrs populations. The amount of pesticides
used by farmers, varietal rotation, and fertilizer
applications are some of the factors that may cause
fluctuation ofleafhopper populations. eventually affecting
tungro incidence. The use of fertilizers has also been
found to enhance the vulnerability of crop stands by
providing ideal conditions for viruses and their vectors
(Thresh. 1982).

Little is known about the relationship of vector
population and disease incidence in crops of genetically
uniform and diverse stands. Information obtained in this
area is important in effectively managing insect vectors
and, consequently, the diseases caused by entities.
especially viruses, transmitted by these vectors.

This study aimed to determine (a) vector populations
in pure and mixed stands of rice cultivars with different
types of resistance, (b) the relationship between tungro
incidence and vector population increase in these pure
and mixed stands, and (c) the relationship between vector
population and weather factors in these pure and mixed


Vector Dynamics in Pure and Mixed Stands
of Rice Cultivars

To determine the relationship between vector
population and disease incidence, green leafhopper
(GLH) populations were assessed in plots of pure and

mixed stands of rice cultivars with different types of
resistance which were primarily set up for experiments
on temporal and spatial progression of tungro in
Barangays Tulungatung, Cawit, and Talisayan in the
western coast of Zamboanga City (Pedroso, 2001). The
cultivars used in pure stands were IR-64, resistant to the
vector and susceptible to the tungro virus; IR-72. resistant
to the virus and intermediately resistant to the vector;
PSBRc 34, intermediately, resistant to both vector and
virus; and PSBRc 18, resistant to the vector and virus.

The mixed stands consisted of:

1. Plot mixtures
Three plots were prepared in each site corresponding
to the three plot mixtures. Each plot measured 12 x 12
meters and was equally divided into sub-plots depending
on the mixtures used. Each sub-plot was randomly
selected and planted to a single rice cultivar of known
resistance. Both plots and sub-plots were randomly
selected. Three mixtures were used based on the
approach of Chin and Husin (1982), as follows:
a. Plot Mixture A (PMA) One half of the 12 x 12
meter plot was each planted with IR-64 and
PSBRc 18
b. Plot Mixture B (PMB) One third of the 12 x 12
meter plot (3 sub-plots) was each planted with
IR-64. PSBRc 18 and IR-72
c. Plot Mixture C (PMC) One fourth of the 12 x
12 meter plot (4 sub-plots) was each planted.
with IR-64. PSBRc 18, IR-72 and PSBRc 34

2. Plot mixtures with varying population densities
Three plots each measuring 12 x 12 sq. meter area
were planted with the same cultivars with different
resistance genes as in pure stands at a population density
of 250.000. 160.000 and 333.333 hills per hectare.
Seedlings of each cultivar were transplanted at 20 x 20
cm. 25 x 25 cm. and 15 x 20 cm spacing, respectively, to
obtain the aforementioned planting densities. Seeds of
each cultivar as in pure stands were sown separately
and transplanted at different population densities.

3. Cultivar mixture in rows within a plot (VR) Four
cultivars (PSBRc 18, IR-72, PSBRc 34 and IR-
64) were used in this experiment.

Exactly 0.5 kg of seeds of each cultivar was
thoroughly mixed before sowing in the seedbed to ensure
that the transplanted seedlings are of different genotypes.

J. Pedroso, A.D. Raymundo and A.C. Sumalde Volume 37 (1) January-June 2001 29

J. Pedroso, A.D. Raymundo and A.C. Sumaide

Volume 37 (1) January-June 2001 29

4. Cultivars m alternate rows (AR) The population of the vector varied between sites and
The same cultivars as min pure stands were used. seasons. Infestation during the dry season was higher
Each cultivar was planted alternately every 1.5 meters than during the wet season except in Barangay Cawit
in a 12 x 12 meter plot so that each cultivar appeared (Table 3).
twice in a plot.
GLH population increased with time. Initial
The assessment of GLH populations was done by population levels of the vector during the wet (10 DAT)
sampling using a 30 cm diameter insect net. and dry seasons (15 DAT) were high in all cultivars.
The 60 rows ofplant in a 12 x 12 meter plot were divided The trend of population and timing of peaks varied among
into four zones consisting of 13 rows per zone. Two cultivars. In some cultivars, peaks of disease infection
rows served as buffer at the end of each zone. Twenty- coincided with peaks of vector populations. The increase
two sweepings were made within the 13 rows of plant. in disease incidence followed the same pattern as that
The number of sweeps was based on the normalpace of increase in GLH population.
of an individual. The leaflhoppers
that were caught were counted using a tally counter. In Barangay Tulungatung, populations of GLH in IR-
Both nymphs and adults from each plot were 64 during the dry season increased rapidly at 22 DAT,
recorded separately. Assessment of the population was decreased at 36, then rose again later. GLH populations
done at weekly intervals by counting the number of GLH in both PSBRc 34 and IR-72 increased likewise at 22
per plot starting at 10 and ending at 65 DAT. Records of DAT, peaked at 36 DAT and declined at 43-57 DAT.
relative humidity, temperature and rainfall were obtained Vector population was high in PSBRc 18 (222) with an
at the Philippine Coconut Authority (PCA) weather early peak at 22 DAT and declined continuously thereafter
station in Zamboanga City. Disease, in terms of (Fig. 1) while it was low in IR-72 and PSBRc 34 (Table
incidence, was assessed at the same time period as that 3).
of recording GLH population. To determine tungro
incidence, the total hills per plot were obtained and hills During the wet season, GLH populations among
with symptoms of disease were counted. A hill was cultivars were far lower than during the dry season (Table
considered infected if one or more tillers showed 3). IR-64 was susceptible to GLH as indicated by the
symptoms of tungro (Chancellor. 1996) Percent highest population (314) which peaked at 43 DAT (Fig.
incidence was determined using the equation: I1). The two pure stands of resistant cultivars (PSBRc
18 and IR-72) had the same GLH peaks at 43 DAT,
number of infected hills/plot while PSBRc 34 had the earliest peak (22 DAT) and
Percent incidence = ------------------------- x 100 thereafter declined.
total number of hills/plot
The highest population of GLH in all pure stands in
Data Gathering and Analysis Barangay Cawit was at 36 DAT during the dry season
and at 36-43 DAT during the wet season. IR-64 was
Percent disease incidence in each variety was susceptible as indicated by the high number ofGLH (Fig.
obtained and correlated with GLH populations. Disease 2). Populations drastically dropped at 22-29 DAT in all
progress curves and vector population curves were cultivars during the dry season but not during the wet
constructed for each variety in both seasons. Rainfall, season. Fluctuations in GLH population were very
temperature and relative humidity were likewise evident among cultivars from 10 to 60 DAT.
correlated with GLH populations.
The initial population of GLH in PSBRc 34 in
RESULTS Barangay Talisayan during the dr); season was high (1400)
compared with the rest of the cultivars. This declined
Green Leafhopper (GLH) Populations in Pure until 57 DAT. Each cultivar differed in its population
Stands of Rice Cultivars peak which was between 29-43 DAT (Fig. 3).

Significant correlations existed between disease More GLH were observed in the field during the
incidence and GLH population in some cultivars and dry season than during the wet season (Table 3). GLH
mixed stands (Tables 1 and 2). In most cases, disease populations in two resistant cultivars (IR-72 and PSBRc
incidence increased with increase in GLH population. 34), continuously increased from 10 DAT to 43 DAT

30 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

and thereafter decreased. In PSBRc 18 and IR-64, GLH
dropped at 22 DAT and slowly increased again at 29
DAT. Both IR-72 and IR-64 had more vectors at 36
DAT while PSBRc 18 and PSBRc 34 were heavily
infested at 43 and 50 DAT, respectively (Fig. 3).

Among pure stands. IR-64 had high GLH population
in the three barangays during the dry and the wet season
followed by PSBRc 18. while IR-72 and PSBRc 34 had
usually less vector populations (Table 3).

Approximately two weeks after the GLH population
initially increased, tungro incidence in IR-64 and PSBRc
18 rose rapidly at 36 DAT during the dry season and a
week earlier during the wet season (Figs. 4 and 5). After
this population peak at 36 DAT. incidence declined at
50-57 DAT. Results indicated that the tungro infection
on cultivars at 29 DAT was due to GLH infestation at 15
DAT. The appearance of vectors in the field at 15-22
was critical to the rapid development of tungro at 36

GLH Populations in Mixed Stands
of Rice Cultivars

Populations ofNephotettix virescens in mixed stands
were comparable to that in pure stands (Table 3). GLH
populations were high at 10-15 DAT in all mixed stands.
The pattern of population peaks was similar to that in
pure stands (Figs. 1 and 2).

In Barangay Tulungatung. the population of GLH in
plots with mixture of two cultivars rapidly rose at 22
DAT then slowly declined at 29 DAT to 36 DAT. It then
started to increase again. reaching its peak at 43 DAT
and decreased thereafter. Plots with mixture of three
cultivars and four cultivars had drastically increased GLH
populations at 22 DAT and 36 DAT, respectively and
slowly declined at 50-57 DAT. GLH populations in the
mixture in alternate rows increased at 43 DAT.

During the wet season, GLH in the mixture of two
cultivars had a similar trend as in the dry season, but
peaked at 43 DAT, while plot with mixtures of three
cultivars had high GLH populations at 36 DAT, which
decreased afterwards. The mixtures with four cultivars
and the mixture of cultivars planted in alternate rows
also had high GLH populations at 22 DAT (Fig. 1).
Mixtures planted in close (15 x 20 cm) and wider spacing
(25 x 25 cm) had similar GLH populations at 36 to 43
DAT. A much higher population was found at 36 DAT
in plots spaced at 20 x 20 cm. However, cultivars planted

within rows had more GLH at 43 DAT. Total population
of vectors varied among mixtures at 57 DAT (Table 3).

During the wet season, plots with closer spacing (15
x 20 cm) had more GLH than widely spaced plots (25 x
25 cm), plots spaced at 20 x 20 cm, and cultivars within
rows. At 15 DAT, GLH increased up to 36 DAT in plots
with closer spacing and thereafter declined. The highest
number of GLH in this mixture was 150 while the mixture
of cultivars within rows, and the mixture with 20 x 20
cm spacing and that of widely spaced mixture reached
their peaks at 43 DAT with low GLH populations and
declined at 50 DAT (Fig. 1).

Tungro incidence in cultivars continuously increased
at 36-43 DAT and declined at 50 DAT. Similar to the
pattern of disease increase in pure stands, the disease
progress curves increased with increasing GLH
population in the mixtures with cultivars within rows and
that planted alternately in rows during both wet and dry
seasons. The peak of infection of each mixture differed
from the peak of GLH population (Figs. 6 and 7).

Infection peaks in some cultivar mixtures coincided
with GLH population similar to that in pure stands.

In Barangay Cawit, the total GLH population during
the wet season was higher than during the dry season
(Table 3). Disease incidence on these mixtures was
likewise higher during the wet season than during the
dry season. It appeared that disease incidence increased
with increase in number of vectors.

Similarly high GLH populations in mixed and pure
stands in Barangay Talisayan were observed during the
dry season except in mixtures that were widely and
closely planted and mixtures planted in alternate rows.
Incidence in these mixtures increased at 29 DAT and
declined (Fig. 3). The same trend of increase in GLH
population was found in these mixtures during the wet
season. Although tungro incidence likewise increased
with increasing GLH population, peaks of infection
differed among these mixtures.

The mixture of cultivars within rows had more GLH
at 43 DAT. which thereafter declined. Those planted with
20 x 20 cm spacing (PDA) was high at 29 DAT, which
also declined afterwards. However, closely planted
mixtures, appeared less damaged during the dry season.
Contrary to the results obtained during the dry season.
plot mixtures with 20 x 20 cm spacing showed less
damage by GLH during the wet season (Fig. 3).

J. Pedroso, A.D. Raymundo and A.C. Sumalde Volume 37 (1) January.June 2001 31

Volume 37 (1) January-June 2001 31

J. Pedroso, A.D. Raymundo and A.C. Sumalde

Average vector population varied among mixtures.
In most cases, regardless of the location, the mixture of
two, three and four cultivars. plots widely and closely
spaced and cultivars planted in alternate rows had higher
GLH populations than the rest of the mixtures. However.
of the three plot mixtures ofvarying cultivar components.
the mixture of three cultivars had consistently high GLH
populations than those in two and four mixtures. Similarly.
the mixture planted in close spacing had high vector
population than those in wide spacing (Table 3).

The spatial incidence of tungro in Barangay
Tulungatung during the dry season showed that PSBRc
18 and cultivars within rows were related to the population
of GLH (Table 2). The rest of the mixtures showed no
significant association despite the seemingly high
correlation coefficients (r) Incidence in some mixtures.
however. w\as not correlated \with the vector population.

Nymphal and Adult Populations of GLH

Peak nymph populations differed in all the sites.
During the dry season, nymphal population was high at
29-36 DAT and declined thereafter, while the adult
population started to increase at 36 to 50 DAT. During
the wet season. the nymphal population was higher at 15
to 29 DAT while the adult population rapidly increased
at 29 to 50 DAT.

Average nymph population was higher (898) during
the dry season than during the wet season (284) while
there were more adults during the wet (429) than during
the dry (35 1) season (Table 4).

GLH Populations and Weather Factors

Fluctuations in GLH populations were not
significantly correlated with meteorological data in this
study. Correlation coefficients (r) between rainfall.
temperature. and relative humidity and GLH populations
in pure and mixed stands were not significant (Table 5).

Incidence in IR-64 appeared to have a slightly higher
correlation coefficient (r) with humidity (r = 0.63). rainfall
(r = 0.52) and temperature (r = -0.68) while in PSBRc
34. the high correlation coefficients (r) was with rainfall
(r = 0.59). However. despite the higher r values, they
were found not significant at a = 0.05. In mixed stands.
correlation coefficients were generally higher in relation
to relative humidity than to rainfall and temperature.


The significant relationship between number ofGLH
and tungro incidence in some pure and mixed stands
indicate that the incidence of the disease can be a function
of the number of GLH.

Among the pure stands, the build-up of GLH
populations in the susceptible cultivar. IR-64 was faster
than that in resistant cultivars. In Barangay Talisayan.
where incidence of the disease was high. this cultivar
showed susceptibility to both the virus and the vector.
On the other hand. PSBRc 18. although considered
resistant, appeared to be susceptible to the vector and
the virus in Barangay Tulungatung where GLH
populations and disease incidence were high. Both IR-
72 and PSBRc 34 were resistant to both virus and the
vector. Chancellor (1996), in his study on leafhopper
ecology, mentioned that incidence was associated with
vector populations during the early part of the epidemic
but not during the later stage when GLH population

Studies on vector population revealed that the
amount of disease in a crop is dependent on the size of
insect population and percentage of infective individual
in the population. A large insect population produces
catastrophic results (Chiykow ski, 1982). Suzuki et al.
(1992) stated that the increase in tungro incidence was
preceded by the population build-up of N. virescens.
Furthermore. in a study of Dahal et al. (1988), cultivars
resistant to tungro generally were observed to sustain
low incidence of the disease in the field and usually had
less GLH population. IR-72. on the other hand, was
effective in reducing leafhopper number and in preventing
the occurrence of tungro disease even under conditions
of high inoculum (Chancellor, 1995).

The variation of GLH populations in mixed stands
clearly showed the interacting effects of resistance genes
of the mixtures to the vector. In Barangay Tulungatung
during the dry season, the high GLH population in
mixtures was comparable to that of the pure stands which
had low disease incidence. Antibiosis, in the resistant
component of the mixture, might have restricted the
feeding of viruliferous insects to the phloem, rather than
the xylem of the hosts (Dahal et al., 1988). Kobayashi
et al. (1992) reported that RTBV resistant accessions
exhibited antibiosis to GLH making it difficult to
distinguish between resistance to virus infection and

32 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

Journal of Tropical Plant Pathology

32 Volume 37 (1) January-June 2001

resistance to the vector. However, studies on wild species
revealed that resistance to RTBV infections in the
accessions tested does not depend on vector resistance.
Data revealed a moderate to low antibiosis to Nephotettix
virescens and resistance to infection by the tungro virus.
The data on leafhopper abundance suggested a degree
of adaptation of the insects on these varieties. The insects
probed repeatedly but did not feed on the phloem, so
that disease incidence remained low (Rapusas and
Heinrichs, 1981). Simulation studies by Holt (1996)
examining the influence of variety type showed that even
a relatively small increase in the proportion of resistant
varieties could have a significant effect in reducing tungro
incidence in some circumstances. Furthermore, results
of survey conducted in the municipality of Polangui,
Albay province and Luzon indicated that farmers clearly
benefited from growing leafhopper-resistant varieties
because of reduced tungro incidence (Chancellor. 1999).
The low GLH populations m Barangay Tulungatung
during the wet season with high tungro incidence might
indicate that such mixtures were effective against the
vector but not against the virus. High GLH population
precedes high infection; however, one or a few GLH
can increase infection.

In most cases, plot mixtures of two, three, and four
cultivars in closer and wider spacings had higher GLH
populations than the rest of the mixtures in the three
barangays during the dry and the wet seasons. In relation
to disease incidence, mixtures in wide (25 x 25 cm) and
close (15 x 20 cm) spacings, cultivars in alternate rows
and mixtures combined with two cultivars had higher
disease incidence and GLH populations. The production
of more tillers due to close and wider spacings provided
a modified environment favorable to the vector and
increases the area of susceptible cultivar. This enhanced
GLH feeding on these cultivars.

Temperature, relative humidity and rainfall had
negligible influence on GLH populations in the field.
Although weather factors could be contributory to vector
development, their effect on GLH populations was not
significant in this study. This agrees with the absence of
a relationship between leafhopper abundance and
weather data reported by Chancellor (1996).


Green leafhopper populations (GLH) varied among
the pure and mixed stands of rice with different types of
resistance during the dry and wet seasons of 1999 in the

three barangays. The build-up was rapid in the
susceptible variety IR-64. Single resistant varieties failed
to reduce GLH population.

GLH population was significantly correlated with
disease incidence in some cultivars. The peak of
infection coincided with the peak of GLH population in
some cultivars.

The population of GLH started to rise at 15 DAT
and rapidly increase thereafter reaching its peak at 36-
43 DAT. The most critical stage of population increase
was at 15 DAT when plants are young and susceptible
to infection. Thus, to prevent the rapid increase of GLH
populations in rice fields, insect control earlier than 10
DAT preferably in the morning is recommended.

Plot mixtures oftwo, three, and four cultivars in close
and wider spacing had higher GLH populations than the
rest of the mixtures in the three barangays for both
seasons. Such mixtures appeared susceptible to the
vectors. In relation to disease incidence, the mixtures in
wide and close spacings, cultivars in alternate rows and
mixtures combined with two cultivars had higher disease
incidence and GLH populations.

Environmental factors such as temperature, humidity
and rainfall had no significant influence on GLH
population under the conditions of the experiment.


BATAY-AN EI, MANCAO SC. 1999. Managmentoftungro
disease by chemical control of the greenleafhoppervector,
pp. 159-166. InRicetungrodiseasemanagement. T.C.B.
Chancellor, O. Azzam andK L. Heong (Eds). IRRI 166pp.

BOTIENBERG IL 1990. Impact ofcropping intensity and
asynchrony on the epidemiology of rice tungro virus in
Malaysia Int J. PtL Prot Trop. 7:103-106.

BROWNING JA. 1974. Relevanceofknowledge about natural
ecosystems for development of pest management
programs for agro-ecosystems Proc. Amer. Phytopathol.
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Chancellor, O.AzzamandK. L Heong(Eds.). IRRI. 166p.

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CHANCELLOR TCB. 1995. The ecology of the leafhoppers 0. Azzam and K. L. Heong (Eds.) NRI. 106 p.
vectors of rice tungro viruses in relation to the dynamics
of the disease. Unpublished Ph.D. thesis. University of HASANUDDIN A, CABAUATAN P.Q, HIBINO H. 1986.
Reading. UK. Studies on tolerance oftungro disease and their possible
use for disease management, pp. 98-106. In
Risk factors for rice tungro disease in endemic areas. pp.
58-65. In: Rice tungro disease epidemiology and vector HEINRICHS EA. 1979. Control ofleafhopperandplanthopper
ecology. Chancellor, T.C.B. P.S. Teng and K.L Heong vectorsof ricevirus. pp. 529-560. In: Leafhopper Vectors
(Eds.) IRRI Discussion Papers Series No. 19, 104 p. and Plant Disease Agents. Maramorosch. K. and Harris,
K.F. (Eds.). New York: Academic Press.
S, TENG PS. 1999. The influence ofvarietal resistance HIBINO H, ISHIKAWA K, OMURA T, CABAUATAN PQ,
and synchrony on tungro incidence in irrigated rice KOGANEZAWAH. 1991. Characterizationofricetungro
ecosystem in the Philippines. pp. 119-127. In Rice tungro bacillitus and rice tungro spherical virus. Phytopathology
disease management. T.C.B. Chancellor, O. Azzam and K. 81:1130-1138.
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CHANCELLORTCB,HEONGKL,COOKAG,ILLAREAL Association of two types of virus particles with penyakit
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of two kinds of rice tungro associated viruses by insect
CHANCELLOR, TCB, HEONG KL, COOKAG. 1997. Role vectors. Phytopathology 69:1266-1268.
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disease. T.C.B. Chancellor and J.M. Thresh (Eds.) NRI Annu Rev. Phytopathol. 34:249-274.
106 pp.
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by leafhopper-borne pathogens, p. 341. In Plant Disease and vector ecology. Chancellor, T.C.B, P.S. Teng and
and Vectors. Ecology and Epidemiology. Maramorosch, K.L Heong (eds.) NRI, 104 pp.
K. and Harris K.F (Eds.) Academic Press. New York.
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DAHAL G. 1997. Rice tungro disease and green leafhopper Annu. Rev. Phytopathol. 34:275-279.
vectors in Nepal: current status and future research
strategies, pp. 84-93. In Epidemiology and management KHUSH G. 1994. Breeding (Planning Report for 1984), pp.
of rice tungro disease. T.C.B. Chancellor and J. M Thresh 338-339. In Proc. Fourth National Rice Institute
(Eds.). NRI. 106pp. Conference, Egypt.

FLORES ZM, VM AGUIERO. 1990. Changes in cultivar 1993. Resistance to rice tungro bacilliform virus (RTBV)
reactions to tungro due to changes in "virulence' to the found in wild oryzae spp. Intl.Rice Res. Newsl. 17:8-9.
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HASANUDDIN A, WIDIARTA, IN, YUDIANTO. 1999. tungrovirus, pp. 459-466. In: Plant Virus Disease Control.
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(Eds.). IRRI. 166 p. contributory factors for its outbreaks in the Philippines,
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34 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

LITSINGER JA. 1989. Second generation insect
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NEMOTO H, HABIBUDDIN H. 1995. Adaptation of
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Bailos, Laguna. 4 pp.

PEDROSO JP. 2001. Epidemiology of tungro disease
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Ph. D. Dissertation, University of the Philippines Los
Bafios. College, Laguna, Philippines, 170pp.

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survey data in the Philippines. Plant Dis. 77:376-382.

H, SOEROTO. 1992. Rice tungro disease vectored by
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THRESH JM. 1982. Cropping practices and virus spread.
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VILLAREAL S. 1999. Leafhopper control by insecticides is
not the solution to the tungro problem. pp. 139-142. In
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Azzam and K. L. Heong (Eds.). Intl. Rice Res. Inst., Los
Bafios, Laguna, Philippines. 166 pp.

J. Pedroso, A.D. Raymundo and A.C. Sumalde

Volume 37 (1) January-June 2001 35

Table i. Correlation coefficients (r) between temporal disease development and GLH population in pure and mixed stands
of rice cultivars during the dry and wet seasons of 1999

Dr Season Wet Dr\ Wet Dry Wet
Season Season Season Season Season

Pure Stand
IR-64 0.89* 0.29 0.21 0.31 0.78 0.68
PSBRc 18 0.20 0.67 0 1 0.24 0.43 0.88
1R-72 0,92* 0.54 0.40 0.34 0.45 0,68
PSBRc 34 0.68 0.79 0.26 0.33 0.04 0.95*

lMied Stand
PMA 0.94* 0.40 0.83 0.56 0.62
PMB 0.21 0.86 0.014 0.69 0.05 0.72
PMC 0.56 0.33 0.57 0.84 0.75 0.28
PDA 0.85 0.72 0.35 0.22 0 04 0.24
PDB 0 80 0.71 0.20 0.79 0.57 0.61
PDC 0.85 0.72 0.51 0.56 0.14 0.05
AR 0.63 0.50 0.49 0.76 0.34 0.51
VR 0.85 0.82 0.90* 0.41 ().89* 0.31

*Sienificant at l 01 05
PMA Plot miiures o1"2 cultivars (IR-64 and PSBRe 18)
1\I1 Plot mixtures of 3 cultivars (IR-64. PSBRc I X and 111R-72)
'.\IC PI'lot mixtures of 4 culti vars (IR-64- PSI3Rc 1 1. IR-72 :and PSBRc 34)
P1).\ Plot mixtures spaced 20x20 cm: 2 cullivars (IR-64 and PSBIRc 1 )
P') Plot mixtures spaced 25x25 cm. 3 cultivars (11R-64. PSl3Rc 18 and IR-72)
IDC Plot mixtures spaced 15x20 im: 4 cultivars (1R-64. P'SlRc I. IR-72 and I'SBRc 34)
KR Cultivar mixtures in alternate ro\\s. 4 cultivars ( lR-64. I'S Re IX. 1R-72 and PSBRc 34)
\ R Cultivar mixtures within rows: 4 culitars (1R-64. PSBRc 1X. IR-72 and PSHRc 34)

Table 2 Correlation coefficients (r) between spatial disease development and GLH population in pure and mixed stands
of rice cultivars duinny hle dry and wet seasons of 1999

Dry Season Wet Dr\ Wet Dry Wet
Season Season Season Season Season

Pure Stand
IR-64 0.80 0.62 0.13 0.79 0.21 0.86
PSBRc 18 0.97* 0.60 0.015 0.08 0.07 0.87*

Mixed Stand
VR 0.94* 0.05 0.1 0.37 0.84 0.97*
AR 0.95* 0.92 01.2 0.23 0.20 0.56

*Significant at a 0.05
I legend:
.R Mixtures planted in alternate roxs: 4 cultivars (1R-64. P'SBRc I X. 1R-72 and PSBRc 34)
VR Mixtures planted within lows: 4 cultixalr (1R-64. I'SBRc I X. IR-72 and PSI Rc 34)

36 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

Table 3. Weekly mean populations of GLH in pure and mixed stands of rice cultivars per plot (88 sweepings/plot) in three barangays during the dry
and wet seasons of 1999 from 10-57 DAT.

Dry Season Wet Season Dry Season Wet Season Dry Season Wet Season

Pure Stand
PSBRc 18 222 105 85 110 253 106
IR-72 138 92 92 182 128 119
PSBRc34 155 71 84 131 465 78
IR-64 198 139 100 207 '694 103

Mixed Stand
PMA 209 48 69 -828 114
PMB 200 62 92 118 1066 134
PMC 174 30 101 166 346 124
PDA 148 27 107 121 165 83
PDB 163 38 68 134 128 99
PDC 155 66 84 139 126 113
AR 165 43 106 125 66 78
VR 169 30 82 157 301 84

PMA = Plot mixture of 2 cultivars (IR-64, PSBRc 18)
PMB = Plot mixture of 3 cultivars (IR-64, PSBRc 18 and IR-72)
PMC = Plot mixture of 4 cultivars (IR-64, PSBRc 18, IR-72, PSBRc 34)
PDA = Plot mixture with population density A 20x20 cm; 2 cultivars (IR-64 and PSBRe 18)
PDB = Plot mixture with population density B 25x25 cm; 3 cultivars (IR-64, PSBRc 18 and IR-72)
PDC = Plot mixture with population density C 15x20 cm; 4 cultivars (IR-64, PSBRc 18, IR-72, PSBRc 34)
AR = Plot mixture planted in alternate rows; 4 cultivars (IR-64, PSBRe 18, IR-72, PSBRc 34)
VR = Plot mixture planted within plots; 4 cultivars (IR-64, PSBRc 18, IR-72, PSBRc 34)

Table 4. Average number of nymphs and adults of green leafhopper (GLH) in pure and mixed stands of rice cultivars in
Barangay Cawit, Zamboanga City during the dry and wet seasons of 1999.

Nymph Adult Nymph Adult

IR-72 651 67 374 51

PSBRc 18 874 -30 299 592

IR- 64 1006 601 186 671

Mixture 1060 405 277 400

Total 3591 1403 1136 1714

Average 898 351 284 429

Table 5. Correlation coefficients (r) between vector populations and weather factors in Barangay Talisayan in pure and
mixed stands during the dry season of 1999.

Rainfall Relative Humidity Temperature

Pure Stand
IR-64 0.52 0.63 -0.68
PSBRc 18 0.39 0.08 -0.38
IR-72 0.08 -0.07 0.24
PSBRc 34 0.59 0.22 -0.52

Mixed Stand
Plant Mixture
PMA -0.005 0.45 0.16
PMB 0.22 0.10 0.15
PMC 0.55 0.52 -0.03
Population Density
PDA -0.18 0.50 0.12
PDB -0.004 0.49 -0.13
PDC 0.03 0.58 -0.05

Cultivar Mixture
Within Rows (VR) -0.28 0.58 -0.05

Cultivar Mixture in
Alternate Rows 0.02 0.40 0.08

"Not Significant at a 0.05

38 Volume 37 (1) January-June 2001 Journal of Tropical Plant Pathology

Wet Season

500 -- IN 4
- PSBRc18
450 -E+- PSBRc34
400 --- IR-72
0 150

10 15 20 25 30 35 40 45 50 55 60 10 15 20 25 30 35 40 45 50 55 60
Days alter transplanting Days after transplanting


450 ----- PMA
400 -- PMB
--- PMC
350 -- AR



0 -- po m re-------PI--72)
10 15 20 25 30 35 40 45 50 55 60 10 15 20 25 30 35 40 45 50 55 60
Days after transplanting Days after transplanting

-4- PDA
500 PDB
450 6 PDC
400 --- VR
0 350
g 250
o 200
-- +-- -- --- -1
10 15 20 25 30 35 40 45 50 55 60 10 15 20 25 30 35 40 45 50 55 60
Days after transplanting Days after transplanting

PMA plot mixtures of two cultivars (IR-64, PSBRc 18)
PMB plot mixtures of three cultivars (IR-64, PSBRc 18, IR-72)
PMC plot mixtures of four cultivars of four cultivars (IR-632. PSBRc 18. IR-72, PSBRc 34)
PDA plot mixtures spaced 20x20 cm; 2 cultivars (IR-64. PSBRc 18)
PDB plot mixtures spaced 25x25 cm; 3 cultivars (IR-64, PSBRc 18, IR-72)
PDC plot mixtures spaced 15x20 cm; 4 cultivars OR-64, PSBRc 18. IR-72, PSBRc 34)
AR cultivar mixtures in altemate rows; 4 cultivars (IR-64 PSBRc 18. IR-72, PSBRc 34)
VR cultivar mixtures within rows; 4 cultivars (IR-62. PSBRc 18, IR-72. PSBRc 34)

Fig. 1 GLH population in pure and mixed stands of rice cultivars in Barangay Tulungatung
during the dry and wet seasons of 1999.

J. Pdros, AD. Rymudo ad AC. Smale Voume37 j) Jnuar-Jue ZU1 .

Dry Season

Volume 37 (1) January-June 2001 zo

J. Pedroso, A.D. Raymundo and A.C. Surnalde

fi "
6 Q







90 i
75 |

60 a

4 -- IR 64
---- PSBRc 34
a 300 e nlR-7r

~ 50

10 15 20 25 30 35 40 45 50 55 60
Das aller mtsantina
S30 AR
s on
o 150

10 15 20 25 30 35 40 45 50 55 60
Days aler transplanlin


o 150
1 300

ot 150 -

10 15 20 25 30 35. 40 45 50 55 0

Days afteriansplning

10 15 20 25 30 35 40 45 50 55 60

-a- Poe
-0- P0

10 15 20 25 30 35 40 45 50 55 60
10 1520 2530 5 40 45055600

10 15 20 25 30 35 40 45 50 55 60
Days ar transplant

PMA plot Rildures o tawo culinvars (IR-64. PSBRc 18)
PMB plot rmiores of thrue cultivas (IR-64. PSBRc 18. IR-72)
PMC plot mnitres ot tour culmas aotfour cullivas R-632. PSBRc 18, IR-72, PSBRc 34)
PDA plot miUses spaced 20x20 cmn 2 cullivars (R-84. PSBRc 18)
PDB plot mulures spaced 2525 cman; 3 auli as OR-4. PSBRc 18. IR- 72)
POC plot mimes spaced 150 cman 4 cullivams (IR-64, PSBRc 18. IR-72. PSBRc 34)
AR culuar m~bies in altemal 4 cullams R-64., PSBRc 18, IR-7. PSBRc 34)
VR clivar mfumse wilhin r~mws 4 cuivags (R-62, PSBRc 18. IR-72. PSBRc 34)

Fig. 2 GLH population in pure and mixed stands of rice cultivars in Barangay Cawit during
the dry and wet seasons of 1999.

"*-Dl -r 20


Wet Seaun

U -i----------
10 15 20 25 30 35 40 45 50 55 60
Days after transplanting

2000 -0-PMA
S1500 15 -- 2PMC

1000 -


0 / \
10 15 20 25 30 35 40 45 50 55 60
Days after transplanting

1200 -e- VR
1050 .--PDB


8 600
Y 450


10 15 20 25 30 35 40 45 50 55 60
Days after transplanting

10 15 20 25 30 35 40 45 50 55 60
Days after transplanting

10 15 20 25 30 35 40 45 50 55 60
Days after transplanting

10 15 20 25 30 35 40 45 50 55 60
Days after transplanting

PMA plot mixtures of two cultivars (IR-64, PSBRc 18)
PMB plot mixtures of three cultivars (IR-64, PSBRc 18. IR-72)
PMC plot mixtures of four cultivars of four cultivars (IR-632, PSBRc 18, IR-72, PSBRc 34)
PDA plot mixtures spaced 20x20 cm; 2 cultivars (IR-64, PSBRc 18)
PDB plot mixtures spaced 25x25 cm; 3 cultivars (IR-64, PSBRc 18. IR-72)
PDC plot mixtures spaced 15x20 cm; 4 cultivars (IR-64, PSBRc 18, IR-72, PSBRc 34)
AR cultivar mixtures in alternate rows; 4 cultivars (IR-64, PSBRc 18, IR-72, PSBRc 34)
VR cultivar mixtures within rows; 4 cultivars (IR-62. PSBRc 18, IR-72 PSBRc 34)

Fig. 3 GLH population in pure and mixed stands of rice cultivars in Barangay Talisayan
during the dry and wet seasons of 1999.

J. Pedroso, A.D. Raymundo and A.C. Sumalde Volume 37 (1) January-June 2001 41

Dry Season

J. Pedroso, A.D. Raymundo and A.C. Sumaide

Volume 37 (1) January-June 2001 41


180 L
140 -
120 i
100 +
80 4
40 -


8 4


15 22 29 36 43 50 57

4 m



200 r

180 +



120 i

100 t

80 ;

60 -

40 4

0 -

10 15 22 29 36 43 50 57


Figure 4. Disease incidence (DI) and green leafhopper (GLH) population in IR-64 and
and PSBRc 18 in Barangay Cawit, Zamboanga City during the dry season
of 1999.

Journal of Tropical Plant Pathology

42 Volume 37 (1) January-June 2001

300 T 30

250 -25
20 w
^ 200 20

W 150 15
w w

i 1001 10

50 -5

0 -0 -
10 15 22 29 36 43 50 57

450 PSBRc 18 23

400 20

350 18
I 300 15
-J I w
L 250 13 0

W200 10 M
I /w
z 150 --8

100 5

501 3

10 15 22 29 36 43 50 57

Figure 5. Disease incidence (DI) and green leafhopper (GLH) population in IR-64
and PSBRc 18 in Barangay Cawit. Zamboanga City during the wet season
of 1999.

J. Pedroso, A.D. Raymundo and A.C. Sumalde Volume 37 (1) January-June 2001 43

J. Pedroso, A.D. Raymundo and A.C. Surnalde

Volume 37 (1) January-June 2001 43

_- Dl


180 -
120 -
60 -

10 15

-+---------,- -I---- -C I I I I-----
22 29 36 43 50 57

Figure 6. Disease incidence (DI) and green leafhopper (GLH) population in cultivars
within rows (VR) and cultivars in alternate rows (AR) in Barangay Cawit,
Zamboanga City during the dry season of 1999.

Journal of Tropical Plant Pathology

------- --+---A-- ---- I I I I I---- --
15 22 29 36 43 50 57

44 Volume 37 (1) January-June 2001



--*--DI 20







10 15 22

29 36 43 50 57------
29 36 43 50 57

15 22
15 22

29 36 43 50 57

Figure 7. Disease incidence (DI) and green leafhopper (GLH) population in cultivars
within rows (VR) and cultivars in alternate rows (AR) in Barangay Cawit,

Zamboanga City during the wet season of 1999.

300 T

200 +

150 t










90 Y

75 9


200 i

150 +





'Associate Professor. Department of Pest Management. College of Agriculture. Central Luzon State
University. Mufioz, Nueva Ecija 3120. Philippines: Research Specialists. Philippine Rice Research Institute,
Maligava. Munoz. Nueva Ecija 3120. Philippines: 'Associate Professor. Department of Plant Pathology, College
of Agriculture and Environmental Science. OARDC, Ohio State University. Wooster, OH. USA and Pen-Urban
Program Leader, Asian Vegetable Research and Development Center, Shanhua, Tainan, Taiwan.*

During the 2000 and 2001 planting seasons, a disease outbreak in onion (Allium cepa)
occurred in Nueva Ecija and other onion growing provinces in Luzon. Symptoms include
sunken necrotic spots with clusters of orange to black masses of conidia arranged in
subcircular form. Leaves were twisted and curled with dieback symptoms. Bulbs of affected
plants were generally slender with elongated necks, roots were short and sparse which led
to collapse of the plants. Isolation and test for pathogenicity, following Koch's postulates,
showed that the disease was caused by Colletotrichum gloeosporioides. Captan, Benomyl and
Mancozeb reduced disease incidence and apparent infection rate in onion.

Key Words: Anthracnose. Colletotrichum gloeosporioides, onion


Onion is usually planted as second crop to rice
during the dry season. It is counmonly grown in Nueva
Ecija in Central Luzon (8,010 ha) and Ilocos Region
(4.299 ha). Nueva Ecija accounted for 60% (51,245
MT) of the country's total onion production while the
Ilocos Region contributed 36% (34,834 MT). The
return of investment is high making onion one of the
most profitable crops in the country. Onion accounts
for 34% of the total vegetable industry's foreign
exchange earnings. In 1996 alone, export of fresh
onion reached 27.227 tons worth US$11.4 million.
Onion production, however, is marked by fluctuations
because of pest and diseases. area harvested and
changes in weather (Dar. 1999).

A significant increase in volume of production
was recorded from 1990 to 1999. but the industry
suffered from heavy losses in 2000 and 2001 due to an
outbreak of a strikingly unusual and uniformly
distributed disease which occurred in Nueva Ecija
and neighboring onion growing provinces in Luzon
causing yield losses of as high as 80 to100% in almost
all of the fields (Alberto et al.. 200 b). Conspicuous
aboveground symptoms of foliar drying often
associated with poor bulb development as well as
distinct twisting and curling of leaves and neck
elongation occurred on most of the onion plants. The

damage to the industry was so high such that it
warranted immediate investigation on the nature.
causes. etiology and management of the disease. The
disease now poses a serious threat to all onion
growing areas of the country (Alberto et al., 2001)

This study was done to identify the disease and its
causal pathogen, including its morphological
description and host range, and to evaluate the
efficacy of various fungicides against the disease.


Isolation and Preparation of Inoculum

All studies were conducted at the laboratory and
greenhouse facilities of the Philippine Rice Research
Institute m Mufioz, Nueva Ecija from 2000 to 2001.
The characteristic symptoms of the disease were
described and noted. Dark acervuli from onion leaves
were collected and transferred to potato dextrose agar
with 1% streptomycin sulfate. The set-up was
incubated at 280C for 72 hours and a portion of the
fungal growth was re-inoculated in plated potato
dextrose agar with 1% streptomycin sulfate to obtain a
purified growth of the pathogen. The fungus was
allowed to sponrlate for 1 week at 28TC in darkness
until abundant conidia were produced on aerial

hyphae and pinkish conidial masses developed
beneath the mat of the mycelium.

mIoculum was prepared by scraping the surface
growth of mycelial mats and spore masses from
10 petri dish cultures, suspending in 100 ml of
sterile distilled water, and then filtering through
two layers of cheesecloth. The inoculum density of
the pathogen was adjusted to 2.7 x 106 conidia/ml.
A sticker solution (Hoestick, 5ml/16 1 of water) was
added to the conidial suspension.

Pathogenicity Test

Forty five day-old seedlings of onion cv. Red
Pinoy (East West Seed Co.). previously grown in a
seedbed, were transplanted into #5 clay pots.
Treatments were: 1) Damaged Leaves 2) Undamaged
Leaves 3) Uninoculated Control. There were 10
seedlings per treatment (1 seedling/pot) replicated
four times. At 30 days after transplanting, conidial
suspension was sprayed on damaged and undamaged
leaves when the plants had 3-4 fuily expanded leaves.
Sterile distilled water was used for the untreated

To maintain about 100% relative humidity, all
inoculated seedlings were covered with dark
cellophane bags inside a glasshouse for 48 hours prior
to exposure to natural conditions outside.. The relative
humidity was maintained by spraying the plants with
water three times a day until symptoms were fully

Re-Isolation and Re-Inoculation

From infected onion plants in the above test, the
pathogen was re-isolated and re-inoculated using the
same methods and cultivar as in previous experiments
to confirm its pathogenicity.

Characterization and Identification

The pathogen was grown on PDA slope in plastic
tubes, sealed and sent to CABI Bioscience, UK
Centre, Bakeham Lane, Egham, Surrey. UK for
positive identification. Mature and immature acervuli
were mounted on cotton blue lactophenol and
examined under the light microscope. The
morphology of acervulus and conidia were described,
recorded and documented.

Host Range and Cross Inoculation

The pathogen isolated from onion was inoculated
to fruits of mango, papaya, and vegetables namely,
bell pepper, tomato and okra, and legumes like string
beans, winged beans and soybeans. The fruits and
vegetables were washed first with water then with
10% chlorox solution and blot-dried. Black acervuli
collected from the prepared cultures were inoculated
on the surface of the fruits, which were subsequently
placed in an incubation chamber lined with moist
filter paper and incubated for 4 to 5 days for symptom
development. For legumes, leaves of 3 week-old
seedlings grown in plastic pots filled with soil and
compost were spray-inoculated with spore suspension
(2.7 x 106 conidia/ml plus sticker, 5ml/161i of water)
of C. gloeosporioides.. The seedlings were then
covered with cellophane bags for 72 hours to maintain
relative humidity close to 100%. After 72 hours, the
bags were removed. Water was sprayed over the
seedlings three times daily to maintain high relative

The pathogen was also isolated from mango and
avocado fruits infected with anthracnose, using the
tissue planting technique for isolation of fungal
pathogens from diseased tissues. The isolated
pathogen was purified in plated potato dextrose agar
(PDA) and re-ioculated on healthy mango and
avocado fruits to confirm its pathogenicity. The
purified isolates from mango and avocado were cross-
inoculated to onion leaves which were incubated in a
moist chamber until symptoms developed.

Fungicide Bioassay

A seed layer of C. gloeosporioides in PDA
medium was made by transferring 0.5 ml from 50 ml
suspension (40.000 conidia/ml) of the pathogen into
Petri dishes. Based on the manufacturer's
recommended rates, 1 liter suspension of each of 17
commercial fungicides (Table 1) were prepared. Four
sterilized paper discs were immersed in each
suspension for 5 minutes and transferred to the surface
of plated agar seeded with C. gloeosporioides. Paper
discs for untreated control were soaked in sterile
water. The set up was incubated at room temperature.
Zones of growth inhibition were measured 48 hours

Volume 37 (1) January-June 2001 47

R.T. Alberto et al

maintain relative humidity close to 100% prior to
exposure to natural conditions outside the glasshouse.
The fungicides were sprayed at intervals of: a) 5 days,
b) 7 days. c) 10 days. and d) 14 days. The
effectiveness of the fungicides and spray intervals
were determined based on percent disease incidence
and apparent infection rate (Vanderplank, 1963).
Three trials were conducted



Infected leaves had shallow sunken necrotic spots
with clusters of orange masses of conidia arranged in
subcircular rings. As the disease progressed. the
orange conidial mass hardened and turned into black
acervuli (Fig. 1. anthracnose symptoms) while the
leaves became twisted and curled (Fig. 1. twister
symptoms). In severely infected plants, dieback
symptoms appeared, associated with short and sparse
roots which led to collapse of the plants. Bulbs of
affected plants were generally slender with elongated

Pathogenicity Test

Symptoms appeared 2 days after inoculation. The
pathogen formed lesions in 148 out of 160
inoculations (92.5%) on damaged leaves and 109 out
of 150 inoculations (72.5%) on undamaged leaves
(Table 2). Lesions were not formed in uninoculated
leaves. Within 3 days of inoculation, whitish shallow
sunken spots developed on the leaves. As the lesions
matured. orange masses of conidia arranged
subcircularly formed at the center of the lesions. The
masses of conidia hardened and turned into black
acervuli while the leaves exhibited curling and
twisting. Lesions enlarged, coalesced, and damaged
large areas of the leaves. Most of the plants were
completely damaged by the disease at 7 days after

Reisolation and Inoculation

From the artificially inoculated plants, C.
gloeosporioides was reisolated and after three
inoculations with the isolates. plants showed
symptoms similar to those previously described.

Cross Inoculation

Cross inoculation of the isolate showed that it

could also infect bell pepper, tomato, mango, and
papaya fruits but not okra (Table 3). Legumes like
string beans, winged beans and soybeans were also
infected. C. gloeosporioides isolated from mango and
avocado did not cause infection in onion.

Characterization and Identification

The disease is caused by an extremely common
and widespread fungal species aggregate called
Glomerella cingulata (Stoneman) Spauld. & Schrenk.
(anamorph. Colletotrichum gloeosporioides (Penzig)
Penzig & Sacc. (IMI384216). This airborne pathogen
has a wide host range and occurs in tropical regions of
Africa, Asia and Latin America and is primarily a
saprobe or parasite on a very wide range of plants
(Maude, 1990).

Acervuli from brown to black normally found in
the necrotic areas were usually setose or glabrous,
sometimes rounded and elongated (Fig. 2). Setae were
few to absent. Conidia were cylindrical with obtuse
ends, sometimes ellipsoid with rounded apex, hyaline,
aseptate and uninucleate, 8-25 x 3-8u. Colonies on
PDA were either grayish white, orange or black with
fructification showing diurnal zonation.

Fungicide Bioassay

The widest zone of growth inhibition of the
pathogen was observed in the treatment with Captan..
This was followed by Benomyl and Mancozeb.
Thiophanate methyl also showed considerable
inhibition on the growth of the pathogen. Difeno-
conazole. Metalaxyl + mancozeb, Chorothanlonil, and
Propones had moderate inhibition effects on the
growth of the pathogen while the rest of the fungicides
had no inhibitory effect at all.

Captan applied as protectant at higher rate
(80g/161i water) was the most effective in reducing
incidence of anthracnose (32.6%) as compared to
Benomyl and Mancozeb at 38.2% and 54.4%,
respectively (Table 4). The apparent infection rate
(0.25) was also lower in Captan-treated plants.
Symptoms in Captan-treated plants were observed 10-
11 days after inoculation, compared to 5-6 days for
Benomyl and 4-5 days for Mancozeb. Spray
applications of fungicides at 5-day intervals

significantly reduced the incidence and apparent
infection rate of the disease. There were no
differences in disease incidence and apparent infection

48 Volume 37 (1) January-June 2001 Journal of Plant Pathology

48 Volume 37 (1) January-June 2001

Journal of Plant Pathology

respectively (Table 4). The apparent infection rate
(0.25) was also lower in Captan-treated plants.
Symptoms in Captan-treated plants were observed 10-
11 days after inoculation, compared to 5-6 days for
Benomyl and 4-5 days for Mancozeb. Spray
applications of fungicides at 5-day intervals
significantly reduced the incidence and apparent
infection rate of the disease. There were no
differences in disease incidence and apparent infection
rates at 10 and 14 day-spray intervals.


The disease was first reported by Ebenebe (1980)
as onion twister in Nigeria. Later it was reported as
anthracnose in Indonesia by Galvan et.al. (1997) and
in Thailand by Black (personal communication).

This is the first time that the occurrence of
anthracnose in onion is reported in the Philippines.
The disease is caused by Colletotrichum
gloeosporioides. The fungus can be easily isolated
from any part of the affected plant including the bulb
and neck regions. It invades most of the parts of the
leaves and the necks. When rain occurs for more than
48 hours, serious leaf damage is apparent as minute
lesions coalesce to form lesions large enough to topple
down the leaves from the site of infection in less than
72 hours after inoculation.

Observations made during 2000 and 2001 showed
that C. gloeosporioides could infect onion foliage at
any growth stage. It caused minimal damage when
less than 12 hours of continuous leaf wetness and high
relative humidity occurred. However, when leaf
wetness and high relative humidity exceeded 24
hours, leaf twisting of leaves and masses of orange
conidia appeared and developed faster into black
acervuli resulting in severe foliar and bulb damage.

Among 17 commercial fungicides tested, Captan,
Benomyl and Mancozeb were most effective against
anthracnose of onion. Among the three, the lowest
infection frequency and apparent infection rate were
observed in Captan-treated plants. Captan was well
tolerated by the plants and it had a pronounced
prophylactic effect against the disease. However, its
curative effect is yet to be determined. Although these
three fungicides were shown to be effective against
the disease, anthracnose still poses a problem to onion
growers because of the dearth of new disease

management technologies and the recent withdrawal
of both Captan and Benomyl in the market. New
technologies must be developed and new fungicides
should be evaluated to counteract the destructive
effects of the disease.


Know your new disease of onion "Anthracnose". Proc..
14th National Rice Research and Development
Convention. Philippine Rice Research Institute,
Maligaya, Munoz, Nueva Ecija, Philippines.

Anthracnose: Serious disease of onion. Proc. Annu.
Conv. Pest Manag. Council Philippines, CSSAC/DA-
RFU 5, Pili, Camarines Sur, Philippines, May 2-6,

DAR WD. 1999. Critical issues and strategies for the
development of major agricultural crops. Proc.Annual
Meeting, Natl. Acad. Sci. Tech. (NAST). Manila Hotel.

EBENEBE AC.1980.Onion twister disease caused by
Glomerella cingulata in Northern Nigeria. Plant
Disease. 64:1030-1032.

AH PERMADI, C KIK. 1997. Screening for
resistance to anthracnose (Colletotrichum
gloeosporioides Penz.) in Allium cepa and its wild
relatives. Euphytica. 95:173-178.

MAUDE RB. 1990. Leaf diseases of onion. In: H.D.
Robinowitch and J.L. Brewster (Eds.). Onions and
Allied Crops. Vol. 11, pp. 173-190. CRC Press. Boca
Raton, Florida.

VANDERPLANK, JE. 1963. Plant Diseases, Epidemics
and Control. Acad. Press, New York. 349pp.


This research was supported by the Integrated Pest
Management Collaborative Research Support Program
(IPM CRSP) which was funded by USAID Grant No.
LAG-G-00-93-00053-00 with Virginia Polytechnic Institute
and State University as management entity; Philippine Rice
Research Institute as lead institution; and University of the
Philippines Los Banos, Central Luzon State University,
Visayas State College of Agriculture, Ohio State
University, Pennsylvania State University and Asian
Vegetable Research and Development Center as
collaborating institutions.

R.T. Alberto et al Volume 37 (1) January-June 2001 49

R.T. Alberto et al

Volume 37 (1) January-June 2001 49

Table 1. Fungicides evaluated for efficacy against Colletotrichum gloeosporioides in onion.

Product Name Chemical Name Rate RR (g/16 li water) Source
(g a.i./ha) Lowest Highest

Captan 50WP Captan 500 40 80 Zeneca Asiatic Agro
Benlate WP Benomyl 500 10 20 Du Pont, Taiwan
Dithane M45 Mancozeb 800 40 75 Rohm & Hass, France
Manzate 200WP Mancozeb 800 30 60 Du Pont, USA
Sweep WP Thiophanate methyl 500 5 20 Cyariarid Agro, Phil.
Parafungus 80WP Mancozeb 800 30 60 Planters Phil.
Score 250EC Difenoconazole 250 5 8 Novartis Agro Phil.
Ridomil MZ 58WP Metalaxyl+Mancozeb 450 6 10 Novartis Agro Phil.
Daconil 2787 75WP Chlorothalonil 750 19.2 28.8 Rhone Poulenc Phil.
Vandozeb Plus WP Mancozeb 800 30 60 Elf Atochem Agri.
Anthracol 70WP Propineb 700 50 65 Bayer Phil.
Redeem 800WP Mancozeb 800 2.5 5 Sanachem Ltd, S.A.
Kocide 101WP Cupric Hydroxide 500 20 60 Griffin Corp., USA
Funguran-OH WP Copper Hydroxide 500 20 60 Urania Agrochem
Previcur N EC Propamocarb hydrochloride 722 6.4 10 Hoechst, Far East
Vigitran Blue WP Copper oxychloride 580 50 80 Hoechst, Phil.
Flint 50WG* Not yet approved 62.5 8 16 Novartis Agro Phil.
*Not yet registered in FPA.

Table 2. Pathogenicity of Colletotrichum gloeosporiodes on onion leaves.

Treatments Number of Leaves Leaves Infected
Inoculated (%)

Inoculated Damaged* 160 92.5
Inoculated Undamaged 150 72.5
Uninoculated Control 154 0

'Leaf surface slightly brush.

Table 3. Pathogenicity of Colletotrichum gloeosporioides on fruits, vegetables and legumes.

Number of Fruits Inoculated %
Fruits Infected

Mango 5 100
Papaya 5 100
Bell Pepper 5 100
Tomato 10 100
Okra 10 0

Number of Leaves Inoculated % Leaves Infected

Wingedbean 168 93.75
Stringbean 180 97.22
Soybean 128 93.75
'Number of plants inoculated: Wingedbean-7. Stringbean-10. Soybean-8

so Volume 37 (1) January-June 2001

Journal of Plant Pathology

Table 4. Incidence (%) and apparent infection rates of anthacnose on Captan, Benomyl and Mancozeb treatments at
different spray intervals.

Fungicide Incidence (%)** (r)* Spray Incidence (%) (r)
Treatment Interval (Days)

Captan 28.8 d 0.27 c 5 46.8c 0.24b
Captan 32.6 e 0.25 c 7 50.2 b 0.35
Benomyl 40.0 d 0.32 bc 10 52.3 0.36
Benomyl 38.2d 0.30 b" 14 54.0a 0.35"
Mancozeb 58.1b 0.36
Mancozeb 54.4 0.33 bc
Control 93.2 a 0.47 a

'Apparent Infection Rate
'Means ofthe same letters are not significantly different at 5% LSD.

a b

Figure 1. (a) anthracnose symptom in onion leaf sheath, (b) twisting, curling and dieback symptoms.

a b c d

Figure 2. (a) black acervuli on the leaf blade, (b) matured acervuli, (c) conidiospores, (d) colonies in PDA with diurnal

R.T. AIato et al Vohane 37(1) January-June 2001 51

Vokfne 37 (1) January-June 2001 51

R.T. Aberto et al



'Senior Agriculturist. former Supervising Agriculturist, Bureau of Plant Industry, Davao National Crop
Research and Development Center. Bago-Oshiro. Davao City, and 3Professor, Department of Plant Pathology.
University of the Philippines Los Balios. College. Laguna, Philippines

The occurrence of papaya ringspot potyvirus in papaya is recorded for the first time
in three municipalities in South Cotabato, namely Polomolok, Tupi, and Tampakan and
more recently in Sta. Cruz, Davao del Sur. Symptoms observed on the leaves collected
from a Solo papaya plantation in Silway 8, Polomolok, South Cotabato last October,
1999 were mild mottle on young leaves and leaf distortion including reduction of the
lamina and chlorosis. Oily streaks which were very severe were also observed on some
of the petioles from the sample trees infected. Watersoaked spots were observed on the
stem of the plants. Ringspot symptoms can be observed on green fruits of Solo papaya
but did not have distinct round marks as observed on the PRSV from other countries
and those from Luzon. Distinct round marks have been observed on the latest infected

fruits of the Cavite type. ELISA tests
antisera gave positive results but not with


Papaya ranks seventh in importance among
the commercial fruits grown in the Philippines.
The most important cultivars grown are Solo,
which is grown mainly for export. while Cavite
Special, Morado and Sinta are used as fresh and
processed fruits. In the Philippines, papaya is
usually consumed as fresh fruit while the green
fruit is used as vegetables or salads. The
markets for export are mainly Hong Kong,
Japan, Saudi Arabia. and United Arab Emirates
(UAE). In 1999. about 5,300 hectares were
planted to papaya with total production of about
71.500 mt and the country exported 1,200 mt of
fresh papaya mainly to Japan valued at about
P65 M (BAS. 2000: Villegas. 2002).

Currently, the papaya industry is confronted
with serious pests that hamper not only
productivity but the growth of the papaya export
sector as well. Several fungal. bacterial and
viral diseases have been reported to infect
papaya in the Philippines (Bajet et al. 1992).
The most common fungal diseases of papaya in
the field are Phytophthora root rots, seedling
damping off and anthracnose. There are several
postharvest diseases. such as anthracnose,
Rhizopus and Fusarium rot, which cause heavy'
losses during transport, storage. and marketing.

conducted on papaya samples with PRSV
the papaya leaf distortion mosaic potyvirus

The most common bacterial disease is papaya
crown rot caused by Erwinia caricae n. sp.
Three viral diseases of papaya have been
recorded in the country. These are papaya leaf
curl and papaya mosaic which were both
reported by Teodoro (1960) and the ringspot
which was reported to be infecting papayas in
Silang, Cavite in 1982 (Opina, 1986). The
etiology of the diseases reported by Teodoro
(1960) was not mentioned while the ringspot
was confirmed to be PRSV in the 1990s
(Eusebio, 1992; Eusebio et. al., 1994; Eusebio
et. al., 1997).

PRSV has spread to the neighboring
provinces of Batangas, Laguna, Rizal and
Quezon (Opina, 1986). In a 1990-1992 survey,
the disease has also been recorded in the Luzon
provinces of Bulacan, Pampanga, Nueva Ecija,
Pangasinan, La Union, Ilocos Sur, Ilocos Norte,
Camarines Sur, Sorsogon, Mindoro and
Palawan (Eusebio, 1992; Eusebio et. al., 1994).
In 1997. it has been confirmed to be present in
Marinduque, Negros Oriental. Aklan and Leyte
(Eusebio et. al. 1997).

There have been suspicions on the
occurrence of PRSV in Mindanao. In 1992,
Espino et. al. reported that seeds obtained from
fruits of Solo papaya grown in Mindanao

reacted to antibodies to PRSV that they
produced despite the absence of symptomatic
plants in the area. This paper reports the
occurrence of PRSV in Mindanao based on
symptomatology and ELISA. Preliminary
results have been reported (Herradura et al..


Since 1998 several reports on the alleged
mcidence of PRSV in Mindanao have been
referred to the Bureau of Plant Industry. As a
result of these reports and referrals, several field
visits were done on papaya plantations in South
Cotabato suspected to exhibit papaya ringspot
symptoms on fruits. In 1999, fruits with
symptoms quite similar to ringspot, intercepted
from a vapor heat treatment facility, were
submitted to the Bureau of Plant Industry in
Davao City for disease diagnosis.


Using indirect ELISA (Eusebio et al. 1992;
Eusebio et al..1997). samples including fruits
collected from Darong, Sta. Cruz. Davao del
Sur were tested using papaya ringspot
potyvirus-Los Bafios (PRSV-LB) antiserum
following the methods described and using the
same solutions and buffers mentioned above.
On the petioles, the oily streaks/water soaked
lesions were dissected out and used in the
ELISA test.

A parallel test was done on a separate
ELISA plate using PRSV-P and papaya leaf
distortion mosaic virus (PLDMV) antisera by
double antibody sandwich (Clark and Adams.
1977) ELISA from Dr. Maoka. Okinawa, Japan.
The plate was coated with IgG fraction of
PRSV-P-126 at 1:1000 dilution using coating
buffer and PLDMV-56 at 1:800 dilution using
the same buffer and incubated for 2 hours at
room temperature and washed with PBST 3
times. Samples \were prepared by grinding the
leaves with PBST at 1:20 dilution dispensed
into different wells. incubated overnight, and
then washed as above. The addition of enzyme
conjugated IgG diluted at 1:800 for P1 WV-P-
126 and 1:600 for PLDMV-56, the sut~trate
solution, washing and evaluation of reactions

were essentially as those described earlier
(Clark and Adams. 1997: Eusebio, 1992;
Eusebio et al. 1994; Eusebio et al., 1997). The
absorbance value at 405 nm was determined
after 30 min to 1 hr in an ELISA reader
(Titertek Multiskan Plus).


Symptoms observed on papaya leaves
collected in October, 1999 were mild mottle on
Young leaves and leaf distortion with leathery
texture on leaflets (Fig. IA). Oily streaks were
also seen on some of the petioles of the sample
plants inspected. On the fruits, characteristic
ringspot symptoms can be observed (Fig. 1B).
PRSV-infected plants in Luzon have been
characterized as exhibiting vein clearing and
chlorotic spots followed by distortion of the
leaves, shoestringing, ringspots on fruits and on
the upper portion of the stem as well as elonga-
ted oily streaks on petioles and stems (Opina.
1986: Eusebio. 1992; Eusebio et al., 1994:
Ramos, 1987). Another inspection done on
January, 2000 revealed that the oily streak
symptoms have progressed on the petioles as
they were much more distinct than during the
initial inspection and thus have been easier to
diagnose. The water soaked spots also
developed on some areas of the stem of the
infected plants (Fig. 1C). Reduction and
chlorosis of leaves were also observed.

Typical ringspot symptoms on the fruits on
both cultivars Morado and Cavite Special were
observed during the first recognition of the
disease in Polomolok, South Cotabato at a
neighboring backyard of the first site of infec-
tion. A total of 1.600 plants have been recorded
to be infected with the disease 300 m away from
this initial site. Disease mapping in Polomolok
and in the municipality of Tupi showed two
backyards with infected plants. Another occur-
rence was recorded in Davao last May. 2000
while the latest was recorded in Tampakan. a
municipality in South Cotabato, last February,
2001 when a number of papaya growers submit-
ted some fruits showing ringspot symptoms.
Plants with ringspots on the fruits and
shoestringing of leaves have been observed
during the last inspection of papaya farms in
Tupi in June, 2001. Figure 2 shows the map of
Southern Mindanao where the disease has been

Volume 37 (1) January-June 2001 53

L.E Herradura et al


Extracts of papaya leaf samples with mottle
symptoms from South Cotabato reacted
positively with PRSV-LB antisera by indirect
ELISA but not with the Japan antiserum. The
same positive results were obtained with the
samples exhibiting oily streaks on the petioles
submitted by the Plant Quarantine Service-Los
Bafios. BPI. Samples from Tupi and Davao del
Sur were also positive for the virus. These
indicate that the disease has spread in South
Cotabato and in Davao del Sur.

Whether the strain present in South
Cotabato is a mild one needs further study
especially since growers reported that
production does not seem to be affected even
when plants are PRSV-infected. However, this
observation was made on plants which were
infected during the later stage of development.

The PRSV antisera from Japan did not react
to extracts of samples collected from South
Cotabato but reacted to extracts of infected
samples collected from Los Baios (data not
shown). This indicates that the antisera from
Japan were recognizing epitopes common to
and exhibited by the Los Bafios isolate of
PRSV. Its negative reaction with the extracts
from South Cotabato indicates the possibility of
the occurrence of a different strain of PRSV in
South Cotabato since the symptoms on the
leaves were not quite similar to the PRSV
symptoms of infected papaya in Luzon.
Absorbances in ELISA were generally much
higher with the Los Bafios samples than with
those from South Cotabato.

The presence of PRSV in Mindanao could
have been the result of either of the following.
(1) A cucurbit-infecting strain of PRSV
mutating to infect papaya as speculated to be a
reason of the occurrence of the disease in
papaya in Australia by Bateson et al., (1994).
Brunt et al.. (1990) reported that PRSV has two
strains, the papaya or P strain that infects
papaya and cucurbits, and the watermelon or W
strain that attacks only cucurbits. There is no
report of the presence of the W strain in the
country. however. (2) The result of migration or
movement of the virus from Luzon and Visavas.
The disease has been noted to spread into new
areas of papaya production. Reports of workers

in other countries have shown that from a
defined location, the disease spreads to
surrounding areas in same island and to other
islands and even across international
boundaries. From the first report in Hawaii
(Gonzalves, 1998; Gonzalves and Ishii, 1980),
papayas in the other Hawaiian islands and
tropical countries along the Pacific rim like
Taiwan. Thailand. Malaysia, and Indonesia in
the West and in Mexico. Central and South
American countries like Venezuela (Dela Rosa
and Lastra. 1983) and the State of Florida
(Conover, 1964) have been reported to be
attacked by the disease (Gonzalves. 1998:
Purciful et al., 1984). In the Philippines, from
the first report in Silang, Cavite, Luzon, the
disease has moved to other provinces in Luzon
island, other Luzon islands like m Mindoro and
Palawan. and to the islands in the Visayas, like
Panay and Leyte, among others (Eusebio et al,
1997). (3) The other reason could be due to
disease introduction through contaminated
seeds. Bayot et al. (1990) have demonstrated
seed transmission of PRSV.

The results confirm for the first time the
presence of PRSV in Mindanao Island,
Philippines particularly in South Cotabato and
Davao del Sur. It was reported previously that
the disease is in Mindanao when seeds of
papaya reacted positive to antibodies of PRSV
(Espino et al., 1992). The presence of the
disease puts a grave threat to the commercial
Solo papaya plantations in the region which
essentially caters to the export market. With the
apparent susceptibility of the Solo cultivar and
with the cropping practices employed in these
plantations, like monoculture using relatively
large areas of land, although separate but are
probably close to each other, the disease could
become widespread and could develop into
epidemic proportions in due time (Magdalita et
al..1988). On the other hand, disease spread was
nil or very minimal in papaya grown with other
crops (Bajet, 1996; Opina and Tomines, 1998).

Eusebio (1992), Ramos (1987) and Opina
(1986) and other workers in the country
observed that PRSV infected plants exhibit
severe symptoms of shoestringing, distinct oily
streaks on petioles and stems and distinct
ringspot on fruits. The symptoms of the PRSV
ELISA positive samples used in this present
study were mottling and streaking on petioles

54 Volume 37 (1) January-June 2001 Journal of Plant Pathology

64 Volume 37 (1) January-June 2001

Journal of Plant Pathology

and stems. However, the ringspots on fruits do
not resemble those observed and reported in
Luzon and other areas, even on the same Solo
cultivar. The symptoms could be due to
differences in the environmental conditions in
the region or to the virus strain or both. PRSV
strains are of different pathogenicity, hence
variations in symptom expression have been
reported. The occurrence of mild strains has
been used as a strategy to manage the disease
through cross protection (Yeh et al., 1986;
Tennant et al.. 1994). Likewise, the negative
results obtained on the papaya samples tested
with antiserum of PLDMV. is a proof and
provides support that PLDMV is still not
present in the country. Result of a 5-year
survey of virus diseases of fruit trees has shown
that PRSV is present in the Philippines but
PLDMV was not and so far has only been
reported to occur in Okinawa and in Taiwan.


BAJET NB. 1996. Management of papaya virus
diseases by intercropping. Terminal Report,
Basic Research Program, University of the
Philippines Los Banos, College, Laguna.

ringspot and other diseases of papaya in the
Philippines: proceedings of a symposium/
workshop. Philippine Phytopathological Society,
Department of Plant Pathology. University of
the Philippines Los Banos.

W, GIBBS A, DALE J. 1994.Papaya ringspot
potyvirus: isolate variability and origin of PRSV
type P (Australia). J. Gen. Virol. 75:3547-3553.

Seed transmissibility of papaya ringspot virus.
Philipp. J. Crop Sci. 15: 107-11.

Viruses of tropical plants. CABI. Wallingford,
Oxon, UK

CLARK MF, ADAMS AN. 1977. Characteristic of
the microplate method of enzyme-link
immunosorbent assay for the detection of plant
viruses. J. Gen. Virol. 34: 475-483.

CONOVER RA. 1964. Distortion ringspot, a severe

disease of papaya in Florida. Proc, Flo. Sta.
Hort. Soc. 77: 440-444.

DE LA ROSA M, LASTRA R. 1983. Purification
and partial characterization of papaya ringspot
vimrs. Phytopathol. Zeitshchr. 106: 329-336.
Indexing papayas for plant viruses pp. 54-58. In
Proc. First Nat. Symp. /Workshp. on Ringspot
and Other Diseases of Papaya in the Philippines,
Bureau of Plant Industry, San Andres, Manila,
February 7-8, 1991.

EUSEBIO AA. 1992. Serological detection of
papaya ringspot virus (PRSV) in the Philippines.
Unpublished BSA Thesis, University of the
Philippines Los Bafios, College, Laguna.

SD. 1994. Enzyme-linked immunosorbet assay
for the detecting of papaya ringspot virus in
Luzon, Philippines. Philipp Agric. 77:383-392.

NB BAJET. 1997. Purification and serological
characterization of papaya ringspot potyvirus.
Philipp. Phytopathol. 33:49-67.

GONSALVES D, ISHI I. 1980. Purification and
serology of papaya ringspot virus.
Phytopathology 70: 1028-32.

GONSALVES D. 1998. Control of papaya rimgspot
virus in papaya: A case study. Annu. Rev.
Phytopathol. 36:415-437.

2000. Detection of PRSV in Southern
Mindanao. Proc. Annu. Cony. Pest Man.
Council Philipp., Baguio City, May 3-6, 2000

VILLEGAS VN. 1988. Epidemiology of
papaya ringspot in the Philippines. Philipp.
Phytopathol. 25: 1-11.

OPINA OS. 1986. Studies on a new virus disease
of papaya in the Philippines. Food Fert.
Technol. Ctr. Bull. 33. Taiwan, R.O.C.

OPINA OS, TOMINES R. 1996. Management of
papaya ringspot. isolation and intercropping of
papaya. Philipp. Phytopathol. 32:51-56

GONSALVES D. 1984. Papaya ringspot virus.
CMI/AAB Desc. Plant Viruses No. 292, (No. 84
Revis., July 1984) 8 pp.

LE Herradura et al Volmane 37(1) January-June 2001 55

LE Herradura et al

Volume 37 (1) January-June 2001 55

RAMOS CS. 1987. Characterization of an
unidentified virus attacking papaya (Carica
papaya L.) in the Philippines. Unpublished
M.S. Thesis, University of the Philippines Los
Bafios, College, Laguna

Differential protection against papaya ringspot
virus isolates in coat protein gene transgenic
papaya and classically cross-protected papaya.
Phytopathology 84: 1359-1366.

TEODORO NG. 1960. Plant diseases. Agr.
Industry. Life 23:20.

VILLEGAS VN. 2002. Conventional and
biotechnology-assisted breeding in developing
ringspot virus resistant papaya. Professorial
lecture, University of the Philippines Los Bafios,
College of Agriculture, 04 March 2002

D. 1986. Control of ringspot virus by seedling
inoculation with mild strain. Food Fert. Technol.
Cntr. Book Series No. 33.

56 Volume 37 (1) January-June 2001 Journal of Plant Pathology

66 Volume 37 (1) January-June 2001

journal of Plant Pathology

Figure 1. Symptom of papaya infected with papaya ringspot potyvirus in South Cotabato. A. Mild mottle and
leaf distorion. B. Ringspots on PRSV infected fruits and C. Oily streaks on petioles.

L.E Herradura et al

Volume 37 (1) January-June 2001 57



:C"",llc~; o IMMl


M"M tO. MA~

Figure 2. Municipalities in South Cotabato and
detected in symptomatic papaya sam
symptoms in the specific municipality


vao del Sur where papaya ringspot potyvirus (PRSV) was
SNumber with asterisk (*) are the plants observed with



2Assistant Scientists and 3Former Plant Pathologist, Ent
.earch Institute, Los Bafios, Laguna, Philippines

Surveys of rice tungro disease were conduct
North Cotabato, Bohol, and the Bicol Region ir
incidence was assessed visually based on typical
indexed serologically by ELISA for the presence
rice tungro spherical (RTSV) viruses. The rel
variables (ecosystem, synchrony of planting,
incidence, double RTBV and RTSV, single RTB'

In a given site, both variety and synchi
incidence, double RTBV and RTSV infection, a
proportion of susceptible varieties in Isabela
incidence and double RTBV and RTSV and sir
than in Nueva Ecija. This difference in visual
attributed to differences in the proportion of fie
these sites. Thus only 43% of the fields in Isabc
to 81% in Nueva Ecija. Disease incidence was g
due to the low proportion of fields planted with
asynchronous planting in most areas. The plant
a high disease incidence and virus infection ir
synchronously planted areas.

Considering the various socioeconomic
planting, the use of resistant varieties remains ti
disease management. Serological indexing of kl
the relationship between variety and synchroi
showed that high tungro incidence is not only as
infection but also with high single RTSV infe
RTSV-infected plants, which did not exhibit
tungro incidence, could have been overlooked.


The rice tungro disease is still considered in the
lippines as an important virus disease with no
.ctical solution. The total area affected by this
ease during a given year u usually only a small
portion of the total area planted. However, an
break of tungro is significant because it results in


LLA2 and 0. AZZAM3

)logy and Plant Pathology Division, International Rice

in farmers' fields in Isabela, Nueva Ecija,
e Philippines from 1995 to 1997. Tungro
nptoms. Leaf samples were collected and
f the rice tungro bacilliform (RTBV) and
mnship between production environment
I variety) and disease variables (visual
ind single RTSV) was examined.

y of planting influenced visual tungro
single RTSV infection. There was a high
Nueva Ecija, but the levels of disease
RTSV infections were higher in Isabela
idence and virus infection levels can be
that are synchronously planted between
were synchronously planted as compared
rally low in the other sites, which can be
sceptible varieties despite the practice of
of susceptible varieties is associated with
synchronously planted areas, but not in

istraints associated with synchronous
e the most practical component of tungro
samples gave a better understanding of
of planting and virus infection. It also
iated with high double RTBV and RTSV
n. Without serology, the importance of
iptoms but contributed to high overall

:ause a complex of two viruses, the rice tungro
:illiform virus (RTBV) and rice tungro spherical
as (RTSV) (Hibino et al., 1978), causes the disease.
e rice green leafhopper (GLH) N. virescens is
isidered as the most efficient vector of the rice
gro viruses (Hibino and Cabunagan, 1986).

Although several control methods against the

cultivation of susceptible varieties (Loevinsohn, 1
Holt et al., 1996), asynchrony of plan
(Loevinsohn. 1984; Holt and Chancellor, 1997),
high vector populations (Lim et al., 1974) v
identified as potential factors influencing tur
epidemics. These factors have been studied separa
but their multiple, complex interactions have yet t(
investigated. In 1993, Savary et al. L
correspondence analysis to characterize tungro disc
epidemics in the Philippines based on history
survey data. Results indicated that high tur
incidence was associated with intermediate plan
dates, whereas absence of tungro was associated \
very early or very late planting dates. In addition
tungro endemic areas, increasingly higher disc
incidence was associated with increasing ve
populations and proportion of viruliferous vect
However, in another study carried out in a tunl
prone locality of Southern Luzon in the Philippi
Chancellor et al., (1995, 1996) showed that ci
planted late have higher tungro disease than tl
planted earlier. Furthermore. within variety grol
vector abundance and source proximity mad<
significant contribution in regression mo
predicting incidence. Unfortunately, these stu
did not address the complex relationship betw
several production environment variables (ecosyst
synchrony of planting, and variety) and sev
variables describing the structure of epidemics in
field (visual incidence, double RTBV and RT
single RTBV. and single RTSV infection) in a holi

In this study, survey data collected from five s
in the Philippines from 1995 to 1997 were use(
analyze the contributions of ecosystem, synchron,
planting, and variety to tungro incidence at each
Associations or independence between (1) site
production environment variables (ecosyst
synchrony of planting, and variety), (2) produce
environment variables and disease variables (vi:
scoring of incidence, single and double infection
RTBV and RTSV), and (3) visual scoring of disc
and virus infections were analyzed by Chi-Square 1
The overall relationships, among the variables
between visual scoring and virus infection v
examined using correspondence analysis.
implication of the results on tungro disc
management and tungro epidemiological studies

Survey Sites

The survey covered six provinces of
Philippines: Isabela, Nueva Ecija, North Cotab
Albay, Camarines Sur and Bohol (Fig. 1).
adjacent provinces of Albay and Camarines Sur v
considered as one site (referred to as Bicol). Tl
sites, which are major rice growing areas, are
considered as tungro "hot spots" (Baria, 1997).
each site, five towns were randomly selected an(
each town, five barangays were subsequently cho
S In each barangay a fanner's field was rando
S selected and this was considered one sampling unit
most cases, the same towns in a province v
covered from 1995 to 1997, but different farm
fields in each barangay were selected over
succeeding years.

;Data Collection

Surveys were conducted only during the
season, when tungro incidence was expected to
high. The following kinds of information v
collected from each farmer's field: date of visit, n,
I of barangay, variety planted, age of crop, ecosyst
synchrony of planting and visual scoring of tur
S incidence. Field surveys were conducted when
crop in more than 75% of the fields was at tille
stage corresponding to 30 to 60 days a
transplanting or seeding.

Fields were classified by ecosystem, i.e. irrig&
if the ecosystem had assured irrigation for at least
crop in a year, and rainfed if it had no assi
irrigation water and water availability depended so
on rainfall. The synchrony of planting in a field
determined by comparing the crop age with all
fields within a 0.75-km radius. A field was consider
S as synchronously planted if the age or develop
stage of the crop during the disease assessment
similar to that of more than 70% of the fields wi
the radius. Otherwise, the field was considered
asynchronously planted.

Tungro incidence was assessed visually
counting the number of infected hills in four rando
selected quadrants of 1m x Im. Assessments v
made at tillering stage when tungro symptoms, wl
is characterized by discoloration of leaves f

60 Volume: Journal of Plant Patho

60 Volume:

Journal of Plant Pathc

anrous shades of yellow to orange, as well as stunting
Ou. 1985). were most conspicuous. The percentage
)f infection by tungro viruses was based on 30 whole
eaf samples taken from each field. Leaves were
ampled by walkmg diagonally across the field and
making a leaf from a hill every 20 steps. A leaf was
sampled regardless of whether it exhibited tungro-like
symptoms or was healthy-looking. Leaf samples for
;ach field were bulked, placed in glycine bags,
abeled, and packed in plastic bags and sent
n-mediately bN air courier to the Virology Laboratory
)f the International Rice Research Institute (IRRI).
rhey were indexed for the presence of RTBV and
ITSV bv enzyme-linked inmunosorbent assay
ELISA) (Bajet et al., 1985).

Data Analysis

The survey data were analyzed mainly by
correspondencee analysis (Hill. 1974: Greenacre. 1984:
Savarv et al.. 1993: 1995). They were categorized.
md contingency tables were built prior to analyses.
)ata categorization, building of contingency tables.
Chi-Square. and correspondence analysis were done
.sing STAT-ITCF version 4.0 (Beaux et al.. 1992).

Data Categorization. The first step in
correspondencee analysis is the categorization and
transformation of quantitative variables into coded
qualitative data so that these can be analyzed
simultaneouslyy with qualitative variables. Classes for
each variable (Table 1) were established in such a
way that the number of fields in each class would be
nore or less balanced and the Chi-Square tests
betweenn variables would be valid (Siegel. 1956:
gibbons. 1976).

The quantitative variables, which were
collectively referred to as "disease variables" were
visual scoring of tungro incidence (VI) and percent
infection with double RTBV and RTSV (BS). single
RTBV (B). and single RTSV (S). The qualitative
variables were: site, ecosystem. synchrony of planting.
and variety. The last three variables were referred to
is "production environment variables". Sites consisted
of the provinces of Isabela (ISA). Nueva Ecija (NE).
Camarines Sur and Albav in the Bicol region (B1C).
Bohol (BOH), and North Cotabato (NC). The
provinces of Camarines Sur and Albay were
consideredd as one site because of (a) comparatively
smaller sample sizes (number of surveyed fields) in
:he provinces and (b) their proximity. Two broad
classes of ecosystems were considered: irrigated (IRE)
and rainfed (RFE). The two classes of planting

synchrony were syncnronous planting (3r) ana
asynchronous planting (AP). Varieties were
;ategorized as susceptible varieties (SV), varieties
lamed by IRRI or IR varieties (OIV), Philippine Seed
3oard Rice (PSBRc series varieties (OPV), and
traditional or locally named varieties (OTV). The
;usceptible varieties were IR 64. PSBRc 12. and
'SBRc 14. IR 64 is a highly vector- and tungro virus-
susceptible variety (Cabunagan et al.. 1996). while
'SBRc 12 and PSBRc 14 are susceptible to tungro
)ased on tests conducted in major rice-growing areas
n the Philippines (PhilRice, 1997). The field reaction
:o tungro of the other IRRI or PSBRc series varieties
:hat were planted during the survey is either resistant
or intermediate (PhilRice. 1997).

Contingency Tables. The next step in
correspondencee analysis is the building of
:ontingency tables (Siegel. 1956: Gibbons, 1976). A
:ontingency table shows the frequency distribution
ind allows the exploration of the relationship between
wo qualitative or coded quantitative variables. If each
field is categorized by R classes of one variable and C
classes of a second variable the data can then be
presented in a table with R rows and C columns. The
entry in any RC cell is the number of fields falling
nto that cell (Tables 2. 3, and 4). For example, the
value of 130 in the NE x IRE cell in the site x
ecosystem contingency table means that there are 130
irrigated fields (IRE) in Nueva Ecija (NE).

Chi-Square Test. A Chi-Square test can be
applied to test the null hypothesis of independence of
:he distribution frequencies of the two variables. Two
variables were considered independent if the ratio of
:he various classes of the first variable remains the
same over all the classes of the second variable. Chi-
Square tests were used to determine the independence
between (1) site and production environment variables
(ecosystem, synchrony of planting, and variety)
(Table 2), (2) production environment variables and
disease variables (visual incidence, double RTBV and
RTSV. single RTBV. and single RTSV infections)
(Table 3), and (3) visual scoring of disease incidence
and virus infections (Table 4).

Correspondence Analysis. The relationships
among variables would be difficult to interpret if
presented in tabular form. Correspondence analysis
generates graphs where axes are used to plot
categorized variables and therefore facilitates the
interpretation of their relationship (Benzecri, 1973:
Hill. 1974: Greenacre, 1984). The procedure for
correspondence analysis is similar to principal

C. Cabunagan et al Bludry-June ~UUI 01

.C. cabunagan et al

inuary-june Luu i U-1

component analysis and involves the computation of
eigenvalues and eigenvectors (Benzecri. 1973: Hill,
1974. Greenacre, 1984).

In both analyses, the sum of the eigenvalues is
called inertia. The term inertia m correspondence
analysis is defined as the total Chi-Square statistic
divided by the total number of observations
(Greenacre. 1984). The total inertia can be decom-
posed by looking at the contribution of eachb ixis For
example. if 71.3% of the total inertia can be explained
b\ axis 1. it means that 71.3% of the amount of
information can be explained by examining the classes
along axis 1. Since the total inertia is the summation
of inertia over classes, the part played by each class or
point in establishing an axis can be quantified. The
contribution of a class to an axis. as shown in Table 5,
measures the contribution of each class to the total
inertia accounted for by an axis. For example, among
the row variables or profiles. class ISA contributes
57.0% of the total inertia, while among the column
variables or profiles: IN3 contributes 20.5% of the
total inertia m axis 1. On the other hand, the
contribution of an axis to a class indicates how well an
axis describes a class. It is also the correlation
between the axis and the class (Greenacre, 1984). In
Table 5, the closer the value is to 1.0 is. the better the
axis describes a class. Values of contribution of axis
to classes near the origin are close to zero. For
example, among the column variables, axis 1 has a
contribution of 0.988 to the inertia of class ISA. while
among the row variables, the same axis has a
contribution of 0.953 to class BS3. This high value
indicates that these classes are practically on axis 1.
Coordinates for the axes are defined based on the
eigenvalues. The sign of the new coordinates indicates
the direction that the classes deviate from the origin.
The proximity of points representing classes indicates
correspondences or associations that can be checked
using chi-square tests. When a series of classes
representing successive levels of coded quantitative
variables that reflects a logical increase (e.g.. VII to
V13: BSI to BS3) is considered, a path linking the
successive classes can be drawn. The movement along
this path may be examined in relation to the positions
or paths of the other classes. Graphs can be generated
with correspondence analysis where coordinates of
classes are plotted along the axes.

The data used for correspondence analysis are
classes of coded variables. specifically. the matrix of
contingency tables shown in Tables 3 and 4.
Correspondence analysis was used to examine (1) the
relationship between production environment and

disease variables (Fig. 3), and (2) the relationship
between visual scoring of disease incidence and virus
infection (Fig. 4). The relationship between
production environment and disease variables was
analysed using correspondence analysis by consider-
ing site, ecosystem, synchrony of planting, and variety
as the rows or the descriptive variables, while the
disease variables were considered as columns or the
variables to be described (Table 3). To examine the
relationship between visual scoring of disease
incidence and virus infection, VI was considered as a
row variable, while BS, B, and S were considered as
column variables (Table 4).


Some Features of the Surveyed Sites

The sites significantly differed in terms of
ecosystem (x' = 28.73, P <0.005), synchrony of
planting (x = 105.83. P <0 005) and variety (x =
202.26. P <0.005) (Table 2). Most of the farmers'
fields in all sites were irrigated (Fig. 2a) and
asynchronously planted, except in Nueva Ecija, where
81% of the fields surveyed were synchronously
planted (Fig. 2b).

Susceptible varieties covered 66% and 41% of the
fields in Isabela and Nueva Ecija, respectively
(Fig.2c). IR 64 was the most popular variety in both
provinces covering 48% of the fields in Isabela and
39% in Nueva Ecija. PSBRc 12 and PSBRc 14, which
are also susceptible varieties, were widely planted in
the two provinces but they were planted in fewer
fields compared to IR 64. In North Cotabato. 52% of
the fields were planted with traditional or locally
named varieties while 29% were planted with other
IRRI varieties. In Bohol, the most popular varieties
were the PSBRc series and traditional or locally
named varieties, with 47% and 29% of the fields
planted with these varieties, respectively. In Bicol,
62% of the fields were planted with PSBRc series
varieties, indicating that these varieties are also
popular in this site.

Relationship Between Production Environment
Variables and Disease Variables

Except for a single RTSV (S) infection, the other
disease variables were statistically independent of the
ecosystem (Table 3), whereas visual incidence (VI),
double RTBV and RTSV (BS) and S were dependent
on the synchrony of planting and variety. This

62 Volume 37 (1) January-June 2001 Journal of Plant Pathology

62 Volume 37 (1) January-June 2001

Journal of Plant Pathology

indicates that asynchronous planting favors BS and S
infections Varieties differed in their susceptibility to
BS as well as to S infections. Susceptible varieties
had a higher proportion of BS and S infections than
the other categories of variety. The ratio of single
RTBV (B) infection is the same in all categories of
variety (x2 = 5.54, P = 0.477), which indicates that
the varieties did not vary with respect to their
susceptibility to B infection.

Correspondence Analysis of the Relationship
Among Site, Production Environment, and
Disease Variables

Axes 1. 2. 3, and 4 accounted for 71.3%. 12.6%,
6.1%, and 4.0%. respectively, of the information or
the total inertia of the data set. The first two axes,
which accounted for most of the information (83.9%)
represented by the data in Table 3, were used to plot
the classes (as shown in Table 1) using their
coordinates along these axes. The resulting graph
(Fig. 3), where axis 1 is horizontal and axis 2 is
vertical, shows the relationship among the classes.
Table 5 shows the relative contribution of axes 1 and
2 and the contribution of the classes to these axes.
This table was used as a basis for data interpretation.
Tungro disease incidence and its relationship with the
other variables considered in the survey can be
generally interpreted along axis 1 because: (1) the
relative contribution of axis 1 to classes VI. BS. and S
is higher than that of axis 2. and (2) axis 1 accounts
for 71.3% of the information or the total inertia of the
data set. This indicates that classes associated with
low visual incidence and low tungro virus infections
are mainly grouped on the left side of the graph.
whereas those associated with high visual incidence
and tungro viruses infection are located on the right
side of the graph (Fig. 3).

The classes with a high contribution to an axis are
those that have a major role in the information
provided by an axis. Table 5 shows that among the
sites, ISA has a high contribution to the total inertia of
axis 1 (57.0) whereas. NE and NC have high
contributions to the total inertia of axis 2 (17.5 and
43.7, respectively). It also shows that both the
ecosystem classes IRE and RFE have low
contributions to the total inertia (0.4 and 3.0,
respectively). These classes are close to the origin,
and the Chi-Square test shows that they are not
associated with the disease variables, except S-
infection (Table 3). Classes for the synchrony of
planting, AP and SP contributed 14.5 and 15.9.
respectively. to the axes and Chi-Square test also

shows that all the disease variables except B-infection
are dependent on the synchrony of planting (Table 3).
Among the variety classes, SV has the highest
contribution to the total inertia (16.7), followed by
OIV (7.0). Most classes of VI and BS have a high
contribution to the axes.

Nueva Ecija (NE) is closely associated with
synchronous planting (SP) and Isabela (ISA) with
susceptible varieties (SV) and asynchronous planting
(AP). Synchronous planting (SP) is associated with
low levels of visual disease incidence (VI1) and virus
infection (BS1, Sl, and B1), whereas asynchronous
planting (AP) Fig. 3 is more closely associated with
medium to high levels of visual disease incidence
(VI2 and VI3) and medium to high levels of virus
infection (S2, S3, B2, and BS3). On the other hand,
susceptible varieties are associated with high levels of
visual disease incidence (VI3) and high virus infection
(S3 and BS3).

Relationship Between Visual Scoring of Disease
Incidence and Virus Infection

Visual incidence (VI) is positively related to
double RTBV and RTSV (BS), single RTBV (B) and
single RTSV (S) infections (Table 4). Visual scoring
of disease incidence overestimated low levels of B
infection, where high incidence (VI3) was observed in
several fields despite a low level of B infection (B1).

Correspondence analysis of the relationship
between visual incidence and BS, B, and S infections
is shown in Fig. 4. Axes 1 and 2 accounted for 91.1%
and 8.9%, respectively, of the total inertia. The
movement of increasing visual incidence and
increasing virus infection is mainly along axis 1. The
paths of increasing VI and BS are similar in shape,
and differ from the paths of increasing B and S
infections. The paths of VI and BS have the same
direction of movement along axis 1 and start in the
same area. indicating strong correspondences.
However, the paths diverge in the later part, so that
the path of BS is longer than that of VI The path of S
started and ended near that of VI1. This indicates that
at low levels of disease incidence, visual scoring is
closely associated with low levels of BS and S
infection, but the degree of this association decreases
as the disease intensifies. The path of increasing B is
different from the paths of the increasing levels of the
other disease variables.

The analytical approaches used in this study
showed that synchrony of planting and variety is

R.C. Cabunagan et al Volume 37 (1) January.June 2001 63

R.C. Cabunagan et al

Volume 37 (1) January-June 2001 63

important factors in the rice tungro pathosystem. It
confirmed earlier studies (Loevinsohn 1984: Holt el
1. 1996. Holt and Chancellor. 1997) which showed
that susceptible varieties and asynchronous planting
favor the occurrence of rice tungro epidemics. The
added value of this study is that it was able to show
the interaction between variety and synchrony of
planting and compare their respective contribution to
visual scoring of disease incidence and virus infection.
The high tungro incidence in Isabela could not be
solely associated with the planting of susceptible
varieties because Nueva Ecija which had the lowest
disease incidence also had a high proportion of
susceptible varieties (IR 64. PSBRc 12 and PSBRc
14). The difference in disease incidence level between
these two sites can be associated with planting
synchrony, i.e. 81% of the fields in Nueva Ecija and
only 43% of the fields in Isabela were planted
synchronously. Disease incidence was generally low
in the other sites, which can be associated with the
very low proportion of fields planted to susceptible
varieties, although asynchronous planting was also
practiced. These results suggest that, in addition to
other possible factors, the interaction between variety
and synchrony of planting is a critical factor in rice
tungro disease incidence. The planting of susceptible
varieties can result in high tungro incidence and
double RTBV and RTSV and single RTSV infections
in asynchronously planted but not in synchronously
planted areas.

Synchrony and timing of planting are known to
affect tungro disease incidence, specifically in relation
to the population of insect vectors and availability of
inoculum source. Asynchronous planting was found to
favor vector population development and maintenance
of virus sources throughout the year and consequently
favored the spread of tungro (Loevinsohn, 1984;
Loevinsohn and Alviola, 1991). Tungro epidemics
occurred in asynchronously planted areas in
Indonesia. Bangladesh. and India (Koganezawa.
1998). The outbreak of tungro in Malaysia in 1982
was partly attributed to asynchronous planting (Chen
and Jatil. 1993). On the other hand, synchronous
planting was found to be effective in eradicating virus
sources and in minimizing the population density of
vectors in South Sulawesi. Indonesia (Cabunagan et
al., 1989). It was considered to be as effective as a
fallow period in reducing tungro disease incidence in
Indonesia (Sama et al.. 1991) and Malaysia (Chen and
Jatil, 1993). Despite the success of synchronous
planting in controlling tungro disease in some areas.
its large-scale implementation has socio-economic
constraints. Synchronous planting may increase

tractor and labor hire rates due to concentrated
demand, and requires extensive irrigation network.
close coordination among government agencies. and,
most importantly, cooperation among farmers
(Loevinsohn, 1984). Asynchronous planting remains a
common feature of many rice-growing areas of the
Philippines because of inadequate irrigation water.
limited tractors during land preparation, and
uncertainty of labor during transplanting (Loevinsohn.

This study underscores the contention that the use
of resistant varieties remains to be the most practical
component of tungro disease management strategy,
particularly in most rice-growing areas where
synchronous planting is difficult to implement.
Varieties that have been planted so far are resistant to
the vector but susceptible to the tungro viruses
(Hibino et al., 1987: Koganezawa and Cabunagan.
1997). Vector-resistant varieties escape tungro
infection in the field under light to moderate tungro
and vector pressure but succumb to infection when
there are strong sources of inoculum and vectors are
abundant (Cabunagan, et al., 1987). As shown in this
study, such varieties cannot be effective in asynchro-
nously planted areas where inoculum sources and
vectors are abundant throughout the year. Moreover,
changes in field response to tungro by vector-resistant
varieties have been reported in the Philippines (Dahal
et al., 1990), Thailand (Inoue and Ruy-Aree, 1977),
and in Indonesia (Manwan et al., 1985) after a few
years of intensive cultivation because of possible
shifts in GLH virulence. New sources of resistance to
tungro viruses have been identified (Koganezawa and
Cabunagan, 1997) and used in the breeding program
at IRRI. Advanced breeding lines with resistance to
rice tungro viruses have been identified (Angeles el
al., 1998) and tested in multi-location field trials in the
Philippines, Indonesia and India (Cabunagan et al.,
1998). Concerted efforts must be directed towards the
development and deployment of varieties, which are
resistant to tungro viruseS.

Serological tests by ELISA gave a better
understanding of the relationship between variety and
synchrony of planting and virus infection. There was a
close association between visual scoring of disease
incidence not only with double RTBV and RTSV
infection but also with single RTSV infection. This
indicates that single RTSV infection contributes to
tungro incidence aside from double RTBV and RTSV
infection. Although the results support the assumption
that the visual scoring of disease incidence can
estimate incidence in the field, they showed the added

64 Volume 37 (1) January-June 2001 Journal of Plant Pathology

64 Volume 37 (1) January-June 2001

Journal of Plant Pathology

value of serological indexing in epidemiological
studies by giving a more precise estimate of the
incidence. RTSV infected plants which did not exhibit
symptoms but contributed about 35% to the overall
tungro incidence would have been overlooked. RTSV
may be seen as an independent, damagmg virus
disease of its own as previously reported in the
Philippines (Bajet et al. 1986) and in Indonesia
(Sama et al., 1991). In highly-susceptible varieties, it
is capable of causing a 40% vield loss (Hasanuddin
and Hibino. 1989). In this study, RTSV had a more
important role in tungro epidemics than RTBV. The
occurrence of RTSV was associated with synchrony
of plantmg and variety and levels of RTSV infection
higher than RTBV infection were observed in the
surveyed sites. The importance of RTSV in tungro
epidemics was also pointed out by Satapathy et al.,
(1997), who reported the role of RTSV resistance in
reducing tungro disease spread.

The analytical approaches based to analyze the
survey data set in this study confirm the importance of
synchrony of planting and the use of resistant varieties
for effective and sustainable rice tungro disease
management. In addition, through the use of
serological indexing by ELISA, the study shows the
respective contributions of tungro viruses to disease
incidence and the added value of such tests in tungro
disease epidemiological studies.


The authors are grateful to X.H. Truong, J. Fernandez,
M.J. Du, and B. Zaragoza for the conduct of the survey in
their respective areas and for E. L. Coloquio and P.
Domingo for their assistance in the ELISA test of leaf
samples at IRRI.


TCB. 1998. Advanced breeding lines with resistance to
rice tungro viruses. Int. Rice Res. Notes 23:17-18.

Enzyme-linked immunosorbent assay to diagnose rice
tungro. J. Plant Prot. Trop. 2:125-129.

HIBINO H. 1986. Occurrence and spread of rice
tungro spherical virus in the Philippines. Plant Dis. 70:

BARIA AR. 1997. Status of rice tungro disease in the
Philippines: Guide to current and future research. pp
76-83. In: Chancellor, T.C.B., Thresh J.M. (Eds.),
Epidemiology and Management of Rice Tungro
Disease. Nat. Res. Inst. Chatham, United Kingdom.

VERNEAU M. 1992. STAT-ITCF User's Manual.
Institute Technique des C6r6ales et des Fourrages, Paris,

BENZECRI JP. 1973. L'Analyse des Donn6es, tome 2:
L'Analyse des Correspondences. Dunod, Paris.

1987. Resistance of rice plants to Nephotettix virescens
in relation to rice tungro-associated viruses. pp. 66-76.
In: Proc. Workshop on Rice Tungro Virus, Maros
Research Institute for Food Crops, Maros, Indonesia,
pp. 66-76.

1989. Rice tungro (RTV) and its vector leafhopper
development in synchronized-planting areas. Int. Rice
Res. Newsl. 14: 27.

SUBRAMANIAN N. 1998. Multi-location evaluation
of promising advanced breeding lines for resistance to
rice tungro viruses. Int. Rice Res. Notes 23:15-16.

1996. Evaluation of rice germplasm for resistance to
tungro disease. pp. 66-76.In: Chancellor, T.C., Teng,
P.S., Heong, K.L. (eds.), Rice Tungro Disease
Epidemiology and Vector Ecology. Int. Rice Res. Inst,
P. O. Box 933, 1099 Manila, Philippines,

Spread of rice tungro virus disease between fields. pp
27-28. In: Raccah, B. (Ed.), Sixth International Plant
Virus Epidemiology Symposium, Jerusalem, 23-29
April 1995, Phytopathological Society of Israel,
Agricultural Research Organization, Bet Dagan.

MAMBANUA MGM. 1996. Risk factors for rice
tungro disease in endemic areas. pp 58-65. In:
Chancellor, T.C.B, Teng, P.S. and Heong, K.L. (eds.).

RC. Cabunagan et al Volume 37 (1) January-June 2001 65

Volume 37 (1) January-June 2001 65

P-C. Cabunagan et al


02 BS3
,2(91.1%) ^ '^
v11 \ B1 .., .0,. ^VI3 ..-^
00 ail ~\ ^ ^-^^'
v ^ '"* '><^^ -*"
^^ -.....^>- ^
^ BS2 ^
-0.4 -I ------\---------------------l-------i-------|-------l
-0.4 -0.2 0. 0.2 0.4 0.6 0.8 1.0
0 Axis 1 (8.9%)
Figure 4. Correspondence analysis for tnngro incidence based on visual scoring and tungro infection
based on serological assay. Values in parentheses refer to the amount of information
accounted for by an axis. VI = proportion of hills with tungro disease based on visual
scoring; BS. B, and S = proportion of leaf samples infected with RTBV and RTSV. RTBV
alone, and RTSV alone, respectively.

Volume 37 (H January-June 2001 71

TIONTCO ER, THRESH JM. 1997. Effect of
introduced sources of inoculum on tungro disease
spread in different varieties. pp 11-21. In: Chancellor.
T. C.B. and Thresh, J.M (eds.), Epidemiology and
Management of Rice Tungro Disease, Nat. Res. Inst.,
Chatham, United Kingdom.

characterization of rice tungro epidemics in the
Philippines from historical survey data. Plant Dis.

GEBBINCK HW. 1995. Use of categorical

information and correspondence analysis in plant
disease epidemiology. pp 11-21. In: Advances in
Botanical Research Vol. 21. Incorporating Advances in
Plant Pathol. Acad. Press, Ltd., London.

SIEGEL S. 1956. Nonparametric Statistics for the
Behavioral Sciences. McGraw-Hill Book Company,
Inc., Tokyo.

F. 1990. Population dynamics of the green leafhopper.
Nephotettix virescens Distant (Hemiptera:
Cicadellidae) in synchronised and staggered
transplanting areas of paddy fields in Indonesia. Res.

R.C. Cabunagan et al Volume 37 (1) January-June 2001 67

Volume 37 (1) January-June 2001 67

R.C. Cabunagan et al

O Isabela

N /ueva Ecija

-Camarines Sur

Uo vvu-lIII- vtI \.I Ulluuai llm u fV I UInllI Ul rladll raillUIULIV











20 -



Isabela Nueva Ecija North
Isabela Nueva Ecija North


* Local and unnamed varieties

o Other PSBRc varieties

* Other IRRI varieties
* Susceptible varieties



Figure 2. Proportion of fields classified according to ecosystem (a), planting synchrony (b), and varieties (c) in the surveyed
sites. Acronyms of varieties are explained in text.

R.C. Cabunagan et al Volume 37 (1) January-June 2001 69

77'- El Synchronous

1 2 a Asynchronous

100 -

80 -


40 -



R.C. Cabunagan et al

Volume 37 (1) January-June 2001 69

0.2 Bicol

Ecija VI
yn. BS3
-yV. f Isabela
planting B Susceptibl sabela
iainfed B1. e
BS1 OtherPSRc varieties
0.0 I| va-etle S3 .-'
\ Bo OTradJ local Ibigated S2 '
\ varieties \
axis 2 (12. %) .-
Ilther Y A V13
IRRI vieties g..,,-lSfatVing

V12 2'"
-0.2 BS2

North /

-0.4 .


-0.6 -I I--
-0.4 0.0 0.2 0.4 0.6 0.8 1.0
0.2 axis 1 (71.3%)

Figure 3. Correspondence analysis for rice tungro disease epidemics at five sites in the
Philippines. Values in parentheses refer to the amount of information accounted for
by an axis. VI = proportion of hills with tungro disease based on visual scoring; BS,
B, and S = proportion of leaf samples infected with RTBV and RTSV, RTBV alone,
and RTSV alone, respectively.

70 Volume 37 (1) January-June 2001 Journal of Plant Pathology

70 Volume 37 (1) January-June 2001

Journal of Plant Pathology


0.2 BS3

S2 (91.1%)
V10.0 B1 ... SVI3 -
0.0 -

. ,B3. .'" /S 3

-0.-.- ...- B2 -'

SBS2 /

-0.4 -1 I I

-0.4 -0.2 0. 0.2 0.4 0.6 0.8 1.0
Axis 1 (8.9%)
Figure 4. Correspondence analysis for tungro incidence based on visual scoring and tungro infection
based on serological assay. Values in parentheses refer to the amount of information
accounted for by an axis. VI = proportion of hills with tungro disease based on visual
scoring; BS. B, and S = proportion of leaf samples infected with RTBV and RTSV. RTBV
alone, and RTSV alone, respectively.

R.C. Cabunagan et al Volume 37 (11 January-June 2001 71

Volume 37 (1) January-June 2001 71

R.C. Cabunagan et al

No. of
Description Code fields Range or class description

Quantitative Variables

Proportion of lulls m a sampling area VII 202 0% < IN < 1%
with tungro disease based on visual VI2 117 1% < IN < 5%
scoring VI3 123 5% < IN < 100%

Proportion of leaf samples with nece BS I 271 0% < BS < 1%
tungro bacillifonn virus (RTBV) and BS2 80 1% < BS < 20%
rice tungro spherical virus (RTSV) BS3 91 20% < BS < 100%

Proportion of leaf samples with B1 326 0% < B < 1%
RTBV alone B2 97 1% < B < 20%
B3 19 20% < B < 100%

Proportion of leaf samples with Sl 190 0% < B < 1%
RTSV alone S2 155 1% < B < 20%
S3 97 20% < B < 100%

Qualitative Variables

Site ISA 67 Isabela province
NE 135 Nueva Ecija province
BIC 34 Bicol region (Albay ond Camarines Sur)
BOH 70 Bohol province
NC 136 North Cotabato province

Ecosystem IRE 3X3 Generally irrigated
RFE 59 Generally rainfed

Planting Synchrony AP 232 Asynchronous or staggered planting
SP 210 Synchronous or simultaneous planting

Variety SV 112 Susceptible varieties (IR64, PSBRc 12 and
PSBRc 14)
OlV 99 Other IRRI varieties'
OPV 116 Other PSBRc series varieties2
OTV 115 Traditional, locally named, and unnamed varieties

'IRRI varieties are those that were developed hby the International Rice Research Institute until 1990.
- Since 1990. varieties developed in the Philippines were coded as I'SBRc regardless of the institution or breeding center that developed them. PSB stands tor
'lPhilippines Seed Hoard and Re stands otr Rice

Table 1. Description of variables and classes.

72 Volume 37 (1) January-June 2001

Journal of Plant Pathology

Table 2 Comparison of the different surveyed sites in the Philippines with respect to production environment factors'

Ecosystem Synchronm of Planting Variety
(p-value) (p-value)

ISA 55 12 28.73 38 29 105.83 44 6 13 4
NE 130 5 ( 001) 25 110 (0.001) 55 35 31 14
BIC 34 0 33 1 2 5 21 6
BOH 52 18 49 21 4 13 33 20
NC 112 24 87 49 7 40 18 71

ISA Isabela province. NE Nueva Ecil, province. BIC Hicol region 13011 Bohol province. NC North Cotahato province. IRE general\ irrigated.
RFI- generally rainted. AP asynclronous planting. SP synchronous planting. SV susceptible varieties. OV \ other IRRI varieties. OPV other
PS3Rc series varieties. and ()T\ traditional. locally named, and unnamed varieties.

Table 3. Relationship between site. production environment variables, and disease variables'.


Disease variables

VIl VI2 V13

7 BSI BS2 I3S3 2

BI 32 B3 x

S1 S2 S3 -2

15 7 45 91 60 2 9 56 21351 48
90 29 16 (()001) 10( 19 8 (A.)00) 111
18 6 10 24 2 8
25 24 21 51 15 4
S4 51 31 X 86 15

Production environment

168 101 114 6.00 229 70 84 3 65 281
34 16 9 (0.050) 42 10 7 (0.161) 45
73 62 97 55.97 128 43 61 10.44 165
129 55 26 (0.001) 143 37 30 (0.005) 161
42 19 51 2769 48 16 48 62.23 85
52 25 22 (0.001) 63 31 5 (0.001) 71
53 41 22 78 19 19
55 32 28 82 14 19

19 0 46.31 3 38 26 54.65
20 4 (0.001) 66 37 32 (0.001)
30 3 1 21 9 4
61 9 0 38 22 10
76 46 14 62 49 25

83 19 3.08 166 122 95 19.22
14 0 (0.214) 24 33 2 (0().0(1)
53 14 4.06 87 85 60 7.18
44 5 (0.131) 103 70 37 (0.028)
25 2 5.54 30 43 39 27.53
22 6 (0.477) 40 43 16 (0.o01)
91 20 5 63 32 21
79 30 6 57 37 21

'ISA- Isabela province, NI' Nueva F[cija province. BIC Bicol region. BOll 13ohol province. NC North Cotahato province. IRE generally irrigated.
RIE eenerallv rained. AP asvnchronous planting. SP synchrtonous planting SV susceptible varieties. ()IV other IRRI varieties. OPV other
PSBRc series varieties, and ()TV traditional, locally named, and unnamed varieties VI refers to the proportion of hills with tungro disease based on visual
scoring, whereas BS. B. and S refer to the proportion of leaf samples infected with RTBV and RTSV. RTBV alone, and RITS\ alone, respectively.

Volume 37 (1) January-June 2001 73

R.C. Cabunagan et al






le 4. Relationshlup between tingro incidence assessed based on visual scoring and tungro vis infection assessed based
on serological indexing of leaf samples .

Virus infection levels2


11 163 26 13 123.35 6(
12 72 31 14 (0.001) 78 3
13 36 23 64 84 3

11 refers to the proportion ol-hills with tungro disease based on visual scoring, vwl
'BL' and RT.SV. RTBV alone, and RTSV alone, respectively.

value) (p-value)

22 47.13
6 (0.011) 50 38 29 (0.001)
4 27 50 46

is BS, B. and S refer to the proportion ol leaf samples infected with

Table 5 Results of correspondence analysis showing the relative
contribution of classes to axes'.

contribution of axes to classes and the reciprocal

Relative contribution of axes2 Reciprocal contribution to axes (%)3
Axis 1 Axis 2 Total Axis 1 Axis 2 Total

Rows (Descriptive variables)
ISA 0.978 0.008 0.986 57.0 2.5 59.5
NE 0.665 0.241 0.906 8.5 17.5 26.0
NC 0.150 0.761 0.911 1.5 43.7 45.2
BOH 0.331 0 0.331 1.8 0 1.8
BIC 0.051 0.439 0.490 0.2 7.5 7.7
IRE 0.250 0.042 0.292 0.2 0.2 0.4
RFE 0.249 0.043 0.292 1.5 1.5 3.0
AP 0.661 0.196 0.857 5.4 9.1 14.5
SP 0.661 0.194 0.855 6.0 9.9 15.9
SV 0.935 0.042 0.977 13.3 3.4 16.7
OIV 0.367 0.136 0.503 2.3 4.7 7.0
OPV 0.420 0.002 0.422 1.7 0 1.7
OTV 0.338 0 0.338 0.7 0 0.7

Columns (Variables to be described)
VII 0.544 0.273 0.817 6.0 17.1 23.1
VI2 0.339 0.365 0.704 2.0 12.1 14.1
V13 0.878 0.028 0.906 20.5 3.7 24.2

BSI 0.947 0.006 0.953 12.4 0.4 12.8
BS2 0.054 0.518 0.572 0.3 14.5 14.8
BS3 0.953 0.023 0.976 43.1 5.9 49.0
B1 0.027 0.683 0.710 0.1 9.6 9.7
B2 0.100 0.484 0.584 0.4 12.3 12.7
B3 0.036 0.702 0.738 0.2 23.6 23.8

S1 0.828 0.001 0.829 8.1 0.1 8.2
S2 0.318 0.013 0.331 1.9 0.4 2.3
S3 0.621 0.004 0.625 5.1 0.2 5.3

'ISA = Isabela province, NE Nueva Ecija province, BIC = Bicol region, BOH = Bohol province, NC = North Cotabato province, IRE = generally irrigated,
RFE = generally rainted, AP = asynchronous planting, SP synchronous planting, SV = susceptible varieties, OIV = other IRRI varieties, OPV = other
PSBRc series varieties, and OTV = traditional, locally named, and unnamed varieties. VI refers to the proportion of hills with tungro disease based on visual
scoring, whereas BS. B. and S refer to the proportion of leaf samples infected with RTBV and RTSV, RTBV alone, and RTSV alone, respectively.
'The contribution of an axis to a class indicates how well an axis describes a class. The closer the value is to 1.0, the better the axis describes a class.
'The contribution of a class to an axis measures the contribution of each class to the total inertia accounted for by an axis.

R.C. Cabunagan et 81 Volume 37 (1) January-June 2001 75

R.C. Cabunagan et al

Volume 37 (1) January-June 2001 75



POTYVIRUSES. DEV Villamor, LE Herradura
and NB Bajet, Bureau of Plant Industry, Davao City
and UPLB, College, Laguna

Banana bract mosaic (BBrMV) and abaca mosa
(AbaMV) potyviruses were purified following
modification of the procedure of Thomas et
(Phytopathology 87:698-705). SDS-PAGE analyv
of the dissociated virons revealed two (BBrMV) ai
three (AbaMV) major protein bands with estimate,
sizes of 31 and 39 kDa and 31. 34 and 39 kD
respectively. Those bands are likely to be co
proteins of the two viruses as they reacte
respectively, to BBrMV and to maize dwarf mosa
virus strain B antisera in western blot. The reaction
MDMV-B As to the AbaMV dissociated proteins
western blot further confirms that AbaMV
serologically related to MDMV-B. When purifii
viruses were used as antigens to produce the
respective antiserum. the antiserum react
specifically to banana and abaca plants infected wi
BBrMV and AbaMV in indirect ELISA. The
antisera detected the same bands corresponding to tl
dissociated virions of BBrMV a.d AbaMV in weste
blot indicating a comparable reaction with the
corresponding foreign antiserum.

Bajet, UPLB. College. Laguna

An ELISA survey of corn (Zea mays L.) was done 1
assess the occurrence of virus or viruses attackir
corn plants at the UPLB Central Experiment Static
from December 2000 to February 2001. Random.
selected symptomatic samples were collected ar
tested using three antisera: maize stripe tenuivin
(MStpV), maize mosaic rhabdovirus (MMV) ar
maize dwarf mosaic potvvirus (MDMV). Resul
show that MStpV was the most commonly detected

DN 5, PILI, CAMARINES SUR, May 2-5, 2001


to rice stripe virus (RStpV). The results showed th;
MStpV is related to RStpV and RGSV. SDS-PAG
analysis of the proteins extracted from the MStp\
positive samples showed protein of approximately 1
kDa This protein species is most likely the noncaps:
protein of MVtpV.

JP Pedroso, AD Raymundo, AC Sumalde and NB
Bajet, Western Mindanao State University,
Zamboanga City and UPLB, College, Laguna

The study on vector dynamics revealed that green
leafhopper (GLH) populations varied among the pui
and the mixed stands of rice with different types (
resistance during the dry and the wet seasons of 199'
The population build-up was rapid in the susceptib
cultivar, IR-64. Pure stands of resistant cultivars ha
high GLH as in the susceptible cultivar.

The mixture of two cultivars (IR-64 and PSBRC 18
three (IR-64, PSBRC 18 and IR-72) and four cultivai
(IR-64,PSBRC 18. ir-72 and PSBRC 34) planted i
close (15 X 20 CM) and wider spacing (25 x 25 cr
had high GLH populations. Incidence of the disease
was also high in these mixtures especially in areas c
high inoculum source. It appears that divers
genotypes constituting these mixtures had no effect i
suppressing development of the virus and reducing
GLH populations.

GLH population was significantly correlated wit
disease incidence in IR-64, IR-72, PSBRC 18. mixture
of two cultivars (IR-64 and PSBRC 18) and fou
cultivars (IR-64, PSBRC 18, IR-72 and PSBRC 34
planted within rows in some sites and seasons
However, vector population in other cultivar mixture
had no correlation with incidence.

Nephotettix virescens rapidly increased at 15 DAT an

prevent early increase ot UiLH population tor
subsequent disease infection, spraying of insecticides
earlier than 10 DAT is recommended.

environmentall factors such as temperature, humidity
nd rain fall had no significant influence on GLH

EGGPLANT. NL Opina, RT Alberto, SE
lantiago, RL Tiongco, S Miller and RG
vaghirang, IPB, UPLB, College, Laguna; PhilRice,
luiioz, Nueva Ecija; and Ohio State University, USA

The influence of eggplant cultivar and cultural
practices on the management of bacterial wilt on
ggplant was evaluated in San Jose, Nueva Ecija.
Eggplant varieties significantly affected the incidence
if bacterial wilt in mulched, unmulched, cultivated
nd uncultivated plots. Grafted eggplant had the
west bacterial wilt incidence and was comparable to
he highly resistant check, Eg 203. No significant
differences on bacterial wilt infection were observed
in eggplant grown under different cultural practices.

)f the 59 Ralstonia solanacearum isolates evaluated
or aggressiveness using Eg 203, Abar and Casino
ultivar, 5 isolates had 33% bacterial wilt infection on
he resistant cultivar Eg 203. SAS cluster analysis
;rouped the isolates into 8 clusters. Most of the
*irulent isolates belonged to Clusters 1 to 5 while the
ess virulent isolates were in Clusters 6 to 8. No
:orrelation was observed between aggressiveness,
iovar and origin.

n screening for resistance. Eg 2043 had the lowest
averagee bacterial wilt infection both under greenhouse
nd field conditions.

:uambot, USMARC-USM, Cotabato

promisingg inbred lines and hybrids from CIMMYT
nd USM were tested to identify, select and develop

various entries for resistance to Philippine corn downy
nildew. Out of 1,913 entries/lines tested, 1,741
ntries/lines were from CIMMYT and the other 172
ame from USM. It was noted that 1,402 entries/lines
ut of the 1,913 entries/lines or 73.29% showed a
degree of resistance to infection based on their
action to the disease. Plants showing intermediate
action and susceptible response were also noted in
hle field. Three hundred twenty three (323) entries or
6.88% had intermediate infection while 188 or
1.83% exhibited susceptible reaction.

)ISEASES. AD Raymundo and NM Basio, UPLB,
college Laguna

Wn experiment on the spread of bunchy-top in an
isolated one-hectare abaca field was undertaken in
Iabaco, Albay, Likewise, to better understand the
dynamics of bunchy-top and mosaic virus diseases,
computer simulation was done using STELLA
software and GIS modeling.

The spread of bunchy-top is quite slow. In the one-
lectare field of abaca with a hill of 5 diseased abaca
eedlings serving as initial focus or source of
noculum, it took almost one year before adjoining
tills became infected. At the start, spread was
extremely slow. As the number of infected plants,
serving as additional sources of inoculum, increases,
pread and intensification became faster.

, STELLA model of bunchy-top, which included the
dynamics of disease linked to population dynamics of
he insect vector, shows that the disease can spread
aster under optimum conditions. This model can be
utilized in predicting bunchy-top under varying
conditions .

, GIS predictive model of both the bunchy-top and
mosaic provided information on disease increase and
pread based on previous data.

The rates of increase and spread of the diseases
derived from the spatio-temporal analysis and from
he computer simulation can be used in devising
trategies for eradication as regards spread of
operation, location of eradication sites, and
deployment of personnel.

S1[dCt DI raij~r~ nuary-June 2001 77

inuary-June 2001 77

stract oj rapemb

WILT. NL Opina, GA Romo, RL Tiongco and
Miranda, IPB, UPLB, College, Laguna

Bacterial wilt caused, by Ralsonia solanacearu
one of the emerging important diseases of crucifix
the Philippines.

A total of nine representative biovar is(
of R. solanacearum from white potato (WP 20-b
2), tomato (T116-biovar 3 and T6-biovar 4), pi
(P2-biovar 3 and P20-biovar 4), eggplant I
biovar 3 and E5-biovar 4), ampalaya (Am 2-biox
and Chinese cabbage (Ccl-biovar 3) were 1
against Chinese cabbage and pechay comm
varieties. Results showed that the two varieti
Chinese cabbage namely Corazon and Esper
showed resistant reaction to T116, P2 and \
while the Black Behi cultivar of pechay
moderately resistant to WP 20 and P2 isolates.
Chinese cabbage cultivars appear to be more res
to bacterial wilt than pechay. Biovar IV isola
pepper from Quezon was the most virulent.

Of the seven cabbage varieties screened for resist
to biovar isolates of R. solanacearum, three cul
namely, Express 60. Yr Sumnmer 50 and K-K (
were rated resistant to all isolates.

In Chinese cabbage, Shirane and Hidaka
rated resistant to T116 (B3) and WP 2C
moderately resistant to CC1 and T6 isc
respectively. Cultivar B 189C 1 was resistant to V
and Ccl.

Out of fifteen pechay varieties only Chinese p
#22 and GC 89-396 were moderately resistant
the rest were either moderately susceptible
susceptible to Ccl R. solanacearum isolate.

Of the twelve varieties of Chinese cabbage eva
for resistance to bacterial wilt in the field. 7 were
resistant, 1 moderately resistant, 1 mode
susceptible and 3 susceptible. The resistant
were 63-1565, CC 99-291, CC63-1565, CC 9<
Hall 095, CC 00-287 and CC 99-289.

PM Maedalita, ATD Ocampo, LC Valencia, V

UPLB, College, Laguna and Kasertsart University
The zygotic embryos of Solo papaya were isc
and induced to form somatic embryos that
is squashed and used for transient and s
in transformation. In conjunction with this, a
construct pCP-LBP containing the coat protein
of the Philippine PRSV isolate was made and use
Is transformation. An optimized protocol
ar microprojectile bombardment was also established
er this protocol, a pressure level of 1,000-1, 2 kPa
8- distance of 12.5 cm was identified as the
3) condition for transforming somatic embryos
Ad transient gus expression is high using
al conditions. The distribution of microprojectiles c
of target tissues is random for all transformation el
a, A total of 18,200 immature zygotic embryos
!0 isolated and 7,845 (43%) produced somatic emt
as All the somatic embryos were bombarded wit
he plasmid DNA of the coat protein gene ol
nt Philippine PRSV isolate. A total of
of bombardments were made. The bombarded ti
were subjected to a selection medium containing
strength MS added at first with 150 mg L-', their
ce 300 mg L- kanamycin. A modification of the or
.rs selection procedure of Fitch et al. (1990)
ss, incorporated in this work. Bombarded embryos
directly placed either onto the generation mi
containing high kanamycin (300 mg L-') or nc
;re all, then transgenic plants will be selected by
ut analysis. These modifications will promote
:s, regeneration of putative transgenic plantlets 6-8
20 after bombardment which is much faster than
the standard method. Kanamycin-survivor em
were regenerated on a full strength De Fossard
ay with 0.25 gm each of BAP and NAA and 10liM
ile The transformed embryos consisting of 48 indi,
or transformation events are presently regenerating
small plantlets three months after micropro
la Cueva, EM de la Cruz, NL Opina, GC Molii
and MP Natural, IPB, UPLB, College, Laguna

Sugarcane plants with typical symptoms of 1
stunting disease were collected from sug,
growing areas in the country. Two saps extr
methods, namely, the use of sterile pliers and tl

Th Volui Journal of Plant Pat

78 Volul

Journal of Plant Pat

tcuum pump attached to a sterne tlasK were F1
)yed. The bacterium was successfully isolated C
the two extraction methods. At early stage of So
h on M-SC agar slants, the different isolates of (A
bacterium exhibited gram-positive reaction. T;
:ver, as the isolates grew older the reaction M
ne gram variable. All isolates exhibited non-acid
characteristics. The cells were irregular in shape, D
straight to slight curved rods, with a coryneform ve
ub-shaped morphology. The cells were very ja
with sizes ranging from 0.96 to 3.84 by 0.19 to ol
pun. Based on electron micrograph, cells contain bc
Iflagellated septa and motility was not observed us
10-day old agar or broth cultures examined tn
phase contrast microscope. The cells occurred ef
r or in pairs and oftentimes exhibited V-forms h3
observed under wet mounds. w

of the isolates induced the characteristic O
toms of RSD. All inoculated test plants had ef
er internodes relative to the control when di
sted 6 months after inoculation, tr
isolates from different sugarcane growing areas tri
hologically and culturally conform with the al
option of Gillaspie and Teakle (1989). cl
rmatory tests of the isolates using dot blot ra
moassay and polymerase chain reaction using
tic primers revealed that the bacterium was
mia xyli subsp, xyli. Il
onan and VM Escopalao, University of R
emr Mindanao, Kabacan, N. Cotabato pi
icides which appeared to possess eradicative yi
;rty against white root rot disease of rubber in the d(
ry were: flutriafol, tridemorph, improdione, fii
ozeb, clorothalonil and copper fungicide, at
ed rubber seedlings completely recovered and p,
morphs of Rigidoporus lignosus on the collar and M
roots dried up after drenching with fungicide in
on at 2-week intervals for three months, st
vise, four Trichoderma species (T. harzianum, T. d
lokoningii, T vinrde, Trichoderma sp.) and C
:illium sp. revealed mycoparasitic activity in p;
oiling the disease. It was further noted that at
rmorphs and basidiocarp of the fungus failed to w
op and the affected plants recovered. in

onan and CO Gonzales, University of Southern
anao, Kabacan, N.Cotabato

oconazole at the rate of 5ml/lter of water was
effective in controlling Curvularia leaf blight of
-uit. Moreover, based on a disease rating index
ied 15 days after the second application, it was
Than benomyl, the standard fungicide check
Methyl thiophanate at the rate of 35 g/l,
norph at 10.8 ml and fosetyl-Al at 35 g were
ive against Curvularia leaf blight while copper
ixide at 25 g and cooper oxychloride at 41 g/l
rated less effective against the disease.

the other hand, tridemorph was found very
ive against Sclerotium stem rot ofjackfruit. The
se is very systemic yet strikingly, sample plants
d with tridemorph were already free from any
toms of active lesions 15 days after the last
nent application. Difenoconazole was affective
st stem rot while captain at the rate of 38 g/l,
othalonil at 19 g/l and metalaxyl at 50 g/1 were
as less effective against the disease.

TEMS. EB Gergon, SR Francisco, AM
zar, and S Miller, PhilRice, Mufioz, Nueva
and Ohio State University, USA

hull burning (RHB) on the soil surface is widely
iced by many onion growers in Nueva Ecija,
)pines mainly to reduce weeds and increase
This practice, however, has not been
nented. A replicated experiment in farmers'
to determine the effects of RHB on onion yield
on pests in rice-onion cropping systems,
:ularly weeds and soil-born root-knot nematodes,
idogyne grammicola and to assess its economic
&t in onion production was undertaken. Results
ed that RHB reduced weed densities by 50% and
titial population densities of M graminicola four
after burning up to one month after transplanting.
anized rice hulls also enhanced soil nutrients,
:ularly P and exchangeable K, as shown in soil
sis at harvest. Onion yield increased by 28.9%
15 cm thick rice hulls were burned and
ised by 44.2% when thickness was increased to
n as compared to no RHB. Yield and cost effect

icr OT rapers iry-June ~Uui I~

iac or papers

ary-june zuul /.

analysis showed that this technology can be adopted
by farmers.

Estioko, Philippine Coconut Authority, Albay
Research Center, Guinobatan, Albay

The prevalence of the cadang-cadang disease in some
parts of the country has caused problems in the
international trade of coconut products. Earlier studies
revealed the presence of the coconut cadang-cadang
viroid or CCCVd in different parts of an infected palm
at varied concentration levels. This study was done to
verify possible CCCVd contamination of some export
products. The effects of various physical and
chemical elements on the CCCVd isolate were
likewise determined especially those that are applied
in food and non-food processing.

Using an improved molecular hybridization assay
(MHA) that was previously developed for field
indexing, no CCCVd was detected in the meat and
water (soil and liquid endosperm) of young and
mature coconuts in diseased palms but the viroid was
present in the husk (pericarp). Thus, products derived
from coconut endosperm should be exempted from the
ban (e.g. desiccated coconut, coconut in powder/slice
form, makapuno jelly, nata de coco, vinegar and
wine). In the case of fresh young coconut of "buko",
wherein the husk could be infected, the chance for the
diseased nut to be exported is negligible since it is
relatively small and scarified on the outer surface.
Otherwise, the source palms have to be tested or
certified CCCVd-free. For the by-products of mature
coconut husk (e.g. coco peat or coir fiber), these could
be considered free of CCCVd as the husk is composed
of practically dead tissues and the viroid could not
survive additional exposure to processing conditions.

CCCVd was found to be insensitive to
microwave/ionizing radiation, low temperature
pasteurization, freezing/thawing. and phenol/sodium
metabisulfite treatment. It was inactivated/degraded
by incubation at 100 "C for 45 min, by autoclaving at
118 OC for 15-30 min, and by treatment with enzyme
RNase, alkali solution (e.g. 10% KOH) and at
least 1% formalin.

As Mindanao is free from cadang-cadang, all coconut

products from this island (whether unprocessed)
should be automatically be spared from restriction.
For those coming from cadang-cadang infected areas,
not all coconut products are contaminated with
CCCVd as revealed by this study. Furthermore, there
are various physical and chemical elements applied to
food and non-food products processing that can
inactivate/degrade the viroid. However, in situations
requiring health certification, the CCCVd diagnostic
technique applied in this study is recommended for
use in quality standard testing.


AGLAONEMA. TO Dizon and LA Reyes, IPB,
UPLB, College, Laguna

Mycelial growth of Colletotrichum sp., the cause of
Colletotrichum leaf spot in Aglaonema, was inhibited
by mancozeb, ampicillin, benomyl and fosetyl-Al at
recommended rate using agar plate test.

A reduction in lesion size, as the length of exposure of
plant to benomyl, fosetyl-Ai and mancozeb increased,
was observed under screenhouse test. Of the two
methods of fungicide application used, spraying was
effective with benomyl and mancozeb, while
drenching with fosetyl-Al. Of the three fungicides
tested, fosetyl-Al caused the highest percent lesion
size reduction.

Villanueva, J de Dios, M Fajardo, U Duque and
HX Truong. Philippine Rice Research Institute,
Mufioz, Nueva Ecija

Fusarium moniliforme, causal organism of bakanae
disease, was isolated from rice seeds collected in
Panay Island and Nueva Ecija. Fungal DNA of
isolates was extracted and screening for universal and
ITS primers was done. Pathogenicity test and seed
infection of the isolates were also conducted.

With MR primers, F. moniliforme isolates across
location showed four different genetic profiles.
Isolates from Panay Island caused plant elongation
while Nueva Ecija isolate- infected plants exhibited
stunted growth. DNA fungal profiles showed more

80 Volume 37 f 1) January-June 2001 Journal of Plant Pathology

80 Volume 37 (1) January-June 2001

Journal of Plant Pathology

variation than the pathogenicity profile across
location Understanding of the rice seed-pathosystem
is the key to disease managernm't.

Alcachupas, IPB, UPLB, 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" 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 (Chenopodium
quinoa) to its original systemic host (Lagenaria
siceraria). The virus also induced necrotic lesion on
C. amaranticolor-inoculated bottlegourd, showed
slight mosaic symptoms at the early stage, and later
developed a well-defined mosaic with green blisters,
leaf outgrowths and deformed leaf. Inoculated
cucumbers exhibited dark green and vein clearing
symptoms 2 weeks after inoculation. The virus was
not able to infect Nicotiana glutinosa, Chenopodium
murale, Datura metel. Citrullus vulgaris and C.
moschata. D. stramonium exhibited a very mild
mosaic symptom.

The negative strain of partially purified extracts of
PTA (phosphotungstate) showed rigid rod virus
particles of the tobamovirus group. Indirect Enzyme-
Linked Immunosorbent Assay (ELISA) gave positive
reactions to TMV antiserum only but not to NMV,
WMV-2, ZYMV and PRSV antisera suggesting its
relatedness to TMV or tobamovirus.

Based on the results of bioassays, electromicroscopy,
and ELISA tests, the virus was determined to be a
strain of cucumber green mottle mosaic virus

Santiago and RQ Bermundo, Bureau of Postharvest
Research and Extension. Mufioz. Nueva Ecija

Aspergillus flavus was isolated from peanut seeds.
The isolates were tested for their ability to produce

aflatoxin using the agar plug method. In addition,
thirty fungal isolates were taken from healthy and
diseased peanut seeds samples collected from
different areas in the provinces of Ilocos Norte, Ilocos
Sur, Isabela, Nueva Ecija, Quirino, Pangasinan,
Cagayan, Bukidnon, La Union and Agusan Sur.
These fungal isolates were tested for their antagonistic
activity against aflatoxin-forming A. flavus. Fungal
antagonists against A. flavus were also isolated from
corn seeds.

One out of seven isolates of A. flavus from peanut
produced aflatoxin. In the test for non-volatile
compounds, seven fungal isolates suppressed the
growth of A. flavus isolates from peanut. In the test
for volatile compounds, five isolates from peanut and
five from corn inhibited the growth of A. flavus from

Tiongco, HR Rapusas, UG Duque, JJ Tagubase
and AE Villanueva, Philippine Rice Research
Institute, Mufioz, Nueva Ecija

Field surveys were conducted over a two-year period
in a 50-ha contiguous area in Midsayap, North
Cotabato to investigate the factors affecting the
occurrence and abundance of major rice pests. Crop
establishment, abundance of major insect pests, and
disease incidence differed markedly over time. Green
leafhoppers, white stemborer, rice black bug, and
tungro disease were endemic in the area. A trend in
increased tungro incidence was obtained with
increased catches of migrating vectors but not with
vector abundance as indicated sweep net collections.
The variety grown has an important influence on
tungro incidence and rate of spread in the endemic
area. Rice black bug and white stemborer were low in
number and did not cause significant damage to the

JP Rillon, GC Santiago, AE Villanueva, UG
Duque, MS de la Cruz and AR Martin, Philippine
Rice Research Institrite, Mufioz, Nueva Ecija

Hybrid rices and parentals were
resistance to major insect pests and
greenhouse and field conditions.

evaluated for
diseases under
Identified pest

Abstract of Papers Vohune 37(1) JanuaryJune 2001 81

Volufne 37 (1) January-June 2001 81

Abstract of Papers

distant parents could be used in the development of
hybrids and identify pest resistant F1 hybrids for
mmercial production. The greenhouse evaluation
ncentrated on bactenal leaf blight (BLB), blast, and
igro. In the field, entries were evaluated for
distancee to BLB, sheath blight (ShB) and stem borer.
lien diseases like sheath rot (ShR), bacterial leaf
eak (BLS) and narrow brown spot occurred, the
:ries were evaluated accordingly.

varietal breakdown. Selected lines from three
ferent resistance donors Oryza barthii, 0.
ipogon and Uti mera used to study the background
'ects of resistance breakdown. DNA genotyping
ing random amplified polymorphic DNA (RAPDs),
crosatellites (simple sequence repeats, SSRs),
distance gene analogues (RGAs) and amplified
.gment length polymorphism (AFLPs) were done on
Sdonor sources, recurrent parents and the derived

- -- -- E ,.- 4 L- y U.U


newly identified genes through inoculation E

es and retention of all the desirable straits of the recipient variety.
B through a series of backcrossing.

B rice varieties were inoculated with nine races of DEVELOPMENT OF AN IMPROVED EMBR
trial blight (BB) using the clip inoculation CULTURE PROTOCOL FOR COCONUT. E]
hod. Very few were susceptible to the nine races Rillo, CA Cueto, WR Medes and MaBBA Ubald
one of the varieties was resistant to all races. Philippine Coconut Authority, Albay Research Cen
?/ly released varieties had better resistance since Guinobatan, Albay
st of these had resistance to several races and are
erally resistant to predominant races. Based on the Comparative studies of four embryo culture protc
ilts, crosses wure made to transfer reported genes showed that Eeuwens (Y3) formulation was r
>ortant in most rice growing areas, particularly Xa- suitable than Murashige & Skoog's (MS) medium
gene that was found effective to Philippines races coconut embryo culture. UPLB and CPCRI protc
BB. A series of backcrossing has been started and gave better growth and'development of seedlings
etic analysis to find new genes will commence ARC protocol. Seedlings obtained from !
>n generating populations for analysis. medium showed higher ex vitro survival than the c
three media. Based on the results of Study
"'ybrrd" protocol, basically a Y3 mec
RFORMANCE OF ELITE LINES WITH supplemented with higher iron and si
CTERIAL BLIGHT RESISTANCE concerictl.ion, combination of ARC and U]
VELOPED USING DNA MARKER-AIDED vitanuin sources, Ig/L AC and 7g/L agar,
LECTION. MC Abalos, RC San Gabriel, JBAS conceptualized. Results using LAGT and 1V
Idulao, YAS Dimaano and RTabien:, Philippine embryos showed that myo-inositol and BAP-F
e Research Institute, Mufioz, Nueva Ecija could be omitted with. at detrimental effect to gr(
and c :velopment in vitro and ex vitro. Results o1
ent developments in DNA research are now being hybrid protocol confirmed the need for a least
d in crop improvement. These are important too:s passage in solid state and reduction of sugar du
t complement the classical methods of variety the growth and development phase. Results
elopment. DAS technology can be used to hasten showed improvement in terms of earlier leaf and
action of desirable plants and the direct transfer of formation, higher percentage of cultures
ned genes. It can facilitate generation advance, simultaneous shoot and root formation, hi
crossing, and progeny test. DNA markers were percentage of transplantable seedlings within
d to transfer bacterial blight (BB) genes from months and higher recovery rate, and ex vitro surv
ine IRBB5-21 having Xa-5, and Xa-21, and The treatments are being tested on makapuno emb
i9183 with gene from wild rice, 0. minute to three and an "optimized hybrid" protocol for coc
>ular varieties, PSB Rcl4, BPI Ri-10 and IR64. embryo culture is being developed.
ies of backcrossing selection and generation
'ance were done to identify elite lines.
the initial yield trials, three lines with yields of CONTROL OF GOOSEWEED IN IRRIGATE
2 to 7.13 t/ha out yielded the check PSB Rc28 and RICE: AA Eusebio and AK Watson, IRRI, Los
72. The same lines together with two more lines Bafios, Laguna
ificantly outyielded the checks in an advanced

isolate caused almost 100% mortality on.all growth provided by this biocontrol agent as that of 2.4-
stages of the target weed species from seedlings to dichlorophenoxy acetic acid (2,4-D), the standard
flowering plants. It has a restricted host range and herbicide for control of S. zelanica, Application of
readily infects its host at relatively low spore this fungus proved to be effective in small field
concentration even in the absence of free moisture on experiments but the advantage of applying this weed
the leaf surface. The pathogen, which was readily control measure over any other weed control methods
mass-produced in liquid culture, when applied as a in relation to economics, productivity, and
conidial suspension equally infects the four different sustainability was not established in this experiment.
ecotypes of S. zeylanica under greenhouse conditions. Further research, especially in larger, on-farm field
In field experiments same level of control was trials, is required to confirm these findings.

84 Volume 37 (1) January-June 2001 Journal of Plant Pathology


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

2. Manuscripts must be reports of original research, excerpt of meritorious reviews, and should have not
been published elsewhere. The decision of the Editorial Board to accept or reject manuscript is final.

3. The manuscript should be typed on one side of 8 1/2 x 11 inch paper, double spaced throughout.

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

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

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

7. Literature citation should be in alphabetical order. Do not cite unpublished work; it should appear as
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Res, Amer J Bot

8. Tables should be numbered consecutively, and each type on a separate page. They must have descrip-
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11. See latest Journal of Tropical Plant Pathology for more details on the format of papers to be submitted to
the journal.

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