EVALUATION OF RICE DIFFERENTIAL VARIETIES AGAINST RAIPUR BROWN PLANTHOPPER Nilaparvata lugens (Stal) (HOMOPTERA : DELPHACIDAE) STRAIN

Transcription

1 EVALUATION OF RICE DIFFERENTIAL VARIETIES AGAINST RAIPUR BROWN PLANTHOPPER Nilaparvata lugens (Stal) (HOMOPTERA : DELPHACIDAE) STRAIN M.Sc. (Ag.) Thesis by PRITANSHA BHAGAT DEPARTMENT OF ENTOMOLOGY COLLEGE OF AGRICULTURE INDIRA GANDHI KRISHI VISHWAVIDYALAYA RAIPUR (C.G.) 2014

2 EVALUATION OF RICE DIFFERENTIAL VARIETIES AGAINST RAIPUR BROWN PLANTHOPPER Nilaparvata lugens (Stal) (HOMOPTERA : DELPHACIDAE) strain Thesis Submitted to the Indira Gandhi Krishi Vishwavidyalaya, Raipur by Pritansha Bhagat IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in Agriculture (Entomology) Roll No ID No JULY, 2014

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5 ACKNOWLEDGEMENT First of all I would like to thank and praise Almighty God the most beneficent and merciful, for all his love and blessing conferred upon mankind. First and foremost I would like to place on record my ineffable indebtedness to my Hon ble guide and Chairman of my Advisory Committee, Dr. Sanjay Sharma, Principal Scientist and Professor, Department of Entomology, IGKV, Raipur, for illuminating guidance, unique supervision, kind sympathetic attitude, constant encouragement, valuable and fruitful suggestions, constructive criticism and constant efforts during the entire course of investigation and preparation of manuscript. I am sincerely thankful to him for his meticulous efforts devoted towards my research work even in the midst of his busy schedule. I own profound debt to Dr. V. K. Kosta, Head of the Department of Entomology, for timely inspecting and encouraging us during the course of work in the midst of his busy schedule and for kind sympathetic attitude during the whole degree programme. I extend my heartiest thank to members of my advisory committee Dr. V. K. Koshta, Professor and Head, Department of Entomology and Dr. D. K. Rana, Senior Scientist and Professor, (Department of Entomology), Dr. Girish Chandel, Professor, (Department of Plant Molecular Biology and Biotechnology), Dr. R.R. Saxena, Professor, (Department of Agril. Statistics, Mathematics and Computer Science) for their critical suggestions and regular encouragement during course of my investigation. I am highly indebted to the teachers of my Department Dr. A.K. Dubey, Dr. Rajeev Gupta, Dr. S.K. Shrivastva, Dr. (Smt.) Jaya Laxmi Ganguli, Dr. R.N. Ganguli, Dr. H.K. Chandrakar Dr. Y.K. Yadu, Dr. V.K.Dubey, Shri Gajendra Chandrakar, Shri Vikas Singh, Shri Navneet Rana and Smt. Sonali Deol for their constant co-operation, suggestion, encouragement and help during my investigation. I am highly obliged to Hon ble Vice Chancellor, Dr. S. K. Patil, Dr. M. P. Thakur, Director Extension Services, Dr. J. S. Urkurkar, Director Research Services, Dr. S. Patel, Dean, College of Agriculture, Raipur and Dr. S. S. Shaw, Director of Instructions, IGKV, Raipur for providing necessary facilities to conduct the present investigation.

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7 CONTENTS Chapter Title Page I INTRODUCTION 1-5 II REVIEW OF LITERATURE Screening of rice differential varieties carrying different resistant gene through standard seed box screening Location of vascular bundle in cross- section stem of different rice differential 2.2 Assessment of feeding activity of BPH on differential varieties carrying different resistant gene through honey dew test Assessment of feeding activity of BPH on differential varieties carrying different resistant gene through probing mark test Survival test of BPH nymph on different varieties of rice plant carrying different resistant gene through nymphal survival method Status of egg parasitization of brown planthopper in rice differential varieties III MATERIAL AND METHODS Mass rearing of BPH Screening of rice differential varieties carrying different resistant gene through standard seed box screening technique Location of vascular bundle in cross- section stem of different rice differential IV Assessment of feeding activity of BPH on differential varieties carrying different resistant gene through honey dew test Assessment of feeding activity of BPH on differential varieties carrying different resistant gene through probing mark test Survival test of BPH nymph on different varieties of rice plant carrying different resistant gene through nymphal survival method Status of egg parasitization of brown planthopper in rice differential varieties 3.5 Statistical analysis 43 RESULTS AND DISCUSSIONS Screening of rice differential varieties carrying different resistant gene through standard seed box screening technique Location of vascular bundle in cross- section stem of different rice differential Internodal position Nodal position Correlation between average plant damage score and anatomical studies with respect to location of vascular

8 V bundle in stem of rice varieties Assessment of feeding activity of BPH on differential varieties carrying different resistant gene through honey dew test Assessment of feeding activity of BPH on differential varieties carrying different resistant gene through probing mark test Correlation studies between honey dew excretion values & no. of probe marks as feeding behaviour of BPH Correlation studies between average plant damage score and honey dew excretion values Correlation studies between average plant damage score and probed marks Survival test of BPH nymph on different varieties of rice plant carrying different resistant gene through nymphal survival method Status of egg parasitization of brown planthopper in rice differential varieties Correlation studies between nymphal Survival and egg parasitization status of BPH Correlation studies between average plant damage score and per cent nymphal survival Correlation studies between average plant damage score and egg parasitization SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR THE FUTURE RESEARCH WORK 5.1 SUMMARY Screening of rice differential varieties carrying different resistant gene through standard seed box screening Location of vascular bundle in cross- section stem of different rice differential Assessment of feeding activity of BPH on differential 82 varieties carrying different resistant gene through honey dew test Assessment of feeding activity of BPH on differential varieties carrying different resistant gene through probing mark test Survival test of BPH nymph on different varieties of rice 83 plant carrying different resistant gene through nymphal survival method Status of egg parasitization of brown planthopper in rice 84 differential varieties 5.2 CONCLUSIONS SUGGESTIONS FOR THE FUTURE RESEARCH 86 WORK ABSTRACT 87 REFERENCES

9 LIST OF TABLES Table Title Page No. 3.1 Material used for experiment Standard for rating damage by brown planthopper 4.1 Plant damage score of Planthopper Special Screening (PHSS) Lines against Raipur BPH population 4.2 Location of vascular bundle in cross section stems of twenty different rice differential varieties (Internode position) 4.3 Location of vascular bundle in cross section stems of twenty different rice differential varieties (Node position) 4.4 Correlation between average plant damage score and location of vascular bundle in cross section stem of different rice varieties 4.5 Average feeding rate (honeydew excretion value) of BPH on differential varieties carrying different resistance gene 4.6 Average feeding marks (probing marks) of BPH on differential varieties carrying different resistance gene 4.7 Correlation between honey dew excretion values and no. of probe marks as feeding behaviour of BPH. 4.8 Correlation between average damage score and honey dew excretion values 4.9 Correlation between average plant damage score and probe marks by BPH feeding 4.10 Percent survival of BPH on differential varieties carrying different resistance gene 4.11 Status of BPH egg parasitisation in rice differential varieties carrying different resistance gene 4.12 Correlation between percent nymphal survival and egg parasitization of BPH Correlation between average damage score and per cent nymphal survival 4.14 Correlation between average plant damage score and per cent egg parasitisation

10 LIST OF FIGURES Figure Title Between Page 3.1 Layout of screening of rice genotypes for BPH resistance Average plant damage score of rice differentials Comparative measurement of distance VB E & P E (Internode position) 4.3 Comparative measurement of distance VB E & P E (Node position) 4.4 Regression equation on VB E distance (Internode) for average plant damage score 4.5 Regression equation on P E distance (Internode) for average plant damage score 4.6 Regression equation on VB E distance (Node) for average plant damage score 4.7 Regression equation on P E distance (Node) for average plant damage score 4.8 Feeding rate (honey dew excretion value) of BPH on set of rice differentials 4.9 Feeding marks (probed marks) by BPH on set of rice differentials 4.10 Comparative feeding behaviour ( honey dew excretion area & no. of probes) by BPH on set of rice differentials 4.11 Regression equation of no. of probed marks for honey dew excretion values 4.12 Regression equation of honey dew excretion values for average plant damage score 4.13 Regression equation of no. of probed marks for average plant damage score 4.14 Per cent survival of BPH nymphs on different rice differentials Per cent parasitization and unparasitization of hopper eggs Comparative assessment of per cent nymphal survival & egg parasitisation of BPH 4.17 Regression equation of egg parasitisation for per cent nymphal survival 4.18 Regression equation of per cent nymphal survival for average plant damage score

11 LIST OF PLATES PLATE NO. PARTICULARS BETWEEN PAGE 1 BPH adult female BPH adult male Mass rearing of BPH Stages of BPH on rice plants Seed box screening test for BPH resistance Anatomical investigation of rice stem in relation with BPH feeding Honey dew excretion test on set of rice differentials Assessment of feeding activity of BPH through probing mark test Nymphal survival test on set of rice differentials Egg parasitization of BPH on set of rice differentials 42-43

12 LIST OF ABBREVIATIONS ABBREVIATION DESCRIPTION % Per At the rate of µm micro metre b/n between BPH Brown plant hopper 0 C Degree Celsius CD cm et al. Fig. G ha hrs i.e. Kg m ha mm Mt No. PHSS P E SEm Sr. No. viz. VB - E Critical difference Centimeter And other Figure Gram Hectare Hours That is Kilogram Million hectare Millimeter Metric tone Number Plant hopper special screening Phloem to epidermis Standard error of mean Serial Number Namely Vascular bundle to epidermis

13 Introduction

14 CHAPTER-I INTRODUCTION Rice (Oryza sativa, L.) is the leading food crop for humans, providing dietary energy and protein for half of the world s population especially in the Asian region.total rice production, however, is expected to face serious challenges and is likely to be more unstable in the near future (Timmer, 2010). A world food crisis happened in , and this caused the price of food in international markets to increase drastically (Timmer, 2010). In addition, it has been predicted that the world population in 2020 will increase 29.7% to 7,593 billion people (Mullin, 1999), which could cause a huge demand for this crop. Therefore, it will be necessary for annual rice production to double to keep up with worldwide demand. Rice (Oryza sativa L.) is one of the most important cereal crop in the world, which has occupied an area of million hectares, with a total production of 476 million tones and productivity kg/ha (Anonymous, 2012). India is the second largest producer of rice after china which has an area of over million hectares with the production of million tones and productivity 2207 kg /ha (Anonymous, 2012). Rice productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the total global production of rice is lost annually owing to the damage caused by biotic factors, of which nearly 21% is attributed to the attack of insects pests (Brookes and Barfoot, 2003). Among the biotic factors, brown planthopper (BPH) Nilaparvata lugens (Stal.) is one of the major biotic constraint of rice productivity causing huge yield losses every year in rice grown throughout tropical, subtropical and temperate areas in Asia (Park et al., 2008).

15 Oerke (2005) has claimed that potential rice yield loss due to pests is between 65% to 80% production. The brown planthopper (BPH), Nilaparvata lugens (Stal), is one of the major phloem feeding insects which causes serious damage to the plant. They injure the plants through direct sucking of the plant sap, and they also transmit viruses, causing plant wilting or hopper burn when it becomes serious (Catindig et al., 2009). Previous to the green revolution, periodic outbreaks of BPH occurred in Japan and Korea. These outbreaks have been documented in records for more than 400 years (Mochida et al., 1977). The brown planthopper (BPH) Nilaparvata lugens (Stal) was the major rice pest of the green revolution in the 1970s and 1980s. In tropical Asia outbreaks were sporadic and restricted to small areas. However, BPH has become very serious in many tropical countries within the last 50 years and since the green revolution. In the 1970s, BPH caused extensive damage to the rice crop in Asia (Dyck and Thomas, 1979), mainly, because of the unpredictability of infestations and the dramatically severe damage caused. BPH populations can increase dramatically after insecticide applications (Shepard et al., 1995), because the widespread misuse of insecticides kills the important BPH natural enemies in rice (Kenmore et al., 1984). Moreover, the excessive use of chemical fertilizer increases the fecundity and survival of BPH, further increasing population. Host-plant resistance is an important strategy to reduce the damage caused by BPH and increase rice productivity. Twenty-one major genes for BPH resistance have been identified by using standard evaluation methods developed at the International Rice Research Institute (IRRI) to distinguish resistance or susceptibility of rice genotypes to BPH biotypes/populations (Jena & SukMan,

16 Plate 1 : BPH adult female (a) macropterous (fully-winged forms) and (b) brachypterous (truncate-winged forms) Plate 2 : BPH adult male (a) macropterous (fully-winged forms) and (b) brachypterous (truncate-winged forms)

17 2010.). Although many resistance loci have already been discovered, not all can be used to protect the rice plant from BPH attack (Jairin et al., 2007). Development of novel control strategies can be facilitated by comparison of BPH feeding behaviour on varieties exhibiting natural genetic variation, and then elucidation of the underlying mechanisms of resistance (Ghaffar et al., 2011). The mouthparts of BPH, like other phloem feeding insects, consist of a stylet bundle which forms the piercing and sucking organ (Sogawa,1982 ; Seo et al., 2009). BPH feeds on the plant by inserting the stylet bundle with an accompanying salivary sheath into the plant locating the phloem tissue and then regulating the ingestion of the pressurised plant sap (Sogawa,1982 ; Seo et al., 2009). The colour of BPH varies from yellowish brown to dark brown. Length of macropterous male ranges 2.3 to 2.4 mm (3.8 to 4.2 mm, including fore wing), female ranges 2.8 to 3.2 mm (4.4 to 4.8 mm, including fore wing), brachypterous male ranges 2.0 to 3.1 mm, female ranges 2.7 to 3.5 mm, post-tibial spur with teeth (Okada, 1977). BPH adult male and female are shown in plate-1 & plate-2.variations of the macropterous form of wings and the spur at the apex of the hind tibia are the characteristic of genus Nilaparvata. Insects on plant surfaces display complicated behaviours, many of which cannot be distinguished because of technological limitations (Sogawa, 1982; Backus, 1988; Habibi et al., 2008). Host-searching behaviour is an important process by which insects seek resources to acquire food and mates, and for oviposition and establishing nesting sites and refugia (Bell, 1990). It can be divided into several phases including habitat location, and host location, acceptance, suitability and regulation. Chemicals play an important role in these processes (Vinson, 1976). Primary metabolites including asparagine and phytosterols stored

18 prior to damage are reported to be involved in host-searching behavior in the BPH, because high asparagine and low phytosterol levels appear to attract BPHs (Shigematsu et al., 1982; Jung and Im, 2005). In contrast, the hydrocarbon- and carbonyl-containing fractions of the rice surface wax can reduce BPH settling and probing, and compel the insect to move from the stem to the leaves (Woodhead and Padgham, 1988). Secondary metabolites including volatile rice compounds play a vital role in the location of hosts by BPHs (Liu et al., 2002; Qi et al., 2011), which prefer to settle on BPH-susceptible rice varieties rather than on rice containing BPH resistance genes (Qiu et al., 2010). Rice plants pyramided with BPH resistance genes Bph6 and Bph12 have been reported to be more repellent to BPH than those having only Bph6 or Bph12 (Qiu et al., 2012). Development of resistant rice cultivars through host plant resistance is generally considered to be the most economic and effective way for controlling BPH population. Till date, 26 BPH resistance genes have been identified in wild species Oryza eichengiri, Oryza australiensis, Oryza officinalis,oryza glaberima, Oryza rufipogon, Oryza minuta and Indian cultivars (Zhang, 2007; Fujita et al., 2008). Many rice varieties with resistance to plant hopper have been developed and released to farmer for commercial cultivation, however the situation become alarming when the resistance of these new varieties diminished because of apparent selection for virulent biotypes of the pests (Khush, 1979). BPH populations on rice have been categorized into four biotypes (Khush et al., 1985). The population in the east and Southeast Asia is reported as biotype 1, while biotype 2 originated in Indonesia and Vietnam as dominant biotype (Khush, 1979). Biotype 3 was produced in the laboratory at IRRI (Pathak and Khush, 1979)

19 and in Japan whereas biotype 4 is found only in South Asia. The variation in virulence pattern was evident among BPH population in India. Monitoring of virulence and characterization of population is important for the development of insect pest resistant variety. Therefore, the general target in this project was to exploit the different phenotypic screening methods to identify the virulence characteristics of Raipur BPH strain against the rice differential varieties carrying different resistance genes which contribute to the development of plant resistance characteristics. Keeping in this view, the present investigation entitled Evaluation of rice differential varieties against Raipur brown planthopper Nilaparvata lugens (Stal) (Homoptera: Delphacidae) strain. was conducted in the Glass House, Department of Entomology, College of Agriculture, IGKV, Raipur during the year with the following objectives : 1. Screening of rice differential varieties carrying different resistance gene through standard seed box screening technique. 2. Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through honey dew test. 3. Survival test of BPH nymph on different varieties of rice plant carrying different resistance gene through nymphal survival method.

20 Review of Literature

21 CHAPTER-II REVIEW OF LITERATURE The studies have been carried out by different scientists on the various aspects of host plant resistance and antibiosis to Nilaparvata lugens (Stal.) on rice. For the sake of convenience and clarity the literature pertinent to the present studies was reviewed and included in this chapter. The brown plant hopper, Nilaparvata lugens (Stal.) belongs to the family Delphacidae, sub order Homoptera and order Hemiptera.Through out the world 14 determined and 2 undetermined species are reported as the member of genus Nilaparvata (Mochida et al., 1977), out of which Nilaparvata lugens is recognized as serious pest of rice (Nasu,1964). The first outbreak of brown plant hopper was observed in Myanmar, at the Kyaukse farm in the 1970 during wet season on the rice cultivars IR 20, IR 22, IR 24 and C4-64. Nine local cultivars i.e. Panthista, Sapasi, Ngakywe, Bahatni, Pawsanhmwe, Aungreya, Seingyi, Pyidawaye and Lothotgyi were highly resistant to the Kyaukse population of brown plant hopper. The occurrence and evolution of prolific biotypes of brown plant hopper is a constant threat to the stability of the pest resistant rice varieties presently under cultivation (Pathak and Saxena, 1980). The term BPH biotype refers to the populations of Nilaparvata lugens which differs in their ability to feed on and destroy varieties with a specific major gene for resistance (Diehl and Bush, 1984). Several wild species of Oryza are highly resistant to all the known biotypes of brown plant hopper, whitebacked planthopper, and leafhopper (Jena and Khush, 1990).

22 Seven differential donors tested at Raipur with local population of brown planthopper showed variation in reactions as compared to IRRI BPH population. Similarly, twenty one IR varieties exhibited differences when the results were examined against three BPH biotypes at IRRI. Thus concluded that the Raipur BPH population is different from that of at IRRI. Similarly, 15 Pattambi BPH donors also behaved dissimilarly towards Raipur BPH population too less virulent than that of Raipur (Pophaly and Rana, 1994). 2.1 Screening of rice differential varieties carrying different resistance gene through standard seed box screening technique. Sogawa and Pathak (1970) reported that occurrence of asparagines in minute quantities in Mudgo variety of rice considered to be primary cause of resistance against BPH. Mohammad and Alam (1978) screened 4324 varieties from the International Rice Research Institute collection. Twenty varieties (all Indica types and mostly from India and Sri Lanka) were resistant or moderately resistant. These varieties generally exhibited antibiosis and were not preferred by the insect. Norris and Kogan (1980) reported that a wide array of chemical substances including inorganic chemicals, primary and intermediary metabolites and secondary plant substances are known to impart biochemical resistance in a host plant to a wide variety of insect pests. Seshu and Kauffman, (1980) provided significant information on biotype variation in brown plant hopper and sources of genetic resistance to the different biotypes in the various countries as results from International Rice Brown Plant hopper Nursery screening in several Asian countries over a 5-year period ( ).

23 Biotype differences of lower order within the common insect populations in East and Southeast Asian countries are also evident. Differential reactions within areas in India were also noted. Within each of the group of varieties of Bph 1, bph 2, Bph 3, and bph 4 genes were identified at IRRI, certain differences are evident from the reactions in other countries and, based on those, a set of differential varieties has been proposed. PTB33, Suduru Samba, Sinna and Sivappu were resistant at almost all test sites. Several improved breeding lines derived from PTB33 were promising in all the regions of Asia and Solomon Island. Velusamy and Chelliah, (1984) tested 50,423 rice accessions against BPH at IRRI during 1967 to Out of these 555 accessions were found resistant. Thirty accessions have been reported as highly resistant out of 45 accessions, evaluated at Coimbatore. Sujatha et al., (1987) stated that phenol, silica, phosphorus potassium, sulphur and iron contents were positively correlated with resistance against BPH while the protein, nitrogen, zinc and manganese contents were negatively correlated with BPH resistance in rice. The presence of toxic substance or absence of required nutrients affects the biology of insect which is termed as antibiosis. Reddy and Misra (1995) screened 30 rice cultivars for resistance to Nilaparvata lugens (Stal.) in U.P., India. Rice cultivars culture 1, IR 28 and IR 8 showed a marked promise against the pest but were significantly less resistant than the resistant check Ptb33. Nanda et al. (2000) reported that resistant rice variety ARC 6650 had low level of amino acid percentage (5.27 %) and total starch content (5.75 %) as

24 compared to 8.12 % amino acid and 7.98 % total starch content in susceptible TN1 30 day old potted plants. Shen et al., (2002) investigated the resistance of 38 elite rice lines to brown planthopper (BPH), Nilaparvata lugens in the laboratory during in China, five elite lines were found to be resistant to BPH. Urre et al., (2004a) tested one hundred sixty eight promising rice genotypes against brown planthopper in laboratory conditions, at IGKV, Raipur. Out of which, 7 rice genotypes were found resistant, 26 as moderately resistant and rest of the genotypes as susceptible to brown planthopper. Soundararajan et al.,(2004) used the standard seedbox screening test (SSST) to measure the levels of resistance on standard checks, parents and DH lines of the cross IR64/Azucena for resistance to BPH at seedling stage. In this experiment, the damage scoring of Ptb33, TN1, IR 64, Azucena and DHLs were recorded as 3.7 ± 0.7, 8.3± 0.7, 5.7 ± 0.7, 9.0± 0.0 and 7.1± 0.1, respectively. Bhimrao et al., (2005) tested one hundred twenty one promising rice genotypes against brown planthopper in laboratory conditions at IGKV, Raipur. Out of these 3 rice genotypes were found resistant, 20 as moderately resistant and rest of the genotypes as susceptible to brown planthopper. Sue et al., (2006) carried out screening of rice genotypes along with resistant check Rathu Heenati and susceptible check TN1 and found that Kaharamana was resistant to biotype 1 of BPH with score 1.63, while was susceptible with score They also stated that Kaharamana was less resistant than Rathu Heenati whose resistance score was 0.4.

25 Atone (2008) tested five hundred seventy eight rice genotypes against BPH out of these, only two genotypes i.e. R and IR were found as highly resistant, 26 resistant and 19 moderately resistant. Siddegowda, (2009) conducted screening trials over a period of thirteen years ( ), 645 and 2186 entries/cultivars were screened against brown planthopper under Planthopper Screening (PHS) and National Screening Nursery (NSN1) trial, in green house and field. The study revealed that 158 donors possessing different levels of resistance to BPH. The rice cultivars IET 7575, IET 8116, IET 8110, IET 9912, IET 9873 and BPT 2217 were identified as resistant varieties, whereas IET 7575 and IET 8116 were released as BPH tolerant varieties for cultivation in Cauvery command area. The entry number KAUM , KAUM 95-1, RP 4656-IR B, RP and CR AC were found promising under retesting. MTU-2077, IET-8110 and MTU-2070 varieties were identified as resistance to BPH in Tungabhadra project area. Harini et al., (2010) in the present study, an attempt was made to validate and fine-map the genetic locus associated with brown planthopper (BPH) resistance locus from IR , a derivative of O. sativa, O. minuta cross, using simple sequence repeats (SSR markers). AF6 segregating population 300 individuals were developed by crossing IR with a highly susceptible variety, Mahsuri scoring 4.5 and 9.0 (on the scale 1-9) to BPH in greenhouse screening experiments. Madurangi et al., (2010) evaluated the nature of BPH resistance in seventeen Oryza nivara (WRAC 01, 02, 04, 07, 11, 12, 14, 19, 21, 22, 24, 25, 35, 41, 46, 62, and 9864) accessions collected from different locations in Sri Lanka. The variety Ptb 33(resistant), Bg 379/2, Bg 300(moderately resistant) and Bg 380

26 (susceptible) were used as check varieties. According to the results WRAC 04, 41, 25 and Ptb 33 recorded as resistant (score 0-3), WRAC 46, 35, 24, 22, 21, 14, 7, 2,1, 9864 and Bg 379/2 as resistant to moderately resistant (score ), WRAC 11, 12 and Bg 300 as moderately resistant (score ), WRAC 19 and 62 as moderately resistant to moderately susceptible (score ), while no checked accessions were recorded as susceptible to the BPH indicating potential of using Oryza nivara as a source of BPH resistance. Santhanalakshmi et al., (2010) evaluated 106 F3 families along with their parents Swarna and Ptb33 for resistance to brown planthopper of Indian biotype using the standard seed box technique of IRRI. The donor parent, Ptb33 was found to be highly resistant to the Indian biotype, while the popular variety Swarna was susceptible to BPH. Among the 106 F3 families screened for resistance, the percentage of resistant progeny was between per cent of total progeny. Kumar et al., (2012) conducted studies on new sources for resistance against rice brown planthopper (BPH) under glass house conditions. Among the 191 National Screening Nursery (NSN) entries evaluated, Swarnadhan was found to be highly resistant, CN 1870 was resistant and HKR 05 22, JGL-16267, UPR , CR , WGL-309, CR , NDR-359 and IR-64 were moderately resistant against brown planthopper. Tripathi, (2012) screened five hundred eighty seven rice genotypes against Nilaparvata lugens, out of these 20 were categorized as highly resistant, 82 as resistant, 150 as moderately resistant and rest of the other as moderately susceptible and susceptible to BPH. Among all the genotypes screened, the genotype

27 Ganjeikalli had the least plant damage score (0.54) followed by R (0.63). DRR annual progress report (2014) reported the reaction of evaluation for 16 differentials of Plant hopper special screening trial (PHSS), across 7 locations in 7 valid tests. It is revealed that PTB 33 and RP were identified as promising in all the seven green house tests against BPH Location of vascular bundle in cross-section stem of different rice differential. Nitta et al., (2000) investigated the varietal differences in the number and cross area of large vascular bundles (LVs) at the neck internode in 18 rice cultivars. There were two kinds of LVs in Chinese japonica (CJ), japonica-indica hybrid (JI), tall indica (TI) and semi-dwarf indica (SDI) types, i.e. large (L1) and small (L2) bundles. The number of L2 varied with the cultivar group. The total number of LVs was in the order SDI > TI > JI > CJ > JP (japonica panicle weight type) > JN (japonica panicle number type). The ratio of number of LVs to the number of primary rachis branches (V/R ratio) was nearly 1.0 in JP and JN. However, the V/R ratios in JI, TI and SDI were higher than 1.0 because the number of L2 was added. Total or phloem areas of L1 were larger in TI and SDI than in JP and JN. The whole area of LVs and phloem areas were in the order SDI > TI > JI > CJ > JP > JN because of the difference in the numbers of L2. The whole area of LVs and phloem areas of JP (cv. Koganemasari) and SDI (cv. Gui zhao 2) were increased by N application at the panicle formation stage. Sarwar and Prodhan, (2000) carried out anatomical investigations of 8 rice cultivars (Oryza sativa L.). Four from each of traditional (local) and modern (high

28 yielding) cultivars were carried out to study the variation among cultivars. The epidermis is single layered having cuticle of varying thickness. The number of hypodermal layer is more in modern cultivars compared to traditional ones. The vascular bundles are of two types, outer (small) and inner (large) in all cultivars except Madhabshail-boro where additional median (medium) type is present in between outer and inner bundles.the number, position and arrangement of vascular bundles vary along the cultivars. The outer vascular bundles are embedded in hypodermis and found to push outwards forming outgrowth of different shapes in all cultivars except BR7 and BR 3 where the wavy appearance in outer circumference of the stem is present. Some of inner vascular bundles are attatched or nearer to the hypodermis while rest are away from it. The hollow pith is found to be smaller in modern cultivars compared to traditional ones.the higher number of mechanical tissues (hypodermal cells and inner vascular bundles) in modern cultivars imparts resistance to lodging. Fukuyama, (2001) examined 86 accessions of annual, perennial and intermediate growth habit variants for variation in the numbers of rachis branches in the panicle and vascular bundles in their subtending peduncles. Accessions of annual habit, which regenerate from seed and are adapted to shallow and temporary swamps, developed fewer rachis branches (mean = 6.0) than those of perennial habit (mean = 7.2) which largely regenerate vegetatively and are adapted to stable deep water habits. In both cases variation within growth habit groupings was narrow. Variation in vascular bundle numbers, which has not been reported previously, was similar (10.1 to 10.3), but more variable within annuals. As a result the V/R ratio (vascular bundles : rachis branches) was higher in annuals (mean = 1.71) than

29 among perennials (mean = 1.46). Corresponding measures for both indica and japonica of cultivated rice (O. sativa) varied narrowly and were substantially greater for both rachis branches (mean = 11.6 and 13.8, respectively) and vascular bundles (mean = 19.1 and 14.8, respectively), with V/R ratios of 1.67 for indica and similar to accessions of O. rufipogon of annual habit, and 1.07 for japonica and lower than accessions of O. rufipogon of both perennial and annual habit. Accessions of O. rufipogon from the India and Indochina regions were significantly lower in rachis branch, but not vascular bundle numbers than accessions from China; with the V/R ratio higher among accessions from India than found in other geographic regions of origin. DongFeng et al., (2004) studied that number of vascular bundles, area of one vascular bundle, area of all the vascular bundles and the area of all the phloem of the heavy-panicle type of rice were higher than those of the middle-panicle type of rice. The vascular bundle in the branches of both the heavy and the middle-panicle type of rice recorded similar sink load and sink filling percentage. The developed vascular bundles in the panicle were basic structural factors for good grain filling. The undeveloped vascular bundles in the low position of the panicle might be the cause of the low grain filling in the crop. Huang et al., (2004) observed vascular bundles in the lower secondary branches of hybrid rice cv. Liangyoupeijiu were less developed than those in the upper primary branches. No cell differentiation was observed in sieve elements in the lower secondary branches, and the total area occupied by phloem and sieve tubes was lower than that in the upper primary branches. The soluble sugar content in the lower secondary branches was higher than that in the upper primary branches during

30 the maturity stage. The seed setting percentage in inferior rice grains was lower than in superior rice grains due to the decreased translocation of carbohydrates and other materials. Liang Xue et al., (2009) investigated the effects of ploidy changes on the stem structure, by using flow cytometry (FCM), they distinguished six different ploidy materials (haploid, diploid, and triploid of line FC311 and haploid, diploid, and tetraploid of line FC322), in rice (Oryza sativa L. subsp. Japonica). Also, compared the anatomical stem structure of various ploidy rice plants with paraffin sections. The results show that the increase in number and size of vascular bundle, and area of stem wall and vascular bundle in internodes were frequently associated with increase in ploidy level of the tested plants, indicating a significant gene dosage effect. However, there were no obvious correlations between different ploidy levels and the following traits, such as, layer number and development of collenchyma cell under epiderm in vascular bundle sheath of outer circle and inner circle, starch grains in ground tissue cells, the ratio of total area of vascular bundle and area of stem wall. The structure of two layers of collenchyma cell was rarely observed in the 1st internode of FC322 tetraploid plant. ZhiBin et al., (2009) used the RILs (recombinant inbred lines) population derived from the cross between Qishanzhan (indica) and Arkihikari (japonica) to study the relationship between vascular bundle and panicle characteristics. The results showed that phloem area of large vascular bundle in top second stem was significantly correlated with spikelets per panicle. A significant correlation among number of large and small vascular bundles, phloem area of large vascular bundle in top second stem and primary and secondary branches was found. The xylem area of

31 large vascular bundle and the area of large vascular bundle in top second stem had significant correlation with seed setting rate of primary and secondary branches and seed setting percentage, which indicated that large amount of large and small vascular bundle and large areas of phloem and xylem in top second stem are beneficial to improvement of panicle characteristics. ChenFei et al., (2013) investigated the feeding quality and the related stem morphological traits of rice (Oryza sativa) straw, eight japonica rice cultivars (Wuyujing 3, Yandao 830, Nanjing 44, Nanjing 46, Nanjing 47, Nanjing 5055, Wuxiangjing 14 and Zhendao 10) and one indica rice cultivar (Liangyoupeijiu) were evaluated in Nanjing under a common management schedule. At harvest, 50 tillers (with panicles removed to determine one-panicle weight) were used to investigate the feeding quality parameters such as nonstructural carbohydrates (NSC), crude protein (CP), acid detergent fiber (ADF) contents and in vitro dry matter digestibility (IVD-MD). The top third node of stem was observed for morphological characteristics such as the stem diameter (CD), wall thickness (WT), the percentage of mechanical tissues area (MTA), vascular bundle area (VBA) and parenchyma area in stem and the rate between WT and CD. The feeding qualities of cultural varieties were significantly (P<0.01) different, and NSC content and IVDMD of small ear type Wuyujing 3 and the super rice cultivar Nanjing 44 were high, while those of the late maturing varieties Nanjing 46 and Nanjing 47 were low. CD was significantly (P<0.01) positively correlated with NSC content and IVDMD of rice straw, and also with the one-panicle weight (P<0.01), which could be used as a selection trait for more digestible rice straw.

32 2.2 Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through honey dew test. Pophaly and Rana (1995) evaluated the feeding rate of BPH on some selected resistant varieties ranged between 0.96 mm 2 / female in Chapdo to mm 2 /female in Hinga variety, which exhibited 0.65 plant damage score. The standard seedling test showed Rathu Heenati (bph3) and Balamwee (Bph9) were found moderately resistant, whereas Swarnalata (BPh6) and ARC (bph5) were found resistant. The honeydew excretion test confirmed these results. Sadasivan and Thayumanavan (2003) stated that lectin, Galanthus nivalis agglutinin (GNA) get ingested by insect binds to the midgut epithelial cells. Results indicated that GNA bind to form glycopeptides in BPH gut there by inhibiting the digestive enzymes. As a result, insect takes out its stylet and tries again and again for further intake of food. Western analysis revealed the presence of GNA in the honeydew, gut preparation of BPH and the crude homogenate of the whole body. Lectin binding concentrate on the luminal surface of the epithelial cells,these factors caused negative feeding response offered by plant itself as a defense mechanism by way of release of chemicals in the plant itself as a stimulant imposed by BPH stylet insertion for sucking the sap or due to absence of proper nutritional value required by BPH in the plant itself. Soundararajan et al. (2004) carried out an experiment to study the feeding behaviour as a antibiosis parameter of brown plant hopper Nilaparvata lugens on standard checks. The parents and DH lines of the cross IR64/Azucena for resistance to BPH were evaluated. The honeydew area on Ptb 33, TN1, IR 64, Azucena and

33 DHLs were found 36± 4.4, 682± 31.8, 448.3± 19.8, 614± 36.8 and ± 6.9 in mm2, respectively. Bhimrao et al., (2005) carried out BPH feeding test on resistant cultivars viz. R , RGL-5613 and IR-64. Feeding rate was ranged between to mm2 per female in 24 hours, which was much lower than the feeding on TN1 ( mm 2 /female). Reddy et al., (2005) studied on antibiosis mechanism of resistance against BPH in seven resistant (INRC-8815, 7069, 8754, 8712, 7197, 6153 and 7040) and three moderately resistant (INRC-7143, 6165 and 7318) rice genotypes along with a resistant control (Ptb 33) and a susceptible control (TN1) in green house. BPH fed on resistant and moderately resistant genotypes excreted less amount of honeydew compared to the insects fed on susceptible TN1. Maheshwari et al., (2006) studied mechanisms of resistance i.e. antixenosis and antibiosis, against Nilaparvata lugens in 5 rice genotypes CORH2 (IR 58025A/C20R), IR 58025A (IR4843A/PUSA ), C20R, C10, IET 15423, TN1, Ptb 33 and IR R. They observed that honeydew excretion of BPH was low on resistant genotypes than in the susceptible TN1. The resistant genotype IET recorded minimum (2.80cm 2 ) honeydew excretion and the susceptible TN1 recorded maximum (5.39 cm 2 ). Comparatively less honeydew excretion and more feeding marks recorded in all resistant genotypes than the susceptible TN1. Alagar et al., (2007) carried out the study to assess reaction of different rice genotypes in response to brown planthopper (BPH) Nilaparvata lugens (Stal.). The resistant genotypes Ptb 33, ADT 45 and ASD 7 and the moderately resistant

34 genotypes CO 43 and KAU 1661 recorded the lowest feeding rate as compared with the susceptible genotype TN1. Alagar et al., (2008) studied the feeding behaviour of brown plant hopper (BPH) Nilaparvata lugens on some selected rice genotype. The maximum number of feeding marks were observed on ARC (43.80), which was 4.52 times higher than TN1 and it was followed by ARC 6650 (24.80) and KAU 1661(24.00). W 1263 recorded the lowest feeding rate of mm 2 followed by ARC 6650 (129.70mm 2 ), IR 72(144.17mm 2 ) and KAU 1661 (177.48mm 2 ). Singh et al., (2008) used mutants of rice, IR64 to isolate new sources of resistance to the brown planthopper, Nilaparvata lugens (Stal) (Hemiptera: Delphacidae). In their experiment it was found that honeydew excretion was greater on D1131 but slightly lower on D518 that of IR64. Ghaffar et al., (2011) compared the BPH feeding behaviour on 12 rice varieties over a 12 h period using the electrical penetration graph (EPG) and honeydew clocks. Seven feeding behaviours (waveforms) were identified and could be classified into two phases. The first phase involved patterns of sieve element location including non penetration (NP), pathway (N1+N2+N3), xylem (N5) and two new feeding waveforms, derailed stylet mechanics (N6) and cell penetration (N7). The second feeding phase consisted of salivation into the sieve element (N4-a) and sieve element sap ingestion (N4-b). Production of honeydew drops correlated with N4-b waveform patterns providing independent confirmation of this feeding behaviour. BPH produced lower numbers of honeydew drops and had a shorter period of phloem feeding on resistant rice varieties, but there was no significant difference in the time to the first salivation (N4-b). These qualitative differences in

35 behaviour suggest that resistance is caused by differences in sustained phloem ingestion, not by phloem location. Cluster analysis of the feeding and honeydew data split the 12 rice varieties into three groups: susceptible, moderately resistant and highly resistant. Cruz et al., (2011) determined the effects of adaptation to one resistant variety, IR62 assumed to possess the Bph3 gene on (1) resistance against a series of varieties with similar biotypical responses (presumed to contain the same major resistance genes), and (2) a differential variety with the bph4 gene that occurs at the same chromosome position as Bph3. They also examined the effects of high soil nitrogen on the effectiveness of Bph3. Feeding, planthopper biomass, and development times were reduced in a wild BPH population when reared on IR62 compared with the susceptible standard variety TN1. However, nitrogen application increased the susceptibility of IR62. After 13 generations on IR62, BPH had adapted to the plant s resistance. Virulence of the adapted BPH against the variety Rathu Heenati supports the idea that Bph3 is present in IR62. Across similar IR varieties (IR60, IR66, IR68, IR70, IR72, and IR74) feeding, planthopper biomass, and development rates were generally higher for IR62-adapted than for non-adapted BPH; however, contrary to expectations, many of these varieties were already susceptible to wild BPH. Fitness was also higher for IR62-adapted BPH on the variety Babawee indicating a close relation between Bph3 and bph4. The results indicate that the conventional understanding of the genetics behind resistance in IR varieties needs to be readdressed to develop and improved employment strategies for resistance management.

36 Ponnada et al., (2011) studied the feeding and probing behaviour of BPH on six selected highly resistant advanced rice breeding lines i.e. F81501 DodkiD:1015 X2141 Kabari K: 2388, 3397 Hathipauw H: 332X 1528 E.E.17,2023 Jai Bajrang J: 432 X 2340 Kankadiya K: 446- II, 1940INDCRX 3397 Hathipauw H: 332, F98 let X 692 Chatri C: 62-II1, 5 IET8116 X 1704 Amakoyali A: 719 along with a resistant check (Ptb-33) and susceptible check (TN-I). After standard screening evaluation the damage score ranged between 0.1 to 0.5, 2.4 and 9.0, respectively. All selected highly resistant lines exhibited significantly lowest feeding and highest probing marks. Feeding values ranged between 4.0 to mm 2 per female and probe number between 30.4 to 42.9 per female on test seedlings. Yongfu et al., (2011) studied that rice varieties Swarnalata and B5, which showed high levels of antibiosis and tolerance to BPH, thus were highly resistant in the seedling bulk test; Mudgo and T12, which showed moderate resistance to the insects, had a high level of tolerance and moderate antibiosis to BPH. Varieties Rathu Heenati, ARC 10550, and ChinSaba were identified to be susceptible to BPH, showing a moderate level of tolerance and no antibiosis. In comparison to the evaluation methods of BPH resistance, the honeydew excretion and survival rate could be used to detect the antibiotic level, and the RH, RW, or leaf yellowing days could be employed as indicators to evaluate the rice varieties tolerance. The results should help in understanding BPH-resistance categories of rice varieties and for resistance breeding. Luo et al., (2012) studied the feeding behaviour and oviposition preference of brown plant hoppers (BPH), Nilaparvata lugens (Stal), on rice varieties TN1 (susceptible), IR36 (with resistant gene bph2), Hanyou 3 (drought-

37 tolerant) were determined under drought stress simulated with polyethylene glycol (PEG6000) in laboratory. The number of BPH eggs per plant within 72 h was negatively correlated with the concentration of PEG6000 on TN1 and IR36 (P=0.001 and P=0.008, respectively). The amount of honeydew per female adult within 48 h reduced on TN1 and Hanyou 3 with the increasing concentration of PEG6000, while on IR36 it was very low and no significant difference was found under different concentrations of PEG Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through probing mark test. The result of probing behaviour indicating that the resistant varieties received more number of probing punctures than the susceptible one (Reddy, 1979; Reddy and Kalode, 1985 and Veronica, 1985). The electronic measurement system greatly facilitates the behavioural study of feeding and probing by piercing and sucking insects on susceptible and resistant plants (Kawabe et al., 1981; Velusamy and Heinrichs, 1986). In laboratory studies with six varieties of rice adult brachypterous female of Nilaparvata lugens (Stal.) made more probing marks, produced less honeydew and gained less body weight on resistant than on susceptible varieties (Bagui, 1989). Lu et al., (1999) compared the probing behaviour of different biotypes of Nilaparvata lugens (Stal.) on TN1 (Susceptible variety) and IR-26 and ASD7 (two resistant varieties of rice). There were no obvious differences among the probing frequencies of biotype 2 on these three varieties. Under no-choice feeding conditions, the probing frequencies of biotype 1 on TN1 increased significantly and that of biotype 3 on ASD 7 increased significantly.

38 In all the seven selected resistant rice genotypes, the average probing marks per seedling ranged from 19 to 32, although, in resistant check (ptb33) variety, the probe marks was 29 per seedling, which was significantly at par with six resistant rice genotypes (Urre et al., 2004b). Sable (2010) investigated the probing behaviour of BPH on sixteen resistant rice genotypes. The probing frequency ranged from to which was significantly higher than susceptible check TN1 (10.33). Rana and Dubey (2010) studied the probing marks behaviour of BPH on 23 selected resistant rice donors. The probing marks ranged from to which was significantly higher than susceptible check TN1 (10.33). 2.3 Survival test of BPH nymph on different varieties of rice plant carrying different resistance gene through nymphal survival method. Zhang and Gu (1985) had determined the effects of various levels of certain amino acids in rice plants on the development of Nilaparvata lugens (Stal.) in laboratory studies at China. They stated that young nymphs, especially those in the 1st and 2nd instar, developed rapidly in the presence of adequate amounts of free tyrosine, glycine and lysine in rice plants. The concentrations of free glycine and lysine in plants were negatively correlated with nymphal survival rate. Gao et al., (1990) observed the brown planthopper survival on different rice varieties. They found that insect survival on Xiu-Shui 620 and IR64 was 50 per cent of that on Xiu-Shui 48. Senguttuvan et al., (1991) studied the relative impact of resistance mechanism antibiosis against Nilaparvata lugens in rice varieties under laboratory conditions. The antibiosis in Ptb33 and IR 64 (highly resistant and resistant variety)

39 were expressed as increased nymphal duration, decreased nymphal survival, while the reverse condition was noticed on the susceptible TN1. Velusamy et al., (1995) evaluated three wild rice species and six cultivated rice varieties to determine their mechanisms of resistance to Nilaparvata lugens (Stal.). Wild rice species Oryza officinalis, O. punctata and O. latifolia, cultivated rice Rathu Heenati, Babawee, ARC 10550, Swarnalata, Ptb33 and the susceptible Taichung Native (TN1) were included in the study. N. lugens caged on resistant wild rice had slow nymphal development as compared to N. lugens on cultivated resistant varieties. Nanda et al., (1999) conducted laboratory experiments to study the antibiosis mechanism of resistance in 10 rice varieties to BPH (Nilaparvata lugens Stal). They found more nymphal survival on the susceptible control TN1 (82.0 to 92.0%) as compared to other varieties viz. Mudgo, ASD 7, Rathu Heenati, Babawee, ARC 6650, Utri Rajpan, Udaya, Pratap and Ptb33 (1.0 to 5.0%) at different crop ages. Nanda et al., (2000) observed that the results on the development of nymphs on the resistant genotypes. It is suggested that the insect surviving on resistant genotypes had to face the problem of inadequate or unsuitable nutrition which may due to presence of higher total sugars and non reducing sugars. Greater nymphal survival, on susceptible TN1 proved the availability of good quality food and reverse can be found in resistant genotypes. Low level of total free amino acid and total starch content could be considered as contributing factors in varietal resistance of varieties to brown planthopper. Shuguang et al., (2000) studied the effects of different rice varieties on the biological properties of brown plant hopper (BPH). The results indicated that rice

40 variety mainly affects the nymphal survival rate and the adult oviposition. No significant difference was observed in nymphal development and adult longevity when BPH was reared on the tested nine different varieties. The biological parameters of BPH population always change with rice varieties, so it is considered that the resistance of rice variety to BPH is very complicated. Tanaka et al., (2000) studied virulence of the brown planthopper, Nilaparvata lugens strains, which immigrated into Japan between 1997 and 1999, was examined on five rice varieties, Mudgo (carrying a resistance genes, Bph 1), IR26 (Bph1), ASD7(Bph 2), Norin PL10(Bph 3) and Babawee (Bph 4). Newly emerging brachypterous females N. lugens were released on test rice plants at tillering stage, and they defined the females that became heavily swollen or survived for five days as virulent. Between 45 and 87% of the females were virulent to ASD7, although such high virulence had not been detected before Between 49 and 98% of the females were virulent to Mudgo and IR26. Virulence to rice varieties carrying Bph1 in the N. lugens population has continued to become stronger since the period in which changes in virulence were first found. In contrast virulence of the N. lugens strains to the Norin PL 10 and Babawee was still at a low level, with 5 to 27% of the females being virulent. The results indicate the resistance of Bph 1 has probably broken down and resistance of Bph 2 may be becoming ineffective for the N. lugens population immigrating into Japan. Soundararajan et al., (2002) reported the results on the reduction in the survival rate of BPH on resistant genotypes which might be due to presence of

41 antibiosis factors i.e. presence of feeding deterrents such as soluble salicic acid, malic acid, itaconic acid and benzoic acid in the resistant genotypes. Soundararajan et al., (2003) conducted pot experiments on 15 day old seedlings of 104 rice double haploid (DH) lines to determine their antibiosis effect on the growth and adult longevity of brown plant hopper (BPH) Nilaparvata lugens. The highest growth index of 7.75 was observed on the susceptible control TN1. The mean growth index of BPH was DH 398 showed a maximum growth index of 7.35 followed by DH 116 (7.34). Reddy et al., (2005) studied on the antibiosis mechanism of resistance against BPH in seven resistant (INRC-8815, 7069, 8754, 8712, 7197, 6153 and 7040) and three moderately resistant (INRC-7143, 6165 and 7318) rice genotypes along with a resistant control (Ptb33) and a susceptible control (TN1). The BPH caged with resistant and moderately resistant genotypes had prolonged nymphal development, slow growth and low survival compared to the insects caged with the susceptible control TN1. The nymphal survival on resistant genotypes INRC 8815 was only 32.5 per cent and was on par with resistant check Ptb33. The survival was below 50 per cent in INRC 7069 and INRC 8754 as compared to the 90 per cent survival on susceptible check TN1. Maheshwari et al., (2006) studied the mechanisms of resistance i.e. antixenosis and antibiosis against Nilaparvata lugens in 5 rice genotypes CORH2 (IR 58025A/C20R), IR 58025A (IR4843A/PUSA ), C20R, C10, IET 15423, TN1, Ptb33 and IR R. Comparatively the nymphal development was prolonged in resistant genotypes by 3.8 to 6.4 days compared to 9.40 days in susceptible TN1.

42 Alagar and Suresh, (2007) reported KAU 1661 for lowest nymphal settlement of 4.3, which was 44.3% lower than TN 1 (7.7 nymphs per plant). Among the genotypes tested for oviposition, ASD 16 recorded significantly lowest number of nymphal emergence (122.0 nymphs per plant), which was 61.1% lower than TN 1. CO 46 recorded the highest number of nymphal emergence (210.4), which was 1.49-fold lower than TN 1. Singh et al., (2008) used mutants of rice IR64 to isolate new sources of resistance to the plant hopper Nilaparvata lugens (Stal.) (Hemiptera : Delphacidae). In their experiment it was found that nymphal survival and adult female weight did not differ among rice cultivars. Seo et al., (2009) conducted a nymphal survivorship test and electrical penetration graph (EPG) study on susceptible and resistant rice varieties with four different BPH populations, which were collected in the early 1980s (S-BPH), 2005, 2006, and The S-BPH showed low survival rates on resistant rice varieties carrying Bph1 and bph2. However, recent wild BPH populations seemed to have high resistance breaking ability according to elevated survival rates on most other resistant rice varieties, except Gayabyeo (Bph1 + bph2) and Rathu Heenati (Bph3). Ahmad and Adam, (2011) studied the survival ratio of male to female was : The female lived for a maximum 20 days. The trend of oviposition showed a peak around the 10 days of the female life. The highest no. of produced per female per day was The intrinsic rate of increase in egg production per female per day was and the daily finite of increase was female per day with a mean generation time of days. The net reproductive rate of the population was The population doubling time was days.

43 Kumar et al., (2012) studied antibiosis mechanism of resistance against BPH in thirty rice entries with different level of resistance. Per cent nymphal survival, growth index values, population build-up, average nymphal emergence and adult longevity were significantly lower while nymphal duration was found longer as compared to susceptible check (TN1) on different resistant entries studied Status of egg parasitisation of brown plant hopper in rice differential varieties. Bentur (1982) studied the biology and control potential of three species of egg parasites, viz., Anagrus sp., A. optabilis (Mymaridae) and Oligosita sp. (Trichogrammatidae), and a nymphal/adult parasite Gonatopus sp. of rice plant hoppers. Developmental duration from oviposition to adult emergence noted for these parasites indicated that males of mymarids, in general, developed faster (10 11 days) than females (12 13 days) at C prevailing during October, whereas Oligosita females developed more slowly (14 15 days). However, both A. optabilis and Oligosita developed three days faster at C prevailing during April. Fecundity in terms of number of eggs parasitized per female varied from 12 3 to Fowler et al., (1991) carried out an experiment where rice plants infested with eggs of Nilaparvata lugens (Stal.) or Nephotettix spp. In laboratory cultures,were used to trap egg parasitoids in rice fields at two sites over a period of four days in Sri Lanka. Levels of egg parasitism in per plant varied from 0 to 54% in Nilaparvata lugens and 45 to 100% in Nephotettix spp. Egg predation was normal cause of mortality, but attacked by a species of

44 Panstenon (Hymenoptera: Pteromalidae) killed upto 18% Nilaparvata lugens eggs. N. Lugens eggs were parasitized by Anagrus sp, A. optabilis (Perkins) (Hymenoptera : Mymaridae) and Oligosita sp. (Hymenoptera: Trichogrammatidae). Overall egg parasitism of Nilaparvata lugens was positively related to host egg density at the spatial scale of the rice plant but unrelated at the tiller or batch scale. Claridge et al., (1999) studied that egg mortality is known to be an important factor in brown planthopper (BPH) (Nilaparvata lugens) population dynamics in tropical Asia, but few quantitative data are available on the role of egg parasitoids. Rice plants previously infested in the laboratory with BPH eggs were exposed to natural parasitism and predation in experimental fields for periods of 5 days. Egg batches were dissected from both experimental and control plants after field exposure at seven fortnightly intervals throughout the rice growing season, and BPH nymphs and adult parasitoids were allowed to emerge. Parasitism by species of Oligosita (Hymenoptera, Trichogrammatidae) and Anagrus (Hym., Mymaridae) varied between 18 and 61% in the dry, and from 1 to 65% in the wet seasons. There was generally a trend from low to higher rates through wet seasons, contrasting with more uniform higher levels through dry seasons. Reduction in BPH survival as a result of egg parasitism varied between 29 to 91%. Generally parasitism was density-independent. Atmaja et al., (2000) stated that egg parasitoids of BPH have good potential to reduce BPH populations by Anagrus sp., Oligosita sp., and Gonatocerus spp. Parasitism caused by these parasitoids in the field was 64, 55, and 40%, respectively. All parasitoids have alternative hosts, namely: Sogatella

45 furcifera, Nephotettix spp., Laodelphax striatellus, Empoasca fabae, Typhlocyba pamoria [ Typhlocyba pomaria], Dikrella cruentata, and Circulifer tenellus. Conservation of egg parasitoids of BPH by application of host extract and sugar liquid on the rice plants and allowing the growth of weeds in the round abouts of small dikes in rice fields, especially Paspalum sp., Digitaria sp., and Echinochloa sp. is recommended. Barrion et al., (2001) studied the role of non-rice habitats in maintaining the population of natural enemies (Anagrus) of the brown planthopper, Nilaparvata lugens was investigated in China during Plants laden with eggs of the brown plant hopper were exposed 2 days every week in the rice fields and grassy areas dominated by Digitaria spp. Only 3 species of Anagrus were identified from the exposed eggs of the brown planthopper in both ricefields and Digitaria-dominated grassy areas. These were dominated by A. nilaparvatae (98%). Chiappini (2002) studied a new species of mymarid, Anagrus elegans, as egg parasitoid of the brown plant hopper, Nilaparvata lugens, and the whitebacked plant hopper, Sogatella furcifera, in the South Asian region. Qian et al., (2002) studied the hopper egg parasitoids, which could parasite eggs of the brown planthopper (Nilaparvata lugens), at community level. The communities were made up of 19 hopper egg parasitoids, which belonged to 2 families, Mymaridae and Trichogrammatidae. Three species, Anagrus nilaparvatae, A. longitubulosus [A. perforator] and A. paranilaparvatae [A. optabilis], were the dominant species, had high percentage in the community. The communities were positively related to the number of the brown plant hopper in rice field during

46 the early period, fluctuated in a certain range during the middle period, and increased then decreased greatly before harvesting, in both early and late rice growth. The percentages of BPH eggs with hopper egg parasitoids during the early, middle, and late period of rice growth were about 76, 70, and 50%, respectively. Chandra, (2005) studied searching, drilling and oviposition by the mymarid egg parasitoid, Anagrus flaveolus Waterhouse in laboratory, using eggs of brown planthopper, Nilaparvata lugens (Stal). Observations revealed that oviposition by the female wasp in host eggs embedded inside the plant tissue was accomplished by repeated attempts to drill through the leaf sheath tissue with ovipositor, an act in which the parasitoid achieved success after relentless labour. Therefore, one of the contributory factors affecting the field parasitism of this species appears to be the penetrability of rice leaf sheath. Xiang et al.,(2008) studied the effect of Nilaparvata lugens (Stal) infestation duration and density on the host preference and performance of Anagrus nilaparvatae. The results showed that the parasitoid preferred N. lugens eggs on the plants infested with 10 gravid N. lugens females for 1 d to those plants infested with 10 gravid females for 2 or 3 d. It was also found to prefer N. lugens eggs on plants infested with 10 or 20 adult females after 24 h of infestation to those plants infested with 5 or 80 females. The parasitoid's offspring had lower survival rates, fecundities, female ratios, indexes of capacity for population increase, and longer developmental durations on plants when they were infested with high N. lugens density (80 adult females per plant). However, the performance of the parasitoid on plants infested with low N. lugens density (5 female adults per plant) was similar to those on plants

47 with intermediate N. lugens density (10 or 20 adult females per plant). Low preference of the parasitoid for N. lugens eggs on plants with heavy or light infestation levels may be correlated with low host suitability and detectability, respectively. The result implies an important role of herbivore-induced rice volatiles in the host preference of the parasitoid A. nilaparvatae, by which the parasitoid perceives the host and its suitability.

48 Materials and Methods

49 CHAPTER - III MATERIALS AND METHODS The present investigation entitled Evaluation of rice differential varieties against Raipur brown plant hopper Nilaparvata lugens (Stal) (Homoptera: Delphacidae) strain. was carried out in the Glass House, Department of Entomology, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.) during PHSS (Planthopper Special Screening) entries provided by DRR under AICRIP programme were tested under glasshouse conditions by adopting standard evaluation technique. The materials used and the techniques employed for conducting various experiments are presented under following heads. Table 3.1 : Materials used: S R Gene List of differential varieties No. Designation Cross 1 bph2 ASD 7 (ACC 6303) Karsamba Red 2 Bph3+Bp Rathu Heenati (ACC Rathu Heenati h ) 3 bph4 Babawee(ACC 8978) - 4? Milyang 63 Tongil/IR //YR TN1 S.Check 6 Bph6 Swarnalatha(ACC ) 7 bph7 T 12 (ACC 56989) - 8 bph8 Chinsaba (ACC ) 9 bph9 Pokkali - 10 TN1 S.Check 11 Bph1+ IR 64 IR /IR Bph18 IR IR *3/O.australiensis B 13 bph2 IR 36 IR //4*IR

50 117/O.nivara///CR ? MUT NS 1-15 TN1 S.Check 16? OM 4498 IR 64/OMCS 2000//IR 64 17? RP bph2+bp Ptb33 h3+ 19 BPH20/2 IR TN1 S.Check 3.1 Mass rearing :- The mass screening in the greenhouse is used to discard susceptible lines and identify possible resistant cultures. Mass-rearing of BPH is essential for mass screening of varieties. The insect is mass-reared on the susceptible variety Taichung Native 1 (TN), susceptible variety that provide food and sites for oviposition. The original colony is started by caging a pair of adults (virus-free insects from areas harboring the grassy stunt virus) on the rice plant. Two methods of mass-rearing are employed- 1) Rearing on old potted plants and 2) Rearing on seedling mats. But here first technique is preferred. Pathak and Khush (1977) at IRRI described this method, and most countries use it. To get the regular supply of insect for various studies, the brown plant hopper (BPH), was mass reared initially at 30 5 C on potted TN1 (Taichung Native) variety and the population was maintained throughout the year in the air cooled glass house. Brown plant hopper population were reared on 40 to 45 days old TN1 plants, inside the rearing cage of 75x75x75 cm size consisting of wooden frame with small window on front side and fine wire mesh on top sides. Cages were

51 View of rearing cage Plant ready for feeding Plant inside the cage Single plant with female BPH Single plant with nymph population Plate 3 : Mass rearing of brown plant hopper

52 mounted on cemented platform with a water level of 7.5 cm height to avoid the entry of ants and other crawling insects and also to maintain humidity in glass house. Potted TN1 plants were placed inside the rearing cages for egg laying along with at least 3-4 pairs (male and female) of brown plant hopper per hill. After 2-3 days, the females started egg laying inside the leaf sheath of paddy plants. Later on, after emergence of nymphs from plants, released brown plant hopper pairs were transferred to another TN1 plant with the help of aspirator for egg laying. When newly emerged nymphs reached to second instar, they were used for screening of rice genotypes. (The mass rearing of BPH shown in plate-3). Likewise, the multiplication process of brown plant hopper insect continued for mass screening, probing mark test, honeydew test and nymphal survival. (Different stages of BPH on rice plant is shown in plate - 4 ). 3.2 Screening of rice differential varieties carrying different resistance gene through standard seed box screening technique in glasshouse condition. In early screenings, it was observed that test lines planted at either end of a tray was more likely than others to escape insect attack. Various methods and designs for planting test rows were evaluated and a modified layout that minimized the chances of escape (Kalode et a1., (1975) was devised. The test and check genotypes were pre-germinated in petridishes (10 cm diameter).this method involved the infestation of 7 to 10 days old seedlings of test entries grown in puddled soil in wooden trays (50 x 40 x 7 cm). Each tray accommodated 20 test rows, each with 20 seedlings; 2 middle rows of a resistant check, and 4 susceptible border rows of TN1. (The layout for screening of rice genotype is represented in fig.no.3.1). The wooden trays were placed in water in 7.5 cm deep trays to maintain humidity suited to the insects and to keep away ants.

53 Plate 4: Stages of brown planthopper on rice plants: (a) eggs, (b) new ly hatched nymphs, (c) 4th-5th nymphal stages and (d) female and male adults

54 Test genotype Resistant check (Ptb33) Susceptible check (TN1) Fig.3.1: Layout of screening of rice genotypes for BPH resistance

55 About 7-10 days after seeding at the one and the two-leaf stage, the seedlings are infested by scattering a large number of insects on them. The heavily infested plants from the mass-rearing cage are gently tapped over the seedlings. The insects should be on the test varieties as uniformly as possible. Generally second and third instar nymphs are used for infestation. Approximately 8-10 insects/seedling constitutes an optimum population to differentiate the resistant and susceptible lines. The insect's preference or non preference for the test varieties is recorded. But the final rating for resistance is based on the extent of damage to the different test varieties suffer. Up to 1975, a scoring system with a scale of 0 to 5 was used. In 1976, IRRI revised the scoring system (IRTP 1975). At present, plant damage is rated on the standard scoring system of 0-9 (Table 1). The final damage rating is taken when about 90% of the plants of the susceptible check variety are killed usually about 7 to 10 days after infestation. (Seed box screening test for BPH resistance is shown in plate-5). Table 3.2: Standard for rating damage by brown planthopper (revised by IRRI- IRTP 1975; Pathak and Khush 1977). Grade of damage Rating a Symptom 0 HR No visible damage 1 R Partial yellowing at first leaf 3 MR First and second leaves partially yellow 5 MS Pronounced yellowing and some stunting 7 S Wilting and severe stunting 9 HS All test plants dead a HR = highly resistant; R = resistant; MR = moderately resistant; MS = moderately susceptible; S = susceptible; HS = highly susceptible.

56 Three days old seedlings Plant growing in box Seedlings ready for nymph release Nymph population released in seedlings Infested seedlings of different rice genotypes Comparative infestation of BPH in different rice genotypes Plate 5 : Seed box screening test for BPH resistance

57 3.2.1 Location of vascular bundle in cross- section stem of different rice differentials:- Twenty rice cultivars were used for this anatomical investigation. The plant samples were collected at tillering stage (30-45 days after transplanting) from the 3 rd node and internode (from the top) and fixed separately in Craf III and FFA.(Johansen 1940; Sass,1958). For anatomical investigation hand sectioning method was followed. All sections were cut using a new double-sided razor blade. First, a leveling cut was made for the purpose of forming a right angle with the axis of the stem to ensure that the sections made were cross sections. After each cutting, the cut section that collected on the blade were transferred to a drop of water on a slide and covered with a cover slip by lowering it an angle onto the drop of water containing the sections. The sections were mounted in 50% glycerine and were examined under trinoculor microscope, followed by these steps :- a) Turned the revolving nosepiece to engage 10X objective. b) placed the specimen on stage. c) turned the X-knob and Y-axis knob to move the specimen in light path. d) adjusted the brightness with the light intensity knob. e) turned the coarse and fine adjustment knobs to bring the specimen into focus. f) adjusted the interpupillary distance and dioptre. g) center the field iris diaphragm & adjusted the aperture iris diaphragm & field iris diaphragm. i) after readjusting focus, field & aperture iris diaphragm,observation is started. Anatomical investigations on PHSS lines were carried out to study the distance between the epidermis to phloem located in vascular system of rice stem. This observation have been taken from the internodal and nodal position both, to study

58 the variation in cross section of stems of both positions in same variety. And also to study the variations among different varieties. The aim of this study was to further explore the interactions between the BPH insects feeding and rice plants in an attempt to elucidate the mechanisms involved in rice resistance to the BPH. The mouthparts of BPH, like other phloem feeding insects, consist of a stylet bundle which forms the piercing and sucking organ (Sogawa,1982; Seo et al.,2009). BPH feeds on the plant by inserting the stylet bundle with an accompanying salivary sheath into the plant locating the phloem tissue and then regulating the ingestion of the pressurised plant sap (Sogawa,1982; Seo et al., 2009). (Anatomical investigations of rice stems in relation with BPH feeding is shown in Plate -6). The vascular tissue system itself is comprised of several different tissues with complex functions. There is a description of it :- 1) phloem tissue and the 2) xylem tissue in the vascular tissue system. 1) PHLOEM TISSUE: The phloem tissue is comprised of phloic fibers, sieve tube members, and companion cells. The companion cells are extrodinarily distinct in the phloic tissue. The phloic fibers are found primarily above the phloem, although the outer edge of the rice stem is known to sometimes have a continuous ring of fiber cells just below the epidermis consisting of phloic fibers as well as "other" fibrous schlerenchyma tissue. 2) XYLEM TISSUE: The xylem tissue is comprised of xylary fibers, protozylem vessel members, metaxylem vessel members, and xylem parenchyma cells. The primary function of the vessel members is to transport water and solutes, such as minerals and ions.

59 Observation under trinocular microscope Plate 6: Anatomical investigation of rice stems in relation with BPH feeding

60 Variations in cross- section of stems:- The internal anatomy of the nodal region of the stem of rice plant differs from that of internodal position. The diaphragm and the air channel can be described as part of the arenchymous center. The cross sections of the stem in both the nodal region, just below the panicle and through the panicle, the anatomy of the stem changes, as vascular bundle distribution changes, as there is the absence or presence of an aerenchymous center, and as the crosssections look the least or the most like cross-section through an internodal region. 3.3 Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through honey dew test. The honeydew excretion is widely used to assess feeding activity and consequently a reliable index for resistance and susceptibility of a crop variety to homopteran pests (Auclair, 1959; Liu et al., 1994). Many techniques have been developed to measure the feeding response of Nilaparvata lugens on resistant and susceptible rice plants (Paguia et al., 1980; Pathak et al., 1982; Begum and Wilkins, 1998). The filter paper technique was used for the present study. The honey dew excretion method or feeding test was done as the method suggested by Sogawa and Pathak (1970). As honeydew excretion by planthoppers reflects feeding activity (Paguia et al., 1980; Park and Song, 1988), honeydew excretion can be used as an index of feeding quantity (Wada et al., 1994; Tanaka, 1999). The experiment was undertaken by quantifying the area of honeydew excreted by the insect on filter paper after 24 hrs of confinement on the plants of rice genotypes and check varieties i.e. TN1 and Ptb33, respectively. For this white Whatman No. 1 filter papers (10 cm diameter) were dipped in a solution of bromocresol green (2mg/ lit. Ethanol) indicator and allowed to dry in sunlight

61 Susceptible variety Plate 7: Honey dew excretion test on set of rice differentials

62 thereby filter paper turned to yellowish orange colour as described by Pathak and Heinrichs, (1982). The treated filter papers were placed on an inverted petridish (10 cm diameter) at the base of each plant through a slit made in centre. Thereafter, each plant was covered with inverted glass funnel (75 mm) along with two days old female which were starved for two hours prior to release for the test, allowed to feed for 24 hours. Feeding activity was investigated at 40 days old potted plants. Four replicates were maintained for each variety and each replication contains one female on single tiller of plant. (Honey dew excretion test on set of rice differentials is shown in plate- 7). Immediately upon contact with honeydew secreted by female, blue spots appeared on the treated filter papers. As the concentration of the honeydew increases, the spots turned white in the center with the blue edges. Bromocresol green indicates phloem-based honey dew as blue-rimmed spots (indicate susceptible plants) and xylem- based honeydew as transparent (indicate resistant plants). The spots were traced on a transparency sheet and later on measured by keeping on millimetre (mm) square graph. The amount of feeding by the insect on the different rice genotypes as well as susceptible and resistant checks were expressed in terms of honeydew excretion per female in mm 2 unit Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through probing mark test. Probing mark test was carried out according to methodology suggested by Natio (1964). For this purpose, seeds of different rice genotypes and check varieties i.e. TN1 and Ptb33 were germinated separately in petridishes. Germinated seeds were sown in wooden trays containing well puddled soil. After seven days, the

63 Magnified view of probed marks Plate 8 : Assessment of feeding activity of BPH through probing mark test

64 seedling of each variety was removed from trays and washed thoroughly with water and then transferred individually into 15 cm long test tubes containing a few drops of water. One female (two days old) was introduced individually into each test tube and test tubes were plugged with sterilized cotton swab. The female was allowed to make punctures on the seedling for overnight (12 hrs). Thereafter, the seedlings were taken for staining in another tube containing 1.0 per cent erythrosine dye aqueous solution. Insect probing marks stained thereby counted visually after 30 minutes of staining. Three replicates were maintained for each rice genotypes and each replicate contains one seedling. (Assessment of feeding activity of BPH through probing mark test is shown in Plate- 8). 3.4 Survival test of BPH nymph on different varieties of rice plant carrying different resistance gene through nymphal survival method. The nymphal survival test shows the difference for survival of nymphs on different varieties of rice plants. The well germinated seeds of selected rice genotypes were sown in earthen pots filled with fertilizer enriched puddled soil. After 40 days, the plants were covered by the mylar tube with ventilating windows. Then 10 nymphs (first and second instar) were released in such tubes then the open end of the tube covered by the muslin cloth and tied with rubber band. For each variety four replications were kept. (Nymphal survival test is shown in plate - 9). The plants were observed daily for nymph and emergence of adult. The percent nymphal survival calculated by using following formula (Heinrichs et al., 1985). Per cent nymphal survival = Number of adult emerge X 100 Number of nymphs released

65 View of nymphal survival test Single plant with developing nymphs Plate 9: Nymphal survival test on set of rice differentials

66 3.4.1 Status of egg parasitisation of brown planthopper in different donar differentials. A parasitoid is an organism that feeds on a single host organism. Parasitoids of rice insect pests are insects, which are parasitic only in their immature stages. The adult parasitoid is usually free living and may feed on nectar, honeydew, or body fluids of the host insect. In the life cycle of a typical egg parasitoid, the egg, larva, and pupa stages are spent inside the host egg. The adult parasitoid emerges from the host egg and mates, and then the female parasitoid lays eggs inside other host egg. The study of egg parasitoids of rice hoppers is made difficult by the extremely small sized of the parasitoids and by the nature of hopper oviposition. The delicate host eggs are inserted inside the plant material and cannot be identified or counted without dissection of the rice plant. Complete dissection of the plants allowed us to determine the status of the egg parasitisation in different donar differentials. Studies on egg parasites were carried out using individual rice seedling (40-45 days old). Two gravid females of BPH were confined to individual rice seedlings of twenty varieties of Plant hopper Special Screening trial (PHSS) for 24 hrs. for oviposition. After 9 to 10 days plants were dissected to count numbers of egg parasites. Oviposited stalks/stems have a whitish patch from which they can be identified. Cut stems and dissect them under a microscope for observing parasitized eggs. Unparasitised eggs are creamy white while parasitised eggs are lemon yellow or orange red in colour based on the species of parasitoid. In the present

67 Female of Anagrus sp. Plate 10 : Egg parasitization of BPH on set of rice differentials

68 investigation 2 species of egg parasites are investigated ie. Anagrus sp. and Oligosita sp. (Egg parasitization of BPH is shown in Plate - 10). 3.5 Statistical analysis :- All the observations on seed box screening, honeydew test and nymphal survival were recorded and tabulated in a systematic manner. The final observations were statistically analyzed in complete randomized block design (CRD) with necessary transformation. For sub objectives there was no statistical design of the experiment and results were taken out simply from mean data. Standard statistical procedure were followed as per Gomez and Gomez (1985).

69 Results and Discussion

70 CHAPTER-IV RESULTS AND DISCUSSION This chapter deals with the brief description of results obtained under different objectives of the experiment entitled Evaluation of rice differential varieties against Raipur brown planthopper Nilaparvata lugens (Stal) (Homoptera: Delphacidae) strain. The findings of the present study are compared with the previous findings of the relevant aspects in justified manner to draw a concrete conclusion. The results and discussion are presented here under different headings and sub headings: 4.1 Screening of rice differential varieties carrying different resistance gene through standard seed box screening technique Location of vascular bundle in cross- section stem of different rice differentials 4.2 Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through honey dew test Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through probing mark test. 4.3 Survival test of BPH nymph on different varieties of rice plant carrying different resistance gene through nymphal survival method Status of egg parasitisation of brown plant hopper in rice differential varieties. 4.1 Screening of rice differential varieties carrying different resistance gene through standard seed box screening technique. By following the standard evaluation technique, a set of twenty PHSS (Planthopper special screening) entries were evaluated against Raipur BPH population. All these differentials showed different reactions to Raipur BPH

71 population. The rice genotype Ptb33 showed minimum average plant damage score (0) followed by RP (0.1) and Rathu Heenati (0.68). (Table 4.1) Maximum average plant damage score (9) was observed in genotypes, ASD 7 (ACC 6303), Milyang 63, Chinsaba (ACC 33016), Pokkali, TN1(PHSS10), IR36, MUT NUS 1, OM 4498, IR and TN1(PHSS20). As per the standard evaluation of genotypes tested against Raipur brown plant hopper strain, three rice genotypes (Rathu Heenati (ACC 11730), RP and Ptb 33 were categorized as highly resistant (HR), while five genotypes (Babawee (ACC 8978), Swarnalatha (ACC 33964), T 12 (ACC 56989), IR 64, IR B ) as resistant (R). The genotypes ASD 7 (ACC 6303), Milyang 63, Chinsaba(ACC 33016), Pokkali, IR 36, MUT NS 1, TN1(PHSS15), OM 4498, IR and TN1(PHSS20) were categorized as highly susceptible (HS) to Raipur BPH population. (Table no. 4.1 & Fig no. 4.1). The present findings are in accordance to the DRR annual progress report (2014) which published the reaction of evaluation for 16 gene differentials of Plant hopper special screening trial (PHSS), across 7 locations in 7 valid tests it is revealed that PTB 33 and RP were promising in all the seven green house tests conducted at the different locations of the country against different population of BPH. Likewise Santhanalakshmi et al., (2010) evaluated that the genotype Ptb33 was highly resistant to the Indian biotype, while the variety Swarna was found susceptible to BPH.

72 Kumar et al., (2012) conducted studies on new sources for resistance against rice brown planthopper (BPH) under glass house conditions where 191 National Screening Nursery (NSN) entries evaluated. Swarnadhan was found highly resistant, CN 1870 was resistant and HKR 05 22, JGL-16267, UPR , CR , WGL-309, CR , NDR-359 and IR-64 were moderately resistant against brown planthopper. Several other workers like Mohammad and Alam, 1978; Velusamy and Chelliah, 1984; Shen et al., (2002); Urre et al., (2004a) and Bhimrao et al., (2005) have screened 4324, 50423, 38, 168 and121 entries respectively and stated 20, 555, 5, 7 and 3 varieties under resistant category. The results from International Rice Brown Plant hopper Nursery screening in several Asian countries over a 5-year period ( ) provided significant information on biotype variation in brown plant hopper and sources of genetic resistance to the different biotypes in the various countries. A major distinction between the insect populations in South Asia (Bangladesh, India, and Sri Lanka) and those in the rest of Asia is evident. Biotype differences of lower order within the common insect populations in East and Southeast Asian countries are also evident. Differential reactions within areas in India were also noted. Within each of the group of varieties of Bph 1, bph 2, Bph 3, and bph 4 genes were identified at IRRI, certain differences are evident from the reactions in other countries and, based on those, a set of differential varieties has been proposed. PTB33, Suduru Samba, and Sinna,Sivappu were resistant at almost all test sites. Several improved breeding lines derived from PTB33 were promising in all the regions of Asia and Solomon Islands.

73 Table 4.1 : Average plant damage score of Planthopper Special Screening (PHSS) lines against Raipur BPH, Nilaparvata lugens (Stal.) population. PHSS Lines Designation Parentages Average plant damage score PHSS1 ASD 7 (ACC Karsamba Red 6303) 9 PHSS2 Rathu Heenati Rathu Heenati (ACC 11730) 0.68 PHSS3 Babawee(ACC ) PHSS4 Tongil/IR //YR675- Milyang PHSS5 TN1 S.Check 9 PHSS6 Swarnalatha(ACC ) 1.5 PHSS7 T 12 (ACC 56989) PHSS8 Chinsaba (ACC ) 9 PHSS9 Pokkali - 9 PHSS10 TN1 S.Check 9 PHSS11 IR 64 IR /IR PHSS12 IR B PHSS13 IR 36 PHSS14 PHSS15 PHSS16 MUT NS 1 - TN IR *3/O.australiensis 2.37 IR //4*IR /O.nivara///CR S.Check OM 4498 IR 64/OMCS 2000//IR 64 PHSS17 RP Remark HS HR R HS HS R R HS HS HS R R HS HS HS HS HR PHSS18 Ptb33 0 HR PHSS19 IR HS PHSS20 HS TN1 S.Check 9 * Plant damage score based on 0-9 scale. ** Average plant damage score based on 3 replications.

74 Damage score (0-9 scale) Set of twenty rice differentials Average plant damage score Fig.4.1: Average plant damage score of rice differentials

75 Genes conveying resistance to brown plant hopper in PTB33 seems to be different in South Asia from those in the rest of Asia as evident from the differential reactions of the semi dwarf selections derived from that variety. ( Seshu and Kauffman,1980) Location of vascular bundle in cross- section stem of different rice differentials. Anatomical variation with respect to vascular bundle location varies among the rice differentials. After cutting the cross sections of these rice differentials, distance between the i) vascular bundle and epidermis, ii) phloem and epidermis, was taken under trinocular microscope. The purpose of this observation is to gain the knowledge about their anatomical differences with respect to vascular bundle location in internodal and nodal position of rice stem Internodal position:- Among the internodal position observations, the set of gene differentials exhibited average value of distance from vascular bundle to epidermis, varied from µm to µm. Maximum distance was observed in Ptb33 ( µm). Followed by RP (99.02 µm), Rathu Heenati (ACC 11730) (93.96 µm), Babawee(ACC 8978) (92 µm), Swarnalatha (ACC 33964) (84.3µm) and IR 64 (80.87µm). (Table no. 4.2) Minimum distance was observed in PHSS 20 ie. TN1 (57.65µm), followed by IR (58.14 µm), OM 4498 (58.21 µm), MUT NS 1 (59.46 µm), IR 36 (60.95 µm), Pokkali (66.09 µm), Chinsaba (ACC 33016) (71.08 µm), Milyang

76 63 (76.39 µm), ASD 7(ACC 6303) (76.51 µm), IR B (77.13 µm) and T12 (ACC 56989) (79.77 µm). (Table no. 4.2) Along with these, distance from phloem to epidermis was also measured. Maximum distance was observed in variety T 12 (ACC 56989) ( µm),followed by Swarnalatha (ACC 33964) (125.1 µm), IR 64 ( µm), ASD 7 (ACC 6303) ( µm), RP ( µm). Minimum distance was observed in PHSS 20 ie.tn1 (67.53 µm), followed by Pokkali (79.41 µm), IR (95.57 µm), OM 4498 ( µm), IR B ( µm), MUT NS 1 ( µm), Chinsaba (ACC 33016) ( µm), Babawee (ACC 11730) ( µm), IR 36 ( µm), Rathu Heenati(ACC 11730) ( µm), Ptb 33 ( µm) and Milyang 63 ( µm). (Table 4.2 & fig. no. 4.2).

77 Table 4.2: Location of vascular bundle in cross section stems of twenty different rice differential varieties (Internodal Position) Sr.N Designation VB E P E o. ( µm ) ( µm ) 1 ASD 7 (ACC 6303) Rathu Heenati (ACC 11730) Babawee(ACC 8978) Milyang TN Swarnalatha(ACC 33964) T 12 (ACC 56989) Chinsaba (ACC 33016) Pokkali TN IR IR B IR MUT NS TN OM RP Ptb IR TN (VB = Vascular bundle, E= Epidermis, P= Phloem, µm = micrometre)

78 Nodal position :- Among the nodal position the set of gene differentials exhibited average value of distance from vascular bundle to epidermis, varied from µm to µm. Among the distance between vascular bundle and epidermis, maximum distance was observed in genotype Ptb 33 (94.38 µm), followed by RP (93.26 µm), Rathu Heenati (ACC 11730) (93.23 µm), Babawee(ACC 8978) (92.34 µm) and Swarnalatha (ACC 33964) (90.92 µm). Minimum distance was observed in PHSS20 ie. TN1 (52.62 µm), followed by IR (59.99 µm), and OM 4498 (60.14 µm) (Table no.4.3). The distance between phloem (located inside vascular bundle) and epidermis was also observed under trinocular microscope. Maximum distance was observed in Ptb 33 ( µm), followed by RP ( µm), Rathu Heenati (ACC 11730) ( µm), Babawee (ACC 8978) ( µm), Swarnalatha (ACC 33964) ( µm) and IR 64 ( µm). (Table no. 4.3 ) Minimum distance was observed in PHSS20 ie.tn1 ( µm) followed by IR ( µm), OM 4498 ( µm), and MUT NS 1 ( µm). Anatomical studies observed among the set of rice differentials have depicted in (Table no. 4.3 & fig.no.4.3). Eames and McDaniels, 1947, Sharman (1942) and Esau (1953), makes a general comment that the complex arrangement of vascular bundle (in grass haulm) is related to the variable orientations of the different traces of the same leaf. The results obtained are little bit similar to the works done by Nitta et al., (2000), who investigated the varietal differences, in the number and cross area of large vascular bundles (LVs) at the neck internode in 18 rice cultivars.

79 Table 4.3 : Location of vascular bundle in cross section stems of twenty different rice differential varieties (Nodal Position) Sr.No. Designation VB E P E (µm) (µm) 1 ASD 7 (ACC 6303) Rathu Heenati (ACC 11730) Babawee(ACC 8978) Milyang TN Swarnalatha(ACC 33964) T 12 (ACC 56989) Chinsaba (ACC 33016) Pokkali TN IR IR B IR MUT NS TN OM RP Ptb IR TN (VB = Vascular bundle, E= Epidermis, P= Phloem, µm = micrometre)

80 VB E ( µm ) P E ( µm ) Fig.4.2: Comparative measurement of distance b/n VB-E & P-E (Internode) VB E (µm) P E (µm) Fig.4.3: Comparative measurement of distance b/n VB-E & P-E (Node)

81 Brown planthopper (BPH), Nilaparvata lugens (Stal), is one of the major phloem feeder which causes serious damage to rice plant. They injure the plants through direct sucking plant sap from phloem, there may be chance of escape due to unreachability of neonates to proper feeding site in deeply situated vascular bundle of Ptb33 but it is available superficially in TN Correlation between average plant damage score and anatomical studies with respect to location of vascular bundle in stem of rice varieties. Among intermodal position, there was highly significant negative correlation (r = -0.87) between average plant damage score and vascular bundle to epidermis distance and significant negative correlation (r = -0.52) from phloem to epidermis at 5 % level of significance. Sarwar and Prodhan, (2000) carried out anatomical investigations of 8 cultivars of rice (Oryza sativa L.) and concluded that the number, position and arrangement of vascular bundles vary along the cultivars. Among the nodal position, there was highly significant negative correlation (r = -0.91) between average plant damage score and vascular bundle to epidermis distance. There was highly significant negative correlation ( r = ) between average plant damage score and phloem to epidermis distance was observed. Short distance of vascular bundle form epidermis facilitated the pest insect for maximum feeding (Table no. 4.4 & fig. 4.5, 4.6, 4.7).

82 Table 4.4 : Correlation between average damage score and location of Sr. No. Designation 1 ASD 7 (ACC 6303) 2 3 Rathu Heenati (ACC 11730) Babawee(ACC 8978) 4 Milyang 63 5 TN1 6 Swarnalatha(ACC 33964) 7 T 12 (ACC 56989) 8 Chinsaba (ACC 33016) 9 Pokkali 10 TN1 11 IR IR B 13 IR MUT NS 1 15 TN1 16 OM RP Ptb33 19 IR vascular bundle in cross sections stem of different rice varieties Average damage score Internodal position VB E ( µm ) P E ( µm ) Nodal position VB E (µm) P E (µm) TN Correlation -0.87** -0.52* -0.91** -0.81* (VB = Vascular bundle, E = Epidermis, P = Phloem, µm = micrometre)

83

84 4.2 Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through honey dew test. All the twenty rice genotypes carrying different bph resistant genes, exhibited average honeydew excretion values varied from mm 2 to mm 2 per female in 24 hrs. The genotype Ptb33 had the lowest honeydew excretion value which was found significantly lower than the rice genotype TN1. The genotype Ptb33 showed the lowest honeydew excretion value (19.67 mm 2 ) in 24 hrs per female followed by Rathu Heenati (ACC 11730) (25.00 mm 2 ), RP (27 mm 2 ), Swarnalatha (ACC 33964) (28.00 mm 2 ), T 12(ACC 56989) (29 mm 2 ), IR64 (29.33 mm 2 ), ASD 7 (ACC 6303) (30.67 mm 2 ), MUT NS 1 (31 mm 2 ), Babawee(ACC 8978) (35 mm 2 ), IR B (37.33 mm 2 ), IR 36 (38.67 mm 2 ) and Chinsaba (ACC 33016) (43 mm 2 ), these were statistically at par among themselves. The honeydew excretion of BPH on given set of rice differentials is shown in Plate-7. Among the set of gene differentials tested, genotype TN1 had the highest average honeydew excretion value mm 2 in 24 hrs per female followed by, Pokkali, IR , OM 4498 showed the honey dew excretion values, mm 2, 58.00mm 2, and 51 mm 2, respectively. These all were statistically at par among themselves. The honeydew excretion of BPH on resistant genotypes is shown in Table no. 4.5 & fig. no. 4.8.

85 Table 4.5: Average feeding rate (honeydew excretion value) of BPH on Sr.No. differential varieties carrying different resistance gene Designation * Honeydew excretion in 24hrs (mm 2 /f) 1 ASD 7 (ACC 6303) (5.58) a 2 Rathu Heenati (ACC 11730) (5.05) a 3 Babawee(ACC 8978) 35 (5.96) a 4 Milyang (7.13) 5 TN (7.60) b 6 Swarnalatha(ACC 33964) 28 (5.34) a 7 T 12 (ACC 56989) (5.43) a 8 Chinsaba (ACC 33016) (6.60) a 9 Pokkali (7.74) b 10 TN (8.20) b 11 IR (5.46) a 12 IR B (6.15) a 13 IR (6.26) a 14 MUT NS (5.61) a 15 TN (8.09) b 16 OM (7.18) b 17 RP (5.24) a 18 Ptb (4.49) 19 IR (7.65) b 20 TN (7.76) b SEm± 0.75 CD 2.15 (Figures in the parentheses are square root transformed value) *Average of four replications

86 The present findings are in agreement with findings of Soundararajan et al.,(2004), they reported feeding behavior as a antibiosis parameter of BPH (Nilaparvata lugens Stal) on TN1 and Ptb33, parents and DH lines of the cross IR64/Azucena for resistance to BPH. The honeydew area on Ptb33, TN1, IR 64, Azucena and DHLs were found 36± 4.4, 682± 31.8, 448.3± 19.8, 614± 36.8 and ± 6.9 in mm 2, respectively. Bhimrao et al., (2005) carried out BPH feeding test on resistant cultivars viz. R , RGL-5613 and IR-64. Feeding rate was ranged between to mm2 per female in 24 hours, which was much lower than the feeding on TN1 ( mm 2 /female). Likewise, Reddy et al.,(2005) concluded that BPH fed on resistant and moderately resistant genotypes, excreted less amount of honeydew compared to the insects fed on susceptible TN Assessment of feeding activity of BPH on differential varieties carrying different resistance gene through probing mark test. Among the set of rice differentials, carrying different bph resistant gene, average probed marks values per seedling were ranged from to In light of Statistical interpretations numbers of probes received by Ptb33 was significantly higher than TN1(PHSS15). The genotype Ptb33 showed the maximum probed marks (38), followed by Rathu Heenati(ACC 11730) (32.33), RP (32.00), Swarnalatha(ACC 33964) (31.67), T 12(ACC 56989) (29.33) and IR64 (28.33), these were statistically at par among themselves. The probed marks by BPH on given set of rice differentials is shown in table no.4.6. The genotype TN1 (PHSS 15) had the minimum no. of probed marks (13) followed by OM 4498 which had shown average no. of probed marks These

87 were statistically at par among them. The average probed marks by BPH on given set of gene differentials is shown in fig no The rice genotype Ptb33 had the highest (38.00) average probing marks, which exhibited the mean honeydew excretion value of 19.67mm 2 followed by Rathu Heenati and RP , had the probed marks (32.33), (32.00) and honeydew excretion value of mm 2 and 28 mm 2 respectively. Similar results have found on the other rice differentials which showed that the genotype had the higher probed marks, was excreted lower honey dew. The comparative feeding behaviour (honey dew excretion area and no. of probed marks) by BPH is presented in fig no The present findings are in agreement with findings of Maheshwari et al., (2006) who studied that the resistant genotype IET recorded minimum (2.80cm 2 ) honeydew excretion and the susceptible TN1 recorded maximum (5.39 cm 2 ). Comparatively less honeydew excretion and more feeding marks recorded in all resistant genotypes than the susceptible TN1. Likewise, Ponnada et al., (2011) concluded that highly resistant lines exhibited significantly lowest feeding and highest probing marks. Statistically numbers of probes received by all resistant genotypes tested were significantly high as compared to susceptible check TN1, as prompted by several workers like Reddy (1979), Reddy and Kalode (1985) and Veronica (1985), resistant varieties receive higher number of probes than that of susceptible variety TN1. Piercing the stylets and holding it for long time depend upon the nutritional fulfilment of sap being drained by insect through stylets (Velusamy and Heinrichs, (1986). Female of BPH made more probing marks on resistant variety than on susceptible (Bagui, 1989).

88 Table 4.6 : Average feeding marks (probing marks) of BPH on differential Sr.No. varieties carrying different resistance gene Designation **Average probing marks 1 ASD 7 (ACC 6303) (4.14) b 2 Rathu Heenati (ACC 11730) (5.73) a 3 Babawee(ACC 8978) (4.98) b 4 Milyang (4.88) b 5 TN (3.72) c 6 Swarnalatha(ACC 33964) (5.67) a 7 T 12 (ACC 56989) (5.46) a 8 Chinsaba (ACC 33016) (5.12) 9 Pokkali (4.06) 10 TN (3.89) c 11 IR (5.37) a 12 IR B (4.81) b 13 IR (4.26) b 14 MUT NS (4.78) b 15 TN (3.67) c 16 OM (3.89) c 17 RP (5.70) a 18 Ptb (6.20) 19 IR (4.10) b 20 TN (3.89) c SEm± 0.35 CD 1.02 (Figures in the parentheses are square root transformed value) **Average of three replications

89 ASD 7 Rathu Heenati Babawee Milyang 63 TN1 Swarnalatha T 12 Chinsaba Pokkali TN1 IR 64 IR IR 36 MUT NS 1 TN1 OM 4498 RP 2068 Ptb33 IR71033 TN1 no. of probes & honey dew area ASD 7 Rathu Heenati Babawee Milyang 63 TN1 Swarnalatha T 12 Chinsaba Pokkali TN1 IR 64 IR IR 36 MUT NS 1 TN1 OM 4498 RP 2068 Ptb33 IR71033 TN1 Average no. of probes ASD 7 Rathu Heenati Babawee Milyang 63 TN1 Swarnalatha T 12 Chinsaba Pokkali TN1 IR 64 IR IR 36 MUT NS 1 TN1 OM 4498 RP 2068 Ptb33 IR71033 TN1 honey dew area Fig.4.8: Honey dew excretion by BPH on set of rice differentials Set of twenty rice differentials Fig.4.9: Probed marks by BPH on set of rice differentials Average no. of probes Fig.4.10: Comparative feeding behaviour of BPH on set of twenty rice differentials no. of probes Set of twenty rice differentials Honey dew area

90 Lu et al. (1999) investigated the probing behaviour of different biotypes of Nilaparvata lugens on TN1 (susceptible variety) and ASD 7 (resistant variety). The probing frequency of biotype 1 on TN1 decreased significantly and that of biotype 3 on ASD 7 increased significantly. As per the various pioneer workers in this field, negative feeding response offered by plant itself as a defence mechanism by way of release of chemicals in the plant itself as a stimulant imposed by BPH stylet insertion for sucking the sap or due to absence of proper nutritional value required by BPH in the plant itself. Role of such chemicals were studied by Sadasivan and Thayumanavan (2003) who stated that lectin, Galanthus nivalis agglutinin (GNA) get ingested by insect binds to the midgut epithelial cells. Results indicated that GNA bind to form glycopeptides in BPH gut there by inhibiting the digestive enzymes. As a result, insect takes out its stylet and tries again and again for further intake of food. Western analysis revealed the presence of GNA in the honeydew, gut preparation of BPH and the crude homogenate of the whole body. Lectin binding concentrate on the luminal surface of the epithelial cells. Reddy et al., (2004) reported that significant increase in phenolic content was observed after BPH infestation in resistant culture INRC (0.157 to mg g-1 tissue). Whereas, in BPH susceptible TN1 the total phenols reduced from 0.15 to mg g-1 as it was reported by earlier workers, that injuries to plants are liable to increase the toxic phenolic compounds in injured cells, the effective toxins probably being quinone (Goodman et al., 1967).

91 Soundararajan et al., (2002) attributed feeding rate to the planthopper to the capability to differentiate the resistance and susceptible genotypes of rice. The presence of oxalic acid and salicylic acid act as feeding deterrent to BPH. The present study indicated that, feeding was lower on all the resistant genotype than on TN1. The insect had to make more feeding marks on the resistant genotypes in an attempt to locate a suitable feeding site than in susceptible one (Alagar et al., 2008). The resistant genotypes elicited a feeding response from the insect gut but could not sustain prolonged feeding, which probably due to presence of certain biochemicals in the host plant Correlation studies between honey dew excretion values and no. of probe marks as feeding behaviour of BPH. There was highly significant negative correlation (r = -0.85) between honey dew excretion values and no.of probed marks done by brown planthopper. (Table 4.7 ). As the no. of probe marks will increase, the value of honey dew excretion by BPH will decrease. For illustration, the rice genotype Ptb33 showed maximum no. of probe marks (38), followed by Rathu Heenati(ACC 11730) (32.33) and RP (32) which correlatively had minimum value of honey dew excretion ie mm 2, 25 mm 2 and 27 mm 2, respectively. (Fig no. 4.11) It is very clear that susceptible host has received less probe marks and excrete more honeydew because of the presence of required nutritional value or the absence of harmful biochemical in the plant itself, whereas, in resistant host the more probe marks and less honeydew excretion might be the indication of unsuitability of nutrition in the plant or presence of certain plant biochemical which checks the feeding and proves the presence of antibiosis mechanism of resistance.

92 Table 4.7 : Correlation between honey dew excretion values and no. of probe marks as feeding behaviour of BPH. Sr.No. Designation * Honeydew excretion in 24hrs (mm 2 /f) 1 ASD 7 (ACC 6303) (5.58) 2 Rathu Heenati (ACC 11730) (5.05) 3 Babawee(ACC 8978) 35 (5.96) 4 Milyang (7.13) 5 TN (7.60) 6 Swarnalatha(ACC 33964) 28 (5.34) 7 T 12 (ACC 56989) (5.43) 8 Chinsaba (ACC 33016) (6.60) 9 Pokkali (7.74) 10 TN (8.20) 11 IR (5.46) 12 IR B (6.15) 13 IR (6.26) 14 MUT NS (5.61) 15 TN (8.09) 16 OM (7.18) 17 RP (5.24) 18 Ptb (4.49) 19 IR (7.65) 20 TN (7.76) **Average probing marks (4.14) (5.73) (4.98) (4.88) (3.72) (5.67) (5.46) (5.12) (4.06) (3.89) (5.37) (4.81) (4.26) (4.78) (3.67) (3.89) (5.70) (6.20) (4.10) (3.89) SEm± CD Correlation -0.85**

93 4.2.3 Correlation studies between average damage score and honey dew excretion values. There was highly significant positive correlation (r = 0.76) between the average plant damage score and honey dew excretion values of brown planthopper. As the average plant damage score will increase, the value of honey dew excretion by BPH will also increase. For illustration, the rice genotype Ptb33 showed minimum average plant damage score (0) followed by RP (0.1) correlatively exhibited the minimum honey dew excretion values mm 2, 27mm 2, respectively. Mamimum average plant damage scored by genotype TN1 (9) exhibited maximum honey dew excretion (66.67 mm 2 ) correlatively. (Table no. 4.8 & fig. no. 4.12). The present findings are in agreement with the findings of Alagar et al., (2008) who observed maximum number of feeding marks on ARC (43.80), which was 4.52 times higher than TN1 and it was followed by ARC 6650 (24.80) and KAU 1661(24.00). W 1263 recorded the lowest feeding rate of mm 2 followed by ARC 6650 (129.70mm 2 ), IR 72(144.17mm 2 ) and KAU 1661(177.48mm 2 ). Likewise, Yongfu et al., (2011) who studied that rice varieties Swarnalata and B5, showed the high levels of antibiosis and tolerance to BPH. Mudgo and T12, showed moderate resistance to the insects, had a high level of tolerance and moderate antibiosis to BPH. Varieties Rathu Heenati, ARC 10550, and ChinSaba were identified to be susceptible to BPH, showing a moderate level of tolerance and no antibiosis. In comparison to the evaluation methods of BPH resistance, the honeydew excretion rate could be used to detect the antibiotic level.

94 Table 4.8 : Correlation between average damage score and honey dew excretion values Sr.No. Designation Average plant damage score 1 ASD 7 (ACC 6303) 2 Rathu Heenati (ACC 11730) 3 Babawee(ACC 8978) 4 Milyang 63 5 TN1 6 Swarnalatha(ACC 33964) 7 T 12 (ACC 56989) 8 Chinsaba (ACC 33016) 9 Pokkali 10 TN1 11 IR IR B 13 IR MUT NS 1 15 TN1 16 OM RP Ptb33 19 IR TN *Average honey dew excretion value (mm 2 ) (5.58) (5.05) 35 (5.96) (7.13) (7.60) 28 (5.34) (5.43) (6.60) (7.74) (8.20) (5.46) (6.15) (6.26) (5.61) (8.09) (7.18) (5.24) (4.49) (7.65) (7.76) SEm± 0.75 CD 2.15 Correlation 0.76**

95 4.2.4 Correlation studies between average damage score and probe marks: There was highly significant negative correlation ( r = ) between the between the average plant damage score and probe marks by feeding of brown planthopper. As the average plant damage score will increase, the no. of probe marks by BPH will decrease. For illustration, the rice genotype Ptb33 showed minimum average plant damage score (0) followed by RP (0.1) and Rathu Heenati(ACC 11730) (0.68) correlatively had maximum no. of probe marks ie. 38, 32.33, and 32, respectively. Mamimum average plant damage scored by genotype TN1 (9) had correlatively minimum no. of probe marks (13). (Table no.4.9 & fig. no. 4.13)

96 Table 4.9 : Correlation between average damage score and probe marks by BPH feeding:- Sr.No. Designation Average plant damage score 1 ASD 7 (ACC 6303) 2 Rathu Heenati (ACC 11730) 3 Babawee(ACC 8978) 4 Milyang 63 5 TN1 6 Swarnalatha(ACC 33964) 7 T 12 (ACC 56989) 8 Chinsaba (ACC 33016) 9 Pokkali 10 TN1 11 IR IR B 13 IR MUT NS 1 15 TN1 16 OM RP Ptb33 19 IR TN **Average probing marks (4.14) (5.73) (4.98) (4.88) (3.72) (5.67) (5.46) (5.12) (4.06) (3.89) (5.37) (4.81) (4.26) (4.78) (3.67) (3.89) (5.70) (6.20) (4.10) (3.89) SEm± 0.35 CD 1.02 Correlation -0.85**

97 No. of probed marks Honey dew excretion values No. of probed marks Fig.4.11: Regression Equation of no. of probed marks for honey dew excretion values y = x R² = Honey dew excretion values Series1 Linear (Series1) Fig.4.12: Regression Equation of honey dew excretion values for average plant damage score y = 2.848x R² = Average plant damage score Series1 Linear (Series1) Fig.4.13: Regression Equation of no. of probed marks for average plant damage score y = x R² = Average plant damage score Series1 Linear (Series1)

98 4.3 Survival test of BPH nymph on different varieties of rice plant carrying different resistance gene through nymphal survival method. In a given set of twenty rice differentials, the nymphal survival test was carried out on 40 days old plant. All these genotypes exhibited average nymphal survival values varied from to per cent. The genotype Ptb33 had the lowest nymphal survival per cent which was found significantly lower than the rice genotype TN1 (PHSS20). The genotype Ptb33 showed the minimum nymphal survival value (35.00 %), followed by Rathu Heenati (ACC 11730) (40.00 %), RP (42.50 %) and Swarnalatha(ACC 33964) (45.00 %), these were statistically at par among themselves. Statistically second minimum nymphal survival value was observed in IR 64 (47.5%), followed by IR B (50.00%), Babawee(ACC8978) (52.50 %), T12 (ACC 56989) (55.00 %) and Chinsaba (ACC 33016) (60.00 %), these were statistically at par among themselves. The nymphal survival per cent of BPH on given set of rice differentials is shown in table no The genotype TN1 (PHSS 20) showed the maximum nymphal survival value (87.50 %), followed by OM 4498 (80.00%), these were statistically at par among themselves. Nymphal survival per cent value shown by twenty rice differentials is depicted in fig. no The results were in conformity with the findings of several workers viz. Gao et al., (1990) who investigated the brown planthopper survival on different rice varieties. They found that insect survival on Xiu-Shui 620 and IR64 was 50% of that on Xiu-Shui 48. Similarly, Senguttuvan et al., (1991) studied antibiosis in Ptb33

99 and IR 64 (highly resistant and resistant variety) were expressed as increased nymphal duration, decreased nymphal survival, while the reverse condition was noticed on the susceptible TN1. Only small proportion of BPH nymphs develop as an adult when subjected to stay and fed on resistant variety. Velusamy et al., (1995) evaluated three wild rice species and six cultivated rice varieties to determine their mechanisms of resistance to Nilaparvata lugens (Stal.). Nanda et al., (1999) observed more nymphal survival on the susceptible control TN1 (82.0 to 92.0%) as compared to other varieties viz. Mudgo, ASD 7, Rathu Heenati, Babawee, ARC 6650, Utri Rajpan, Udaya, Pratap and Ptb33 (1.0 to 5.0%) at different crop ages. Likewise, Reddy et al., (2005) studied that BPH caged with resistant and moderately resistant genotypes had prolonged nymphal development, slow growth and low survival compared to the insects caged with the susceptible control TN1. Kumar et al., (2012) studied the per cent nymphal survival, growth index values, population build-up, average nymphal emergence and adult longevity were significantly lower while nymphal duration was found longer as compared to susceptible check (TN1) on different resistant entries.

100 Table 4.10 : Percent survival of BPH on differential varieties carrying different resistance gene Sr.No. Designation **Nymphal survival % 1 ASD 7 (ACC 6303) 62.5 (14.44) 2 Rathu Heenati (ACC 11730) 40.0 (11.48) a 3 Babawee(ACC 8978) 52.5 (13.19) b 4 Milyang (14.75) c 5 TN (16.93) d 6 Swarnalatha(ACC 33964) 45.0 (12.22) a 7 T 12 (ACC 56989) 55.0 (13.48) b 8 Chinsaba (ACC 33016) 60.0 (14.14) b 9 Pokkali 72.5 (15.45) c 10 TN (16.66) d 11 IR (12.53) 12 IR B 50.0 (12.88) b 13 IR (15.31) c 14 MUT NS (16.13) 15 TN (16.68) d 16 OM (16.40) d 17 RP (11.83) a 18 Ptb (10.75) 19 IR (15.84) c 20 TN (17.19) d SEm± 0.59 CD 1.68 (Figures in parentheses are arc sine transformed value) **Average of four replications

101 nymphal survival % Fig.4.14: Per cent survival of BPH nymph on set of rice differentials Set of twenty rice differentials nymphal survival %

102 Wild rice species Oryza officinalis, O. punctata and O. latifolia, cultivated rice Rathu Heenati, Babawee, ARC 10550, Swarnalata, Ptb33 and the susceptible Taichung Native (TN1) were included in the study. N. lugens caged on resistant wild rice had slow nymphal development as compared to N. lugens on cultivated resistant varieties. Likewise, Nanda et al. (1999) conducted laboratory experiments to study the antibiosis mechanism of resistance in 10 rice varieties to BPH (Nilaparvata lugens Stal) They found more nymphal survival on the susceptible control TN1 (82.0 to 92.0%) as compared to Mudgo, ASD 7, Rathu Heenati, Babawee, ARC 6650, Utri Rajpan, Udaya, Pratap and Ptb33 (1.0 to 5.0%) at different crop ages. Low nymphal survival on resistant genotypes/variety was also reported by Soundararajan et al., (2003), Reddy et al., (2005), Maheshwari et al., (2006), Alagar and Suresh (2007). The results on reduction in survival rate of BPH on resistant genotypes might be due to presence of antibiosis factors i.e. presence of feeding deterrents such as soluble salicic acid, malic acid, itaconic acid and benzoic acid in the resistant genotypes as reported by Soundararajan et al., (2002). Results on the development of nymphs on the resistant genotypes suggested that the insect surviving on resistant genotypes had to face the problem of inadequate, unsuitable nutrition which may due to presence of higher total sugars and non reducing sugars. Greater nymphal survival, on susceptible TN1 proved the availability of good quality food and reverse can be found in resistant genotypes. Low level of total free amino acid and total starch content could be considered as the contributing factors in varietal resistance of the varieties to brown planthopper (Nanda et al., 2000).

103 Zhang and Gu (1985) had determined the effects of various levels of certain amino acids in rice plants on the development of Nilaparvata lugens (Stal.) in laboratory studies at China. They stated that young nymphs, especially those in the 1st and 2nd instar, developed rapidly in the presence of adequate amounts of free tyrosine, glycine and lysine in rice plants. The concentrations of free glycine and lysine in plants were negatively correlated with nymphal survival rate Status of egg parasitisation of brown planthopper in rice differential varieties. Egg parasitisation was also observed in the set of twenty rice differentials. Egg parasitoids of BPH have potential to reduce BPH populations, are Anagrus sp., and Oligosita sp. Level of parasitisation in per plant of twenty different rice genotypes has been seen under glass house condition, which varied from 0 % to %. Maximun egg parasitisation was observed in genotype Ptb 33 (89.48%). Anagrus sp., Oligosita sp., were seen in this genotype. Second maximum egg parasitisation was observed in Rathu Heenati(ACC 11730) (83.88), followed by RP (52.95 %) and Swarnalatha (ACC 33964) (48.55 %). Per cent parasitisation of all twenty rice differentials are shown in (table no.4.11 & fig. no. 4.15) Minimum egg parasitisation was observed in TN1 (PHSS10) (3.94%), followed by OM 4498 (10.04%), (table no.) Two species of BPH egg parasitoids have been observed ie. Anagrus sp. and Oligosita sp. Maximum BPH egg parasitisation was done by Anagrus sp. as compared to parasitisation done by Oligosita sp. (Table no. 4.11)

104 Table 4.11 : Status of BPH egg parasitisation in rice differential varieties carrying different resistance gene Composition of egg parasite Sr. Designation Unparasitiz Parasitized species No. ed egg % egg % Anagrus spp. Oligosita spp. 1 ASD 7 (ACC 6303) Rathu Heenati (ACC 11730) Babawee(ACC 8978) Milyang TN Swarnalatha(ACC 33964) T 12 (ACC 56989) Chinsaba (ACC 33016) Pokkali TN IR IR B IR MUT NS TN OM RP Ptb IR TN (Data don t need any transformation)

105 120 Fig.4.15: Per cent parasitization& unparasitization of hopper eggs Unparasitized egg % Parasitized egg % Fig.4.16: Comparative assessment of per cent nymphal survival & egg parasitization of BPH Ave. Survival % Parasitized egg %

106 Similar findings were reported by Fowler et al., (1991) on the rice plants infested with eggs of Nilaparvata lugens (Stal.) or Nephotettix spp. In laboratory cultures,were used to trap egg parasitoids in rice fields at two sites over a period of four days in Sri Lanka. Levels of egg parasitism in per plant varied from 0 to 54% in Nilaparvata lugens and 45 to 100% in Nephotettix spp. Nilaparvata lugens eggs were parasitized by Anagrus sp., A. optabilis (Perkins)(Hymenoptera: Mymaridae) and Oligosita sp. (Hymenoptera: Trichogrammatidae). Atmaja et al., (2000) studied that egg parasitoids of BPH have good potential to reduce BPH populations; these are: Anagrus sp., Oligosita sp., and Gonatocerus spp. Parasitism caused by these parasitoids in the field was 64, 55, and 40%, respectively. Qian et al., (2002) studied the hopper egg parasitoids, which could parasite eggs of the brown planthopper ( Nilaparvata lugens), at community level. The communities were made up of 19 hopper egg parasitoids, which belonged to 2 families, Mymaridae and Trichogrammatidae. Three species, Anagrus nilaparvatae, A. longitubulosus (A. Perforator) and A. Paranilaparvatae (A. Optabilis), were the dominant species, had high percentage in the community. The percentages of BPH eggs with hopper egg parasitoids during the early, middle, and late period of rice growth were about 76%, 70 %, and 50%, respectively. Natural parasitism is mostly dependent on the density of their host insect,it may be possible that parasitism may be higher in the susceptible cultivars and viceversa in resistant cultivars.

107 4.3.1 Correlation studies between nymphal survival and egg parasitisation status of BPH. There was highly significant negative correlation (r = -0.83) was observed between nymphal survival and egg parasitisation status of brown planthopper (table no. 4.12). On increase of BPH nymphal survival percent the egg parasitisation per cent of BPH will decrease, correlatively. For illustration, the rice genotype Ptb33 showed minimum per cent of nymphal survival (35%) followed by Rathu Heenati (ACC 11730) (40.00%) correlatively had maximum egg parasitisation per cent ie % and % respectively. Mamimum per cent nymphal survival showed by the genotype TN1(PHSS10) (82.50 %) had correlatively minimum egg parasitisation per cent (3.94 %). (Comparative assessment of per cent nymphal survival & egg parasitisation of BPH is shown in fig no. 4.16). In resistant cultivars number of eggs per egg mass was very low as compared to susceptible cultivar. This variation may be due to the statistical values. The results are in agreement with Xiang et al., (2008) who studied that parasitoid's offspring had lower survival rates, fecundities, female ratios, indexes of capacity for population increase, and longer developmental durations on plants when they were infested with high N. lugens density. (Table no & fig.no. 4.17)

108 Table 4.12: Correlation between percent nymphal survival and egg parasitization of BPH. Sr.No. Designation Nymphal survival (%) Total egg parasitisation (%) 1 ASD 7 (ACC 6303) 62.5 (14.44) Rathu Heenati (ACC 11730) 40.0 (11.48) Babawee(ACC 8978) 52.5 (13.19) Milyang (14.75) TN (16.93) Swarnalatha(ACC 33964) 45.0 (12.22) T 12 (ACC 56989) 55.0 (13.48) Chinsaba (ACC 33016) 60.0 (14.14) Pokkali 72.5 (15.45) TN (16.66) IR (12.53) IR B 50.0 (12.88) IR (15.31) MUT NS (16.13) TN (16.68) OM (16.40) RP (11.83) Ptb (10.75) IR (15.84) TN (17.19) 0.00 SEm± 0.59 CD 1.68 Correlation -0.83**

109 4.3.3 Correlation between average plant damage score and per cent nymphal survival: There was highly significant positive correlation ( r = 0.89 ) was observed between average plant damage score and per cent nymphal survival. On increase of average plant damage score the per cent nymphal survival of BPH will also increase. For illustration, the rice genotype Ptb33 showed minimum average plant damage score (0) followed by Rathu Heenati (0.68) correlatively showed the minimum nymphal survival per cent % and %, respectively. Maximum average plant damage scored by genotype TN1 (9) had the maximum per cent nymphal survival (87.5 %) correlatively. The susceptible variety provides better condition for the survival and development of nymph as compared to resistant varieties. (Table no.4.13 & fig. no. 4.18)

110 Table 4.13 : Correlation between average damage score and per cent nymphal survival: Sr.No. Designation 1 ASD 7 (ACC 6303) 2 Rathu Heenati (ACC 11730) 3 Babawee(ACC 8978) 4 Milyang 63 5 TN1 6 Swarnalatha(ACC 33964) 7 T 12 (ACC 56989) 8 Chinsaba (ACC 33016) 9 Pokkali 10 TN1 11 IR IR B 13 IR MUT NS 1 15 TN1 16 OM RP Ptb33 19 IR TN1 Average plant damage score Nymphal survival % 62.5 (14.44) 40.0 (11.48) 52.5 (13.19) 65.0 (14.75) 85.0 (16.93) 45.0 (12.22) 55.0 (13.48) 60.0 (14.14) 72.5 (15.45) 82.5 (16.66) 47.5 (12.53) 50.0 (12.88) 70.0 (15.31) 77.5 (16.13) 82.5 (16.68) 80.0 (16.40) 42.5 (11.83) 35.0 (10.75) 75.0 (15.84) 87.5 (17.19) 9 SEm± 0.59 CD 1.68 Correlation 0.89**

111 4.3.4 Correlation between average plant damage score and per cent egg parasitisation :- The highly significant negative correlation ( r = ) was observed between the average plant damage score and per cent egg parasitisation. As the average plant damage score will be higher, the per cent egg parasitisation of BPH will decrease. For illustration, the rice genotype Ptb33 showed minimum average plant damage score (0) followed by Rathu Heenati(ACC 11730) (0.68) correlatively had maximum per cent level of BPH egg parastisation % and %, respectively. As per potential of parasitisation of the egg parasitoid, some of the eggs were left unparasitized in a big egg cluster laid in the susceptible variety. The genotype TN1(PHSS10) had maximum damage score(9), showed correlatively minimum per cent of egg parasitisation (3.94 %). (Table no. 4.14)

112 Table 4.14 : Correlation between average plant damage score and per cent egg parasitisation: Sr.No. Designation Average plant damage score Total egg parasitisation % 1 ASD 7 (ACC 6303) Rathu Heenati (ACC 11730) Babawee(ACC 8978) Milyang TN Swarnalatha(ACC 33964) T 12 (ACC 56989) Chinsaba (ACC 33016) Pokkali TN IR IR B IR MUT NS TN OM RP Ptb IR TN Correlation -0.75**

113 Per cent nymphal survival Per cent egg parasitisation Fig.4.17: Regression Equation of egg parasitisation for per cent nymphal survival y = x R² = Per cent nymphal survival Series1 Linear (Series1) Fig.4.18: Regression Equation of per cent nymphal survival for average plant damage score y = 3.741x R² = Average plant damage score Series1 Linear (Series1)