BIOCHEMICAL COMPONENTS IN RELATION TO PESTS INCIDENCE OF PIGEONPEA SPOTTED POD BORER (MARUCA VITRATA) AND BLISTER BEETLE (MYLABRIS SPP.

Legume Res., 31 (2) : 87-93, 2008 BIOCHEMICAL COMPONENTS IN RELATION TO PESTS INCIDENCE OF PIGEONPEA SPOTTED POD BORER (MARUCA VITRATA) AND BLISTER BEETLE (MYLABRIS SPP.) P. Anantharaju and A.R. Muthiah Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore 641 003, India. ABSTRACT The present study was carried out in pigeonpea [Cajanus cajan (L.) Millsp.] to identify the resistant sources for spotted pod borer and blister beetle. The study of spotted pod borer resistance or tolerance was carried out in open field conditions without spraying any insecticide. The screening was done on seven parents and twelve F 1 s hybrids based on biochemical components. Considering the resistance to spotted pod borer and blister beetle, under unsprayed field conditions, the highest grain yield has been recorded by LRG 41 with lowest yield loss. The hybrid LRG 41 x ICPL 87119 registered the highest yield coupled with lowest yield loss. Hence the parent LRG 41 and the cross LRG 41 x ICPL 87119 are potential sources for future breeding programs. Biochemical basis of resistance may be due to low amount of total free amino acid, crude protein content and high amount of total phenolics in the pigeonpea genotypes against spotted pod borer and blister beetle. INTRODUCTION Pigeonpea grows well in tropical and subtropical environments. Pigeonpea is damaged by over 200 insect species at various growth stages (Lateef and Reed, 1990). Among the pests, Spotted pod borer (Maruca (testulalis) vitrata Geyer.) and flower feeder Blister beetle (Mylabris spp.) are the major ones causing drastic reduction in the yield of pigeonpea. Several biochemical factors are known to be associated with insect resistance in pigeonpea. Knowledge on genetic nature of resistance in the host plant can be of great value in breeding programs by providing a quantitative basis for recombination and selection and for identifying those gene products and resistance factors which are most likely to be responsible against the genetic plasticity of the pests. Breeding for resistance is a powerful tool in crop protection. Once we develop resistant genotypes, they can be used for a long time, without increase in the cost of production and environmental pollution and it is safe for human health. Genetic resistance in plants is one of the most effective and economic means of controlling pests in an eco-friendly way. Resistant plants are the first line defense against pests. A successful breeding programs for pest resistance depends upon the sound knowledge on genetics of resistance. Breeding for resistance has been very successful in reducing damage caused by many pests (Maxwell and Jennings, 1980), whereas the use of chemicals can create hazards to human health and produce undesirable side effects on non-target insects, animals and plants. Hence, it is desirable to develop genotypes resistant to spotted pod borer and blister beetle in order to step up the production and productivity in this important pulse crop. Efforts to develop pest resistant varieties have met with little success; and there is still limited understanding of insect- host relationship. Hence there is an urgent need to develop pigeonpea varieties tolerant to both field and storage pests as a component of integrated pest management. MATERIAL AND METHODS The twelve hybrids along with their parents (four lines and three testers) were raised in two conditions i) Protected condition- recommended all packages of practices ii) Unprotected condition- recommended all packages of practices (except plant protection schedules) as per crop production guide (Anonymous, 1999) were followed throughout the crop period in three

88 LEGUME RESEARCH replications using a randomized block design, at Millet Breeding Station, Tamil Nadu Agricultural University, Coimbatore during June, 2003. Each genotype was sown in ridges and furrows in a single row of 5m length with a spacing of 60 x 45 cm. A total of 10 plants per row were uniformly maintained, out of which five randomly selected plants were utilized for observation. The study of spotted pod borer resistance or tolerance was carried out in open field conditions without spraying any insecticide. The screening is done on seven parents and twelve F 1 s hybrids. The five important characters viz., seed yield per plant (both protected and unprotected conditions), total number of webbings leaves, flowers and young pods (only under unprotected condition) was counted in five randomly selected plants for each entry and the mean was computed. The biochemical components like crude protein content, total free amino acids and total phenolic content under the insecticide free field conditions were recorded on randomly selected competitive plants in each row representing each genotype. The present study on blister beetle damage was carried out under unprotected field conditions. The damage due to blister beetle was scored on randomly selected five plants per genotype. The total number of flowers (normal flowers + damaged flowers) and damaged flowers were counted on flower bearing branch upto 25 cm length from the top; three branches per plant were taken for the count and the mean scoring was expressed in percentage. Pest Incidence (PI) (%) = Total number of damaged flowers 100 Total number of flowers The seeds obtained from each of the six randomly selected plants for a particular genotype was air dried, weighed and expressed in grams, both protected and unprotected conditions and the yield loss calculated using following formula: Yield loss (%) = Yield under Yield under protected unprotected condition condition 100 Yield under protected condition The data on the pest in the field experiments were transformed to values and analysis of variance was carried out by randomized block design (Panse and Sukhatme, 1958) and mean were separated by Duncans Multiple Range Test (Gomez and Gomez, 1984). The data were transformed into Arc sin (data in %) (Duncans, 1951) was applied for comparing mean. Nitrogen content in the seeds of pigeonpea was estimated by micro kjeldahl method utilizing distillation set (Jackson, 1973). The protein content in seeds was calculated by multiplying the nitrogen content with the factor 6.25. One gram of plant sample (500mg young flower buds and 500mg young pods 30 days after fertilization) was taken and estimated for total free amino acid by Ninhydrin-Citrate-Glycerol method (Lee and Takahasi, 1966). The mean values were expressed in mg/g and utilized for statistical analysis.one gram of plant sample (500mg young flower buds and 500mg young pods 30 days after fertilization) was taken and estimated by Folin-Ciocalteau method (Bray and Thorpe, 1954). The mean values were expressed in mg/g and utilized for statistical analysis. Correlation coefficients were worked out to assess the relationship between biochemical components and pests incidence. The y variables (dependent characters) and x variables (independent characters) where Y - Pest incidence, X 1 - Crude protein content, - Total free amino acids and X 2

X 3 X 4 - Total phenolic content - Yield loss Correlation coefficients were worked out to assess the relationship between seed yield per plant and biochemical components. The y variables (dependent characters) and x variables (independent characters) where Y X 1 X 2 X 3 - Seed yield per plant, - Crude protein content, - Total free amino acids and - Total phenolic content RESULTS AND DISCUSSION Experiments conducted separately under protected and unprotected field conditions resulted in the identification of spotted pod borer resistant genotypes. Maruca, the spotted borer, causes severe loss to both flowers and young pods. The number of webbings in flowers and young pods is a criterion to assess the relative resistance of a genotype. The assessment of damage to the flowers and young pods has been the most reliable method to determine of pod borer resistance. The scoring of parents for Vol. 31, No. 2, 2008 89 spotted pod borer damage in the present study revealed that LRG 41 to be the most tolerant parent. Among the cross combinations, LRG 41 x ICPL 332 recorded the lowest spotted pod borer damage. Blister beetle incidence produce conspicuous damage in pigeonpea at the flowering stage. Present studies on blister beetle damage revealed that, LRG 41 and ICPL 87119 recorded minimum incidence of the pest under insecticide free field conditions. Among the hybrids LRG 41 x ICPL 87119 and LRG 41 x ICPL 332 showed very low incidence of this pest. Considering the damage caused by the Maruca and blister beetle in the parents and hybrids under unprotected conditions, the two genotypes LRG 41 and ICPL 332 will serve as potential sources for incorporating resistance in future breeding programmes. Under unsprayed conditions, the highest grain yield has been recorded in LRG 41 with lowest yield loss. Among the crosses, LRG 41 x ICPL 87119 and CO 6 x ICPL 87119 registered the highest yield along with lowest yield loss. Table 1. Performance of seven parents with regard to insect pests incidence (spotted pod borer and blister beetle) screened under insecticide free conditions in relation to biochemical components Varieties Scoring of Crude protein Total free Total phenolic Yield insect damage content (%) amino acids content (mg/g) loss # (mg/g) $ $ (%) Spotted pod Blister beetle borer (No. of damage webs/ 6 plants (%) CO5 (L 1 ) 35.67 (5.97)* 47.12 (43.35)** 24.39 0.997 1.14 99.66 CO6 (L 2 ) 18.33 (4.27)* 36.09 (36.92)** 21.73 0.680 2.78 70.91 VRG17(L 3 ) 45.00 (6.70)* 43.71 (41.38)** 21.91 0.760 1.95 85.81 LRG41(L 4 ) 13.00 (3.58)* 19.07 (25.89)** 21.35 0.456 7.40 41.06 APK1(T 1 ) 27.33 (5.22)* 27.18 (31.41)** 21.80 0.760 1.33 95.03 ICPL87119(T 2 ) 52.00 (7.21)* 24.32 (29.53)** 23.40 0.857 2.26 69.03 ICPL332(T 3 ) 13.00 (3.60)* 44.21 (41.67)** 19.73 0.343 3.04 58.76 Grand mean 29.19 (5.22)* 34.53 (35.73)** 22.04 0.69 2.84 73.89 CV (%) 5.18 2.48 4.72 5.33 2.43 2.19 CD (5%) 0.481 1.578 1.849 0.065 0.122 2.877 SEd 0.220 0.724 0.849 0.030 0.056 1.320 Figures inparentheses indicates square root values (*) and Arc sin values (**) # Estimated from dry seeds $ Estimated from young flower buds and pods

90 LEGUME RESEARCH Several biochemical factors are known to be associated with insect resistance in pigeonpea. The biochemicals such as low sugar, amino acids, proteins and high phenols contents can offer resistance to most of the pod borer species in pigeonpea and can form a basis for selection of pigeonpea genotypes. The variety CO5 which possessed the highest protein content (24.39 per cent) total free amino acids (0.997mg/ g) and the lowest phenolic content (1.14mg/g) suffered the highest yield loss of 99.66 per cent due to the incidence of Maruca and Blister beetle (Table 1). The parent LRG 41 possessed the highest phenolic content of 7.40mg/g showed the lowest yield loss of 41.06 per cent by the pests. The parent ICPL 332 possessed the lowest protein content (19.73 per cent) and total free amino acids (0.343mg/g), but the phenolic content in this variety is lower (3.04mg/g) compared to that of LRG 41 and this variety registered an yield loss of 58.76 per cent (Table 1). In the case of hybrids, LRG 41 x ICPL 332 recorded the highest total phenolic content of 4.66mg/g, the lowest crude protein content of 19.14 per cent and lowest content of total free amino acids (0.30mg/g). It suffered the lowest yield loss of 46.08 per cent damage by the pests. In contrary, the hybrid CO 5 x ICPL 87119 possessed the highest protein content (23.24 per cent) and higher total free amino acids (0.87mg/ g), but a lower phenolic content (1.56mg/g), which registered the highest yield loss of 95.00 per cent damage by Maruca and Blister beetle (Table 2). The biochemical constituents of pigeonpea seed govern its nutritive value. There is wide variability present in pigeonpea for different constituents as reported by Singh et al. (1984). Table 2. Performance of twelve crosses against spotted pod borer and blister beetle damage under insecticide free field condition Crosses Scoring of Crude protein Total free Total Yield insect damage content (%) amino phenolic loss # acids content (%) (mg/g) $ (mg/g) $ Spotted pod Blister beetle borer (No. of damage webs/ 6 plants (%) CO 5 x APK 1 48.0 (6.92 )* 57.56 (49.35 )** 21.35 0.88 1.23 94.69 CO 5 x ICPL 87119 48.0 (6.92 )* 52.86 (46.64 )** 23.24 0.87 1.56 95.00 CO 5 x ICPL 332 72.0 (8.48 )* 47.58 (43.61 )** 23.07 0.99 3.13 90.04 CO 6 x APK 1 40.0 (6.29 )* 32.50 (34.75 ) ** 21.86 0.72 2.05 82.69 CO 6 x ICPL 87119 47.0 (6.85 )* 35.37 (36.46 )** 21.57 0.77 2.41 65.97 CO 6 x ICPL 332 30.0 (5.42 )* 44.17 (41.64 )** 20.33 0.34 3.49 67.34 VRG 17 x APK 1 60.0 (7.72 )* 49.67 (44.81 )** 21.73 0.76 1.64 87.97 VRG 17 x ICPL 87119 74.0 (8.58 )* 39.34 (38.84 )** 20.92 0.80 2.11 77.77 VRG 17 x ICPL 332 54.0 (7.34 )* 37.51 (37.76 )** 19.95 0.57 3.11 67.10 LRG 41 x APK 1 35.0 (5.87 )* 27.33 (31.51 )** 20.98 0.59 3.68 83.10 LRG 41 x ICPL 87119 68.0 (8.24 )* 14.23 (22.16 )** 20.93 0.66 3.84 53.48 LRG 41 x ICPL 332 15.0 (3.83 )* 24.20 (29.46 )** 19.14 0.30 4.66 46.08 Grand mean 49.2 (6.87 )* 38.53 ( 38.08)** 21.25 0.68 2.74 75.96 CV (%) 8.10 3.48 3.68 3.10 4.18 5.02 CD (5%) 0.943 2.242 1.322 0.036 0.194 6.451 SEd 0.454 1.081 0.637 0.017 0.093 3.100 Figures in parentheses indicates square root values (*) and Arc sin values (**) # Estimated from dry seeds $ Estimated from young flower buds and pods

Vol. 31, No. 2, 2008 91 Biochemical constituents present in the host plant exert profound influence on growth, development, survival and reproduction of insects in various ways (Painter, 1958). Informations about the effect of biochemical constituents on the incidence of spotted pod borer and blister beetle has been studied in detail in the present investigation. The estimation of crude protein, total free amino acids and total phenolic content in different entries of the present study showed their definite influence on the incidence of spotted pod borer and blister beetle. Protein quality is of prime importance in the pigeonpea products used for human food (Salunkhe et al. 1986). Hulse, (1977) found that the protein content of pigeonpea seed samples ranged between 18.5 and 26.3 per cent. The genotypes CO 5 and ICPL 87119 recorded maximum amounts of crude protein but on the other hand, they were susceptible to the pests. Insects prefer genotypes with more protein content. In the present study LRG 41 recorded minimum protein content, suffered less insect damage and gave more yields under insecticide free field conditions. Eventhough LRG 41 and VRG 17 showed almost similar protein content, the phenolic content in LRG 41 was significantly very high, hence less insect damage was noticed in LRG 41. Hardwick (1965) and Sahoo and Patnaik (2002) also reported that genotypes with more crude protein contents were susceptible to insect attack. Hence, breeders should aim at reasonable level of protein content with acceptable level of tolerance to insect pests. Pigeonpea had the lowest values for free amino acids among several legumes examined by Eggum and Beames, (1983). Protein fractions play an important role in determining the overall amino acid composition of seed proteins. The parent CO 5 has been found to possess the maximum amount of total free amino acids, which is more prefered by the blister beetle and spotted pod borer and it is highly susceptible to both the pests. Whereas the lowest content of total free amino acids is found in ICPL 332, which had the least pests incidence. The amino acids content was low in resistant pigeonpea genotypes viz., in LRG 41and ICPL 332 as compared to the susceptible genotypes CO 5 and APK 1. Similar findings were also reported by Sahoo and Patnaik (2002). Hence the breeders may aim at tolerant genotypes with reasonable level of total free amino acids. Phenolic, the aromatic compounds, which are wide spread in plant kingdom and occur in all most all parts of the plants. Among the parents, LRG 41 showed the maximum amount of total phenolic content, which imparts resistance to spotted pod borer and blister beetle. The crosses LRG 41 x ICPL 332 recorded the highest amount of total phenols with lower incidence of the pests. The associations of biochemical components in relation to the incidence of the pests were worked out and results are presented in Table 3. The relationship between spotted pod borer incidence and the crude protein content was positive (0.273). A highly significant positive association (0.609) of total free amino acids with the incidence of spotted pod borer was also observed. Total phenolic content is negatively Table 3. Correlation coefficients of the spotted pod borer and blister beetle incidence with the biochemical constituents of young flower buds and pods for selected seven varieties and their twelve crosses Characters Crude Total Total Yield protein free amino phenolic loss content acids content (g) (%) (mg/g) (mg/g) # $ $ Spotted pod 0.273 0.609** -0.378 0.363 borer (Damage in young pods and flower buds) Blister beetle 0.232 0.379-0.594** 0.655** damage (Flowers) Seed yield -0.276-482* 0.663** - plant -1 (Insecticide free conditions) *Significant at 5 % level # Estimated from dry seeds **Significant at 1 % level $ Estimated from young flower buds and pods

92 LEGUME RESEARCH correlated (-0.378) with the spotted pod borer incidence (Table3). Similarly positive association of crude protein content (0.232) and total free amino acids (0.379) with the incidence of blister beetle was observed in the present study. A highly significant negative correlation (-0.594) of total phenolic content with the incidence of blister beetle is also revealed from the present study. Under insecticide free conditions, the seed yield was positively and significantly correlated (0.663) with the total phenolic content (Table 3). Significant yield loss was associated with blister beetle damage whereas statistically nonsignificant association was observed in the yield loss with the damage of spotted pod borer (Table 3). Existence of positive correlation between crude protein content, total free amino acids and incidence of pests (spotted pod borer and blister beetle) suggests that the lines with better protein or amino acid content do suffer most, when we aim at high quality genotypes, there is possibility of increased due to pest attack. The negative association between phenolic contents and pests incidence revealed that by increasing phenolic contents, resistance could be achieved. Total phenolic contents responsible for imparting resistance has also been reported by Sithananthan et al. (1980), Dass and Odak (1987), Mohan et al. (1987), Ananthakrishnan et al. (1990), Verulkar and Singh (2000) and Sahoo and Patnaik (2002). From the results, it is inferred that low amount of crude protein, free amino acids and high phenolic contents can offer resistance to insects pests in pigeonpea and can form a basis for selection of pigeonpea genotypes for pests resistance. Several screening techniques are available for a number of insect pests but more knowledge on other aspects of host plant resistance viz., genetics of resistance, basis and mechanisms of resistance is required to be understood in a better way, to develop durable insect resistant varieties of pigeonpea. Breeding for resistance is a powerful tool in crop improvement and once it is achieved, it will not only reduce the cost of production but also substantially prevents the environmental pollution. REFERENCES Ananthakrishnan, T.N.et al. (1990). Proc. Indian Acad. Sci. (Anim. Sci.)., 99: 39-52. Anonymous. (1999). Crop Production Guide. Department of Agriculture, Government of Tamil Nadu. pp.73-78. Bray, H.G. and Thorpe, W.V. (1954). Methods Biochemical Anal., 1: 27-52. Dass, S.B and Odak, S.C. (1987). Crop Improv., 14 (1) : 64-68. Duncans, D.B. (1951). Va.J.Sci., 2: 171-189. Eggum, B.O and Beames, R. M. (1983). In: Seed protein: Biochemistry, Genetics, and Nutritive value. (W.Gottschalk and H.P.Muller. (eds.) ) Martinus Nijhoff/ Dr.W.Junk.Publishers, The Hague, The Netherlands, pp.499-531. Gomez, K.A. and Gomez, A.A. (1984). Statistical Procedures for Agricultural Research. Wiley International Science Publication, John Wiley Sons, New York, pp. 207-215. Hardwick,D.F.(1965). Memorial Ent. Soc. Canada, 40: 350-356. Hulse, J.H. (1977). In: Nutritional Standard and Methods of Evaluation for Food Legume Breeder. (Hulse, J.H. et al.) International Research Center, Ottawa, Canada, pp. 88-100. Jackson, M.L. (1973). Soil Chemical Analysis. Prentice Hall of India, Pvt. Ltd., New Delhi, pp.183-197. Lateef, S.S. and Reed, W. (1990). In: Insect Pests of Tropical Food Legumes (Singh, S.R., ed.). Johnwiley & Sons. Chichester, UK, pp. 193-242. Lee, Va. Pin and Takahasi, T. (1966). Analytical Biochem., 14: 71-77 Maxwell, F.G and Jennings, P.R. (1980). In: Breeding Plants Resistant to Insect (Maxwell, F.G and Jennings, P.R. ed.). John Wiley Sons, New York. Mohan, S. et al. (1987). Curr. Sci., 56: 723-735. Painter, R.H. (1958). Annu. Rev. Entomol., 3: 267-290.

Vol. 31, No. 2, 2008 93 Panse, V.G. and Sukhatme, P.V. (1958). Statisical Methods for Agricultural Workers. Indian Council of Agricultural Research.New Delhi, p. 327. Sahoo, B.K. and Patnaik, H.P. (2002). J. Res. Orissa Univ. Agric. Tech., 20 (2): 16-20. Salunkhe, D.K. et al. (1986). Food Sci. Nutri., 23 (2): 130-145. Singh,U. et al. (1984). J. Food Sci. Technol, 21: 367-372. Sithananthan, S, et al. (1980). In: Proc. Intl. Workshop on Pigeonpea, December, 15-19,1980, Vol.2. Patancheru, Andhra Pradesh, India,pp. 329-335. Sithananthan, S. V. et al. (1983). Intl. Pigeonpea Newsl., March,1983: 68-69. Verulkar, S.B. and Singh, D.P. (2000). In: Natl. Symp. Management of Biotic and Abiotic Stresses in Pulse Crops, 26-28, June, 1998, IIPR, Kanpur, pp.75-78.