Mechanism and Farmers' Perception of Mixed Variety Planting
Mechanism and Farmers' Perception of Mixed Variety Planting
Mechanism and Farmers' Perception of Mixed Variety Planting
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MECHANISM AND FARMERS’ PERCEPTION OF MIXED<br />
VARIETY PLANTING FOR THE MANAGEMENT<br />
OF RICE TUNGRO DISEASE<br />
YUJI SHIBATA<br />
SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL<br />
UNIVERSITY OF THE PHILIPPINES LOS BAÑOS<br />
IN PARTIAL FULFILLMENT OF THE<br />
REQUIREMENTS FOR THE<br />
DEGREE OF<br />
MASTER OF SCIENCE<br />
(Plant Pathology)<br />
JULY 2006
The thesis attached hereto, entitled “MECHANISM AND FARMERS’<br />
PERCEPTION OF MIXED VARIETY PLANTING FOR THE MANAGEMENT<br />
OF RICE TUNGRO DISEASE” prepared <strong>and</strong> submitted by YUJI SHIBATA in partial<br />
fulfillment <strong>of</strong> the requirements for the degree <strong>of</strong> MASTER OF SCIENCE (PLANT<br />
PATHOLOGY) is hereby accepted.<br />
MARIO V. PERILLA IL-RYONG CHOI<br />
Member, Guidance Committee Co-Chair, Guidance Committee<br />
________________________ ________________________<br />
Date signed Date signed<br />
EDNA Y. ARDALES<br />
Chair, Guidance Committee<br />
______________________<br />
Date signed<br />
Accepted as partial fulfillment <strong>of</strong> the requirements for the degree <strong>of</strong> MASTER OF<br />
SCIENCE (PLANT PATHOLOGY).<br />
VIRGINIA R. OCAMPO<br />
Director, Crop Protection Cluster<br />
_____________________________<br />
Date signed<br />
ERNESTO V. CARPIO<br />
Dean, Graduate School<br />
University <strong>of</strong> the Philippines Los Baños<br />
______________________________<br />
Date signed<br />
ii
BIOGRAPHICAL SKETCH<br />
The author was born on August 26, 1976 as the third <strong>of</strong> child <strong>of</strong> Kouichi Shibata<br />
<strong>and</strong> Fumie Shibata in Showa, Saitama, Japan. He received his elementary education from<br />
Houshubana elementary school <strong>and</strong> his secondary education from Edogawa junior high<br />
school in Showa <strong>and</strong> Kasukabe high school in Kasukabe <strong>and</strong> graduated in 1994.<br />
In 1997, he enrolled at the Tokyo University <strong>of</strong> Agriculture (TUA) taking up<br />
Bachelor <strong>of</strong> Science in Agriculture major in International Development <strong>of</strong> Agriculture<br />
where he graduated in 2001. He developed an interest in plant viruses <strong>and</strong> tropical<br />
agriculture in Southeast Asia since he joined the Laboratory <strong>of</strong> Tropical Crop Protection<br />
during his junior <strong>and</strong> senior years at TUA.<br />
After college, he decided to pursue his MSc degree in Plant Pathology at the<br />
University <strong>of</strong> the Philippines Los Baños (UPLB) in 2001. In July, 2004 he was granted a<br />
MS-Thesis Research Scholarship <strong>and</strong> worked at the Entomology <strong>and</strong> Plant Pathology<br />
Division (EPPD) as a MS-Research Scholar at International Rice Research Institute<br />
(IRRI).<br />
He is a member <strong>of</strong> the Philippines Phytopathological Society (PPS)<br />
iii<br />
YUJI SHIBATA
ACKNOWLEDEMENT<br />
For their invaluable support <strong>and</strong> contributions in the completion <strong>of</strong> this MSc.<br />
Thesis, the author would like to express his sincerest gratitude <strong>and</strong> appreciation to the<br />
following individuals <strong>and</strong> organizations.<br />
Dr Edna Y, Ardales, Chairman <strong>of</strong> the Guidance Committee, Crop Protection<br />
Cluster, UPLB, for her kind encouragement, <strong>and</strong> meaningful suggestions in improving<br />
the manuscript.<br />
Dr Il-Ryong Choi, Co-Chair <strong>of</strong> the Guidance Committee, for the direction <strong>and</strong><br />
guidance in the conduct <strong>of</strong> the experiment, his constructive comments, suggestions, <strong>and</strong><br />
deep insight in the preparation <strong>of</strong> the outline <strong>and</strong> manuscript.<br />
Dr Mario V. Perilla, member <strong>of</strong> the Guidance Committee, for his suggestions <strong>and</strong><br />
criticism in the improvement <strong>of</strong> the manuscript.<br />
Mr. Rogelio C. Cabunagan, in-house adviser at IRRI, for his unselfish <strong>and</strong><br />
continuous support during the conduct <strong>of</strong> the experiment <strong>and</strong> the improvement <strong>of</strong> the<br />
manuscript.<br />
Mr. Mario Izon, Panfilio Domingo, Esquirion Baguioso, for their expertise <strong>and</strong><br />
unlimited support during the conduct <strong>of</strong> the experiment.<br />
Mrs. Violeta Bartolome <strong>and</strong> Adoracion Resurreccion for their expertise <strong>and</strong> kind<br />
assistance in the statistical data analysis <strong>and</strong> the improvement <strong>of</strong> the manuscript.<br />
All the research scholars in the department especially Mr. Neggussie Shoatatek<br />
Zenna for the valuable discussions, his brilliant brotherhood <strong>and</strong> his moral support.<br />
Drs. Nobuya Kobyashi, Kumi Yasunobu, Yoshinobu Nitta, Yol<strong>and</strong>a F. Chen,<br />
Yuichiro Furukawa, <strong>and</strong> Yuka Sasaki, <strong>and</strong> Yasukazu Hosen for their warm<br />
encouragement <strong>and</strong> meaningful suggestions in the preparation <strong>of</strong> the survey <strong>and</strong> for<br />
improving the manuscript.<br />
All his friends not only in the Philippines but also in the world, for their<br />
continuous friendship despite <strong>of</strong> the distance.<br />
Dr. Luis Rey I. Velasco, Chancellor <strong>of</strong> UPLB, for his respectful support <strong>and</strong><br />
sponsorship during the graduate study at UPLB.<br />
To my parent for their underst<strong>and</strong>ing <strong>of</strong> his study <strong>and</strong> inspirational <strong>and</strong> moral<br />
support through out the study.<br />
iv
TABLE OF CONTENTS<br />
TITLE PAGE i<br />
APPROVAL PAGE ii<br />
PAGE<br />
BIOGRAPHICAL SKETCH iii<br />
ACKNOWLEDGEMENT iv<br />
TABLE OF CONTENTS v<br />
LIST OF TABLES ix<br />
LIST OF FIGURES xi<br />
LIST OF APPENDIX TABLES xiii<br />
ABSTRACT xiv<br />
INTRODUCTION 1<br />
REVIEW OF LITERATURE 8<br />
Rice Tungro Disease 8<br />
Economic Importance 8<br />
The Rice Tungro Viruses 9<br />
Symptom 10<br />
Transmission 11<br />
Disease Development in the Field 12<br />
Disease Management 14<br />
Resistance to Rice Tungro Disease 17<br />
Varietal Mixtures as RTD Management Option 18<br />
Importance <strong>of</strong> Farmers’ <strong>Perception</strong> Survey 23<br />
v
PAGE<br />
Farmers’ Preference for Grain Quality 25<br />
MATERIALS AND METHODS 26<br />
Plant Materials 26<br />
Insect Vector 26<br />
Viruses 27<br />
Assay Procedure for RTBV <strong>and</strong> RTSV infection. 27<br />
Biological Characterization <strong>of</strong> Matatag 9 Resistance to Rice Tungro Disease 28<br />
Forced Test Tube Inoculation 28<br />
Relative Quantification <strong>of</strong> RTBV <strong>and</strong> RTSV in Matatag 9 <strong>and</strong> IR64 29<br />
Preparation <strong>of</strong> samples 29<br />
Optimum assay condition 29<br />
Characterization <strong>of</strong> virus replication 30<br />
Biological Characterization <strong>of</strong> Matatag 9 Resistance to Green Leafhopper 30<br />
GLH Preference <strong>and</strong> Tungro Viruses Transmission 30<br />
Adult Insect longevity 32<br />
Nymphal Mortality 32<br />
Growth <strong>and</strong> Development <strong>of</strong> Nymph 33<br />
Performance Evaluation <strong>of</strong> Varietal Mixture components 35<br />
Comparative Morphological <strong>and</strong> Yield Characteristics <strong>of</strong> Matatag 9 <strong>and</strong> IR64 35<br />
Effect <strong>of</strong> mixed planting <strong>of</strong> resistant <strong>and</strong> susceptible varieties on the incidence<br />
<strong>of</strong> rice tungro disease<br />
Tray Inoculation <strong>and</strong> Experimental Design 36<br />
<strong>Mixed</strong> seed planting layout 37<br />
Interplanting (row mixture) layout 39<br />
vi<br />
36
PAGE<br />
Serial Inoculation in Resistant <strong>and</strong> Susceptible Varieties 42<br />
Farmers’ <strong>Perception</strong> Survey on the Use <strong>of</strong> <strong>Mixed</strong> Seed <strong>Planting</strong> 43<br />
Description <strong>of</strong> Survey Site 43<br />
Survey Procedure 43<br />
Grain Quality Evaluation <strong>of</strong> <strong>Mixed</strong> Seed <strong>Planting</strong> 45<br />
Rice Grains Used 45<br />
Milling Quality 45<br />
Grain Size, Shape <strong>and</strong> Appearance 46<br />
Amylose Content 47<br />
Gelatinization Temperature 47<br />
Gel Consistency 47<br />
RESULTS AND DISCUSSION 49<br />
Biological Characterization <strong>of</strong> Matatag 9 Resistance to Rice Tungro Disease 49<br />
Rates <strong>of</strong> Infection with RTSV <strong>and</strong> RTBV under Different Inoculum<br />
Levels<br />
Temporal Change in Apparent Rates <strong>of</strong> Infection with RTSV <strong>and</strong><br />
RTBV<br />
Temporal Change in The Accumulation <strong>of</strong> Tungro Viruses 51<br />
Biological Characterization <strong>of</strong> Matatag 9 Resistance to Green Leafhopper 57<br />
Settling preference 57<br />
Adult Insect longevity 59<br />
Nymphal Mortality 59<br />
Growth <strong>and</strong> Development <strong>of</strong> GLH Nymph 61<br />
Performance Evaluation <strong>of</strong> Varietal Mixture Components 65<br />
Comparative Morphological <strong>and</strong> Yield Characteristics <strong>of</strong> Matatag 9 <strong>and</strong> IR64 65<br />
vii<br />
49<br />
49
Effect <strong>of</strong> mixed planting <strong>of</strong> resistant <strong>and</strong> susceptible varieties on the incidence<br />
<strong>of</strong> rice tungro disease<br />
PAGE<br />
Serial-Inoculation in Resistant <strong>and</strong> Susceptible varieties 70<br />
Farmers’ <strong>Perception</strong> Survey on the Use <strong>of</strong> <strong>Mixed</strong> Seed <strong>Planting</strong> 75<br />
Respondents’ pr<strong>of</strong>ile. 75<br />
Farm pr<strong>of</strong>ile 75<br />
Farmers’ awareness <strong>and</strong> knowledge <strong>of</strong> rice tungro disease 80<br />
Farmers’ awareness <strong>of</strong> rice tungro disease control 81<br />
Awareness <strong>of</strong> mixed seeds planting 83<br />
Grain Quality Evaluation <strong>of</strong> <strong>Mixed</strong> Seed <strong>Planting</strong> 91<br />
Milling Quality 91<br />
Grain Size, Shape <strong>and</strong> Appearance 92<br />
Amylose Content <strong>and</strong> Gelatinization Temperature 95<br />
Gel Consistency 96<br />
Sensory evaluation <strong>of</strong> cooked rice 96<br />
SUMMARY AND CONCLUSIONS 100<br />
RECOMMENDATIONS 105<br />
LITERATURE CITED 106<br />
APPENDIX 124<br />
viii<br />
68
LIST OF TABLES<br />
TABLE PAGE<br />
1 Ratios <strong>of</strong> seed mixtures used for mixed variety planting 36<br />
2 Combination <strong>of</strong> Matatag 9 <strong>and</strong> IR 64 for serial transmission 42<br />
3 Classification for grain size (a) <strong>and</strong> shape (b) 46<br />
4 Numerical scale for scoring gelatinization temperature (GT) 47<br />
5 Infection rate (%) <strong>of</strong> RTBV <strong>and</strong> RTSV at time <strong>of</strong> initial sampling<br />
<strong>and</strong> 60 DAI: 6-day-old seedling<br />
6 Infection rate (%) <strong>of</strong> RTBV <strong>and</strong> RTSV at time <strong>of</strong> initial sampling<br />
<strong>and</strong> 60 DAI: 21-day-old seedling<br />
7<br />
Average seedling infection (%) <strong>and</strong> average number <strong>of</strong> alighted<br />
insect per 5 seedlings <strong>of</strong> IR64 <strong>and</strong> Matatag 9 at different<br />
observation times from the GLH preference test.<br />
8 Effect <strong>of</strong> antibiosis factor on GLH behavior <strong>and</strong> other life history<br />
traits<br />
9 Comparison <strong>of</strong> tiller number during vegetative stage 67<br />
10 Comparison <strong>of</strong> agronomic traits <strong>of</strong> mixture components at harvest 67<br />
11 Comparison <strong>of</strong> physical characteristics <strong>of</strong> grain 67<br />
12 Pr<strong>of</strong>ile <strong>of</strong> farmer respondents <strong>of</strong> the survey in Ajuy, Iloilo,<br />
Philippines<br />
13 Varieties planted for the past 4 seasons among 52 respondents in<br />
Ajuy, Iloilo, Philippines<br />
14 Percentage <strong>of</strong> farmers reporting major pests <strong>and</strong> diseases on rice<br />
farming in Ajuy, Iloilo, Philippines<br />
15 Farmers’ experience <strong>of</strong> RTD incidence in their farm <strong>and</strong> its damage<br />
seriousness ranked by farmers<br />
16 Farmers’ perceptions <strong>of</strong> causes <strong>and</strong> spread <strong>of</strong> RTD 81<br />
ix<br />
52<br />
53<br />
58<br />
60<br />
76<br />
77<br />
79<br />
80
TABLE PAGE<br />
17 Farmers’ control measures for rice tungro disease <strong>and</strong> perception <strong>of</strong><br />
insecticide effectiveness<br />
18 Farmers’ awareness <strong>of</strong> the availability <strong>of</strong> tungro resistant variety<br />
<strong>and</strong> experience <strong>of</strong> resistant breakdown<br />
19 Major reasons to the termination <strong>of</strong> the mixed seed planting <strong>and</strong> its<br />
adaptation constraints in farmers’ field in Ajuy<br />
20 Grain length <strong>and</strong> width <strong>of</strong> milled rice harvested from plots planted<br />
to different seed mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S<br />
(susceptible IR64) analyzed by Cervitec 1625 Grain Inspector<br />
21 Degree <strong>of</strong> chalkiness <strong>of</strong> milled rice harvested from plots planted to<br />
different seed mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S<br />
(susceptible IR64) analyzed by Cervitec 1625 Grain Inspector<br />
22 Amylose content <strong>and</strong> gelatinization temperature <strong>of</strong> milled rice<br />
harvested from plots planted to different mixture ratio <strong>of</strong> R<br />
(resistant Matatag 9) <strong>and</strong> S (susceptible IR64)<br />
23 Viscosity analysis <strong>of</strong> cooked milled rice harvested from plots<br />
planted to different mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S<br />
(susceptible IR64) by the Rapid Visco Analyzer.<br />
x<br />
82<br />
83<br />
85<br />
93<br />
93<br />
95<br />
96
LIST OF FIGURES<br />
FIGURE PAGE<br />
1 Experimental set-up for settling preference experiment. Five 10day-old<br />
seedlings each <strong>of</strong> test variety planted in a pot were<br />
r<strong>and</strong>omly placed inside a plastic cage.<br />
2 Experimental set-up for growth <strong>and</strong> development <strong>of</strong> GLH nymph<br />
test among four test varieties.<br />
3 Example <strong>of</strong> seed layout <strong>of</strong> T2 (75:25 for Matatag 9 <strong>and</strong> IR64) seed<br />
mixtures for tray inoculation: R indicates position <strong>of</strong> resistant<br />
variety, Matatag 9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety,<br />
IR64.<br />
4 Seed layout <strong>of</strong> T2. R indicates position <strong>of</strong> resistant variety, Matatag<br />
9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety, IR64.<br />
5 Seed layout <strong>of</strong> T3. R indicates position <strong>of</strong> resistant variety, Matatag<br />
9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety, IR64.<br />
6 Seed layout <strong>of</strong> T4. R indicates position <strong>of</strong> resistant variety, Matatag<br />
9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety, IR64.<br />
7 The site (shaded red) <strong>of</strong> the survey in the municipality <strong>of</strong> Ajuy,<br />
Iloilo Province, Philippines<br />
8 Infection rate <strong>of</strong> RTBV <strong>and</strong> RTSV in three varieties with 1, 3, <strong>and</strong><br />
5 viruliferous GLH by the forced test tube inoculation.<br />
9 Temporal pattern <strong>of</strong> RTBV <strong>and</strong> RTSV accumulation in Matatag 9<br />
<strong>and</strong> IR64. Both 6-day-old <strong>and</strong> 21-day-old seedlings were used at<br />
inoculation.<br />
10 Symptom caused by RTD on Matatag 9 (left) <strong>and</strong> IR64 (Right).<br />
Matatag 9 did not show typical RTD symptom as compared with<br />
IR64 which showed yellowing <strong>and</strong> stunting symptom.<br />
11 Temporal changes in the number <strong>of</strong> viruliferous GLH on the<br />
seedlings <strong>of</strong> IR64 <strong>and</strong> Matatag 9.<br />
12 Morphological appearance <strong>of</strong> Matatag 9 (left) <strong>and</strong> IR64 (right) at<br />
the 107 DAT in the screenhouse.<br />
xi<br />
31<br />
34<br />
38<br />
39<br />
40<br />
41<br />
44<br />
50<br />
55<br />
56<br />
58<br />
66
FIGURE PAGE<br />
13 Growth comparison between Matatag 9 <strong>and</strong> IR64 at 15 days<br />
interval after transplanting.<br />
14 Relative rates <strong>of</strong> infection with RTBV+RTSV (RBS), total RTBV<br />
(RTB), <strong>and</strong> total RTSV (RTS) observed in mixed seed plantings<br />
<strong>and</strong> interplantings. The relative rates were computed based on<br />
those observed in the pure st<strong>and</strong> <strong>of</strong> IR64 (100% S).<br />
15 Duration <strong>of</strong> virus retention ability through 5 days serial-inoculation<br />
with different combinations <strong>of</strong> resistant <strong>and</strong> susceptible varieties.<br />
16 Farmers’ awareness <strong>of</strong> the mixed seed planting for rice tungro<br />
disease control (A), their belief <strong>of</strong> control effectiveness (B), <strong>and</strong><br />
experience <strong>of</strong> the mixed seeds planting in their field (C).<br />
17 Milling yield <strong>of</strong> the rough rice harvested from plots planted to<br />
different seed mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S<br />
(susceptible IR64).<br />
18 Percentage <strong>of</strong> whole grain <strong>and</strong> broken rice from rough rice<br />
harvested from plots planted to different seed mixture ratio <strong>of</strong> R<br />
(resistant Matatag 9) <strong>and</strong> S (susceptible IR64) analyzed by Cervitec<br />
1625 Grain Inspector<br />
19 Grain appearance <strong>of</strong> rough rice (paddy) <strong>and</strong> milled rice from each<br />
treatment.<br />
20 Viscosity curves <strong>of</strong> cooked milled rice harvested from plots<br />
planted to different mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S<br />
(susceptible IR64) by the Rapid Visco Analyzer<br />
xii<br />
66<br />
69<br />
71<br />
84<br />
91<br />
92<br />
93<br />
97
LIST OF APPENDICES<br />
Appendix PAGE<br />
1 Questions for socioeconomic survey in Ilo-ilo. 124<br />
xiii
ABSTRACT<br />
YUJI SHIBATA, University <strong>of</strong> the Philippines Los Baños, July 2006.<br />
<strong>Mechanism</strong> <strong>and</strong> Farmers’ <strong>Perception</strong> <strong>of</strong> <strong>Mixed</strong> <strong>Variety</strong> <strong>Planting</strong> for the<br />
Management <strong>of</strong> Rice Tungro Disease.<br />
Major Pr<strong>of</strong>essors: Dr. Edna Y. Ardales <strong>and</strong> Dr. Il-Ryong Choi<br />
To characterize the resistance <strong>of</strong> Matatag 9 against rice tungro disease (RTD),<br />
Matatag 9 <strong>and</strong> an RTD-susceptible variety IR64 were evaluated for their reactions to<br />
tungro viruses <strong>and</strong> the insect vector (green leafhopper, GLH). The results indicated that<br />
low RTD incidence in Matatag 9 was mainly due to the resistance against GLH, although<br />
the contribution <strong>of</strong> moderate resistance against tungro viruses may not be excluded. The<br />
agronomical characteristics <strong>of</strong> Matatag 9 were found to be similar to those <strong>of</strong> IR64,<br />
therefore, it is suitable for mixed planting. <strong>Mixed</strong> seed planting was more effective in<br />
reducing RTD incidence than interplanting, suggesting that the r<strong>and</strong>om spatial<br />
distribution <strong>of</strong> resistant plants is critical for the reduction <strong>of</strong> RTD in fields. Survey<br />
conducted among the farmers in an RTD endemic area indicated that besides the impact<br />
on disease reduction, the grain quality is an important criterion for them to adopt the<br />
mixed seed planting. The quality <strong>of</strong> grains produced through mixed seeds planting <strong>of</strong><br />
Matatag 9 <strong>and</strong> IR64 appeared to be comparable to that <strong>of</strong> IR64. Collectively, these<br />
suggest that the mixed seed planting between Matatag 9 <strong>and</strong> IR64 may be able to <strong>of</strong>fer<br />
both yield stability <strong>and</strong> high grain quality in RTD-endemic areas.<br />
xiv
INTRODUCTION<br />
Rice plants are farmed mainly for the grain used for human consumption in over<br />
one hundred countries around the world (Maclean et al. 2002). The farmers <strong>of</strong> Asia till 90<br />
percent <strong>of</strong> the total world’s rice fields <strong>and</strong> this accounts for 92 percent <strong>of</strong> the global rice<br />
production. Moreover, Asia is home to 59 percent <strong>of</strong> the world’s population <strong>and</strong> the rice<br />
consuming population is growing at 2 percent a year. In humid <strong>and</strong> sub-humid Asia, rice<br />
is the primary staple food (Khush, 1997), <strong>and</strong> directly supplies approximately 21 percent<br />
<strong>of</strong> human per capita energy <strong>and</strong> 15 percent per capita protein each year. Together with<br />
wheat <strong>and</strong> maize, rice provides over 50 percent <strong>of</strong> all <strong>of</strong> the calories that are annually<br />
consumed by the entire human population (Maclean et al. 2002). The population is<br />
expected to increase by 58 percent over the next 35 years. Recent projections indicate<br />
that the dem<strong>and</strong> for rice production in 2025 is expected to be 765 million that is 70<br />
percent more rice than is produced today (Maclean et al. 2002). Consequently the annual<br />
output must increase by over 5 million tones a year just to keep pace with population<br />
growth (Maclean et al. 2002).<br />
Cultivation <strong>of</strong> modern varieties <strong>of</strong> rice was widely accepted by farmers in Asia to<br />
earn more income. This replaced the local varieties, eventually resulting in low diversity<br />
<strong>of</strong> cultivated ones <strong>and</strong> made the modern varieties more vulnerable to pests <strong>and</strong> diseases.<br />
Narrow spacing in planting rice also resulted in high density crops <strong>and</strong> in turn altered the<br />
microclimate under the short canopy <strong>of</strong> rice st<strong>and</strong>s making it more favorable for the
development <strong>of</strong> not only the beneficial, but also harmful flora <strong>and</strong> fauna in the rice<br />
ecosystem (Azzam <strong>and</strong> Chancellor, 2002; Sogawa, 1976; Thresh, 1982).<br />
Tungro identified as a leafhopper-borne disease was first observed in the<br />
Philippines at the experimental farm <strong>of</strong> the International Rice Research Institute (IRRI) in<br />
1963 (Rivera <strong>and</strong> Ou, 1965). Rice tungro disease (RTD) is a destructive rice disease<br />
widely distributed in almost all South <strong>and</strong> South-east Asia <strong>and</strong> some parts <strong>of</strong> East Asia<br />
countries like Japan, China, Thail<strong>and</strong>, Philippines, India <strong>and</strong> Indonesia.<br />
Substantial yield losses have been reported when the RTD outbreak occurred. For<br />
example, in 1971 the estimated yield loss due to RTD was 456,000 tons <strong>of</strong> rough rice in<br />
Central Luzon <strong>and</strong> Cotabato where IR5, IR8, IR22 <strong>and</strong> IR24 were widely cultivated. In<br />
1993 in Davao del Norte, a total <strong>of</strong> 2265 tons amounting to about US$ 40,000 were lost<br />
due to the RTD (Baria, 1993). Shinkai (1977) reported that a total yield loss due to rice<br />
waika virus, which is phylogenetically similar to rice tungro spherical virus (RTSV), was<br />
approximately 10,000 tons in 1973 in Kyushu, Japan.<br />
RTD epidemic is unpredictable <strong>and</strong> once it occurs no control measures can be<br />
applied. In the late 1960’s <strong>and</strong> early 1970’s, severe epidemics caused by RTD resulted in<br />
major production losses in Bangladesh, India, Indonesia, Malaysia, Philippines <strong>and</strong><br />
Thail<strong>and</strong>. In the Philippines specifically, major epidemics occurred in 1957, 1962, 1969-<br />
71, 1983-1984, <strong>and</strong> 1987 (Koganezawa, 1998). Although several control methods against<br />
RTD were proposed <strong>and</strong> applied, epidemics reoccur periodically. Extensive cultivation <strong>of</strong><br />
susceptible varieties was identified as one <strong>of</strong> the potential factors influencing the disease<br />
epidemics (Holt et al. 1996; Cabunagan et al. 2001). Thus, in the above countries,<br />
2
varietal resistance to RTD is an important breeding objective for rice improvement<br />
(Khush <strong>and</strong> Virmani, 1985). The use <strong>of</strong> resistant varieties is considered as the most<br />
economical <strong>and</strong> environmentally sound strategy for management <strong>of</strong> RTD. Also, the use<br />
<strong>of</strong> resistant varieties has been very effective for incidence <strong>of</strong> RTD could be drastically<br />
reduced the following year after its introduction (Sogawa, 1976).<br />
Screening rice germplasm for RTD resistance started as early as 1963 at IRRI <strong>and</strong><br />
breeding program was started in 1966-1967 (Khush, 1977). Several resistant varieties,<br />
Peta, Intan, Sigadis, TKM6, HR21, Malagkit Sungsong, Gam Pai30-12-15, Ptb18,<br />
Pankhari203 <strong>and</strong> BJ1 were identified <strong>and</strong> used as donor parents. In the initial stage,<br />
selected resistant varieties had only vector resistance because <strong>of</strong> the screening methods<br />
adapted in the breeding programs. The screening <strong>of</strong> breeding materials has been done<br />
generally in the field where tungro incidence is low <strong>and</strong> vector resistant varieties are<br />
always selected (Hibino et al. 1987). Lack <strong>of</strong> appropriate diagnostic techniques made the<br />
analysis <strong>of</strong> resistance to tungro viruses difficult. Vector resistant varieties escape RTD<br />
infection under light to moderate vector pressure but succumb to infection under high<br />
vector pressure (Cabunagan et al. 1987). Reaction also changes with change in virulence<br />
<strong>of</strong> GLH vector in the field (Dahal et al. 1990). The differentiation between resistance to<br />
the tungro viruses <strong>and</strong> resistance to the GLH vector is important in the development <strong>of</strong><br />
varieties with durable resistance. In fact, resistance to the tungro viruses is nowadays<br />
regarded as crucial to any long-term management <strong>of</strong> the RTD problem in South <strong>and</strong><br />
Southeast Asia.<br />
3
With the new underst<strong>and</strong>ing <strong>of</strong> the tungro viruses <strong>and</strong> the availability <strong>of</strong><br />
serological techniques such as enzyme linked immunosorbent assay (ELISA) in the early<br />
1980’s, research on RTD resistance has shifted to virus resistance, which may be more<br />
effective <strong>and</strong> durable (Khush et al. 2004). Since the start <strong>of</strong> the mass screening for RTD<br />
resistance at IRRI in 1963 about 30% <strong>of</strong> the total IRRI Rice Germplasm Center<br />
Accession (IRGC Acc) so far has been screened for RTD resistance. The results revealed<br />
that most <strong>of</strong> the resistant accessions originated from Bangladesh, India, Pakistan, <strong>and</strong><br />
Indonesia (Cabunagan <strong>and</strong> Koganezawa, 1993). Many IRGC accessions have been<br />
reported as resistant to RTSV infection (Hibino et al. 1990; Koganezawa <strong>and</strong> Cabunagan,<br />
1997). Although sources for resistance to rice tungro bacilliform virus (RTBV) infection<br />
have yet to be found in rice germplasm at IRRI, several varieties show symptomatic<br />
resistance or tolerance (Hibino et al. 1990; Koganezawa <strong>and</strong> Cabunagan, 1997). The term<br />
“tolerance” refers to varieties that show no or mild symptom <strong>and</strong> no marked yield loss<br />
when infected, regardless <strong>of</strong> virus concentration. Multiplication <strong>of</strong> RTBV is suppressed<br />
in some tolerant varieties such as Utri Merah, Balimau Putih <strong>and</strong> Utri Rajapan, <strong>and</strong><br />
sometimes virus concentration is too low to be detected by ELISA (Takahashi et al.<br />
1993; Cabunagan et al. 1993).<br />
Recently, varieties resistant to tungro viruses (e.g., the Matatag lines) have been<br />
developed (Angeles et al. 1998; Cabunagan et al. 1999) <strong>and</strong> deployed in selected areas<br />
where rice tungro was endemic, in the Philippines. As there are only limited resistance<br />
sources against the tungro viruses, it is important to develop a deployment strategy that<br />
would exert minimal selection pressure on the viruses so as to minimize the occurrence <strong>of</strong><br />
4
esistance-breaking strains due to monoculture, thus prolong the lifespan <strong>of</strong> these<br />
resistant varieties.<br />
In the history <strong>of</strong> rice production, wide adoption <strong>of</strong> modern varieties with<br />
resistance to specific pathogens in a large area <strong>of</strong>ten results in genetic uniformity which<br />
predisposes the crop system to disease epidemics. In order to avoid stepping in such<br />
pathway <strong>of</strong> rice cultivation with the tungro virus resistant varieties, it is critical to deploy<br />
resistant varieties based on underst<strong>and</strong>ing <strong>of</strong> the pathogen population structure as well as<br />
to maintain genetic diversity in the fields. It was suggested that genetic diversity in<br />
agricultural settings reduces selection pressure against pathogens to slow down pathogen<br />
evolution <strong>and</strong> provides greater disease suppression when used over large areas (Leung et<br />
al. 2003). Although there have been a number <strong>of</strong> reports on the application <strong>of</strong> variety<br />
mixtures to suppress the disease <strong>of</strong> small grain crops caused by fungal pathogens (Finckh<br />
et al. 2000; Mundt, 2002 <strong>and</strong> references therein), only few attempts were made to<br />
examine the effects <strong>of</strong> variety mixture on the incidence <strong>of</strong> viral disease (Hariri et al.<br />
2001; Pedroso, 2001; Power, 1991).<br />
Experiments were conducted in Iloilo, Philippines to assess the effects <strong>of</strong> variety<br />
mixed planting <strong>of</strong> resistant (Matatag 9) <strong>and</strong> susceptible (IR64) varieties on the occurrence<br />
<strong>of</strong> RTD <strong>and</strong> the frequency <strong>of</strong> RTD components in this endemic area. The advantages <strong>of</strong><br />
the seed mixtures in RTD suppression <strong>and</strong> yield stability in the tungro endemic area <strong>of</strong><br />
Iloilo were observed in the study (Cabunagan et al. 2004, Cabunagan <strong>and</strong> Choi, 2005).<br />
However, more performance data on the seed mixtures under high disease pressure are<br />
needed to evaluate if this control measure is still effective <strong>and</strong> can provide stable yield.<br />
5
There is no clear explanation <strong>of</strong> RTD reduction in the previous study, therefore, it<br />
is necessary to determine the mechanism for the reduction <strong>of</strong> RTD in mixed planting <strong>of</strong><br />
resistant <strong>and</strong> susceptible varieties. There are two types <strong>of</strong> resistance to rice tungro<br />
disease: (1) resistance to the vector- the leafhopper cannot feed <strong>and</strong> reproduce effectively<br />
on resistant plant <strong>and</strong> thus cannot transmit tungro viruses, (2) resistance to the virus- the<br />
virus cannot survive <strong>and</strong> multiply inside the plant. Thus, it is important to biologically<br />
characterize the mechanism <strong>of</strong> resistance <strong>of</strong> the resistant component Matatag 9 <strong>and</strong> how<br />
this relates to tungro viruses transmission.<br />
In addition, in order to transfer successfully the new tungro disease management<br />
scheme to the farmers, it is important to evaluate farmers’ perception on the use <strong>of</strong> mixed<br />
seeds planting <strong>of</strong> resistant <strong>and</strong> susceptible varieties for the management <strong>of</strong> rice tungro<br />
disease because the first step to improve rice farmers’ RTD management capabilities is to<br />
gain their acceptance <strong>of</strong> the new management scheme. Surveys provide a useful way to<br />
canvas the ideas <strong>and</strong> opinions <strong>of</strong> farmers about pest <strong>and</strong> disease management while at the<br />
same time documenting their current practices.<br />
As a component <strong>of</strong> the mixed seed planting, resistant variety Matatag 9 was<br />
chosen because it has more or less similar plant type, eating quality <strong>and</strong> maturity date to<br />
the susceptible IR64 so that farmers could still cultivate IR64 even in RTD endemic areas.<br />
To answer the question "is the grain quality <strong>of</strong> the mixed seeds comparable to the pure<br />
IR64 which is preferred by the farmers <strong>and</strong> millers" grain quality characteristics <strong>of</strong> the<br />
mixtures with the pure Matatag 9 or pure IR64 were compared in this study. Specifically,<br />
6
data on milling potentials, physical attributes <strong>and</strong> physicochemical characteristics were<br />
examined.<br />
Underst<strong>and</strong>ing the mechanism <strong>of</strong> resistance <strong>of</strong> Matatag 9, specifically how mixed<br />
seed planting <strong>of</strong> resistant Matatag 9 <strong>and</strong> susceptible IR64 reduces RTD incidence,<br />
farmers’ perception <strong>of</strong> the technology <strong>and</strong> the grain quality <strong>of</strong> the mixture compared with<br />
pure IR64 would enable us to predict the sustainability <strong>and</strong> farmers adaptation <strong>of</strong> the<br />
variety mixture system for their management <strong>of</strong> RTD.<br />
The objectives <strong>of</strong> the study are:<br />
1. To determine how mixed planting <strong>of</strong> resistant <strong>and</strong> susceptible varieties influence<br />
the incidence <strong>of</strong> RTD<br />
2. To determine how planting types <strong>and</strong> disease pressure affect RTD incidence in<br />
mixed planting <strong>of</strong> resistant <strong>and</strong> susceptible varieties.<br />
3. To evaluate farmers’ perception on the use <strong>of</strong> mixed planting <strong>of</strong> resistant <strong>and</strong><br />
susceptible varieties for RTD management<br />
7
REVIEW OF LITERATURE<br />
Rice Tungro Disease<br />
RTD is one <strong>of</strong> the most destructive diseases <strong>of</strong> rice in South <strong>and</strong> Southeast Asia,<br />
where epidemics <strong>of</strong> the disease have occurred since the mid-1960s (Azzam <strong>and</strong><br />
Chancellor, 2001). In the Philippine dialect Ilocano tungro means degenerated growth.<br />
RTD is also known by various names in different countries; mentek or penyakit habang<br />
in Indonesia, penyakit merah in Malaysia, yellow-orange leaf in Thail<strong>and</strong> <strong>and</strong> leaf<br />
yellowing in India. Waika caused by a virus closely related to RTSV one <strong>of</strong> the viruses<br />
causing RTD was observed in southern Japan. RTD is also reported in Bangladesh, Sri<br />
Lanka, Nepal, southern China <strong>and</strong> Vietnam (Azzam <strong>and</strong> Chancellor, 2001).<br />
Economic Importance<br />
RTD is a serious virus disease <strong>of</strong> rice in South <strong>and</strong> Southeast Asia since 1960 (Ling<br />
<strong>and</strong> Tiongco, 1979). It has caused considerable losses in rice production in many<br />
countries in Asia. When the disease occurs in epidemic proportion, it can cause severe<br />
crop damage <strong>and</strong> reduce yield greater than 85% if cultivars grown are susceptible, <strong>and</strong><br />
infected at an early growth stages.<br />
It has been reported that in 1993 alone, the disease has devastated 80,000 hectares<br />
in India (Chowdhury, 1997), about 199,000 hectares in Indonesia (Hasanuddin et al.
1997) <strong>and</strong> 18,655 hectares in Malaysia, <strong>and</strong> the total damage was estimated to be nearly<br />
US$10 million (Chen <strong>and</strong> Jalil, 1997).<br />
In the Philippines, RTD is a continuing problem in Central Luzon, Bicol <strong>and</strong><br />
Mindanao, particulary in Cotabato <strong>and</strong> Davao (Baria, 1993). Serrano (1957) reported that<br />
the stunt disease, which is now considered as RTD, was extremely destructive in the<br />
1940’s throughout the major rice-growing regions <strong>of</strong> the Philippines <strong>and</strong> caused a yield<br />
reduction <strong>of</strong> 1.4 million tons equivalent to 30% overall loss annually. RTD epidemics<br />
were occasionally reported in 1960’s to 1989 by the Municipal Agricultural Offices<br />
throughout the country. Outbreak also occurred in Mindanao in 1993 to 1998 <strong>and</strong> in<br />
Negros Occidental in 1998, affecting 900 to 2,700 hectares <strong>and</strong> 700 hectares <strong>of</strong> rice fields,<br />
respectively.<br />
The Rice Tungro Viruses<br />
RTD is a composite disease caused by rice tungro bacilliform virus (RTBV) <strong>and</strong><br />
rice tungro spherical waikavirus (RTSV). RTBV belongs to the genus Badnavirus <strong>of</strong> the<br />
family Caulimoviridae <strong>and</strong> RTSV belongs to the genus Waikavirus <strong>of</strong> the family<br />
Sequiviridae (Mayo <strong>and</strong> Pringle, 1997). Virus particles <strong>of</strong> RTBV are bacilliform, 100-300<br />
nm in length <strong>and</strong> 30-35 nm in width. RTBV has a circular 8-kbp double str<strong>and</strong>ed DNA<br />
genome <strong>and</strong> a single capsid protein (Hull, 1996). Virus particles <strong>of</strong> RTSV are polyhedral,<br />
about 30 nm in diameter. RTSV has 12kb single str<strong>and</strong>ed RNA genome <strong>and</strong> three capsid<br />
proteins (Shen et al. 1993). It appears that RTBV depends on the helper components<br />
9
produced by RTSV for its own transmission <strong>and</strong> is mainly responsible for the severe<br />
RTD symptoms.<br />
Symptoms<br />
RTD symptoms are <strong>of</strong>ten confused with insect infestation <strong>and</strong> nutritional disorders<br />
such as nitrogen <strong>and</strong> iron deficiency (Satapathy et al. 2001). Rice plants infected with<br />
RTBV <strong>and</strong> RTSV show symptoms such as stunting, yellow or yellow-orange<br />
discoloration <strong>of</strong> the leaves, <strong>and</strong> reduced tillering (Hibino et al. 1978). Discolored leaves<br />
may show irregularly shaped dark-brown blotches. The leaves, especially the younger<br />
ones, may show striping or mottling <strong>and</strong> interveinal chlorosis. The first symptom on<br />
young seedlings at one week after inoculation is incomplete emergence <strong>of</strong> the youngest<br />
leaf <strong>and</strong> interveinal chlorosis (Sogawa, 1976). The plant gradually displays more<br />
prominent red discoloration, which usually develops from tip to the base <strong>of</strong> the leaves,<br />
interveinal stripes, <strong>and</strong> small brown irregular rusty spots (Hasanuddin <strong>and</strong> Hibino, 1989).<br />
Symptoms <strong>of</strong> interveinal <strong>and</strong> systemic necrosis usually appear in Oryza glaberrima <strong>and</strong><br />
O. barthii when they are infected with tungro viruses (Rao et al. 1978; Kobayashi et al.<br />
1992). Plants infected with RTBV alone show similar but milder symptoms than that<br />
caused by double infection. Plants infected with RTSV alone show no obvious symptoms<br />
except very mild stunting. In infected plants, RTBV is localized in the vascular bundles<br />
<strong>and</strong> RTSV in the phloem tissues (Sta Cruz et al. 1993).<br />
10
Transmission<br />
The rice tungro viruses; RTBV <strong>and</strong> RTSV are transmitted in a non-circulative,<br />
semi-persistent manner by Nephotettix virescens, N. nigropicutus, N. cincticeps, Recilia<br />
dorsalis, <strong>and</strong> some other Nephotettix spp. N. virescens is the most efficient vector <strong>of</strong> RTD.<br />
The other species are considered as minor vectors (Hibino, 1996). It is generally known<br />
that tungro viruses are not transmitted through any other routes such as pollen, seeds, soil<br />
<strong>and</strong> plant sap (Nish et al. 1975). Females <strong>of</strong> N. virescens transmit RTD more efficiently<br />
than males (Lamey et al. 1967; John, 1968). It is reported that the minimum acquisition<br />
<strong>and</strong> inoculation feeding periods for N. virescens are 5 min <strong>and</strong> 7 min, respectively (Ling,<br />
1972). N. virescens retains RTD infectivity for 2 to 6 days but loses infectivity after<br />
molting (Ling, 1972). Hibino et al. (1978) reported that green leafhopper (GLH) can<br />
retain RTBV for 4-5days <strong>and</strong> RTSV for 2-4days. They readily acquire RTSV from plants<br />
infected with RTSV alone, but do not acquire RTBV from plants infected with RTBV<br />
alone. GLH can acquire RTBV only when exposed to RTSV-infected plants before<br />
feeding on RTBV-infected plants (Hibino, 1983; Hibino et al. 1979). It is speculated that<br />
through feeding on plants infected with RTSV, GLH gain a helper component <strong>and</strong> thus<br />
the ability to acquire RTBV, <strong>and</strong> may retain the helper activity for the acquisition <strong>of</strong><br />
RTBV for seven days (Hibino et al. 1987). RTSV-infected plants may serve as the source<br />
<strong>of</strong> helper factor for transmission as early as 36 hr after inoculation <strong>and</strong> as the virus source<br />
72 hr after inoculation (Chowdhury et al. 1990). Dahal et al. (1990b) reported that<br />
viruliferous GLH usually feed from the xylem on leafhopper-resistant cultivars, <strong>and</strong><br />
11
transmit predominantly RTBV alone, whereas GLH feed mainly from the phloem on<br />
susceptible cultivars <strong>and</strong> can transmit both RTBV <strong>and</strong> RTSV efficiently.<br />
Disease Development in the Rice Field<br />
Several factors such as plant age, cultivars, abundance <strong>of</strong> initial virus inoculum,<br />
population <strong>of</strong> vector, cultural practices, <strong>and</strong> environmental factors may affect the<br />
development <strong>of</strong> RTD in the field (Shukla <strong>and</strong> Anjaneyulu, 1981b <strong>and</strong> 1981c; Anjaneyulu,<br />
1986; Cabunagan et al. 2001).<br />
Ling <strong>and</strong> Palomar (1966) reported a reduction in the percentage <strong>of</strong> rice plants<br />
showing symptoms with increasing plant age at the time <strong>of</strong> inoculation. Gibbs <strong>and</strong><br />
Harrison (1976) also reported plants are most susceptible to infection when young <strong>and</strong><br />
<strong>of</strong>ten most suitable as host for vectors, whereas plants tend to be more resistant to<br />
infection as they grow, thus, making multiplication <strong>of</strong> viruses <strong>and</strong> spread <strong>of</strong> disease<br />
slower during this stage.<br />
Extensive cultivation <strong>of</strong> susceptible varieties is considered as the main factor<br />
favoring the disease epidemics (Loevinsohn, 1984; Holt et al. 1996). Hibino (1996)<br />
pointed out that the continuous use <strong>of</strong> cultivars <strong>of</strong> the same genetic background in wider<br />
scale farming <strong>of</strong>ten led to RTD epidemic because <strong>of</strong> the establishment <strong>of</strong> favorable<br />
condition for the development <strong>of</strong> viruses <strong>and</strong> the survival <strong>of</strong> insect vectors.<br />
Lapis (1991) reported the variability <strong>of</strong> inoculum is one <strong>of</strong> the factors making<br />
RTD occurrence unpredictable. When the outside source <strong>of</strong> inoculum is higher<br />
12
particularly during the wet season, the effect <strong>of</strong> introduced sources <strong>of</strong> inoculum was more<br />
difficult to detect (Thresh, 1976). Moreover, virus transmission increased as the number<br />
<strong>of</strong> source plants increased (Chowdhury et al. 1994).<br />
The dispersal <strong>of</strong> the RTD by the vector seems to be also affected by the levels <strong>of</strong><br />
humidity (Kondaiah et al., 1976). RTD incidence is generally higher during the wet than<br />
during dry season, although it is unclear if this is due to a higher number <strong>of</strong> GLH<br />
(Tiongco et al. 1993).<br />
The levels <strong>of</strong> vector population may correlate with those <strong>of</strong> RTD incidence. It was<br />
reported that the chance for RTD to spread in the field was on average highest at 6 weeks<br />
after transplanting when the first generation large nymphs <strong>of</strong> N. virescens were most<br />
abundant (Suzuki et al. 1992). In another study conducted by Savory et al. (1993) showed<br />
that in RTD-endemic area, the higher RTD incidence was associated with increasing<br />
vector populations <strong>and</strong> proportion <strong>of</strong> viruliferous vector. However, high levels <strong>of</strong> vector<br />
population do not always result in high incidence <strong>of</strong> RTD. Dispersal <strong>of</strong> plant viruses by<br />
insects is also affected by other biological factors such as the number <strong>of</strong> virus inoculum<br />
sources, the number <strong>and</strong> activity <strong>of</strong> viruliferous insects, the readiness for insect to<br />
become viruliferous, the length <strong>of</strong> time for which the insect remains viruliferous, <strong>and</strong> the<br />
susceptibility <strong>of</strong> host plants (Lapis, 1991).<br />
Cultural practices adapted by farmers <strong>of</strong>ten influence RTD epidemics. Multiple<br />
rice cropping over large areas provides a continuous source <strong>of</strong> plant hosts <strong>and</strong> enables the<br />
year-round development <strong>of</strong> insect pests such as GLH, plant hopper, stem borer, <strong>and</strong> leaf<br />
holder (Litsinger, 1989). The planting <strong>of</strong> single cultivar in the field <strong>of</strong>ten triggers the<br />
13
apid progress <strong>of</strong> disease infection. Other adjustable cultivation practices like crop<br />
spacing <strong>and</strong> transplanting timing also may affect the level <strong>of</strong> RTD. The rate <strong>of</strong> disease<br />
spread was slower in closer spacing than under wider spacing (Shukla <strong>and</strong> Anjaneyulu,<br />
1981b <strong>and</strong> 1981c). In an asynchronously planted field, RTD at harvest correlated<br />
positively with the incidence at 6-10 weeks after transplanting (Hasanuddin et al. 1999).<br />
The result <strong>of</strong> Savary et al. (1993) showed that the low RTD incidence was associated<br />
with either very early or very late planting, whereas high RTD incidence was associated<br />
with intermediate planting. Meanwhile, the studies <strong>of</strong> Chancellor et al. (1996) showed<br />
that RTD incidence was much higher in late plantings than in early plantings.<br />
Disease Management<br />
The occurrence <strong>of</strong> RTD tends to be more serious in areas where farmers adopt<br />
asynchronous <strong>and</strong> continuous cropping pattern. Although the incidence <strong>of</strong> RTD can be<br />
managed to a certain level by various cultural practices to disrupt the disease cycle<br />
(Othman et al. 1999; Sama et al. 1991), the deployment <strong>of</strong> varieties resistant against<br />
tungro viruses has been considered most effective (Koganezawa <strong>and</strong> Cabunagan, 1997).<br />
Under field condition the cause <strong>of</strong> RTD is not easily observable <strong>and</strong> the disease is<br />
difficult to predict <strong>and</strong> control. Hence farmers regarded RTD as a serious threat in rice<br />
production, even in areas without much experience <strong>of</strong> RTD outbreaks. The use <strong>of</strong><br />
resistance varieties <strong>and</strong> the application <strong>of</strong> pesticides to control the insect vectors believed<br />
to carry tungro viruses are most common control measures among farmers (Warburton et<br />
14
al. 1996). However, the complex nature <strong>of</strong> RTD <strong>and</strong> the unpredictability <strong>of</strong> its outbreak<br />
have presented farmers with difficult control decisions, compounded by the absence <strong>of</strong><br />
reliable disease management strategies (Chancellor, 1996).<br />
Cultural control such as synchronized cropping, <strong>and</strong> planting time adjustment are<br />
some <strong>of</strong> the methods that have been recommended to prevent the incidence <strong>of</strong> RTD<br />
Though these approaches have an important role in the disease management, there are<br />
some limitations as to their use, for instance synchronized cropping is very difficult to<br />
implement due to difficulty in water control <strong>and</strong> labor shortage. The choice <strong>of</strong> appropriate<br />
planting time when vector population is low also seems to be not applicable due to a shift<br />
in the pattern <strong>of</strong> vector population growth that was observed in Indonesia following a<br />
change in planting dates (Sama et al. 1991).<br />
Unlike fungal pathogens or insect pests, there is virtually no chemical treatment for<br />
RTD since viruses rely heavily on host components for their multiplication, thus any<br />
chemical treatment with antiviral activity would be likely to have anti-host activity as<br />
well (Fraser, 2000). Hence chemical treatment in RTD control is directed mainly to insect<br />
vectors. However, this approach <strong>of</strong>ten may not be effective because <strong>of</strong> the time lapse<br />
between the insecticide application <strong>and</strong> the eradication <strong>of</strong> vectors from field, which may<br />
give the vectors enough time to transmit the viruses. Besides, farmers in developing<br />
countries usually can hardly afford pesticides, <strong>and</strong> the application <strong>of</strong> any insecticide is not<br />
a sound choice due to ecological concerns. There is, therefore, a continued interest for<br />
natural resistance since it is an affordable yet efficient way to fight diseases (Keller et al.<br />
2000).<br />
15
Resistance to virus in plants could be considered as a form <strong>of</strong> biological control<br />
since it involves various biological interactions between host plants <strong>and</strong> infecting viruses.<br />
The resistance may operate hypothetically, when it interferes in any steps <strong>of</strong> viral<br />
pathogenesis: inhibition <strong>of</strong> infection establishment, suppression <strong>of</strong> replication (Keller et<br />
al. 1998), blockage <strong>of</strong> virus movement <strong>and</strong> transport (Albar et al. 2003; Carrington et al.<br />
1996), <strong>and</strong> suppression <strong>of</strong> symptom expression (Fraser, 1990; Hull, 2002; Valkonen,<br />
2002). In RTD pathosystem, the target <strong>of</strong> resistance may be vector, RTBV, RTSV, or<br />
their combinations.<br />
Use <strong>of</strong> resistant variety is one <strong>of</strong> the most important <strong>and</strong> economical components<br />
<strong>of</strong> RTD management strategy. Plant breeders have long recognized the need for varieties<br />
resistant to RTD <strong>and</strong> its vectors, For example, most breeding lines <strong>of</strong> rice produced in<br />
IRRI since 1969 had at least one parent with resistance to GLH. Therefore, all varieties<br />
developed in IRRI except IR20 are rated as having GLH resistance at the time <strong>of</strong> release<br />
(Khush et al. 2004). The major donors <strong>of</strong> the GLH resistance trait were Ptb 18, Gam Pai<br />
30-12-15, <strong>and</strong> Ptb 33. Such rice cultivars show adequate resistance to the vector <strong>and</strong> may<br />
be able to escape RTD in the field under light to moderate disease pressure (Hibino <strong>and</strong><br />
Cabauatan, 1987). The vector resistant varieties helped to minimize yield losses that are<br />
likely to have occurred because <strong>of</strong> RTD. Despite such efforts, the breakdown <strong>of</strong> vector<br />
resistance was <strong>of</strong>ten observed after a few consecutive seasons <strong>of</strong> intensive cultivation <strong>of</strong><br />
formerly resistant varieties (Dahal et al. 1990a), indicating that resistance to the vector<br />
may not be durable. Therefore it is rather desirable to combine vector <strong>and</strong> virus resistance<br />
to confer durable resistance to RTD on plants.<br />
16
Resistance to Rice Tungro Viurses<br />
During 1980s, a change in the strategy for development <strong>of</strong> RTD resistant varieties<br />
was made to incorporate resistance to the viruses in addition to GLH resistance. Although<br />
the sources for complete resistance to RTBV have not been found in rice germplasm,<br />
several varieties were found to be tolerant to RTBV (Imbe et al. 1995; Cabunagan et al.<br />
1996). Tolerant varieties can have lesser degree <strong>of</strong> symptom expression, while allowing<br />
virus to multiply <strong>and</strong> spread (Fraser, 2000). Cultivars originating from Indonesia such as<br />
Balimau Putih <strong>and</strong> Utri Merah express mild symptoms when infected with both RTBV<br />
<strong>and</strong> RTSV (Hasunuddin <strong>and</strong> Hibino, 1989). It was reported that 3 accessions each <strong>of</strong> wild<br />
rice species O. rufipogon <strong>and</strong> O. <strong>of</strong>ficinalis, <strong>and</strong> 1 accession <strong>of</strong> O. ridleyi were tolerant to<br />
RTBV infection, <strong>and</strong> the tolerance dose not seem to depend on vector resistance. These<br />
wild rice species have been considered as useful resources for developing RTD resistant<br />
variety (Kobayashi et al. 1993). Cytological study indicated that the number <strong>of</strong> cells<br />
infected with RTBV or RTSV was found to be less in these tolerant cultivars/species than<br />
in an RTD susceptible variety Taichung Native 1 (TN1). The tolerance in these varieties<br />
as observed having low concentration <strong>of</strong> RTBV could be attributed to fewer susceptible<br />
cells <strong>and</strong> lower levels <strong>of</strong> RTBV multiplication. Breeding for tolerance which may reduce<br />
yield loss under severe infection pressure is important in RTD management. When<br />
tolerance to RTBV is integrated with RTSV resistance, it might become an ideal solution<br />
to control for RTD (Anjaneyulu, 1996).<br />
17
Many IRGC accessions have been reported as resistant to RTSV infection (Hibino<br />
et al. 1990; Koganezawa <strong>and</strong> Cabunagan, 1997). RTSV is important because it is<br />
required for the transmission by leafhopper vectors <strong>of</strong> RTBV (Hibino et al. 1979; Hibino<br />
<strong>and</strong> Cabunagan, 1986). RTSV could occur as an independent disease (Bajet et al. 1986)<br />
<strong>and</strong> capable <strong>of</strong> causing yield loss <strong>of</strong> as much as 40% on susceptible varieties (Hasanuddin<br />
<strong>and</strong> Hibino, 1989). The deployment <strong>of</strong> varieties with RTSV resistance is likely to have a<br />
significant effect in the reduction <strong>of</strong> RTD spread as suggested by Hibino et al. (1988) <strong>and</strong><br />
subsequently demonstrated by Cabunagan et al. (1990), <strong>and</strong> Satapathy, et al. (1997).<br />
RTD infections in RTSV resistant IR26 were at r<strong>and</strong>om whereas, it occurs in patches in<br />
RTSV susceptible IR64.<br />
Varietal Mixtures as RTD Management Option<br />
New approaches <strong>and</strong> strategies that are more effective <strong>and</strong> durable than those now<br />
available are needed so that crop st<strong>and</strong>s may acquire some <strong>of</strong> the stability <strong>and</strong> resistance<br />
<strong>of</strong> natural vegetation (Thresh, 1982). Host needs to be diverse particularly in the form <strong>of</strong><br />
mixtures (Wolfe et al. 1981) so that they could withst<strong>and</strong> the pressure from virus<br />
genotype with matching virulence (Thresh, 1982).<br />
Wolfe (1985) has reviewed the importance <strong>of</strong> diversity (multilines <strong>and</strong> mixtures)<br />
for controlling diseases <strong>of</strong> economic plants <strong>and</strong> Browning (1989) has established a clear<br />
rationale for the use <strong>of</strong> diversity for controlling disease.<br />
18
Recently, host diversity is gaining attention worldwide specially its long term<br />
effect in managing cereal disease. One important factor in using genotype mixture <strong>of</strong><br />
agricultural crops is to attempt to reduce the selection pressure for pathogens that can<br />
overcome valuable forms <strong>of</strong> disease resistance in crop plants (Garrett <strong>and</strong> Mundt, 1999).<br />
Further, a group <strong>of</strong> crop species at scattered sites may restrict the build-up <strong>of</strong> pests <strong>and</strong><br />
diseases <strong>and</strong> enable the crop to escape severe damage (Harlan, 1976). It may also reduce<br />
the risk <strong>of</strong> yield loss caused by abiotic <strong>and</strong> biotic stress by taking advantage <strong>of</strong> yield<br />
compensation occurring in mixtures grown in variable environments (Mundt, 1999).<br />
With most obligate pathogens, disease was less in mixtures than the mean <strong>of</strong> their<br />
components in monoculture (Smithson et al. 1996). The decrease in powdery mildew<br />
(Erysiphe graminis f. sp. hordei) severity in barley mixtures over their component means<br />
ranged from 8.9% to 80% (Kolster et al. 1989), the decrease in stripe rust (Puccinia<br />
striiformis) severity in wheat mixtures over their component means ranged from 13% to<br />
97% (Finckh <strong>and</strong> Mundt, 1992), the decrease in blast (Pyricularia oryzae) severity in rice<br />
mixtures ranged from 50% (Chin et al., 1982) to 66% (Bonman et al. 1986), <strong>and</strong> the<br />
decrease in the severity <strong>of</strong> several bean diseases including rust (Uromyces<br />
appendiculatus), angular leaf spot (Phaseoisariopsis griseola), anthracnose<br />
(Colletotrichum lindemuthianum) <strong>and</strong> halo-blight (Pseudomonas syringae pv.<br />
phaseolicola) in bean mixtures over their components reached 50%, 12.3%, 6.8% <strong>and</strong><br />
16.4%, respectively (Mundt <strong>and</strong> Leonard, 1986; Pyndji <strong>and</strong> Trutmann, 1992).<br />
Successful genetic diversification experiment was conducted in China. The result<br />
showed that the infection <strong>of</strong> the highly susceptible glutinous rice varieties, Huangkenuo<br />
19
<strong>and</strong> Zinuo, with blast was significantly reduced when inter-planted with the generally<br />
resistant indica hybrid varieties, Xianyou 63 or Xianyou 22 (Zhu et al., 2000).<br />
Much effort has been made to investigate mixed cropping systems, especially with<br />
respect to the ecological processes involved in crop performance (Geno <strong>and</strong> Geno, 2001).<br />
The relation between crop or cultivar mixtures <strong>and</strong> disease severity has been investigated<br />
extensively, but has focused mostly on disease caused by windborne pathogens <strong>and</strong> foliar<br />
pathogens <strong>of</strong> small grains (Mundt, 2002). Effects <strong>of</strong> mixed cropping on soilborne<br />
pathogens have been described less frequently (Abadie et al. 1998; Autrique <strong>and</strong> Pots,<br />
1987). For example, Burdon <strong>and</strong> Chilvers (1976) showed reduced spread <strong>of</strong> Pythium sp.<br />
in a garden cress–ryegrass mixture <strong>and</strong> Vilich (1993) reported a reduction in disease<br />
severity <strong>of</strong> soilborne pathogens in barley–wheat mixtures. In these experiments, disease<br />
suppression was investigated for pathogens specific to one or both <strong>of</strong> the crops included<br />
in the mixed cropping. Study on the effect <strong>of</strong> mixed cropping for the soilborne diseases<br />
(non-specific pathogen) was conducted by Hiddink et al. (2005). Their results showed<br />
that mixed cropping <strong>of</strong> soil with Brussels sprouts <strong>and</strong> barley or with triticale <strong>and</strong> white<br />
clover did not enhance disease suppression <strong>of</strong> soil to three soil-borne diseases<br />
Rhizoctonia solani, Fusarium oxysporum f. sp. lini, <strong>and</strong> Gaeumannomyces graminis var.<br />
tritici.<br />
While the mixtures <strong>of</strong> diverse hosts appeared effective in disease control, the<br />
mechanisms <strong>of</strong> disease reduction are still largely unclear (Burdon et al. 1980). It was<br />
suggested that the barrier effects <strong>of</strong> resistant plants (Trenbath, 1977) <strong>and</strong> the reduction in<br />
20
density <strong>of</strong> susceptible plants per se (Brown, 1975) might contribute to the disease<br />
suppression in variety mixtures.<br />
Garrett <strong>and</strong> Mundt (1999) stressed that host-diversity effects could be influenced<br />
by the spatial pattern <strong>of</strong> initial inoculum. Even if a susceptible variety were present, the<br />
spread <strong>of</strong> a disease was slow when an initial inoculum was likewise low. Studies on<br />
Puccinia coronata on oats showed that mixtures <strong>of</strong> large sizes <strong>of</strong> hosts <strong>of</strong> the same<br />
genotypes can produce a large decrease in disease when the disease was focally<br />
distributed. This suggests that the disease spreads from a small number <strong>of</strong> inoculum<br />
sources rather than from those more evenly dispersed throughout fields at the beginning<br />
<strong>of</strong> an epidemic (Mundt <strong>and</strong> Browning, 1985; Mundt <strong>and</strong> Leonard, 1985).<br />
Wolfe (1984) noted that resistant plants interfere with the movement <strong>of</strong> inoculum<br />
between susceptible individuals, <strong>and</strong> that induced resistance or cross-protection may also<br />
reduce disease development. Burdon (1987) explained that mixing <strong>of</strong> individuals with<br />
different levels <strong>of</strong> resistance initiates a complex series <strong>of</strong> interconnected changes which<br />
affect the ability <strong>of</strong> pathogens to survive <strong>and</strong> reproduce <strong>and</strong>, in consequence, disease<br />
development.<br />
There have been very few studies regarding the effects <strong>of</strong> variety mixtures on<br />
diseases caused by viruses. The outcome <strong>of</strong> mixtures on viral diseases may be<br />
complicated by the level <strong>of</strong> abundance <strong>and</strong> the behavior characteristics <strong>of</strong> vector, since<br />
these usually greatly affect the efficacy <strong>of</strong> the transmission the pathogen (Power, 1990).<br />
Power (1991) found that a 1:1 mixture <strong>of</strong> a susceptible <strong>and</strong> resistant oat (Avena<br />
sativa) cultivars reduced the incidence <strong>of</strong> yellow dwarf to approximately the level<br />
21
observed in the resistant component for all three years <strong>of</strong> the study. The incidence <strong>of</strong><br />
wheat soil-borne mosaic virus was reduced by 33.2 <strong>and</strong> 39.8%, respectively, in 1:1 <strong>and</strong><br />
1:3 mixtures <strong>of</strong> susceptible <strong>and</strong> resistant winter cultivars. Under field condition, the<br />
resistant cultivar did not produce viruliferous zoospores <strong>of</strong> the soil-borne vector (Hariri et<br />
al. 2001). Therefore, the presence <strong>of</strong> the resistant cultivar in the mixture may have<br />
reduced virus transmission between susceptible plants in the mixture (Mundt, 2002).<br />
Homogeneous planting <strong>of</strong> cultivars which may have resistance against viruses <strong>and</strong><br />
vectors over large areas could facilitate them to adapt to such cultivars more easily.<br />
Consequently, the use <strong>of</strong> heterogeneous crop populations was proposed <strong>and</strong> eventually<br />
practiced. Diversity in such heterogeneous environment has been considered to be an<br />
essential characteristic promoting balanced polymorphism in natural ecosystems<br />
(Browning, 1974). Although the use <strong>of</strong> resistant varieties is obviously preferred, the<br />
effectiveness may decline after continuous use (Truong et al. 1999). The genetic<br />
uniformity <strong>of</strong> varieties is very crucial in the development <strong>of</strong> RTD epidemics (Chin <strong>and</strong><br />
Wolfe, 1981), since it is highly likely that crop st<strong>and</strong>s that contain more than one species<br />
<strong>of</strong> plant affect virus spread. The mixing <strong>of</strong> crops containing those that are not compatible<br />
to viruses <strong>and</strong> vectors prevalent in the area may allow virus to spread more slowly in such<br />
mixed crops than in single crop species (Gibbs <strong>and</strong> Harrison, 1976). Aphids in cultivar<br />
mixtures showed higher movement rates <strong>and</strong> shorter tenure times than aphids in pure<br />
st<strong>and</strong>s. This may have reduced transmission rates for this virus, because an aphid must<br />
feed for several hours to inoculate a host plant (Power, 1991).<br />
22
Besides their effect in controlling diseases, mixtures have been shown to stabilize<br />
<strong>and</strong> sometimes increase yields (Finckh <strong>and</strong> Wolfe, 1997). In the U.S., more than 100,000<br />
hectares <strong>of</strong> wheat cultivar mixtures are grown for protection against yield losses <strong>and</strong> to<br />
increase yield stability in general (Finckh et al. 1999). <strong>Variety</strong> mixtures help to buffer the<br />
system against a range <strong>of</strong> environmental stresses <strong>and</strong> show highly stable yield (Chin <strong>and</strong><br />
Husin, 1982). The experiments on blast by Ashizawa et al. (1999) <strong>and</strong> Bonman et al.,<br />
(1986) showed that yields <strong>of</strong> mixtures were larger than the means <strong>of</strong> pure st<strong>and</strong>s <strong>and</strong> the<br />
yield increase due to mixing was high under severe epidemic. A mixed population<br />
provided guaranteed yield.<br />
Importance <strong>of</strong> Farmers’ <strong>Perception</strong> Survey<br />
Technology is one <strong>of</strong> the resources for agricultural production <strong>and</strong> alleviates<br />
various physical, economic <strong>and</strong> social constraints. It can be reached by farmers through<br />
technology transfer. Technology transfer refers to the general process <strong>of</strong> moving<br />
information <strong>and</strong> skills from information or knowledge generators such as research<br />
laboratories <strong>and</strong> universities to clients such as farmers (Bozeman, 2000). The outcome <strong>of</strong><br />
new technology transfer depends on the farmers’ adoption <strong>and</strong> willingness to bring this<br />
into practice, <strong>and</strong> further diffusion into the community. Therefore, it is important to know<br />
how farmers perceive newly developed technologies for better underst<strong>and</strong>ing <strong>of</strong> factors<br />
affecting their decision to adopt or not. <strong>Perception</strong> acts as a filter through which new<br />
23
observations are interpreted. The farmer’s choice <strong>of</strong> action will depend on his evaluation<br />
<strong>of</strong> the technology in terms <strong>of</strong> his own personal perspective.<br />
If scientists have to work with farmers to improve crop production <strong>and</strong> crop<br />
protection, they should recognize farmers’ constraints <strong>and</strong> their existing technical<br />
knowledge (Kenmore, 1991). Farmers in developing countries have substantial difficulty<br />
in managing plant diseases, since their underst<strong>and</strong>ing <strong>of</strong> disease processes is limited.<br />
Consequently disease management in developing countries is more <strong>of</strong>ten likely to be<br />
ineffective (Nagaraju et al. 2002; Nelson et al. 2001; Bentley <strong>and</strong> Thiele, 1999).<br />
A farmer survey is an important process to gather data necessary to assess the<br />
needs <strong>of</strong> intended beneficiaries, their knowledge level <strong>and</strong> perceptions for problems, their<br />
constraints in dealing with the problem, <strong>and</strong> their attitudes <strong>and</strong> practices to manage the<br />
problems (Escalada <strong>and</strong> Heong, 1997). Meanwhile farmer’s depth <strong>of</strong> knowledge about<br />
pests is related to the importance <strong>and</strong> visibility <strong>of</strong> the pest, because pathogens are not<br />
usually easily seen, thus it is difficult for farmers to account for the causes <strong>of</strong> diseases<br />
(Bentley <strong>and</strong> Thiele, 1999).<br />
In a survey conducted by Warburton et al. (1997) they found out that, farmers in<br />
the Philippines are aware that varieties have differing resistance or susceptibility to RTD<br />
<strong>and</strong> majority would change to another variety to prevent RTD from occurring again. In<br />
“hotspot” areas or in areas that experience outbreak <strong>of</strong> RTD farmers give importance to<br />
variety resistance <strong>and</strong> is used as one <strong>of</strong> the criteria for selection <strong>of</strong> varieties to plant. But<br />
most farmers are not aware that continuous planting <strong>of</strong> resistant varieties, especially those<br />
24
esistant to vectors may results in the breakdown <strong>of</strong> their resistance (Dahal et al. 1990a)<br />
<strong>and</strong> the emergence <strong>of</strong> new strains <strong>of</strong> tungro viruses (Cabauatan et al. 1995).<br />
Farmers’ Preference for Grain Quality<br />
IR64 released by IRRI in 1985 is preferred among the farmers, millers <strong>and</strong><br />
consumers in the Philippines because <strong>of</strong> its good eating <strong>and</strong> milling qualities (Khush <strong>and</strong><br />
Virk, 2005), however, this variety is highly susceptible to RTD <strong>and</strong> not recommended for<br />
planting in RTD endemic areas <strong>of</strong> the Philippines since 1994 (Anonymous, 1994).<br />
Rice is normally consumed after it has been milled <strong>and</strong> cooked without<br />
undergoing any other processing that could improve quality. While millers prefer<br />
varieties with high milling <strong>and</strong> head-rice recovery, consumers prefer varieties with good<br />
eating quality. As in other countries where rice is grown <strong>and</strong> consumed, in the Philippines<br />
consumer acceptance determine the commercial success <strong>of</strong> a newly released variety<br />
(Merca et al. 1978).<br />
Grain quality includes not only the traditional physical <strong>and</strong> visual properties <strong>of</strong> the<br />
rice grain but also cooked rice texture as indexed by apparent amylose content, alkali<br />
spreading value (index <strong>of</strong> gelatinization temperature), gel consistency, actual texture<br />
measurement <strong>of</strong> cooked grain <strong>and</strong> protein content (nutritional value) (Juliano <strong>and</strong><br />
Duff,1991).<br />
25
Plant Materials<br />
MATERIALS AND METHODS<br />
Two test varieties Matatag 9 (IR73885-1-4-3-2-1-6) released by IRRI as a stop<br />
gap rice tungro disease (RTD) resistant variety in the Philippines in 2002 (Khush et al.<br />
2004), <strong>and</strong> IR64 (IR18348-36-3-3, released in 1985) which is susceptible to RTD <strong>and</strong><br />
only tolerant to green leafhopper (GLH) were used in this study. Taichung Native1 (TN1)<br />
<strong>and</strong> IR62 (IR13525-43-2-3-1-3-2, released in 1984) were used as susceptible <strong>and</strong> resistant<br />
check varieties, respectively.<br />
Insect Vector<br />
The colony <strong>of</strong> GLH was maintained on susceptible variety, TN1 in 55 x 55 x 25 cm<br />
metal cages. Plants <strong>of</strong> TN1 (25 to 35-day old) were used for feeding <strong>and</strong> egg laying up to<br />
the adult stage <strong>of</strong> the insects. To generate the new population, the insects were introduced<br />
into the insect cage containing pots <strong>of</strong> feeding plants <strong>and</strong> left there for two days to allow<br />
them to lay eggs. Then the plants were transferred to other cages for egg incubation <strong>and</strong><br />
nymph hatching. Supply <strong>of</strong> fresh feeding plants to these cages was done every other day.<br />
Viruliferous GLH were prepared from newly emerged population by feeding on plants<br />
infected with RTSV/RTBV for 4 days.
Viruses<br />
Inocula for RTSV <strong>and</strong> RTBV were maintained by successive transmission <strong>of</strong><br />
viruses in TN1. Source plants were tested for the presence <strong>of</strong> RTSV <strong>and</strong> RTBV by<br />
ELISA before acquisition feeding access by GLH.<br />
Assay Procedure for RTBV <strong>and</strong> RTSV Infection<br />
Inoculated plants were evaluated for infection with RTBV or RTSV by ELISA<br />
(Bajet et al. 1985 <strong>and</strong> Cabunagan et al. 1993) using IgG antibody purified from<br />
polyclonal antisera to purified RTBV or RTSV (Cabauatan <strong>and</strong> Hibino, 1988). Polyvinyl<br />
chloride microtiter plates (96-well, flat-bottomed, Becton Dickinson Labware, USA)<br />
were coated separately each with 95µl <strong>of</strong> 0.05 M sodium carbonate coating buffer (pH<br />
9.6) containing 1.5µg/ml <strong>of</strong> IgG against RTBV or RTSV. The IgG-coated plates were<br />
incubated at 37 ◦ C for 3-4 hours. The plates were then washed using 0.02M phosphate<br />
buffer (pH 7.4) containing 0.15M NaCl <strong>and</strong> 0.05% Tween 20 (PBS-T) 6 times for 5 min<br />
each. Leaf samples were homogenized in PBS-T at 1:10 sap dilution using a leaf <strong>and</strong> bud<br />
press apparatus (Erich Pollahne), then 95µl sap from each sample was loaded into each<br />
well <strong>of</strong> the microtiter plate. The plates were incubated at 4 ◦ C overnight <strong>and</strong> then washed<br />
using PBS-T as described above. Extracts from healthy plants <strong>and</strong> RTD infected plants<br />
were used as negative <strong>and</strong> positive controls, respectively. Ninety five µl <strong>of</strong> alkaline<br />
phosphatase conjugated-IgG in PBS-T (1 μg/ml) was added to the wells, incubated at<br />
27
37◦C for 3–4 h, <strong>and</strong> then washed using PBS-T. Ninety five µl <strong>of</strong> ρ-nitrophenyl phosphate<br />
(Sigma, USA) at 1 mg/ml in 10% diethanolamine was added to each well. Color reaction<br />
was allowed to take place for 30 minutes. The presence <strong>of</strong> RTBV or RTSV in leaf<br />
extracts was determined by measurement <strong>of</strong> the absorbance at 405 nm using an ELISA<br />
reader (BOSCH, Germany). Samples with absorbance values at 405 nm greater than 0.1<br />
were considered positive for RTBV or RTSV infection.<br />
Forced Test Tube Inoculation<br />
Biological Characterization <strong>of</strong> Resistance to<br />
Rice Tungro Disease in Matatag 9<br />
Forty 6-day old seedlings each <strong>of</strong> Matatag 9, IR64, <strong>and</strong> TN1 were used per<br />
treatment. Each seedling was placed in a test tube (18 mm x 150 mm) with cap<br />
containing small amount <strong>of</strong> water. The viruliferous GLH (1, 3, or 5 per tube) was<br />
introduced into the test tube <strong>and</strong> was confined for 24 hours. Inoculated seedlings were<br />
transplanted at 5 seedlings per pot <strong>and</strong> maintained in the greenhouse. Two youngest<br />
leaves were collected from each plant at 21 days after inoculation (DAI) for ELISA to<br />
evaluate the presence <strong>of</strong> viruses. The infection rates with virus were calculated using the<br />
following equation:<br />
Infection rate (%) = 100 x (Number <strong>of</strong> infected seedlings/number <strong>of</strong> inoculated seedlings)<br />
28
The experiment was repeated three times, <strong>and</strong> the rates <strong>of</strong> plants infected with<br />
RTBV, RTSV, <strong>and</strong> both RTSV <strong>and</strong> RTBV were calculated.<br />
Relative Quantification <strong>of</strong> RTBV <strong>and</strong> RTSV<br />
Preparation <strong>of</strong> samples. Three viruliferous GLH were introduced into each test<br />
tube containing a 6-day old seedling <strong>of</strong> Matatag 9 or IR64. GLH was allowed to feed on<br />
the seedlings for 24 hours. Inoculated seedlings were transplanted at 5 seedlings per pot<br />
<strong>and</strong> maintained in the greenhouse. Two youngest leaves per seedling were collected from<br />
20 plants per variety at 7, 14, 21, 28, 35, 42, <strong>and</strong> 49 DAI. All plants from which leaves<br />
are collected were kept until 60 DAI. At 60 DAI, two youngest leaves were collected<br />
again from each plant for ELISA to determine the presence <strong>of</strong> viruses.<br />
Optimum assay condition. In order to determine the optimum dilution rate for<br />
quantitative ELISA <strong>of</strong> tungro viruses, serially diluted extract from TN1 (internal control)<br />
Matatag 9, <strong>and</strong> IR64 infected with RTBV <strong>and</strong> RTSV were tested for their reactivity with<br />
antibody against tungro viruses. Sap samples were prepared from the leaves <strong>of</strong> 5<br />
individual plants <strong>of</strong> Matatag 9 <strong>and</strong> IR64 collected at 21 DAI. Each sample was diluted<br />
with extraction buffer at 10X, 20X, 30X, 50X, 100X, 200X, 400X, <strong>and</strong> 800X with three<br />
replications <strong>and</strong> 95 µl <strong>of</strong> diluted sap was placed into the wells <strong>of</strong> an ELISA plate. Each<br />
plate also included internal control samples diluted at 10, 50, 100, 200 <strong>and</strong> 400X. The<br />
absorbance at 405 nm <strong>of</strong> each sample was read at 15, 30, 45 <strong>and</strong> 60 min after addition <strong>of</strong><br />
29
the enzyme substrate. The average absorbance value from 5 samples with 3 replications /<br />
variety for each dilution rate was calculated <strong>and</strong> the optimum condition was determined.<br />
Characterization <strong>of</strong> virus replication. Samples collected from the leaves <strong>of</strong> 20<br />
plants per variety at 7, 14, 21, 28, 35, 42, <strong>and</strong> 49 DAI were tested by ELISA at 10X <strong>and</strong><br />
the optimum dilution to evaluate the relative accumulation levels <strong>of</strong> RTSV <strong>and</strong> RTBV.<br />
ELISA reading was done at 15, 30, 45 <strong>and</strong> 60 min after addition <strong>of</strong> the enzyme substrate.<br />
Samples from leaves <strong>of</strong> plants at 60 DAI were tested by ELISA at 10X dilution to<br />
determine actual infection rate <strong>of</strong> plants with the respective viruses.<br />
Biological Characterization <strong>of</strong> Matatag 9<br />
Resistance to Green Leafhopper<br />
GLH Preference <strong>and</strong> Tungro Viruses Transmission<br />
Pre-germinated seeds <strong>of</strong> Matatag 9 <strong>and</strong> IR64 were planted in 12-cm diameter pots<br />
with 5 seedlings per pot. Ten-day old seedlings were used for inoculation. Two pots, one<br />
planted with IR64 <strong>and</strong> another with Matatag 9 were placed together inside a plastic cage<br />
(Fig. 1). Inoculation was conducted inside the cage by introducing approximately 50<br />
viruliferous GLH from the center top <strong>of</strong> the plastic cage. The number <strong>of</strong> GLH that<br />
alighted on each potted plant was counted 0.5, 4, 8 <strong>and</strong> 24 hours after release. The<br />
counting <strong>of</strong> GLH was replicated 20 times. Insects were removed 24 hours after<br />
30
infestation from the test plants <strong>and</strong> the inoculated plants were maintained in the<br />
greenhouse.<br />
The number <strong>of</strong> alighted GLH were analyzed by Chi-square test to determine<br />
whether observed ratios <strong>of</strong> GLH attracted to resistant <strong>and</strong> susceptible plants deviated<br />
significantly from the no preference ratio <strong>of</strong> 1:1. Infection rates <strong>of</strong> tungro viruses were<br />
evaluated at 21 DAI by ELISA. Correlations between the number <strong>of</strong> alighted GLH <strong>and</strong><br />
the infection rates for RTBV, RTSV <strong>and</strong> both RTBV <strong>and</strong> RTSV, from each variety were<br />
examined.<br />
Fig. 1. Experimental set-up for settling preference test. Five 10-day old seedlings each <strong>of</strong><br />
Matatag 9 <strong>and</strong> IR64 planted in separate pots were placed inside a plastic cage.<br />
31
Adult GLH Longevity<br />
Twenty 6-day old seedlings each <strong>of</strong> Matatag 9, IR64, TN1 <strong>and</strong> IR62 were<br />
prepared. Each seedling was placed in a test tube with nylon mesh top. Ten newly<br />
emerged adult GLH <strong>of</strong> each sex were prepared for each test variety. Insects were<br />
introduced singly into the test tubes. Seedling was replaced whenever it showed severe<br />
necrosis. The longevity <strong>of</strong> GLH was taken as the number <strong>of</strong> days when the insect was<br />
found alive. This experiment was established in split-plot design with 3 replications.<br />
Mean <strong>of</strong> longevity was calculated for the 10 insects <strong>of</strong> each sex introduced into each<br />
variety using the following equation:<br />
Mean <strong>of</strong> Longevity = Total number <strong>of</strong> days that 10 insects live on each test variety/10<br />
Data analysis was done by the least significant difference (LSD) test for the<br />
means, <strong>and</strong> by the balanced analysis <strong>of</strong> variance (BAOV) included in IRRISTAT to<br />
evaluate the significances among varieties <strong>and</strong> between GLH sexes.<br />
Nymphal Mortality<br />
Ten 6-day old seedlings each <strong>of</strong> Matatag 9, IR64, TN1 <strong>and</strong> IR62 were singly<br />
placed in a test tube. Ten 2 nd -instar nymphs <strong>of</strong> GLH were then introduced into each <strong>of</strong> the<br />
tubes. The mouth <strong>of</strong> the test tube was closed with fine-mesh nylon screen for proper<br />
32
ventilation. The experiment was established in r<strong>and</strong>omized complete block design<br />
(RCBD) with 3 replications. The st<strong>and</strong>ard number <strong>of</strong> GLH (number <strong>of</strong> live insects) was<br />
counted at 6 hrs after infestation. Three days after infestation, the mortality <strong>of</strong> the GLH<br />
nymphs in each test tube was calculated using following equation, <strong>and</strong> mortality <strong>of</strong> the<br />
nymphs in all test tubes <strong>of</strong> each test material was averaged <strong>and</strong> used as the antibiosis<br />
rating.<br />
Mortality (%) = 100x (Number <strong>of</strong> insects that survived/ St<strong>and</strong>ard number)<br />
Data analysis was done by the LSD test <strong>and</strong> BAOV to evaluate the significances<br />
among varieties.<br />
Growth <strong>and</strong> Development <strong>of</strong> GLH Nymph<br />
Pre-germinated seeds <strong>of</strong> Matatag 9, IR64, TN1 <strong>and</strong> IR62 were singly sown in a<br />
pot (12 cm in diameter) covered with 10 x 60-cm cylindrical mylar cage with fine-mesh<br />
nylon screen top <strong>and</strong> side windows. Five 2 nd -instar GLH were introduced into the cage at<br />
21 days after sowing. The experiment was established in RCBD with 20 replications.<br />
Growth was measured by counting the number <strong>of</strong> nymphs that became adults <strong>and</strong> the<br />
time taken to reach the adult stage. The insect growth index for each variety was<br />
calculated as the ratio <strong>of</strong> the percentage nymphs becoming adults to the mean growth<br />
33
period in days (Saxena et al. 1974). Data analysis was done by the LSD test, <strong>and</strong> BAOV<br />
to evaluate the significances among varieties.<br />
Fig. 2. Experimental set-up for the test for growth <strong>and</strong> development <strong>of</strong> GLH nymph.<br />
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Performance Evaluation <strong>of</strong> Varietal Mixture Components<br />
Comparative Morphological <strong>and</strong> Yield<br />
Characteristics <strong>of</strong> Matatag 9 <strong>and</strong> IR64<br />
Forty seeds each <strong>of</strong> Matatag 9 <strong>and</strong> IR64 were prepared. Six day-old seedling <strong>of</strong><br />
Matatag 9 or IR64 was singly transplanted at 20cm x 20cm distance between plants with<br />
4 x 10 arrangements in the screenhouse. The condition for soil nutrition <strong>and</strong> soil water<br />
content was maintained uniform during the experiment. Height was measured at 15, 30,<br />
45, 60, 75, <strong>and</strong> 90 days after transplanting (DAT). Number <strong>of</strong> tillers was counted at 30<br />
<strong>and</strong> 60 DAT. Number <strong>of</strong> days to flowering <strong>and</strong> maturity were recorded. Final height from<br />
the bottom to top <strong>of</strong> longest panicles was measured at harvest time. Data on yield<br />
parameters such as number <strong>of</strong> productive tillers, panicle length, number <strong>of</strong> filled grain per<br />
panicle, grain size (width <strong>and</strong> length) <strong>and</strong> thous<strong>and</strong> grains weight (TGW) were obtained<br />
after harvest. The measurement <strong>of</strong> each yield parameter was done as follows:<br />
• Panicle length: 5 panicles per plant were measured<br />
• Number <strong>of</strong> filled grains per panicle: unfilled <strong>and</strong> filled grains from 3 panicles were<br />
counted.<br />
• Grain size: the width <strong>and</strong> the length <strong>of</strong> 100 hulled grains <strong>and</strong> 100 dehulled grains were<br />
measured.<br />
• Weight <strong>of</strong> 1000 grains: 1000 hulled grains <strong>and</strong> 1000 dehulled grains were weighed.<br />
This measurement was repeated five times.<br />
35
Data from each parameter were analyzed by BAOV <strong>and</strong> the LSD test. Plants at<br />
the outer row <strong>of</strong> the plot were excluded for data analysis.<br />
Effect <strong>of</strong> <strong>Mixed</strong> <strong>Planting</strong> <strong>of</strong> Resistant <strong>and</strong><br />
Susceptible Varieties on the Incidence <strong>of</strong> Rice Tungro Disease<br />
For the mixture treatments, seeds <strong>of</strong> the test varieties <strong>of</strong> Matatag 9 <strong>and</strong> IR64 were<br />
mixed thoroughly as shown in Table 1.<br />
Table 1. Ratios <strong>of</strong> seed mixtures used for mixed variety planting.<br />
TREATMENTS<br />
PERCENTAGE IN MIXTURE<br />
Matatag 9 IR64<br />
T1- (Pure st<strong>and</strong> Matatag 9) 100 0<br />
T2- (Mixture) 75 25<br />
T3- (Mixture) 50 50<br />
T4- (Mixture) 25 75<br />
T5- (Pure st<strong>and</strong> IR64) 0 100<br />
Tray Inoculation <strong>and</strong> Experimental Design<br />
Seeds were soaked overnight. Pre-germinated seeds were sown in a plastic tray<br />
(49cm x 37cm x 12.5cm) with 16 columns <strong>and</strong> 25 seeds per column. When seedlings<br />
were 10-days-old, the tray was placed inside a cage to inoculate plants with 3 or 10<br />
viruliferous GLH per seedling. GLH was allowed to feed on the seedling for 4 hours. All<br />
inoculations were conducted at the same time. Two leaves per plant were collected at 21<br />
36
DAI <strong>and</strong> checked for the presence <strong>of</strong> RTSV <strong>and</strong> RTBV by ELISA. The experiment was<br />
established in Split-Plot design with 3 replications.<br />
<strong>Mixed</strong> Seed <strong>Planting</strong> Layout<br />
Five different seed mixtures were prepared as treatment (Table 1). One tray was<br />
used per treatment. Twenty-five pre-germinated seeds were planted in columns 1 to 16.<br />
Fig. 3 shows an example <strong>of</strong> the planting layout for T2 (75:25 <strong>of</strong> Matatag 9 <strong>and</strong> IR64)<br />
mixture.<br />
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Fig. 3. Example <strong>of</strong> seed layout <strong>of</strong> T2 (75:25 for Matatag 9 <strong>and</strong> IR64) seed<br />
mixtures for tray inoculation: R indicates position <strong>of</strong> resistant variety,<br />
Matatag 9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety, IR64.<br />
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Interplanting (row mixture) Layout<br />
For T1 <strong>and</strong> T5 only Matatag 9 or IR64 was planted in all columns in a tray. For<br />
T2 mixture, every fourth column (e.g. columns 4, 8, 12, <strong>and</strong> 16) was planted with IR64,<br />
the rest were planted with Matatag 9 (Fig. 4). For T3 mixture, every second column (e.g.<br />
columns 2, 4, 6, 8, 10, 12, 14, <strong>and</strong> 16) was planted with IR64, the rest were planted with<br />
Matatag 9 (Fig. 5). For T4 mixture, columns 2, 3, 4, 6, 7, 8, 10, 11, 12, 14, 15, <strong>and</strong> 16<br />
were planted with IR64, the rest were planted with Matatag 9 (Fig. 6).<br />
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Fig. 4. Seed layout <strong>of</strong> T2 mixture. R indicates position <strong>of</strong> resistant variety,<br />
Matatag 9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety, IR64.<br />
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40<br />
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Fig. 5. Seed layout <strong>of</strong> T3 mixture. R indicates position <strong>of</strong> resistant<br />
variety, Matatag 9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety, IR64.
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Fig. 6. Seed layout <strong>of</strong> T4 mixture. R indicates position <strong>of</strong> resistant variety,<br />
Matatag 9 <strong>and</strong> S indicates position <strong>of</strong> susceptible variety, IR64.<br />
Rates <strong>of</strong> plants infected with RTBV, RTSV <strong>and</strong> both RTSV <strong>and</strong> RTBV were<br />
obtained from the respective treatments. Mixture effects on disease incidence were<br />
evaluated by the LSD test <strong>and</strong> BAOV in split plot design.<br />
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Serial Inoculation in Resistant <strong>and</strong> Susceptible Varieties<br />
Six-day old seedlings <strong>of</strong> test materials were placed individually in test tubes <strong>and</strong><br />
single viruliferous GLH was introduced <strong>and</strong> allowed to feed for 24 hours, then transferred<br />
to next rice seedling as scheduled in Table 2. The serial transmission was continued for 5<br />
days. Inoculated seedlings were transplanted at 5 seedlings per pot <strong>and</strong> maintained in the<br />
greenhouse. The infection rates <strong>of</strong> tungro viruses were evaluated at 21 DAI by ELISA.<br />
Table 2. Combination <strong>of</strong> Matatag 9 <strong>and</strong> IR 64 for serial transmission<br />
COMBIATION (C) 1 st DAY 2 nd DAY 3 rd DAY 4 th DAY 5 th DAY<br />
C.1 Matatag 9 Matatag 9 Matatag 9 Matatag 9 Matatag 9<br />
C.2 IR64 Matatag 9 IR64 Matatag 9 IR64<br />
C.3 Matatag 9 IR64 Matatag 9 IR64 Matatag 9<br />
C.4 IR64 IR64 IR64 IR64 IR64<br />
42
Farmers’ <strong>Perception</strong> Survey on the Use <strong>of</strong> <strong>Mixed</strong> <strong>Planting</strong><br />
Survey site description. The survey site was in Barangay Progreso <strong>of</strong> the Ajuy<br />
Municipality in the northeast part <strong>of</strong> the province <strong>of</strong> Iloilo, Philippines (Fig. 7). Ajuy is a<br />
4 th class municipality consisting <strong>of</strong> 34 barangays. Ajuy has a total l<strong>and</strong> area <strong>of</strong> 19,432 ha<br />
<strong>and</strong> 15,042 ha is agricultural l<strong>and</strong>.<br />
Survey procedure. A mini-survey by means <strong>of</strong> a questionnaire was conducted<br />
during the farmers’ forum on “Rice Tungro Disease Management” in Barangay Progreso,<br />
September, 2005. The half-day forum aimed to discuss with the farmers through<br />
exchanges <strong>of</strong> ideas <strong>and</strong> knowledge on RTD <strong>and</strong> its management <strong>and</strong> to introduce the new<br />
concept <strong>of</strong> varietal mixture for RTD management.<br />
The questionnaire used in the survey was almost the same as that used by<br />
Warburton et al. (1997) in a comprehensive survey <strong>of</strong> farmer’s perception about tungro in<br />
the Philippines. The only difference was that some questions were added to elicit<br />
farmers’ perception on the use <strong>of</strong> variety mixture as a management option for RTD. The<br />
questionnaire (Appendix 1) was distributed to about 50 r<strong>and</strong>omly selected farmers who<br />
are the members <strong>of</strong> the irrigator association in Ajuy, Iloilo. Respondents were first<br />
requested to fill up the questionnaires by themselves with a guidance <strong>of</strong> the Chief Officer<br />
<strong>of</strong> Research <strong>and</strong> Institutional Development (RAID) as needed.<br />
Questionnaire was aimed to evaluate the farmers’ socio-demographic<br />
characteristic, farming structure, perceptions <strong>of</strong> biotic constraints upon rice production,<br />
43
awareness <strong>of</strong> RTD <strong>and</strong> its control, <strong>and</strong> awareness <strong>of</strong> mixed variety planting. For each<br />
question, the percentage <strong>of</strong> farmers who gave the same answer was calculated. Those<br />
who did not respond to certain questions were excluded from the calculations. In cases<br />
where multiple responses were obtained, total sample size (52 farmers) was used.<br />
Fig. 7. The site (shaded red) <strong>of</strong> the survey in the municipality <strong>of</strong> Ajuy, Iloilo Province,<br />
Philippines<br />
44
Grain Quality Evaluation <strong>of</strong> <strong>Mixed</strong> Seed <strong>Planting</strong><br />
Rice grains. The rice grains used for evaluation were obtained from the crops<br />
grown during the dry season (DS) 2005 crop in Midsayap, Cotabato. The grains were<br />
sun-dried to 14% moisture content. About 1000 grams seeds were collected from the<br />
different treatments as shown in Table 1 for grain quality evaluation at the Grain Quality<br />
<strong>and</strong> Nutrition Research Center (GQNRC) in IRRI.<br />
Milling quality. About 125 gm <strong>of</strong> rough rice grains (paddy rice) were used for<br />
milling quality determinations. Rough rice grains were dehulled with a Satake Laboratory<br />
Sheller in two passes. The resulting brown rice was weighed to get the percentage <strong>of</strong><br />
hulls. The brown rice was milled in a MacGill Mill Number 2 for 30 seconds with the<br />
prescribed added weight (680 gm) on the pressure cover, followed by a second milling<br />
for another 30 seconds without the weight. About 13-14% <strong>of</strong> the outer layer <strong>of</strong> brown rice<br />
was removed through the milling process. The milled rice sample was collected in a jar or<br />
thick paper bag <strong>and</strong> sealed immediately. The rice was allowed to cool before weighing to<br />
minimize grain cracking during cooling. The weight <strong>of</strong> the total milled rice was recorded.<br />
Whole grains (head rice) were separated from the total milled rice with a rice-<br />
sizing device. The indentation size <strong>of</strong> the device depends on the grain size. Two plates<br />
<strong>of</strong> the same size were used for each run. The resulting head rice was weighed. The<br />
percentage <strong>of</strong> hulls <strong>of</strong> rough rice was calculated as follows:<br />
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Brown Rice (%) = Weight <strong>of</strong> brown rice x 100<br />
Weight <strong>of</strong> rough rice<br />
Total Milled Rice (%) = Weight <strong>of</strong> total milled rice x 100<br />
Weight <strong>of</strong> rough rice<br />
Head Rice (%) = Weight <strong>of</strong> head rice x 100<br />
Weight <strong>of</strong> rough rice<br />
Grain size, shape <strong>and</strong> appearance. Length, width <strong>and</strong> percent chalkiness <strong>of</strong><br />
milled rice were determined using the Cervitec 1625 Grain Inspector (Foss Tecator,<br />
Sweden) from five to ten grams sample <strong>of</strong> milled rice. Table 3-a <strong>and</strong> -b are the<br />
classification criteria used for evaluation. The obtained percentage <strong>of</strong> chalkiness was<br />
classified into five levels 1: 0-10%, 2: 10-25%, 3: 25-50%, 4: 50-75% <strong>and</strong> 5: >75%.<br />
Table 3-a. Classification for grain size<br />
SCALE SIZE CATEGORY LENGTH (mm)<br />
1 Very long More than 7.50<br />
3 Long 6.61 < 7.50<br />
5 Medium or intermedium 5.51 < 6.61<br />
7 Short Less than 5.51<br />
Table 3-b. Classification for grain shape<br />
SCALE SHAPE LENGTH/WIDTH RATIO<br />
1 Slender Over 3.0<br />
5 Medium 2.1 to 3.0<br />
9 Bold 2.0 or less then 2.0<br />
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Amylose content. The simplified procedure <strong>of</strong> Juliano (1971) was used for the<br />
amylose content analysis. Rice varieties were grouped on the basis <strong>of</strong> their amylose<br />
content into waxy (0-2%), very low (3-9%), low (10-19%), intermediate (20-25%) <strong>and</strong><br />
high (>25%).<br />
Gelatinization temperature (GT). The alkali digestibility test (Little et al. 1958)<br />
was used for estimating gelatinization temperature. Six grains <strong>of</strong> milled rice were<br />
incubated in 10 ml <strong>of</strong> 1.7% (0.3035 N) KOH at room temperature or 30°C for 23 hours<br />
<strong>and</strong> the degree <strong>of</strong> spreading was assessed according to the seven-point scale as shown in<br />
Table 4.<br />
Table 4. Numerical scale for gelatinization temperature (GT).<br />
Score Spreading Alkali digestion GT<br />
1 Kernel not affected Low High<br />
2 Kernel swollen Low High<br />
3 Kernel swollen; Collar complete or narrow Low or Intermediate High-intermediate<br />
4 Kernel swollen; Collar complete <strong>and</strong> wide Intermediate Intermediate<br />
5<br />
Kernel split or segregated; Collar complete <strong>and</strong><br />
wide Intermediate Intermediate<br />
6 Kernel dispersed; Merging with collar High Low<br />
7 Kernel completely dispersed <strong>and</strong> intermingled High Low<br />
Gel consistency. The pasting properties <strong>of</strong> milled rice were measured with a<br />
Rapid Visco Analyser RVA-4 (Newport Scientific, Sydney, Australia) according to the<br />
procedure <strong>of</strong> Cagampang et al. (1973). Rice flour (3 gm) was weighed directly into the<br />
aluminum RVA canister <strong>and</strong> mixed with 25ml <strong>of</strong> distilled water. The sample was held at<br />
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50 °C for 1 min, heated to 95 °C at a rate <strong>of</strong> 12 °C/min (i.e. in 3.75 min), held at 95 °C<br />
for 2.5 min, cooled to 50 °C at a rate <strong>of</strong> 12 °C/min <strong>and</strong> held at 50 °C for 2.5 min. Based<br />
on the results rice grains were categorized into three as follows:<br />
1. Very flaky rice with hard gel consistency (length <strong>of</strong> gel, 40 mm or less);<br />
2. Flaky rice with medium gel consistency (length <strong>of</strong> gel, 41 to 60 mm); <strong>and</strong><br />
3. S<strong>of</strong>t rice with s<strong>of</strong>t gel consistency (length <strong>of</strong> gel, more than 61 mm).<br />
48
Rates <strong>of</strong> Infection with RTSV <strong>and</strong> RTBV<br />
under Different Inoculum Levels<br />
RESULTS AND DISCUSSION<br />
Biological Characterization <strong>of</strong> Matatag 9<br />
Resistance to Rice Tungro Disease<br />
Less than 20% <strong>of</strong> Matatag 9 was infected with RTBV at 1 viruliferous GLH per<br />
seedling, whereas higher infections were observed in IR64 (58.5%) <strong>and</strong> TN1 (85.0%)<br />
(Fig. 8). For each test variety higher rates <strong>of</strong> infection with RTBV was observed with<br />
increased insect numbers <strong>and</strong> the infection rates between Matatag 9 <strong>and</strong> IR64 were<br />
considerably different. TN1 consistently showed highest infection. The rates <strong>of</strong> infection<br />
with RTSV, however, were considerably low compared to those with RTBV for all test<br />
varieties irrespective <strong>of</strong> the number <strong>of</strong> insects used for inoculation. The RTSV infection<br />
was higher in IR64 than in Matatag 9.<br />
Temporal Change in Apparent Rates <strong>of</strong><br />
Infection with RTSV <strong>and</strong> RTBV<br />
The rates <strong>of</strong> RTBV infection were higher in IR64 than in Matatag 9 regardless <strong>of</strong><br />
the time <strong>of</strong> sampling, although the rate <strong>of</strong> infection with RTBV in Matatag 9 inoculated at
Fig. 8. Infection rate <strong>of</strong> RTBV <strong>and</strong> RTSV in three varieties with 1, 3, <strong>and</strong> 5 viruliferous<br />
GLH by the forced test tube inoculation.<br />
50
6-days old is still considerably high (Table 5). RTBV infection rates at 60 DAI in<br />
Matatag 9 <strong>and</strong> IR64 inoculated at 6-days old were not much different from those<br />
examined at the time <strong>of</strong> initial sampling (Table 5). Mean RTSV infection rates in IR64<br />
<strong>and</strong> Matatag 9 inoculated at 6-days old were 70 <strong>and</strong> 26.4% at time <strong>of</strong> initial sampling,<br />
respectively <strong>and</strong> similar infection rates were observed at 60 DAI (Table 5).<br />
In order to underst<strong>and</strong> the reaction <strong>of</strong> tungro viruses in Matatag 9 <strong>and</strong> IR64 <strong>of</strong><br />
older age, the rates <strong>of</strong> RTBV <strong>and</strong> RTSV infection were also examined in plants<br />
inoculated at 21-days old. Mean RTBV infection rates <strong>of</strong> IR64 <strong>and</strong> Matatag 9 inoculated<br />
at 21-days old were 90.7 <strong>and</strong> 24.3% at time <strong>of</strong> initial sampling <strong>and</strong> 96.4 <strong>and</strong> 32.9% at 60<br />
DAI, respectively, Mean <strong>of</strong> RTSV infection rate in IR64 <strong>and</strong> Matatag 9 inoculated at 21-<br />
days old were 69.9 <strong>and</strong> 2.1% at time <strong>of</strong> initial sampling <strong>and</strong> 90.7 <strong>and</strong> 5.0% at 60 DAI,<br />
respectively (Table 6). No RTSV infections were detected in Matatag 9 at 7 DAI, 28<br />
DAI, 35 DAI, <strong>and</strong> 42 DAI when the plants were inoculated at 21-days old (Table 6).<br />
Relatively high RTSV infection rate was observed in IR64 at time <strong>of</strong> initial sampling as<br />
well as at 60 DAI (Table 6). Infection rate <strong>of</strong> RTSV was extremely low in Matatag 9<br />
regardless <strong>of</strong> the time <strong>of</strong> sampling when it was inoculated at 21-days old.<br />
Temporal Change in the Accumulation <strong>of</strong> Tungro Viruses<br />
Leaf samples <strong>of</strong> Matatag 9 <strong>and</strong> IR64 inoculated with both RTSV <strong>and</strong> RTBV at 6-<br />
days old were collected weekly for 7 weeks. The optimum dilution rate for relative<br />
quantification <strong>of</strong> RTBV accumulation was set at 50X based on a preliminary experiment<br />
51
Table 5. Infection rate (%) <strong>of</strong> RTBV <strong>and</strong> RTSV at time <strong>of</strong> initial sampling <strong>and</strong> at 60 DAI:<br />
6-day-old seedling<br />
RTBV INFECTION a<br />
RTSV INFECTION b<br />
Initial Sampling 60 DAI Initial Sampling 60 DAI<br />
Time <strong>of</strong> initial<br />
sampling M 9 c<br />
IR64 M 9 IR64 M 9 IR64 M 9 IR64<br />
7DAI - - - - 10 55 5 65<br />
14DAI 65 100 60 90 50 90 40 70<br />
21DAI 45 90 45 85 25 80 25 80<br />
28DAI 80 80 80 85 10 75 10 75<br />
35DAI 75 95 75 90 25 55 25 40<br />
42DAI 55 95 60 90 45 75 35 65<br />
49DAI 60 90 60 100 20 60 20 65<br />
MEAN 63.3 91.7 51.3 90 26.4 70 22.9 65.7<br />
a = Infection caused by RTBV alone plus double infection caused by RTBV <strong>and</strong> RTSV<br />
b = Infection caused by RTSV alone plus double infection caused by RTBV <strong>and</strong> RTSV<br />
c = Matatag 9<br />
52
Table 6. Infection rate (%) <strong>of</strong> RTBV <strong>and</strong> RTSV at time <strong>of</strong> initial sampling <strong>and</strong> at 60 DAI:<br />
21-day-old seedling<br />
RTBV INFECTION a<br />
RTSV INFECTION b<br />
Time <strong>of</strong> initial Initial Sampling 60 DAI Initial Sampling 60 DAI<br />
sampling M 9 c<br />
IR64 M 9 IR64 M 9 IR64 M 9 IR64<br />
7DAI 5 70 20 100 0 40 0 95<br />
14DAI 30 100 40 95 5 84.2 5 90<br />
21DAI 10 85 20 95 5 75 5 95<br />
28DAI 20 95 20 95 0 75 0 85<br />
35DAI 35 95 40 95 0 75 5 95<br />
42DAI 30 100 30 100 0 80 10 90<br />
49DAI 40 90 60 95 5 60 10 85<br />
MEAN 24.3 90.7 32.9 96.4 2.1 69.9 5 90.7<br />
a = Infection caused by RTBV alone plus double infection caused by RTBV <strong>and</strong> RTSV<br />
b = Infection caused by RTSV alone plus double infection caused by RTBV <strong>and</strong> RTSV<br />
c = Matatag 9<br />
53
using five 21 DAI samples <strong>of</strong> each test material. The levels <strong>of</strong> RTBV detected by ELISA<br />
fluctuated in Matatag 9, while those in IR64 slightly increased from 14 DAI up to 42 DAI<br />
<strong>and</strong> sharply increased at 49 DAI (Fig. 9). The relative titer <strong>of</strong> RTBV in Matatag 9 was<br />
constantly higher than that in IR64.<br />
Six-day old seedlings <strong>of</strong> test varieties were also inoculated with RTSV to evaluate<br />
the temporal changes in RTSV accumulation. Optimum dilution rate was determined as<br />
20X according to the results from a preliminary experiment. The level <strong>of</strong> RTSV in both<br />
test materials was lowest at 7 DAI, however, in Matatag 9 it drastically increased <strong>and</strong><br />
peaked at 14 DAI <strong>and</strong> decreased at 28 DAI, then slightly increased at 35 DAI <strong>and</strong> slightly<br />
decreased up to 49 DAI, while the levels <strong>of</strong> RTSV accumulation in IR64 drastically<br />
increased up to 21 DAI, <strong>and</strong> sharply decreased (Fig. 9). The maximum level <strong>of</strong> RTSV<br />
accumulation was higher in IR64 than in Matatag 9. It appeared that overall, the patterns<br />
<strong>of</strong> temporal changes in the accumulation <strong>of</strong> RTSV in the two varieties were similar,<br />
although the replication <strong>of</strong> RTSV may reach at the maximum level earlier in Matatag 9<br />
than in IR64.<br />
The temporal changes in virus accumulation were also examined in plants<br />
inoculated at 21-days old. The optimum dilution rate for evaluation was determined as<br />
30X. The level <strong>of</strong> RTBV accumulation in IR64 sharply increased at 14 DAI then rapidly<br />
decreased at 21 DAI <strong>and</strong> slightly increased again at 28 DAI then decreased thereafter,<br />
while that in Matatag 9 gradually increased up to 21 DAI, then decreased up to 35 DAI<br />
<strong>and</strong> slightly increased thereafter. The accumulation levels <strong>of</strong> RTBV in both varieties<br />
inoculated at 21-days old were considerably lower than those observed in plants<br />
54
inoculated at 6-days old throughout the observation (Fig. 9). It seems that the<br />
accumulation level <strong>of</strong> RTSV in IR64 inoculated at 21-days old was fluctuating<br />
throughout the observation, peaking at 14 DAI. Meanwhile, it was shown that the<br />
accumulation <strong>of</strong> RTSV in Matatag 9 inoculated at 21-day old was hardly detected after<br />
peaking at 14 DAI.<br />
Fig. 9. Temporal pattern <strong>of</strong> RTBV <strong>and</strong> RTSV accumulation in Matatag 9 <strong>and</strong> IR64. Both<br />
6-day-old <strong>and</strong> 21-day-old seedlings were used at inoculation.<br />
55
Although the levels <strong>of</strong> RTSV <strong>and</strong> RTBV accumulation did not show drastic difference<br />
between Matatag 9 <strong>and</strong> IR64, far more severe symptoms were developed in IR64 than in<br />
Matatag 9. Severe stunting <strong>and</strong> yellowing were observed in all infected IR64 whereas<br />
Matatag 9 did not show typical RTD symptoms (Fig. 10).<br />
Fig. 10. Symptoms casued by RTD on Matatag 9 (left) <strong>and</strong> IR64 (Right).<br />
Matatag 9 did not show typical RTD symptom as compared with IR64<br />
which showed yellowing <strong>and</strong> stunting symptom.<br />
56
Settling Preference<br />
Biological Characterization <strong>of</strong> Matatag 9<br />
Resistance to Green Leafhopper<br />
The number <strong>of</strong> viruliferous GLH that alighted on IR64 at 0.5 hr after infestation<br />
was considerably lower than that at 8 hrs, <strong>and</strong> the number decreased slightly thereafter<br />
(Fig. 11). In contrast, the number <strong>of</strong> GLH on Matatag 9 slightly increased from 0.5 hr to<br />
4 hrs after infestation <strong>and</strong> then decreased. Chi-square test showed that no significant<br />
value for deviation on insect count between IR64 <strong>and</strong> Matatag 9 at 0.5 hr after infestation<br />
(x 2 = 1.52 NS , P = 0.22), however, it indicated highly significant preference <strong>of</strong> GLH for<br />
IR64 over Matatag 9 at 4 hrs, 8 hrs, <strong>and</strong> 24 hrs after infestation (x 2 = 63.61 ** , x 2 =<br />
172.75 ** , <strong>and</strong> x 2 = 177.21 ** , respectively. P < 0.01).<br />
Comparison between the rates <strong>of</strong> virus infection in Matatag 9 <strong>and</strong> IR64<br />
simultaneously infested with viruliferous GLH during the preference test indicated that<br />
the infection rates with RTSV, RTBV <strong>and</strong> both RTSV <strong>and</strong> RTBV were higher in IR64<br />
than in Matatag 9 (Table 7), although no significant correlation was found between the<br />
number <strong>of</strong> insects alighting on test varieties <strong>and</strong> the rates <strong>of</strong> virus infections.<br />
57
Fig. 11. Temporal changes in the number <strong>of</strong> viruliferous GLH on the seedlings <strong>of</strong> IR64<br />
<strong>and</strong> Matatag 9.<br />
Table 7. Average seedling infection (%) <strong>and</strong> average number <strong>of</strong> alighted insect per 5<br />
seedlings <strong>of</strong> IR64 <strong>and</strong> Matatag 9 at different observation times from the GLH preference<br />
test.<br />
SEEDLING<br />
NUMBER OF ALIGHTED INSECT/5<br />
INFECTION<br />
SEEDLINGS<br />
<strong>Variety</strong><br />
RTSV<br />
RTBV<br />
RTBV RTSV 0.5 hr 4 hrs 8 hrs 24 hrs<br />
IR 64 96 97 96<br />
10.10 ±<br />
1.59<br />
22.95 ±<br />
1.29<br />
26.10 ±<br />
0.86<br />
23.85 ±<br />
1.02<br />
Matatag 9 11 49 19<br />
8.90 ±<br />
1.40<br />
12.35 ±<br />
1.53<br />
8.75 ±<br />
1.05<br />
7.25 ±<br />
1.05<br />
58
Adult GLH Longevity<br />
Range <strong>of</strong> the adult GLH longevity was from 2 to 41 days on IR64 whereas it was<br />
from 2 to 19 days on Matatag 9. A significantly longer longevity <strong>of</strong> both male <strong>and</strong> female<br />
adult GLH was observed on IR64 than that <strong>of</strong> Matatag 9 <strong>and</strong> resistant check variety IR62,<br />
but there was no significant difference between Matatag 9 <strong>and</strong> IR62 (Table 8).<br />
Meanwhile, the longevity <strong>of</strong> adult GLH on susceptible check variety TN1 significantly<br />
differed from IR64. Significant difference in the longevity between sexes was found only<br />
on TN1.<br />
Nymphal Mortality<br />
A significant difference on nymph mortality <strong>of</strong> GLH was observed among test<br />
varieties at 3 days after infestation (Table 8). Nymph mortality on TN1 was lowest, <strong>and</strong><br />
significantly different from the others. A significantly less nymph mortality was<br />
observed on IR64 than on both Matatag 9 <strong>and</strong> resistant check variety IR62, whereas no<br />
significant difference between Matatag 9 <strong>and</strong> IR62 was found, suggesting that the two<br />
varieties were equally resistant to GLH.<br />
59
Table 8. Effect <strong>of</strong> antibiosis factor on GLH behavior <strong>and</strong> other life history traits<br />
EFFCT OF TEST PLANTS ON GLH MEASURED ON THE<br />
BASIS OF<br />
TEST Adult Longevity Mortality Nymph Becoming Developmental Growth<br />
VARIETY Male Female Mean (%) Adults, (%) Period, d Index<br />
IR 62 4.9 ± 0.25a(a) 5.6 ± 0.38a(a) 5.18a 53.90 c 57b 19.32 ± 0.67b 2.98a<br />
Matatag 9 5.3 ± 0.41a(a) 6.3 ± 0.67a(a) 5.78a 64.32 c 39c 19.39 ± 0.62c 2.05a<br />
IR 64 11 ± 1.55b(a) 10.2 ± 1.38b(a) 10.6b 20.66 b 88a 15.35 ± 0.46a 5.78b<br />
TN 1 25.9 ± 1.99c(b) 16.7 ± 1.36c(a) 21.3c 1.74 a 100a 13.94 ± 0.29a 7.2c<br />
*Mean ± SE, Values followed by a common letter in a column are not significantly different at the 5% level by the LSD test.<br />
*Values followed by same letter in parenthesis are not significantly different between sexes on each variety at the 5% level by the<br />
LSD test.<br />
*Growth index = percent nymphs becoming adults divided by mean developmental period.<br />
60
Growth <strong>and</strong> Development <strong>of</strong> GLH Nymph<br />
The nymph development period <strong>of</strong> Matatag 9 significantly differed from<br />
susceptible check variety TN1 <strong>and</strong> IR64, but was not significantly different from resistant<br />
check variety IR62 (Table 8). More than 80% <strong>of</strong> nymphs became adults on both IR64 <strong>and</strong><br />
TN1, while less then 40% <strong>of</strong> nymphs did on Matatag 9. A highly significant difference<br />
was observed between Matatag 9 <strong>and</strong> IR64 or TN1, whereas there was no significant<br />
difference between TN1 <strong>and</strong> IR64 in terms <strong>of</strong> the rate <strong>of</strong> nymphs becoming adult. In<br />
growth index, the value for Matatag 9 was significantly lower than that <strong>of</strong> IR64 <strong>and</strong> TN1,<br />
indicating that Matatag 9 is resistant to GLH nymph <strong>and</strong> may affect the transmission<br />
behavior <strong>of</strong> GLH.<br />
Matatag 9 with tungro resistance derived from O. rufipogon has been released as<br />
stop-gap variety in the Philippines in 2002 (Khush et al. 2004). The resistance was<br />
evaluated based on the St<strong>and</strong>ard Evaluation System (INGER, 1996) in multilocation trials<br />
(Cabunagan et al. 1999). There was, however, no intensive study on the resistance<br />
mechanism <strong>of</strong> Matatag 9.<br />
Forced test tube inoculation provides a more reliable measure <strong>of</strong> the resistance<br />
status <strong>of</strong> test varieties as insects have no choice but to feed on the single seedling placed<br />
in each tube (Azzam, 2000).<br />
In this study, the rate <strong>of</strong> RTBV infection in Matatag 9 was relatively low when it<br />
was inoculated with 1 GLH/plant. However, as the number <strong>of</strong> GLH increased, the<br />
61
infection rate increased (Fig. 8.). The infection rates <strong>of</strong> RTBV in IR64 <strong>and</strong> in susceptible<br />
check variety TN1 were consistently high regardless <strong>of</strong> GLH number. Therefore, these<br />
results suggest that the level <strong>of</strong> RTBV resistance in Matatag 9 is weak or it may possess<br />
resistance against other disease components which may interact with RTBV during the<br />
disease development. Generally, the infection rates with RTBV <strong>and</strong> RTSV were low in<br />
Matatag 9 except when more than 50% <strong>of</strong> Matatag 9 inoculated at 6-days old were found<br />
infected with RTBV. The temporal changes in both RTBV <strong>and</strong> RTSV accumulation in<br />
Matatag 9 appeared to be nearly similar to those <strong>of</strong> IR64 inoculated at either plant ages.<br />
Relative level <strong>of</strong> RTBV accumulation was higher in Matatag 9 than in IR64 when the<br />
seedling was 6-day-old at inoculation. Meanwhile, the relative level <strong>of</strong> both RTBV <strong>and</strong><br />
RTSV accumulation in the seedlings inoculated at 21-days old was lower than those<br />
inoculated at 6-days old. These results indicate that Matatag 9 can be infected with both<br />
RTBV <strong>and</strong> RTSV <strong>and</strong> that RTBV can infect in relatively high efficiency. It was also<br />
shown that the level <strong>of</strong> RTBV accumulation was higher in Matatag 9 than in IR64 if<br />
young seedlings become infected. Such lower levels <strong>of</strong> virus infection in older plants<br />
could be accounted for by adult plant resistance involving indigenous plant defense<br />
response to the insect <strong>and</strong> plant pathogen. Ling <strong>and</strong> Palomar (1966) <strong>and</strong> Gibbs <strong>and</strong><br />
Harrison (1976) demonstrated that plants may become more resistant to virus infection as<br />
they grow older, while young plants are more vulnerable to virus infection. As plants<br />
grow, physiological structures such as thickness <strong>of</strong> epidermal layer <strong>and</strong> leaf surface<br />
condition generally change <strong>and</strong> such changes may affect the insect feeding behavior.<br />
Thus, even though insects settle on plants for a long time, some insects may not acquire<br />
62
inoculum sufficient for effective virus transmission. Harris <strong>and</strong> Harris (2001) described<br />
that aphids eventually face the problem <strong>of</strong> p-protein fibrils <strong>and</strong> callose clogging which<br />
may prevent the entrance <strong>of</strong> plant sap into the maxillary food canal though aphids are<br />
given sufficient feeding time. In addition, feeding in a virus-infected plant may coat the<br />
cuticular lining <strong>of</strong> the feeding apparatus with ACR-HC-virion complex.<br />
The results in this study showed that once RTBV <strong>and</strong> RTSV are introduced into<br />
Matatag 9, both viruses are likely to replicate in Matatag 9 using their typical replication<br />
pathway though symptom development was deterred in Matatag 9, indicating that<br />
Matatag 9 has tolerance against RTBV. On the other h<strong>and</strong>, Matatag 9 could be considered<br />
as resistant to RTSV based on the infection rate in aged plants, though the accumulation<br />
<strong>of</strong> RTSV in Matatag 9 is still readily detectable.<br />
Matatag 9 was found to be a less preferred host than IR64 by GLH for settling,<br />
implying the contribution <strong>of</strong> antixenosis characteristics to resistance <strong>of</strong> Matatag 9. The<br />
mean GLH count on seedling <strong>of</strong> Matatag 9 did not significantly differ from that <strong>of</strong> IR64<br />
at 0.5 hr after infestation, showing that insects moved to the seedling without recognizing<br />
their preferred host. GLH, thereafter, migrated from Matatag 9 to IR64, the number <strong>of</strong><br />
settled GLH on IR64 significantly increased at 4, 8, <strong>and</strong> 24 hrs after infestation, probably<br />
because they recognized features adverse to their feeding in the host. Moreover, during<br />
the experiment, it was observed that the insects that settled on Matatag 9 became restless,<br />
indicating some interference with their feeding on Matatag 9. Similar observation was<br />
reported by Karim (1978). For many phytophagous insects, visual <strong>and</strong> chemical cues play<br />
an important role in host selection (Dixon, 1985). Insects <strong>of</strong>ten examine the plant surface<br />
63
cues by repeatedly tapping the tip <strong>of</strong> the labium against the plant surface <strong>and</strong> determine if<br />
the plant is suitable for settling or feeding (Bakus, 1985). As the insect identify<br />
appropriate probing site, they insert their stylet into the plant surface then suck the plant<br />
sap whether the potential host contains appropriate nutrition or not (Kawabe <strong>and</strong> Mclean,<br />
1980). Therefore, it takes a certain time for the insects to recognize their preferred host.<br />
Short adult longevity, more than 60% nymph mortality, long nymph<br />
developmental period <strong>and</strong> low percentage <strong>of</strong> nymph becoming adult are indicative <strong>of</strong><br />
antibiosis effects <strong>of</strong> Matatag 9 (Kishino <strong>and</strong> Ando, 1978; Liu <strong>and</strong> Takahashi, 1990).<br />
Moreover, growth index on Matatag 9 was significantly lower than both IR64 <strong>and</strong> TN1,<br />
while IR62 had almost same growth index value (Saxena et al. 1974), supporting<br />
significant involvement <strong>of</strong> antibiosis effect in resistance against RTD in Matatag 9.<br />
Collectively, the results <strong>of</strong> this study strongly suggest that the resistance <strong>of</strong><br />
Matatag 9 is mainly due to insect resistance although the contribution <strong>of</strong> resistance<br />
against tungro viruses in Matatag 9 cannot be ignored.<br />
Virus infection <strong>and</strong> symptom development are the consequence <strong>of</strong> virus-vector-<br />
host complex interaction. Since RTD is a composite disease caused by RTBV, RTSV <strong>and</strong><br />
insect vectors, the infection mechanism <strong>and</strong> symptom development might be more<br />
complex than those by other viruses. Knowing the resistance mechanism not only in<br />
Matatag 9 but also in other rice varieties enables us to formulate more appropriate<br />
combination for the mixed seeds planting. Also, it would definitely contribute to the<br />
design <strong>of</strong> deployment strategies for resistant varieties.<br />
64
Performance Evaluation <strong>of</strong> Varietal Mixture Components<br />
Comparative Morphological <strong>and</strong> Yield Characteristics<br />
<strong>of</strong> Matatag 9 <strong>and</strong> IR64<br />
Both Matatag 9 <strong>and</strong> IR64 similarly grew until harvest without any distinct visual<br />
differences (Fig. 12). Fifty percent flowering was recorded at 66 <strong>and</strong> 77 DAT for IR64<br />
<strong>and</strong> Matatag 9, respectively. Both varieties were ready for harvesting by 107 DAT (Fig.<br />
12). Significant difference in height between the two varieties during the growth period<br />
was observed at 45, 75, <strong>and</strong> 90 DAT (Fig. 13), however, the difference was less than<br />
approximately 5 cm. The numbers <strong>of</strong> tillers at 30 <strong>and</strong> 60 DAT did not show significant<br />
difference between varieties (Table 9). Significant differences were observed in final<br />
height <strong>and</strong> panicle length between varieties while no significant differences were<br />
observed in the number <strong>of</strong> productive tiller <strong>and</strong> the numbers <strong>of</strong> filled/unfilled grain per<br />
panicle at harvest time (Table 10). There were significant differences in grain size (width<br />
<strong>and</strong> length) <strong>and</strong> TGW between varieties (Table 11).<br />
65
Matatag 9 IR 64<br />
Fig. 12. Morphological appearance <strong>of</strong> Matatag 9 (left) <strong>and</strong> IR64 (right) at 107 DAT in the<br />
screenhouse<br />
a<br />
a<br />
a<br />
a<br />
a<br />
b<br />
0.0 15 30 45 60 75 90<br />
Day after transplanting (DAT)<br />
a<br />
a<br />
a a<br />
b b<br />
*Treatment means at different DAT followed by a common letter are not significantly<br />
different at the 5% level by the LSD test.<br />
Fig. 13. Growth comparison between Matatag 9 <strong>and</strong> IR64 at every 15 days interval.<br />
66
Table 9. Comparison <strong>of</strong> tiller number during vegetative stage<br />
TEST VARIETY 30 DAT 60 DAT<br />
Matatag 9 8.06 ± 0.44a 17 ± 0.63a<br />
IR 64 7.56 ± 0.54a 17.13 ± 0.84a<br />
*Mean ± SE, Values followed by a common letter in a column are<br />
not significantly different at the 5% level by the LSD test.<br />
Table 10. Comparison <strong>of</strong> agronomic traits <strong>of</strong> mixture components at harvest<br />
TEST NUMBER OF GRAIN/PANICLE<br />
VARIETY Final Height Panicle Length Productive Tiller Filled Unfilled<br />
Matatag 9 96.74 ± 0.76a 23.39 ± 0.27a 14.19 ± 0.58a 129.83 ± 3.95a 10.88 ± 0.85a<br />
IR 64 91.35 ± 0.90b 24.5 ± 0.33b 14.63 ± 0.94a 116.65 ± 4.88a 17.31 ± 2.43a<br />
*Means followed by a common letter in a column are not significantly different at the 5% level<br />
by the LSD test.<br />
Table 11. Comparison <strong>of</strong> physical characteristics <strong>of</strong> grain<br />
TEST HULLED DEHULLED 1000 GW [g]<br />
VARIETY Width Length Width Length Hulled Dehulled<br />
Matatag 9 2.12 ± 0.01b 9.87 ± 0.05a 1.89 ± 0.01b 7.41 ± 0.04a 23.4 ± 0.24b 18.2 ± 0.24b<br />
IR 64 2.33 ± 0.01a 9.48 ± 0.05b 2.09 ± 0.01a 6.98 ± 0.03b 26 ± 0.00a 20.6 ± 0.24a<br />
*Means followed by a common letter in a column are not significantly different at the 5% level<br />
by the LSD test.<br />
67
Effect <strong>of</strong> <strong>Mixed</strong> <strong>Planting</strong> <strong>of</strong> Resistant <strong>and</strong><br />
Susceptible Varieties on the Incidence <strong>of</strong> Rice Tungro Disease<br />
To evaluate the effect <strong>of</strong> mixed planting patterns, namely mixed seed planting <strong>and</strong><br />
interplanting, the relative rates <strong>of</strong> infection with RTSV, RTBV, <strong>and</strong> both RTBV <strong>and</strong><br />
RTSV in each planting pattern were compared.<br />
Reduction in disease was observed with the increase in the proportion <strong>of</strong> resistant<br />
component among treatments on the mixed seed planting <strong>and</strong> interplanting regardless <strong>of</strong><br />
the viruliferous GLH level except relative total RTBV infection (RTB) at 10 GLH level<br />
(Fig. 14). Disease reduction was more evident in mixed seed planting than in<br />
interplanting at both GLH levels. At 3 GLH level, the effect <strong>of</strong> mixed seed planting on<br />
RTB <strong>and</strong> relative total RTSV infection (RTS) seems to be more significant than that <strong>of</strong><br />
interplanting, while no significant difference on relative rates <strong>of</strong> infection with both<br />
RTBV <strong>and</strong> RTSV (RBS) was observed between planting types. At 10 GLH level, no<br />
significant differences between planting types were observed among RBS, RTB <strong>and</strong> RTS.<br />
The results <strong>of</strong> mean comparison among treatments between planting types showed<br />
that the effect <strong>of</strong> mixed seed planting on both RTB <strong>and</strong> RTS was significantly different<br />
from interplanting in both 75% R: 25% S <strong>and</strong> 25% R: 75% S at 3 GLH level.<br />
Interestingly, the RTB <strong>and</strong> RTS in the interplanting <strong>of</strong> 75% R: 25% S were not<br />
significantly different from those in the mixed seed planting <strong>of</strong> 50% R: 50% S.<br />
68
Infection rate (%) RBS<br />
RTB<br />
Infection rate (%)<br />
RTS<br />
Infection rate (%)<br />
a<br />
a<br />
a<br />
a<br />
a a<br />
b<br />
b<br />
b<br />
3 GLH<br />
c<br />
c<br />
d<br />
c cd<br />
c<br />
b<br />
d<br />
cd<br />
d<br />
e<br />
f<br />
d<br />
d<br />
e<br />
a<br />
a<br />
a<br />
ab<br />
a a<br />
b<br />
ab<br />
ab ab<br />
bc<br />
a b<br />
10 GLH<br />
b<br />
b<br />
c<br />
b ab b<br />
Fig. 14. Relative rates <strong>of</strong> infection with RTBV+RTSV (RBS), total RTBV (RTB), <strong>and</strong><br />
total RTSV (RTS) observed in mixed seed plantings <strong>and</strong> interplantings. The relative rates<br />
were computed based on those observed in the pure st<strong>and</strong> <strong>of</strong> IR64 (100% S).<br />
* Bar having a common letter are not significantly different at the 5% level by the LSD<br />
test.<br />
c<br />
bc<br />
d<br />
c<br />
b<br />
d<br />
69
Moreover, RTB in the interplanting <strong>of</strong> 50% R: 50% S was not significantly different from<br />
that in the mixed seed planting <strong>of</strong> 25% R: 75% S at 3 GLH level. At 10 GLH level,<br />
however, no clear statistical differences between planting types were observed in any<br />
treatments, although there were significant differences among treatments for the RBS <strong>and</strong><br />
RTS but not for RTB.<br />
Serial-Inoculation in Resistant <strong>and</strong> Susceptible Varieties<br />
GLH was able to transmit both RTSV <strong>and</strong> RTBV to 1 st plant regardless <strong>of</strong> variety<br />
but infection rates <strong>of</strong> viruses in Matatag 9 were significantly lower than in IR64 (Fig. 15).<br />
When the 2nd plant is Matatag 9, GLH failed to transmit either RTSV or RTBV (C1 <strong>and</strong><br />
2). On the other h<strong>and</strong>, when the 2nd plant is IR64, GLH was able to transmit either<br />
RTSV or RTBV (C3 <strong>and</strong> 4). A few GLH successfully transmitted RTBV up to 4th plant<br />
(C4).<br />
70
% Infection<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
M9<br />
M9<br />
M9<br />
C1 C3<br />
% Infection<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
IR64<br />
M9<br />
IR64<br />
M9<br />
M9<br />
M9<br />
IR64<br />
C2 C4<br />
BS<br />
B<br />
S<br />
BS<br />
BS<br />
B<br />
S<br />
BS<br />
S<br />
B<br />
S<br />
B<br />
Fig. 15. Duration <strong>of</strong> virus retention ability through 5 days serial-inoculation with different<br />
combinations <strong>of</strong> resistant <strong>and</strong> susceptible varieties. C1 <strong>and</strong> C4 represent monoculture. C2<br />
<strong>and</strong> C3 represent varietal mixtures. C1 <strong>and</strong> C3: 1 st plant is resistant variety Matatag 9. C2<br />
<strong>and</strong> C4: 1 st plant is susceptible variety IR64. C1 <strong>and</strong> C2: 2 nd plant Matatag 9. C3 <strong>and</strong> C4:<br />
2 nd plant IR64.<br />
% Infection<br />
% Infection<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
100<br />
90<br />
0<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
M9<br />
IR64<br />
IR64<br />
IR64<br />
M9<br />
IR64<br />
IR64<br />
IR64<br />
M9<br />
IR64<br />
BS<br />
B<br />
S<br />
BS<br />
BS<br />
B<br />
S<br />
BS<br />
S<br />
B<br />
S<br />
B<br />
71
Development <strong>of</strong> varieties with good agronomical characteristics as well as<br />
appropriate disease <strong>and</strong> pest resistance became one <strong>of</strong> the requirements for<br />
biodiversification since the commercial use <strong>of</strong> variety mixture is increasing <strong>and</strong> some<br />
degree <strong>of</strong> agronomic trait uniformity is desired (Mundt, 2002; Smithson <strong>and</strong> Lenne, 1996;<br />
Garret <strong>and</strong> Mundt, 1999). Mixture components are usually mixed in a replacement<br />
design (Zhu et al. 2005) to avoid a contamination with other mixture components <strong>and</strong> to<br />
maintain market value.<br />
Growth comparison <strong>and</strong> evaluation <strong>of</strong> yield components between Matatag 9 <strong>and</strong><br />
IR64 showed that they have similar agronomical characteristics. Although statistical<br />
analysis showed significant differences in few characteristics, the actual differences<br />
between them were virtually negligible <strong>and</strong> may not influence farmers’ harvest practices.<br />
Thus, Matatag 9 <strong>and</strong> IR64 can be mixed <strong>and</strong> harvested together. Significant difference in<br />
TGW was observed between Matatag 9 <strong>and</strong> IR64. The yields from the mixtures, however,<br />
planted in RTD endemic area were not significantly different from the highest yields<br />
achieved by the monocultures <strong>of</strong> either Matatag 9 or IR64 (Cabunagan <strong>and</strong> Choi. 2005).<br />
Yield difference between monocultures from the field trial was approximately 10%<br />
which was comparable to the result <strong>of</strong> the present study. Smithson <strong>and</strong> Lenne (1996)<br />
pointed out that yield in mixture plots usually lie above the mean <strong>of</strong> their components in<br />
monoculture. Yield increases <strong>of</strong> 1 to 5% are <strong>of</strong>ten provided by a variety mixture in the<br />
absence <strong>of</strong> substantial disease, with larger increases when disease is <strong>of</strong> significance<br />
(Mundt, 2002). In the Philippines, the interplanting approach to manage RTD was<br />
initially attempted, however, a majority <strong>of</strong> farmers were not willing to adopt the laborious<br />
72
practice. Instead, the mixed seed planting was designed, aiming for wider adaptation <strong>of</strong><br />
the variety mixture as one <strong>of</strong> the RTD management strategies among farmers (Cabunagan<br />
<strong>and</strong> Choi, 2005).<br />
The results <strong>of</strong> tray inoculation suggest that the mixed seed planting seems to be<br />
more effective to reduce RTD incidence than interplanting in an area when the disease<br />
pressure is not extremely high. Since there is no significant difference between the two<br />
planting methods at 10 viruliferous GLH, both methods would be less effective likewise<br />
in an area where disease pressure is high. Possible explanation <strong>of</strong> the difference in<br />
effectiveness on disease reduction between the two methods is the difference in spatial<br />
distribution <strong>of</strong> mixture components. As both Matatag 9 <strong>and</strong> IR64 in the mixed seeds<br />
planting are r<strong>and</strong>omly distributed, it generates an inconsistent spatial pattern that may<br />
influence the movement <strong>and</strong> feeding behavior <strong>of</strong> insect vector. For example, when<br />
viruliferous GLH from outside alight on Matatag 9, the insects may be forced to transfer<br />
to other plants due to the antixenosis or antibiosis effects <strong>of</strong> Matatag 9, but the insects<br />
alighted on IR64 would stay longer. Interplanting provides a consistent spatial<br />
distribution <strong>of</strong> mixture components. It seems to be easier for insects to reach their<br />
preferred host in the interplanting arrangement. The insect movement in interplanting<br />
may require shorter distance than that in mixed seed planting since IR64 was planted in<br />
consistent pattern <strong>and</strong> infection rate is higher than mixed seed planting. Thus, one <strong>of</strong> the<br />
potential mechanisms for the reduction <strong>of</strong> RTD in the mixed seed planting is the r<strong>and</strong>om<br />
spatial distribution <strong>of</strong> individual resistant plants.<br />
73
RTBV <strong>and</strong> RTSV can be transmitted during host selection events such as sylet<br />
probing on plant tissue <strong>and</strong> plant fluid ingestion regardless <strong>of</strong> varieties though RTD<br />
infection rate is low in Matatag 9. Insects alighted on IR64 may settle longer, while those<br />
l<strong>and</strong>ed on Matatag 9 may move to other plants, thus virus retention ability could be<br />
decreased. RTBV <strong>and</strong> RTSV can be transmitted with low efficiency to the second IR64<br />
but no virus transmission occurs in Matatag 9. Although GLH can retain RTBV for 4-5<br />
days <strong>and</strong> RTSV for 2-4 days (Hibino et al. 1978), once the insects feed on Matatag 9,<br />
they may lose their virus retention ability. Matatag 9 may contain certain factors which<br />
inhibit the binding between viruses <strong>and</strong> leafhopper cuticula or suppress the activity <strong>of</strong><br />
helper component required for the binding between virus <strong>and</strong> cuticula at retention sites <strong>of</strong><br />
the leafhopper (Harris <strong>and</strong> Harris, 2001).<br />
74
Farmers’ <strong>Perception</strong> Survey on the Use <strong>of</strong> <strong>Mixed</strong> <strong>Planting</strong><br />
Respondents’ pr<strong>of</strong>ile. More than 80% <strong>of</strong> the farmers who attended the forum<br />
were residents <strong>of</strong> Barangay Progreso, the site <strong>of</strong> the 4 season field trial on “<strong>Mixed</strong> seed<br />
planting” in the field <strong>of</strong> Mr. Domingo Bonifacio. The other respondents came from<br />
neighboring barangays. Their age ranged from 27 years to 74 years old with an average<br />
<strong>of</strong> 50.5 years old. Around 90% or more <strong>of</strong> the farmers were married <strong>and</strong> more than half<br />
has a family size <strong>of</strong> 6. The average period <strong>of</strong> formal education for all farmers in the<br />
sample was 8.3 years <strong>and</strong> about 42% were at least Elementary School educated, <strong>and</strong> 48%<br />
were High School educated. About 44% were tenant <strong>and</strong> another 38% either share holder<br />
or 10% share farmer. The average rice farming experience is about 18 years (Table 12).<br />
Farm pr<strong>of</strong>ile. The area being utilized as rice field ranged from 0.25 ha to 12 ha<br />
with an average <strong>of</strong> 2.48 ha (Table 12). As shown in Table 13, majority <strong>of</strong> the farmers<br />
were planting improved varieties such as PSB Rc 14, 10, 82 <strong>and</strong> 28. Except for PSB Rc<br />
10, all the rest are known to have good eating quality <strong>and</strong> thus highly favored by farmers<br />
in the area. Furthermore, most PSB Rc varieties are carrying Xa4 gene for resistance to<br />
bacterial leaf blight, except for PSB Rc 82 which carries Xa4+xa5. NSIC Rc 110 is<br />
newly released tugro resistant variety in 2002. Some farmers planted IR64 from time to<br />
time because <strong>of</strong> its superior agronomical performance. Although these varieties could be<br />
75
Table 12. Pr<strong>of</strong>ile <strong>of</strong> farmer respondents <strong>of</strong> the survey in Ajuy, Iloilo, Philippines<br />
Age (years) Mean 50.45<br />
Min 27<br />
Max 74<br />
PERCENTAGE OF<br />
52 RESPONDENTS<br />
Civil status Married 47 90.4<br />
Single 3 5.8<br />
Widow 1 1.9<br />
Not specified 1 1.9<br />
Family size Mean 5.8<br />
Min 2<br />
Max 14<br />
Education Mean 8.25 EG 30.8<br />
(years) Min 2 HSG 44.2<br />
Max 14 CG 9.6<br />
ED 11.5<br />
HSD 3.9<br />
L<strong>and</strong> ownership Owner 15.4<br />
Tenant 44.2<br />
Share holder 19.2<br />
10% share farmer 19.2<br />
Rice field (ha) Mean 0.25<br />
Min 2.48<br />
Max 12<br />
Farming<br />
experience Mean 18.27<br />
(years) Min 2<br />
Max 50<br />
EG = Elementary Graduate, HSG = High School Graduate, CG = College Graduate,<br />
ED = Elementary Dropout, <strong>and</strong> HSD = High School Dropout.<br />
76
Table 13. Varieties planted for the past 4 seasons among 52 respondents in Ajuy, Iloilo,<br />
Philippines<br />
VARIETY PERCENTAGE OF 52 RESPONDENTS<br />
PSB Rc 14 96.2<br />
PSB Rc 10 65.4<br />
PSB Rc 82 34.6<br />
PSB Rc 28 34.6<br />
IR64 25<br />
PILIT (Gultinous) 17.3<br />
NSIC Rc 110 15.4<br />
PSB Rc 112 13.5<br />
PSB Rc 18 9.6<br />
Mixture 3.8<br />
PSB Rc 64 3.8<br />
PSB Rc 80 3.8<br />
PSB Rc 4 3.8<br />
Matatag 6 1.9<br />
PSB Rc 44 1.9<br />
PSB Rc 12 1.9<br />
PSB Rc 17 1.9<br />
PSBRc 72 1.9<br />
Red Rice 1.9<br />
77
acquired either from seed grower or institutional sector, most farmers (69.2%) obtained<br />
them from other farmers. In addition to the seed purchase, one <strong>of</strong> the mixture components,<br />
Matatag 9, is available in Ajuy (Ely S<strong>and</strong>ig, Chief, RAID, personal communication).<br />
Around 15% <strong>of</strong> the farmers were doing rice production for home consumption, whereas,<br />
the others produced for either only commercial or both.<br />
All farmers reported various pests <strong>and</strong> diseases as their common problems in their<br />
rice farm. RTD was regarded by farmers as the most important disease in the area<br />
followed by bacterial leaf blight <strong>and</strong> rice blast. More than half <strong>of</strong> farmers regarded stem<br />
borer, rice bug, <strong>and</strong> GLH/BPH, “waya waya” in Ilongo, as their major pest problems.<br />
Aside from these, non-insect pests such as rat, giant apple snail, <strong>and</strong> maggot were also<br />
reported by a few farmers (Table 14).<br />
78
Table 14. Percentage <strong>of</strong> farmers reporting major pests <strong>and</strong> diseases on rice farming in<br />
Ajuy, Iloilo, Philippines<br />
PLANT DISEASE AND PEST PROBLEM a<br />
PERCENTAGE OF 52<br />
RESPONDENTS<br />
Plant diseases<br />
Rice Tungro Disease (RTD) 65.4<br />
Bacterial leaf blight 26.9<br />
Rice blast 17.3<br />
Bacterial leaf streak 7.7<br />
Sheath blight 3.8<br />
None 15.4<br />
Insect pest<br />
Stem borer 55.8<br />
Rice bug 51.9<br />
Green Leafhopper (GLH) 48.1<br />
Brown hopper (BPH) 23.1<br />
GLH/BPH (waya waya) b<br />
32.7<br />
Armyworm 19.2<br />
Leaf holder 19.2<br />
Caseworm 7.7<br />
Cutwarm 1.9<br />
Non-insect pest<br />
Rat 5.8<br />
Giant apple snail 5.8<br />
Maggot 1.9<br />
Others c<br />
a<br />
Multiple answers possible<br />
23.1<br />
b<br />
General insect name was used in local language<br />
c<br />
Unidentified insect reported by farmers<br />
79
Farmers’ Awareness <strong>and</strong> Knowledge <strong>of</strong> Rice Tungro Disease<br />
Most farmers (86.5%) were aware <strong>of</strong> RTD <strong>and</strong> more than 70% had experienced<br />
damage by the disease at least either once or twice for the past 5 years. However, the<br />
damage was ranked either least serious or moderately serious (Table 15).<br />
There was a wide range <strong>of</strong> responses among farmers about the causes <strong>and</strong> mode<br />
<strong>of</strong> RTD spread what causes RTD <strong>and</strong> how it is spread from plant to plant. Although,<br />
majority <strong>of</strong> farmers recognized virus as the main causal agent <strong>of</strong> RTD, more than half <strong>of</strong><br />
them mention other factors that were not causal agent such as poor soil, water <strong>and</strong> seeds<br />
(Table 16). Fewer farmers (5.8%), in fact, correctly answered both virus <strong>and</strong> GLH. Some<br />
other farmers (46.2%) purely believed that RTD was not caused by virus.<br />
Table 15. Farmers’ experience <strong>of</strong> RTD incidence in their farm <strong>and</strong> its damage<br />
seriousness ranked by farmers<br />
PERCENTAGE OF 52 RESPONDENTS<br />
Number <strong>of</strong> RTD incidence<br />
Once 36.5<br />
Twice 28.8<br />
Three times 5.8<br />
Four times 1.9<br />
Five times 5.8<br />
Ten times 5.8<br />
None 15.4<br />
Severity <strong>of</strong> RTD incidence<br />
Most serious 13.5<br />
Moderately 38.4<br />
Least serious 34.6<br />
Not serious 11.5<br />
No answer 1.9<br />
80
Table 16. Farmers’ perceptions <strong>of</strong> causes <strong>and</strong> spread <strong>of</strong> RTD a<br />
PERCENTAGE OF 52 RESPONDENTS<br />
Causes<br />
Virus 51.9 (9.6)<br />
Insect (general) 36.5<br />
Poor seeds 36.5<br />
Bacteria 26.9<br />
Fungi 23.1<br />
Poor soil 19.2<br />
Poor water 11.5<br />
Spread<br />
GLH 57.7 (7.7)<br />
BPH 28.8<br />
Water 19.2<br />
Seeds 19.2<br />
Wind 5.8<br />
Others 1.9<br />
a Multiple answers possible<br />
* Value in parenthesis representing the percent farmer answered properly.<br />
Less than half <strong>of</strong> farmers (40.4%) believe that water, seeds or BPH spread RTD <strong>and</strong> more<br />
than 50% consider GLH to spread the disease.<br />
Farmers could differentiate a RTD infected plant from other diseased plants based<br />
on either the change in leaf color, stunting <strong>of</strong> the plant or the presence <strong>of</strong> plenty <strong>of</strong> GLH<br />
in their field.<br />
Farmers’ Awareness <strong>of</strong> Rice Tungro Disease Control<br />
Although almost equal percentage <strong>of</strong> farmers controlled RTD in their rice field by<br />
either using insecticides or using resistant varieties, majority believed that the use <strong>of</strong><br />
81
insecticides could not control RTD or it was not effective (Table 17). About 29% <strong>of</strong> the<br />
farmers answered that they removed tungro infected plant from their field. A few farmers<br />
used RTD control methods such as drying a rice field after harvest, <strong>and</strong> application <strong>of</strong><br />
zinc sulfate, or filling paddy field the water <strong>and</strong> then draining it. Additionally, the farmers<br />
were asked to name the insecticide they used or the resistant variety they planted in their<br />
field. NSIC Rc 110, IR60, <strong>and</strong> Mixtures appeared where varieties were planted.<br />
More than 60% <strong>of</strong> the farmers were aware that resistant varieties are available for<br />
the control <strong>of</strong> RTD <strong>and</strong> more than 70% had experienced breakdown <strong>of</strong> resistance to RTD<br />
<strong>of</strong> the varieties they planted (Table 18).<br />
Table 17. Farmers’ control measures for rice tungro disease <strong>and</strong> perception <strong>of</strong> insecticide<br />
effectiveness<br />
PERCENTAGE OF 52 RESPONDENTS<br />
Control measures a<br />
Spraying insecticide 55.8<br />
Using resistant variety 51.9<br />
Removal <strong>of</strong> infected plant 28.8<br />
Others 11.5<br />
<strong>Perception</strong> <strong>of</strong> insecticide effectiveness<br />
Effective 19.2<br />
Not effective 76.9<br />
No answer 3.8<br />
a Multiple answers possible<br />
82
Table 18. Farmers’ awareness <strong>of</strong> the availability <strong>of</strong> tungro resistant variety <strong>and</strong><br />
experience <strong>of</strong> resistance breakdown.<br />
PERCENTAGE OF 52 RESPONDENTS<br />
Availabity <strong>of</strong> resistant variety<br />
Yes 65.4<br />
No 32.7<br />
No answer 1.9<br />
Experience <strong>of</strong> resistance breakdown<br />
Yes 71.2<br />
No 28.8<br />
No answer 0<br />
Awareness <strong>of</strong> <strong>Mixed</strong> Seeds <strong>Planting</strong><br />
Although 60% <strong>of</strong> the farmers were aware <strong>of</strong> the availability <strong>of</strong> resistant rice<br />
varieties, only 46% <strong>of</strong> the respondents were aware <strong>of</strong> the mixed seed planting (Fig. 16 A),<br />
<strong>and</strong> 79% <strong>of</strong> those who were aware believed that it was effective for controlling RTD <strong>and</strong><br />
only 17% believed otherwise (Fig. 16 B). About 70% <strong>of</strong> those who were aware had tried<br />
mixed seed planting either for 1 or 2 seasons (Fig. 16 C), <strong>and</strong> more than 70% had<br />
observed the effectiveness <strong>of</strong> mixed seed planting for controlling RTD. However, only<br />
about 50% <strong>of</strong> those who were aware <strong>and</strong> had tried mixed seed planting would<br />
recommend the practice to other farmers.<br />
83
Percent <strong>of</strong> respondent<br />
A<br />
No (28)<br />
Yes (24)<br />
B<br />
Fig. 16. Farmers’ awareness <strong>of</strong> the mixed seeds planting for rice tungro disease control<br />
(A), their belief <strong>of</strong> control effectiveness (B), <strong>and</strong> experience <strong>of</strong> the mixed seeds planting<br />
in their field (C).<br />
At present, about 70% <strong>of</strong> the farmers who tried mixed seed planting stopped<br />
applying this method <strong>and</strong> the reason they gave were: that they did not have RTD<br />
incidence in their farm (35.3%) or they consumed all the seeds <strong>and</strong> none was available<br />
for next season planting (29.4%). Twenty-four percent farmers stopped planting because<br />
they believed that mixed seed planting had no effect in controlling RTD (Table 19).<br />
84<br />
C
The reason why 54% <strong>of</strong> the respondents were not aware <strong>of</strong> the mixed seed<br />
planting was either because they really have no knowledge about it (43%) or they have<br />
no access to seeds <strong>of</strong> mixtures (36%) that they could use. However, after hearing the<br />
presentation about mixed seed planting during the forum, more than 80% were willing to<br />
try it in the future.<br />
Table 19. Major reasons for the termination <strong>of</strong> the mixed seed planting <strong>and</strong> its adaptation<br />
constraints in farmers’ field in Ajuy.<br />
REASON / CONSTRAINT a<br />
NUMBER OF<br />
RESPONDENTS<br />
Reason <strong>of</strong> termination n=17 b<br />
No rice tungro 6<br />
Consumed seed 5<br />
No effect 4<br />
No answer 2<br />
Laborious 0<br />
Constraint <strong>of</strong> adaptation a<br />
No knowledge about the mixed seed planting 16<br />
No access to mixed seed 10<br />
Doutful 3<br />
Have not tested yet 2<br />
Do not like<br />
a<br />
Multiple answers possible<br />
b<br />
Farmers who had tried the mixed seed planting<br />
1<br />
c<br />
Farmers who had never tried the mixed seed planting<br />
n=28 c<br />
85
Farmer survey is an important data gathering process for assessing the needs <strong>of</strong><br />
intended beneficiaries to determine their knowledge <strong>and</strong> perceptions <strong>of</strong> a pest problem,<br />
their constraints in dealing with the problem, <strong>and</strong> their attitudes <strong>and</strong> practices in pest<br />
management (Escalada <strong>and</strong> Heong, 1997).<br />
The present survey showed that farmers viewed pests <strong>and</strong> diseases, especially<br />
RTD <strong>and</strong> the stem borer, GLH, <strong>and</strong> rice bug, as primary constraints to rice production in<br />
Ajuy, Iloilo (Table 14). Most farmers were able to distinguish the major rice pests <strong>and</strong><br />
diseases <strong>and</strong> their awareness <strong>of</strong> RTD was considerably high. Relatively high knowledge<br />
<strong>of</strong> RTD <strong>and</strong> its management was observed in this study. The reason is probably that most<br />
farmers had participated in the farmers training on RTD management as part <strong>of</strong> the IRRI-<br />
ILOILO PAO DAPITSAKA 2000-2002 Project in Ajuy, Iloilo. In general, farmers’ in-<br />
depth knowledge about pests is associated with the visibility <strong>and</strong> importance <strong>of</strong> the pest,<br />
since disease pathogens are not easily seen, it is difficult for farmers to account for the<br />
causes <strong>of</strong> diseases (Bentley <strong>and</strong> Thiele, 1999). In fact, although majority <strong>of</strong> farmers<br />
regarded the virus as causal agent <strong>and</strong> GLH as the agent <strong>of</strong> spread, they believed that the<br />
disease incidences were caused by not only the interaction between virus <strong>and</strong> GLH but<br />
also the interaction among the virus <strong>and</strong> other microorganisms or poor soil, water <strong>and</strong><br />
seeds condition (Table 16). There seemed to be confusion with the mechanism <strong>of</strong> RTD<br />
incidence among the farmers. Though fewer knew the causal agent <strong>and</strong> its vector, the<br />
information would not likely be disseminated properly from the farmers who have correct<br />
knowledge to the other farmers. The reason might be that the farmers who knew the<br />
mechanism <strong>of</strong> RTD incidence had difficulties in explaining the details appropriately to<br />
86
other farmers. This phenomenon could be explained by studies concerning beliefs about<br />
the causes <strong>of</strong> human disease in the Philippines. The study revealed that people <strong>of</strong>ten have<br />
complex views <strong>of</strong> the cause <strong>and</strong> effect <strong>of</strong> disease, with several causes <strong>of</strong>ten associated<br />
with one or more effects rather than one cause leading to one effect. Often a combination<br />
<strong>of</strong> a person’s susceptibility, perhaps caused by hunger or fatigue, plus an expected natural<br />
phenomenon such as a sudden heavy rain is thought to result in sickness (Himes, 1971).<br />
Consequently, they associate RTD to human diseases like AIDS or cancer (Warburton et<br />
al. 1997), therefore, their belief that RTD could be spread by air, water <strong>and</strong> soil is<br />
consistent with their perception that it is comparable to human pathogens.<br />
In the case <strong>of</strong> RTD management options, farmers usually rely on spraying<br />
insecticide <strong>and</strong> on using resistant variety in their farm although they are aware that the<br />
resistance may break down over time <strong>and</strong> using insecticide was not effective in<br />
controlling RTD (Tables 17 <strong>and</strong> 18). Some farmers remove (rogue) infected plants from<br />
their field <strong>and</strong> fewer apply other cultural managements such as drying a rice field.<br />
Farmers generally find rouging is time-consuming <strong>and</strong> tedious <strong>and</strong> additional labors may<br />
be required. In addition to the insecticide <strong>and</strong> resistant variety, the study revealed that<br />
some <strong>of</strong> farmers have used a banned insecticide <strong>and</strong>/or an inappropriate resistant variety.<br />
The reason would be the limited information resources among farmers. A Department <strong>of</strong><br />
Agriculture (DA) technician is the main source <strong>of</strong> information on tungro. The<br />
technician’s recommendations <strong>of</strong> RTD control for farmers included using either spray or<br />
granular insecticide, rouging, plowing a whole field, <strong>and</strong> draining a field, but those were<br />
varied from area to area. Learning from other farmers <strong>and</strong> from their own experience<br />
87
were also important (Warburton et al. 1997). In Ajuy, most farmers obtained their seeds<br />
from neighboring farmers while others acquired them from their seed growers or the<br />
institutional sector. There seems to be a lack <strong>of</strong> accessibility <strong>of</strong> information (network<br />
system) in the area.<br />
Over the years, farmers have developed methods <strong>of</strong> managing plant diseases.<br />
However, changes <strong>and</strong> intensification within farming system <strong>of</strong>ten bring increased<br />
disease pressures. RTD affect large areas <strong>and</strong> poses difficulties for farmers because <strong>of</strong> its<br />
unpredictability <strong>and</strong> risk <strong>of</strong> major crop loss. Local knowledge <strong>and</strong> practices <strong>of</strong> farmers<br />
must evolve to cope with these problems. Efficient communication technology <strong>and</strong><br />
technology transfer scheme have to be applied among scientists, extension technicians,<br />
<strong>and</strong> beneficiaries. Farmers’ perception surveys on pest management <strong>and</strong> practices in<br />
Southeast Asia have revealed that most farmers are risk adverse (Rola <strong>and</strong> Pingali, 1993;<br />
Heong et al. 1994). Farmers in the Philippines regarded RTD as a serious problem even<br />
in areas where outbreaks are rare. Also, they ranked RTD highly among diseases despite<br />
the relatively low frequency <strong>of</strong> outbreaks because it was extremely destructive when it<br />
occurred <strong>and</strong> enormously difficult to control (Warburton et al, 1997). People tend to<br />
perceive risks with unknown <strong>and</strong> uncontrollable aspects as greater than those with known<br />
<strong>and</strong> more controllable characteristics even though the probability <strong>of</strong> the event is much<br />
less (Slovic, 1987).<br />
Farmers’ perception <strong>of</strong> diseases <strong>and</strong> their underst<strong>and</strong>ing <strong>of</strong> pathogen ecology are<br />
important factors that influence on their adoptability <strong>of</strong> newly developed control methods<br />
(Nelson, 2001). Knowing the farmers’ perception <strong>of</strong> the mixed seeds planting is<br />
88
necessary for further development <strong>of</strong> technology transfer scheme to implement this new<br />
technology effectively to farmers.<br />
The present study showed less than half <strong>of</strong> respondents had expressed the awareness <strong>of</strong><br />
the new technology “mixed seed planting” (Fig. 16 A). The mixed seed planting had<br />
never been introduced to the farmers <strong>and</strong> this could be the reason <strong>of</strong> much unawareness<br />
<strong>of</strong> this new RTD management option among other respondents. Although there were four<br />
seasons field trial <strong>of</strong> “mixed seed planting” conducted in the field <strong>of</strong> Mr. Domingo<br />
Bonifacio, no farmer field day was ever conducted for other farmers to observe the trial.<br />
Only fewer inquisitive farmers had a chance to observe the trials. There were several<br />
farmers' constraints related to the adaptation <strong>of</strong> the mixed seeds planting such as no<br />
knowledge <strong>of</strong> this new technology, no access to the mixed seeds, or doubts about the new<br />
technology itself. These constraints have to be considered as farmers' inputs associated<br />
with the adaptability <strong>of</strong> mixed seeds planting.<br />
In contrast, among the farmers who were aware <strong>of</strong> the mixed seed planting, belief<br />
<strong>of</strong> control effectiveness was considerably high <strong>and</strong> trials had been performed in their rice<br />
field (Fig. 16 B <strong>and</strong> C), implying a high possibility <strong>of</strong> this new technology adaptation<br />
among farmers though half <strong>of</strong> them, at present, would not recommend this new scheme to<br />
other farmers. Moreover, none <strong>of</strong> them considered this new RTD management scheme as<br />
tedious <strong>and</strong> laborious technology, a fact that should increase the possibility <strong>of</strong> the<br />
farmer's adaptation <strong>of</strong> this control method. In addition, after the new technology was<br />
discussed to the farmers during the forum, more than 80% <strong>of</strong> the farmers were willing to<br />
try the mixed seed planting in the future although farmers are satisfied with their current<br />
89
ice quality. In addition to the grain quality, all respondents expressed their desire for<br />
high eating quality. Farmers want to plant varieties with much IR64-like quality traits.<br />
Farmer involvement in research <strong>and</strong> extension have been practiced in many places<br />
(Nelson, 2001). Involving farmers in the research process increases the chance <strong>of</strong> success<br />
in generating an effective agricultural technology (Rhoades <strong>and</strong> Booth. 1982).<br />
Participatory approaches <strong>of</strong>fer researchers a mechanism to ensure that their work is<br />
relevant to farmers’ needs <strong>and</strong> conditions (Pretty, 1995; Selener, 1997). Thus, in order<br />
for farmers to underst<strong>and</strong> the features <strong>of</strong> mixed seed planting, farmer participatory<br />
research approach will be adequate to diffuse this new technology effectively to farmers.<br />
90
Grain Quality Evaluation <strong>of</strong> <strong>Mixed</strong> Seed <strong>Planting</strong><br />
Milling quality. Rough rice samples from the different treatments had similar<br />
percentage <strong>of</strong> brown rice <strong>and</strong> milled rice with brown rice ranging from 72-74% <strong>and</strong><br />
milled rice from 61-64% (Fig. 17). The highest percentage <strong>of</strong> head rice recovery was<br />
from rough rice harvested in 25% R: 75% S (43.6%) <strong>and</strong> followed by 100% S (pure st<strong>and</strong><br />
IR64), whereas, the lowest percentage was found in 75% R: 25% S.<br />
Analysis <strong>of</strong> the milled rice revealed that percentage <strong>of</strong> whole grain in mixtures<br />
were comparable with 100% S or 100% R (pure st<strong>and</strong> Matatag 9) except for that in 75%<br />
R: 25% S <strong>and</strong> broken rice were also highest in 75% R: 25% S (31%), while, other<br />
mixture treatments had a comparable percentage <strong>of</strong> broken rice to 100% S (Fig. 18).<br />
Fig. 17. Milling yield <strong>of</strong> the rough rice harvested from plots planted to different<br />
seed mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S (susceptible IR64).<br />
91
Fig. 18. Percentage <strong>of</strong> whole grain <strong>and</strong> broken rice from rough rice harvested<br />
from plots planted to different seed mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong><br />
S (susceptible IR64) analyzed by Cervitec 1625 Grain Inspector<br />
Grain size, shape <strong>and</strong> appearance. Grain appearance largely determines market<br />
acceptability <strong>of</strong> milled rice <strong>and</strong> grains from mixtures with very low chalkiness could be<br />
readily accepted by consumers in the Philippines.<br />
Visually all the samples from the different treatments had similar appearance in<br />
terms <strong>of</strong> shape <strong>and</strong> size (Fig. 19) <strong>and</strong> these were confirmed by the average length <strong>and</strong><br />
width <strong>of</strong> the milled grains as measured by Cervitec 1625 Grain Inspector (Table 20). The<br />
grains from mixture treatments were classified into long <strong>and</strong> slender rice grain based on<br />
the size (Table 3-a) <strong>and</strong> shape (Table 3-b) classification <strong>and</strong> its ratio in this study. This<br />
grain type is preferred by millers in the Philippines.<br />
92
Fig. 19. Grain appearance <strong>of</strong> rough rice (paddy) <strong>and</strong> milled rice from each treatment.<br />
93
Table 20. Grain length <strong>and</strong> width <strong>of</strong> milled rice harvested from plots planted to different<br />
seed mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S (susceptible IR64) analyzed by<br />
Cervitec 1625 Grain Inspector<br />
TREATMENTS NUMBER OF<br />
GRAINS<br />
ANALYZED<br />
AVERAGE<br />
LENGTH<br />
(mm)<br />
AVERAGE<br />
WIDTH<br />
(mm)<br />
94<br />
LENGTH/WIDTH<br />
RATIO<br />
100% R 519 6.8 ± 0.3 1.9 ± 0.2 3.6 ± 0.5<br />
75% R : 25% S 348 6.7 ± 0.3 1.9 ± 0.3 3.5 ± 0.7<br />
50% R : 50% S 537 6.7 ± 0.3 1.9 ± 0.2 3.5 ± 0.5<br />
25% R : 75% S 460 6.7 ± 0.3 2.0 ± 0.2 3.4 ± 0.5<br />
100% S 483 6.7 ± 0.3 2.0 ± 0.2 3.4 ± 0.5<br />
Milled grains <strong>of</strong> pure st<strong>and</strong> Matatag 9 were more translucent than those <strong>of</strong> pure<br />
st<strong>and</strong> IR64 as shown by the high percentage (74.5%) <strong>of</strong> grains having only less than 10%<br />
chalkiness as compared to 55.5% in pure st<strong>and</strong> IR64 (Table 21). Milled grains from<br />
mixture treatments had better (high percentage with less than 10% chalkiness) grain<br />
appearance than pure st<strong>and</strong> IR64.<br />
Table 21. Degree <strong>of</strong> chalkiness <strong>of</strong> milled rice harvested from plots planted to different<br />
seed mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S (susceptible IR64) analyzed by<br />
Cervitec 1625 Grain Inspector<br />
NUMBER OF CHALK CHALK CHALK CHALK CHALK<br />
TREATMENT GRAINS ANALYZED 0-10 % 10-25 % 25-50 % 50-75 % > 75 %<br />
100% R 519 74.5 23.9 0.7 0.9 0<br />
75% R : 25% S 348 58 39.7 0.9 0.5 0.9<br />
50% R : 50% S 537 70.6 28.5 0.2 0.5 0.2<br />
25% R : 75% S 460 61.6 36.6 0.6 1 0.2<br />
100% S 483 55.5 42.9 0 0.9 0.7
Amylose content <strong>and</strong> gelatinization temperature. Pure IR64 had higher amylose<br />
content than pure Matatag 9 (Table 22), however, all treatments had amylose content<br />
within the range <strong>of</strong> 20-25% that belongs to the intermediate type which is preferred by<br />
consumers in the Philippines.<br />
The gelatinization temperature <strong>of</strong> starch determines the time required for cooking.<br />
Gelatinization temperature is the temperature at which 90 - 98% <strong>of</strong> the starch granules<br />
swell irreversibly in hot water with loss <strong>of</strong> crystallinity <strong>and</strong> birefringence.<br />
Generally, gelatinization temperature ranged from 55° to 79°C. The gelatinization<br />
temperature <strong>of</strong> rice varieties could be estimated based on the spreading score <strong>and</strong><br />
classified as low (55° to 69 °C), intermediate (70° to 74°C), or high (>74°C). As shown<br />
in Table 22, milled grains from the different treatments had gelatinization temperature <strong>of</strong><br />
intermediate to high intermediate.<br />
Table 22. Amylose content <strong>and</strong> gelatinization temperature <strong>of</strong> milled rice harvested from<br />
plots planted to different mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S (susceptible IR64)<br />
TREATMENT AMYLOSE CONTENT,% GT*<br />
100% R 20.5 HI<br />
75% R : 25% S 20.6 HI<br />
50% R : 50% S 19.7 2HI/I<br />
25% R : 75% S 21.4 2HI/I<br />
100% S 22.2 1HI/I<br />
*HI = high intermediate; I = Intermediate; 1, 2 = number <strong>of</strong> grain in different category from the rest among<br />
6 grains<br />
95
Gel consistency From the viscosity measurements as shown in table 23, it showed<br />
that pure st<strong>and</strong> Matatag 9 cooked rice was harder (low breakdown <strong>and</strong> high set back) than<br />
pure st<strong>and</strong> IR64. The viscosity curve <strong>of</strong> mixed rices showed similarity to that <strong>of</strong> pure<br />
st<strong>and</strong> IR64 (Fig. 19). The hardiness <strong>of</strong> the cooked rice (consistency) <strong>of</strong> all treatments was<br />
very similar although Matatag 9 was a little harder <strong>and</strong> IR64 was s<strong>of</strong>t. The texture <strong>of</strong> the<br />
mixture treatments was s<strong>of</strong>t compared to Matatag 9 but harder than IR64. The appearance<br />
<strong>and</strong> texture <strong>of</strong> the cooked mixed rice (T2-T4) was very similar to IR64.<br />
Table 23. Viscosity analysis <strong>of</strong> cooked milled rice harvested from plots planted to<br />
different mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S (susceptible IR64) by the Rapid<br />
Visco Analyzer.<br />
TROUGH TREATMENTS<br />
100% R 75% R : 25% S 50% R : 50% S 25% R : 75% S 100% S<br />
Peak Viscosity (PV) 2386 2795 2691 2881 2919<br />
Trough 1421 1504 1478 1562 1542<br />
Final Viscosity (FV) 3007 3076 3022 3127 3087<br />
Breakdown 1<br />
965 1291 1213 1319 1377<br />
Setback 2 621 281 331 246 168<br />
Consistency 3<br />
1586 1572 1544 1565 1545<br />
1 2 3<br />
= peak viscosity - trough, = final viscosity - peak viscosity, = final viscosity – trough<br />
Sensory evaluation <strong>of</strong> the cooked rice. The appearance <strong>and</strong> texture <strong>of</strong> the cooked<br />
rice from the T2-T4 treatments was very similar to IR64. Matatag 9 cooked rice texture<br />
was a bit hard while IR64 was obviously s<strong>of</strong>t. The mixture treatments showed s<strong>of</strong>ter<br />
cooked rice than Matatag 9 but were not as s<strong>of</strong>t as IR64.<br />
96
PV<br />
Trough<br />
Fig 19. Viscosity curves <strong>of</strong> cooked milled rice harvested from plots planted to<br />
different mixture ratio <strong>of</strong> R (resistant Matatag 9) <strong>and</strong> S (susceptible IR64) by the<br />
Rapid Visco Analyzer.<br />
In the farmers’ perception survey conducted in Ajuy, grain quality was a great<br />
concern among respondents aside from the impacts <strong>of</strong> mixed seeds planting on disease<br />
reduction. Also grain quality is another important criterion to measure the success <strong>of</strong> the<br />
variety mixture in practice.<br />
The milling quality <strong>of</strong> rice may be defined as the ability <strong>of</strong> rice grain to st<strong>and</strong><br />
milling <strong>and</strong> polishing without undue breakage so as to yield the greatest amount <strong>of</strong> total<br />
recovery <strong>and</strong> the highest proportion <strong>of</strong> head rice to broken. The highest percentage <strong>of</strong><br />
head rice recovery was obtained from rough rice harvested in 25% R: 75% S (43.6%)<br />
followed by 100% S (pure st<strong>and</strong> IR64) <strong>and</strong> percentage <strong>of</strong> whole grain in mixtures were<br />
FV<br />
97
comparable with 100% S or 100% R (pure st<strong>and</strong> Matatag 9) except for that in 75% R:<br />
25% S (Fig. 17 <strong>and</strong> 18), indicating head rice recovery in mixtures increased with the<br />
proportion <strong>of</strong> IR64. Head rice recovery <strong>of</strong> Matatag 9 was lower compared with IR64 thus<br />
mixing it with IR64 improved head rice recovery in mixtures<br />
Grain appearance is largely determined by endosperm opacity or the amount <strong>of</strong><br />
chalkiness either on the dorsal side <strong>of</strong> the grain (white belly), or in the center (white<br />
center). Head rice recovery is dependent upon grain size, shape <strong>and</strong> appearance, bold <strong>and</strong><br />
chalky rice grain like IR5 <strong>and</strong> IR8 have low head rice recovery as compared to long,<br />
slender <strong>and</strong> translucent rice grain like IR36 <strong>and</strong> IR42 which has high head rice recovery.<br />
IR64 also has a high head rice recovery (Khush <strong>and</strong> Virk, 2005). Preference for grain size<br />
<strong>and</strong> shape vary from one group <strong>of</strong> consumers to another. In Southeast Asia the dem<strong>and</strong> is<br />
for medium to medium long grains rice (Khush et al. 1979). The results have showed<br />
milled grains from mixture treatments are long <strong>and</strong> slender rice grain <strong>and</strong> have better<br />
grain appearance than pure st<strong>and</strong> IR64 because <strong>of</strong> high percentage with less than 10%<br />
chalkiness. Thus, milled grain <strong>of</strong> seed mixture will be preferred by millers as well as<br />
consumer in Southeast Asia.<br />
In some varieties the grain tends to break more frequently at the “eye” or pit left<br />
by the embryo when it is milled. Rice samples with damaged eyes have poor appearance<br />
<strong>and</strong> low market value. Similarly, the greater chalkiness is equivalent to the lower market<br />
value (Khush et al. 1979).<br />
Many <strong>of</strong> the cooking <strong>and</strong> eating characteristics <strong>of</strong> milled rice are influenced by<br />
the ratio <strong>and</strong> structure <strong>of</strong> two kinds <strong>of</strong> starches, amylose <strong>and</strong> amylopectin in the rice grain<br />
98
(Sanjiva Rao et al. 1952). Amylose is the linear fraction <strong>of</strong> starch (up to 30%), whereas<br />
amylopectin, the branched fraction, makes up the remainder <strong>of</strong> the starch (~70%).<br />
Amylose is almost absent from the waxy (glutinous) rices. Amylose content,<br />
gelatinization temperature, <strong>and</strong> gel consistency test indicated that mixing <strong>of</strong> IR64 with<br />
Matatag 9 improved the cooked rice texture <strong>of</strong> Matatag 9.<br />
A great majority <strong>of</strong> the rices from Vietnam, Thail<strong>and</strong>, Myanmar <strong>and</strong> the Indian<br />
subcontinent have high amylose content. These rices show high volume expansion <strong>and</strong> a<br />
high degree <strong>of</strong> flakiness. They cook dry, are less tender <strong>and</strong> become hard upon cooling.<br />
Low amylose rices cook moist <strong>and</strong> sticky. All <strong>of</strong> the japonica varieties <strong>of</strong> temperate<br />
regions have low amylose content. Varieties grown in the Philippines, Malaysia <strong>and</strong><br />
Indonesia have intermediate amylose content. Intermediate-amylose rices cook moist<br />
<strong>and</strong> tender <strong>and</strong> do not become hard upon cooling. Intermediate amylose rices are the<br />
preferred type in most <strong>of</strong> the rice-growing areas <strong>of</strong> the world, except where low-amylose<br />
japonicas are grown such as Japan.<br />
99
SUMMARY AND CONCLUSION<br />
To characterize the resistance <strong>of</strong> Matatag 9 against rice tungro disease (RTD),<br />
Matatag 9 <strong>and</strong> an RTD-susceptible variety IR64 were evaluated for their reactions to<br />
tungro viruses <strong>and</strong> the insect vector (GLH). Several characteristics have been<br />
revealed through this study. The level <strong>of</strong> RTBV resistance in Matatag 9 is weak or<br />
due to resistance against other disease components as the number <strong>of</strong> GLH influence<br />
the RTBV infection in Matatag 9. The relative quantification <strong>of</strong> RTBV <strong>and</strong> RTSV in<br />
each <strong>of</strong> the varieties at different plant ages indicated that Matatag 9 can be infected<br />
with both RTBV <strong>and</strong> RTSV <strong>and</strong> that RTBV can infect in relatively high efficiency.<br />
Lower levels <strong>of</strong> virus infection in older plants were observed in this study, this could<br />
be attributed to adult plant resistance involving indigenous plant defense response to<br />
the insect <strong>and</strong> plant pathogen. Both viruses are likely to replicate in Matatag 9 using<br />
their typical replication pathway as RTBV <strong>and</strong> RTSV are introduced into Matatag 9,<br />
though symptom development was deterred in Matatag 9, indicating that Matatag 9<br />
has tolerance against RTBV. On the other h<strong>and</strong>, Matatag 9 could be considered as<br />
resistant to RTSV based on the infection rate in aged plants, though the accumulation<br />
<strong>of</strong> RTSV in Matatag 9 is still readily detectable.<br />
Matatag 9 was found less preferred host than IR64 by GLH for settling,<br />
implying the contribution <strong>of</strong> antixenosis characteristics to resistance <strong>of</strong> Matatag 9.<br />
Short adult longevity, more than 60% nymph mortality, long nymph developmental<br />
period <strong>and</strong> low percentage <strong>of</strong> nymph becoming adult are indicative <strong>of</strong> antibiosis
effects <strong>of</strong> Matatag 9. Moreover, growth index on Matatag 9 was significantly lower<br />
than both IR64 <strong>and</strong> TN1, while IR62 had almost same growth index value, supporting<br />
significant involvement <strong>of</strong> antibiosis effect in resistance against RTD in Matatag 9.<br />
101<br />
Collectively, the results <strong>of</strong> this study strongly suggest that the resistance <strong>of</strong><br />
Matatag 9 is mainly due to insect resistance although the contribution <strong>of</strong> resistance<br />
against tungro viruses in Matatag 9 cannot be ignored.<br />
Matatag 9 <strong>and</strong> IR64 have similar agronomical characteristics, thus, Matatag 9<br />
<strong>and</strong> IR64 can be mixed <strong>and</strong> harvested together. In the Philippines, the interplanting<br />
approach to manage RTD was initially attempted, however, a majority <strong>of</strong> farmers<br />
were not willing to adopt the laborious practice. Instead, the mixed seed planting was<br />
designed.<br />
The results <strong>of</strong> tray inoculation suggest that the mixed seed planting seems to<br />
be more effective to reduce RTD incidence than interplanting in an area when the<br />
disease pressure is not extremely high. Therefore, the mixed seed planting could be<br />
recommended to the farmers as a new RTD management option when the disease<br />
pressure is not extremely.<br />
Possible explanation <strong>of</strong> the difference in effectiveness on disease reduction<br />
between the two methods is the difference in spatial distribution <strong>of</strong> mixture<br />
components. As both Matatag 9 <strong>and</strong> IR64 in the mixed seed planting are r<strong>and</strong>omly<br />
distributed, it generates an inconsistent spatial pattern that may influence the<br />
movement <strong>and</strong> feeding behavior <strong>of</strong> insect vector. On the other h<strong>and</strong>, interplanting<br />
provides a consistent spatial distribution <strong>of</strong> mixture components <strong>and</strong> insects may
easily to reach their preferred host in the interplanting arrangement. Thus, the r<strong>and</strong>om<br />
spatial distribution <strong>of</strong> individual resistant plants was proposed as one <strong>of</strong> the potential<br />
mechanisms for the reduction <strong>of</strong> RTD in the mixed seed planting. However, it is still<br />
necessary to conduct a long term field trial in order to underst<strong>and</strong> the mechanism <strong>of</strong><br />
the mixed seed planting particularly the virus-vector interaction <strong>and</strong> their ecology in<br />
the mixture condition.<br />
102<br />
RTBV <strong>and</strong> RTSV can be transmitted during host selection events such as sylet<br />
probing on plant tissue <strong>and</strong> plant fluid ingestion regardless <strong>of</strong> varieties. Also GLH can<br />
retain RTBV <strong>and</strong> RTSV for 2-4days <strong>and</strong> 4-5 days, respectively, however, once the<br />
insects feed on Matatag 9, they may lose their virus retention ability. Therefore,<br />
Matatag 9 may contain certain factors which inhibit the binding between viruses <strong>and</strong><br />
leafhopper cuticula or suppress the activity <strong>of</strong> helper component required in the<br />
binding between virus <strong>and</strong> cuticula at retention sites <strong>of</strong> the leafhopper.<br />
Farmers’ perception <strong>of</strong> diseases <strong>and</strong> their underst<strong>and</strong>ing <strong>of</strong> pathogen ecology<br />
are important factors that influence their adaptation <strong>of</strong> newly developed control<br />
methods. Knowing the farmers’ perception <strong>of</strong> the mixed seed planting is necessary<br />
for further development <strong>of</strong> technology transfer scheme to implement this new<br />
technology effectively to farmers.<br />
The study investigated rice farmers’ knowledge <strong>and</strong> perception <strong>of</strong> RTD <strong>and</strong> its<br />
management <strong>and</strong> focused in detail on farmers’ perception regarding the use <strong>of</strong> mixed<br />
seed planting <strong>of</strong> resistant <strong>and</strong> susceptible varieties for RTD management. The farmers<br />
have relatively high awareness <strong>and</strong> knowledge <strong>of</strong> RTD <strong>and</strong> its management in Ajuy,
Iloilo, Philippines. In Ajuy main control strategies <strong>of</strong> farmers for RTD were based on<br />
using resistant varieties <strong>and</strong> on spraying insecticide although they are aware that the<br />
resistance may break down over time <strong>and</strong> the use <strong>of</strong> insecticide was not effective in<br />
103<br />
controlling RTD. Only 46% <strong>of</strong> the respondents were aware <strong>of</strong> the mixed seed planting,<br />
however, 79% <strong>of</strong> those who were aware believed that it was effective for controlling<br />
RTD <strong>and</strong> only 17% believed otherwise. About 70% <strong>of</strong> those who were aware had<br />
tried mixed seed planting for 1 or 2 seasons, <strong>and</strong> more than 70% had observed its<br />
effectiveness in controlling RTD. The low awareness <strong>of</strong> the technology “mixed seed<br />
planting” was because it had never been introduced to the farmers, therefore, in order<br />
for farmers to underst<strong>and</strong> the features <strong>of</strong> mixed seeds planting, farmer participatory<br />
research approach will be adequate to diffuse this new technology effectively to<br />
farmers. After the new technology was discussed with the farmers during the forum<br />
more than 80% <strong>of</strong> the farmers were willing to try the mixed seed planting in the<br />
future although farmers are satisfied with their current rice quality. In addition to the<br />
grain quality, all respondents expressed their desire to plant varieties with high eating<br />
quality <strong>and</strong> other much IR64-like quality traits.<br />
In the farmers’ perception survey conducted in Ajuy, grain quality was a great<br />
concern among the respondents aside from the impacts <strong>of</strong> mixed seed planting on<br />
disease reduction. Moreover, grain quality is another important criterion to measure<br />
the success <strong>of</strong> the variety mixture in practice.<br />
In general for non-waxy rice, medium <strong>and</strong> long grain varieties with clear or<br />
translucent kernels <strong>of</strong> intermediate amylose that will remain s<strong>of</strong>t even after cooking
are preferred by most consumers in the Philippines. A variety with a desirable<br />
combination <strong>of</strong> intermediate amylose <strong>and</strong> gelatinization temperature <strong>and</strong> s<strong>of</strong>t gel<br />
consistency is IR64. Its grain quality is considered superior to that <strong>of</strong> other IR<br />
varieties <strong>and</strong> it has been adopted widely in tropical <strong>and</strong> subtropical Asia.<br />
104<br />
Analysis revealed that rough rice harvested from plots planted to different<br />
ratio <strong>of</strong> seed mixture have similar milling yield compared to the pure IR64 <strong>and</strong><br />
Matatag 9, the components <strong>of</strong> the mixture. Visually all samples whether from the<br />
mixtures or from pure Matatag 9 or IR64 also look similar in terms <strong>of</strong> grain shape <strong>and</strong><br />
size as they were long <strong>and</strong> slender. Milled rice appearance <strong>of</strong> IR64 is chalkier than<br />
Matatag 9 but chalkiness was reduced in seed mixtures. Rice grains from mixed seed<br />
planting also had intermediate amylose content comparable to IR64 <strong>and</strong> they have<br />
high intermediate to intermediate gelatinization temperature. The hardiness <strong>of</strong> the<br />
cooked rice (consistency) <strong>of</strong> all treatments was very similar although 100% R<br />
(Matatag 9) was a little harder <strong>and</strong> 100% S (IR64) was s<strong>of</strong>t. The texture <strong>of</strong> the<br />
mixture treatments was s<strong>of</strong>t compared to 100% R but harder than 100% S. The<br />
appearance <strong>of</strong> the cooked mixed rice (T2-T4) was very similar to 100% S (Pure IR64).<br />
Thus, grain quality properties would not be affected when IR 64 was mixed in<br />
different ratio with Matatag 9. Moreover, mixing <strong>of</strong> IR64 with Matatag 9 improved<br />
the cooked rice texture <strong>of</strong> Matatag 9 <strong>and</strong> farmers in tungro endemic areas could plant<br />
mixed seeds <strong>of</strong> susceptible IR64 with resistant Matatag 9 <strong>and</strong> be assured <strong>of</strong> grain<br />
quality comparable to IR 64, the most preferred variety by rice millers <strong>and</strong> consumers<br />
in the Philippines
RECOMMENDATIONS<br />
1. Further study is necessary to investigate the RTSV resistance in Matatag 9.<br />
2. Rice variety that has only insect resistance could be used as a (R) mixture<br />
component as long as it has similar agronomical characteristic <strong>and</strong> grain quality as<br />
the commercial but RTD susceptible component.<br />
3. <strong>Mixed</strong> seed planting could be recommended to the farmers as a new RTD<br />
management option when the disease pressure is not extremely high though it is<br />
still necessary to conduct a long term field trial in order to underst<strong>and</strong> the<br />
mechanism <strong>of</strong> the mixed seed planting particulary the virus-vector interaction <strong>and</strong><br />
their ecology in the mixture condition.<br />
4. Farmer participatory research approach is necessary to diffuse this new<br />
technology effectively to farmers in order to underst<strong>and</strong> the features <strong>of</strong> mixed<br />
seeds planting.<br />
5. Since rice grains from mixed seed planting <strong>of</strong> Matatag 9 <strong>and</strong> IR64 have similar<br />
milling yield, physical attributes, <strong>and</strong> physicochemical characteristics with the<br />
pure IR64 <strong>and</strong> improved the cooked rice texture <strong>of</strong> Matatag9, farmers in RTD-<br />
endemic areas could plant mixed seed <strong>of</strong> susceptible IR64 with resistant Matatag<br />
9 <strong>and</strong> be assured <strong>of</strong> grain quality comparable to IR64 <strong>and</strong> still manage RTD.
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Appendix 1. Questions for socioeconomic survey in Ilo-ilo.<br />
A. Participants’ Pr<strong>of</strong>ile<br />
1) Name_______________________________Age_____________<br />
2) Marital Status ( ) Single ( ) Married<br />
3) Number <strong>of</strong> Children_____ Number <strong>of</strong> household members ______<br />
4) Education<br />
( ) Elementary graduate<br />
( ) High School graduate<br />
( ) College graduate<br />
( ) No education<br />
5) L<strong>and</strong> Ownership ( ) Owner ( ) Tenant ( ) Share Holder<br />
6) Size <strong>of</strong> farm area:________________Hectares<br />
7) Size <strong>of</strong> area planted to rice_________Hectares<br />
B. Farm Pr<strong>of</strong>ile<br />
1) How long have you been farming? _________Years<br />
2) Are you a commercial or self sustaining farmer?<br />
( ) Commercial (Sells harvest)<br />
( ) Self sustaining (Produce for family consumption only)<br />
3) Average gross income per year from rice farm ______P<br />
4) Average rice yield per season<br />
WS________Cavans <strong>of</strong> 50 Kg<br />
DS_________Cavans <strong>of</strong> 50 Kg<br />
5) Name <strong>of</strong> varieties planted for the past 4 seasons<br />
________________ _________________<br />
________________ _________________<br />
6) Where did you acquire the varieties you planted?<br />
( ) Seed grower ( ) DA <strong>of</strong>fice ( ) Research Station<br />
( ) Other farmers ( ) Other Sources____________<br />
7) Common insect problems in your rice farm<br />
________________ _________________<br />
________________ _________________<br />
8) Common disease problems in your rice farm<br />
________________ _________________<br />
________________ _________________<br />
9) Rank the importance <strong>of</strong> the following insect <strong>and</strong> disease problems in rice: 1, 2, 3, 4<br />
<strong>and</strong> 5 with, 1 as most important <strong>and</strong> 5 the least important<br />
( ) Stemborer ( ) Rice blast<br />
( ) BPH ( ) Bacterial leaf blight<br />
( ) Rice bug ( ) Tungro
( ) Leaf folders ( ) Sheath blight<br />
( ) Cut worms ( ) Bacterial leaf streak<br />
C. Awareness <strong>of</strong> tungro disease<br />
1) Are you aware <strong>of</strong> tungro disease? ( ) Yes ( ) No<br />
2) Have you had tungro disease in your rice farm ( ) Yes ( ) No<br />
3) For past 5 years how many times did you have tungro in your rice<br />
farm?________Times<br />
4) How serious was the damage <strong>of</strong> tungro in your farm? _____ Rank 1 to 3: with, 1 as<br />
most serious; 2 moderately <strong>and</strong> 3 as least serious<br />
5) What do you think causes tungro disease?<br />
( ) Poor soil ( ) Poor water ( ) Poor seeds ( ) Fungi ( ) Bacteria<br />
( ) Virus ( ) Insects ( ) Others ____________<br />
6) What spread tungro disease?<br />
( ) Water ( ) Seeds ( ) Wind ( ) Brown planthopper<br />
( ) Green leafhopper ( ) Others _______________<br />
7) How do you distinguish tungro from other diseases?<br />
( ) Change <strong>of</strong> leaf color ( ) Height reduction<br />
( ) Presence <strong>of</strong> green leafhopper<br />
D. Awareness <strong>of</strong> tungro disease control<br />
1) How do you control tungro in your rice field?<br />
( ) Spray pesticides. What pesticides? ___________________<br />
( ) Use <strong>of</strong> resistant varieties. What varieties? ______________<br />
( ) Removal <strong>of</strong> infected plants. ( ) Others______________<br />
2) Do you think tungro could be controlled with the use <strong>of</strong> insecticides? ( ) Yes<br />
( ) No<br />
3) If yes, do you think it is effective? ( ) Yes ( ) No<br />
4) Are you aware <strong>of</strong> the availability <strong>of</strong> resistant varieties?<br />
( ) Yes ( ) No<br />
5) Have you experienced breakdown <strong>of</strong> resistance <strong>of</strong> your varieties to tungro disease?<br />
( ) Yes ( ) No<br />
E. Awareness <strong>of</strong> mixed variety planting<br />
1) Are you aware <strong>of</strong> mixed planting <strong>of</strong> resistant <strong>and</strong> susceptible varieties for tungro<br />
control? ( ) Yes ( ) No<br />
2) If yes, do you think it could control tungro disease?<br />
( ) Yes ( ) No<br />
3) If yes, have you tried using mixed variety to control tungro in your rice farm? ( )<br />
Yes ( ) No<br />
4) How long have you practiced this control method in your rice field?<br />
125
( ) 1 season ( ) 2 seasons ( ) 3 seasons ( ) 4 seasons ( ) more<br />
5) Have you observed the effect <strong>of</strong> mixed variety control method against tungro disease<br />
( ) Yes ( ) No<br />
6) Would you recommend this method to other farmers?<br />
( ) Yes ( ) No<br />
7) Are you still planting mixed variety in your rice farm?<br />
( ) Yes ( ) No<br />
8) If no, Why did you stop using mixed seeds?<br />
( ) No tungro incidence ( ) No observed effect ( ) Too laborious<br />
9) If no, why haven’t you applied this method in your farm?<br />
( ) No access to mixed seeds<br />
( ) Have no knowledge about this method<br />
( ) Still doubtful <strong>of</strong> this method<br />
( ) Others, What?________________<br />
10) Are you planning to apply this method in the future?<br />
( ) Yes ( ) No<br />
11) Are you satisfied with the eating quality <strong>of</strong> the rice you planted?<br />
( ) Yes ( ) No<br />
12) Do you like to plant varieties with same eating quality as IR 64<br />
( ) Yes ( ) No<br />
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