breeding for disease resistant in groundnut

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WELCOME TO SEMINAR

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2 BREEDING FOR DISEASE RESISTANT IN GROUNDNUT

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Contents 3 Introduction Disease condition Disease symptoms Screening methods Source of resistance Genetics of resistance and inheritance Breeding methods Achievements Conclusion

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Introduction The groundnut (Arachis hypogaea L .) is most important oilseed crops in India and it is one of main crop in Gujarat Area (Mha) Production (Mt) Yield (kg/ha) India 6.22 7.34 1180 Gujarat 1.90 2.66 1395 Several biotic and abiotic factors reduce groundnut production and affect its seed quality More than 55 pathogens including viruses have been reported to affect groundnut productivity and quality 4

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5 Fig 1. Groundnut growing areas in India

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6 Fig 2. Area and Production of groundnut growing state

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7 Fig 3. Yield of groundnut growing state

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8 Classification of disease

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9 A. Disease caused by fungi Foliar diseases Seed and seedlings diseases Stem, pod and root diseases B. Diseases caused by bacteria Bacterial wilt Bacterial leaf spot

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10 C. Diseases caused by viruses and Mycoplasma like organism Peanut mottle Peanut strip Peanut clump Groundnut rosette Cowpea mild mottle virus (CMMV) D. Diseases caused by nematodes Root knot Root lesion Kalahasti malady

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Economically Important Diseases of Groundnut 11

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Aspergillus's Crown Rot [ HTML | Flash | PPT | ZIP ] Lesson 2 : Stem and Pod Rot [ HTML | Flash | PPT | ZIP ] Lesson 3 : Early Leaf Spot [ HTML | Flash | PPT | ZIP ] Lesson 4 : Late Leaf Spot [ HTML | Flash | PPT | ZIP ] Lesson 5 : Rust Diseases [ HTML | Flash | PPT | ZIP ] Lesson 6 : Bud Necrosis/Stem Necrosis Aspergillus's Crown Rot Stem and Pod Rot Early Leaf Spot Late Leaf Spot Rust Diseases Bud Necrosis/Stem Necrosis 12

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DISEASE SITUATION IN INDIA 13

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14 Fig 4. Distribution of groundnut diseases in India

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ELS LLS RUST DHARWAD Moderate High High JALGAON Moderate High High JUNAGADH Moderate High High RAICHUR Moderate High High KADIRI High Low Low HANUMANGADH High Low Low Foliar fungal disease ELS - Early leaf spot LLS - Late leaf spot DGR, Junagadh Anonymous (2009 )

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16 SEED AND SEEDLING DISEASES Stem rot and collar rot incidence was low to moderate (15%) at Dharwad, Jalgaon, Junagadh and Hanumangadh during the crop growth. Maximum incidence of stem rot reached upto 18-30% at Kadiri and Raichur. Dry root rot was observed up to 10% at Hanumangadh, Kadiri and Virddhachalam.

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VIRAL DISEASES The incidence of PBND was low ( up to 6%) in farmers fields at Dharwad, Hanumangadh and Jalgaon and was not noticed at Jungadh. At Raichur PBND incidence ranged from 25-37% and less( 2-19%) in farmers fields. The incidence of PSND was 1-22% at Kadiri.

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Sr. No Diseases Causal agent Distribution and economic importance Yield loss (%) Nature 1 Collar rot Aspergillus niger Andhra Pradesh, TamilNadu, Gujarat, Maharashtra 28-50 Seed and s oil born 2 Early leaf spot Cercospora arachidicola Southern and Central India 15-59 Soil and air born 3 Late leaf spot Cercospora personata Southern and Central India 15-59 Soil and air born 4 Rust Puccinia arachidis Southern India 10-52 Air born Table 1: Causal organisms, distribution and crop losses of groundnut diseases 18 DGR, Junagadh Anonymous (2009 ) Conti……

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Sr. No. Diseases Causal agent Distribution and economic importance Yield loss ( %) Survival 5 PBND Peanut bud necrosis bud virus M.P., A.P., Gujarat, Maharashtra 30-90 Thrips ( Thrips palmi ) 6 PSND Tobacco streak virus Anatapur, Karnataka, A. P. 23.6 - 7 Stem rot Sclerotium rolfssi Tamilnadu , Gujarat, Maharashtra, M.P., A.P. 27 Seed and soil born 19

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20 Disease symptoms

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The cotyledon covered with black fungal spores. Aspergillus's Crown Rot Infected pods covered with black sooty spores 21

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Early Leaf Spot Sub-circular dark brown spots are produced on the upper leaflet surface. 22

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Late leaf spot Dark brown to black spores are found on the lower leaf surface 23

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Terminal buds of plants are affected when temperatures are relatively high . Leaflets produced on auxiliary shoots show a wide range of symptoms including reduced size, distortion of the lamina, mosaic, and general chlorosis . Bud Necrosis / Stem Necrosis 24

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An orange colored pustules that appear on the lower leaflet surface. Rust Pod rots are characterized by brownish black lesions on the shell. The lesions enlarge and the entire pod surface becomes discolored. Pod Rot 25

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Rotted pegs and pods are the colour of a brown paper bag, Pegs are shredded and pods are thin and brittle. Coarse strands of fungus may be present Stem rot 26

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Diseases Caused by Fungi 27

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A. Pre-emergence Seed and Seedling Rots Rhizoctonia Damping-Off C.O. Rhizoctonia solani khun 28 Yellow Mold C.O. Aspergillus flavus Seeds and non-emerged seedlings become shriveled and brownish. Seedlings show sunken, elongate, dark brown areas just below the soil surface.

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B. Stem, Root and Pod Diseases Black Rot C.O. Cylindrocladium crotalariae Charcoal Rot C.O. Macrophomia phaseolina 29 Reddish orange fungal bodies are formed When pods are invaded their interior surfaces turn gray

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Diseases caused by viruses 30

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31 Groundnut Rosette C.O. Rosette Virus Cowpea Mild Mottle C.O. Cowpea Mild Mottle Virus (CMMV) Stunted plants with chlorotic twisted Rolling of leaflets edges and stunting

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Peanut Yellow Spot C.O. Peanut Yellow Spot Virus Witches Broom C.O. Mycoplasma like organism 32 Chloratic spot appear along the midribs of young leaflets Leaves are small and chloratic and plant is stuntented

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33 Peanut Clump (PCV) C.O. Peanut Clump Virus Young leaves show mosaic mottling and chloratic ring spot

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34 Diseases Caused by Nematodes

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Root-Knot C.O. Meloidogyne arenaria Kalahasti Malady C.O. Tylenchorhynchus brevelineatus 35 Small brownish yellow lesions appear on the pegs, and on young developing pods. Enlarge roots and pegs

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Diseases Caused by Bacteria 36

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Bacterial Leaf Spot C.O. Unidentified bacterium Bacterial Wilt C.O. Pseudomonas solancearum 37 Small light brown, circular to irregular lesions appear on the lower leaves Sudden wilting and death , but leaves remains green

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38 Screening Methods

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39

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40 Sr. no. Genotypes Leaf spot diseases Mean 2001 2002 2003 1 JL 24 36.7 51.7 46.67 45.02 2 J 45 30.0 31.7 45.0 35.56 3 ICGV 93242 55.0 66.6 71.67 64.41 4 JSSP-9 2.4 5.0 30.0 12.46 5 TMV- 2 47.5 60.0 55.0 54.16 6 ICGV 86590 26.7 30.0 51.67 36.12 7 VRI 2 33.3 41.6 41.67 38.85 8 K 134 35.8 43.8 60.0 46.36 9 Sel 1 - 48.3 73.33 60.81 10 DH 53 2.8 4.3 4.67 3.92 11 DH 56 21.3 11.3 16.67 16.42 12 GPBD 4 - 3.33 4.67 3.98 Table 2. Screening of groundnut genotypes against Leaf spot disease Venugopal et al. (2004) Bangalore

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41 Sr. no. Genotypes Rust disease (%) Mean PBND (%) Mean 2001 2002 2003 2002 2003 1 JL 24 27.5 43.3 - 35.5 2.6 24.74 13.68 2 J 45 17.5 26.6 26.05 5.3 14.65 9.98 3 ICGV 93242 21.7 31.6 26.61 5.3 9.95 7.63 4 JSSP-9 1.3 2.6 1.95 6.6 3.02 4.81 5 TMV- 2 22.5 35.5 28.75 0.6 22.05 11.33 6 ICGV 86590 4.5 8.0 6.25 5.3 13.27 9.29 7 VRI 2 17.8 25.6 21.70 3.3 9.54 6.42 8 K 134 20.8 33.3 27.05 6.6 16.48 11.54 9 Sel 1 - 40.0 40.0 2.6 13.59 8.10 10 DH 53 3.2 3.0 3.1 3.3 4.98 4.14 11 DH 56 4.0 4.0 4.0 4.6 8.14 6.37 12 GPBD 4 - 1.6 1.6 3.3 2.28 3.07 Table 3. Screening of groundnut genotypes against Rust disease and PBND Bangalore Venugopal et al. (2004)

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42 Sr. No. Per cent incidence Mean 2001 2002 2003 2004 1 LGN 1 5.88 20.58 19.56 16.34 2 LGN 2 12.0 10.41 22.91 15.14 3 LGN 69 15.15 0.0 0.0 5.05 4 LGN 74 - - 2.04 2.04 5 LGN 83 - 9.37 12.0 10.68 6 TAG 24 42.48 22.22 26.31 28.84 7 JL 24 - 9.37 12.0 10.68 Table 4. Reaction of promising genotypes against stem rot Makne et al. (2004) Latur

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Table 5. Screening the hybrid plants for PBND, PMV and PSTV by ELISA Disease ELISA reading + control ELISA reading – control ELISA reading Hybrid plants PBNV 3.4 0.07 0.07 PMV 3.4 0.07 3.22 PSTV 3.34 0.07 0.08 + control = ELISA reading when the plant is completely infected. – control = ELISA reading of uninfected plant. Mallikarjuna et al . (2002) Netherland 43

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44 Genetic study of disease

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45 Sr. No. Species Sources Type 1 PI 109839 America. fastigiata 2 PI 162857 America. fastigiata 3 PI 259639 America. fastigiata 4 PI 215696 Peru fastigiata 5 PI 351879 Peru fastigiata 6 PI 381622 Peru fastigiata 7 PI 390595 Peru fastigiata 8 PI 259747 Peru Valencia 9 PI 298115 USA Valencia 10 NC Acc 17090 Peru Valencia 11 EC 76446 (292) Uganda Valencia 12 NCACC 17133 (RF) S. America Valencia 13 DHT 200 Peru Valencia Nigam et al. (1980) ICRISAT, Patancheru Table 6 . Sources of diseases resistant in groundnut for early and late leaf spot, rust, PBND and PSND.

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SR.NO. DISEASES CROSSES GENETIC NATURE OF RESISTANT REMARKS REFERENCES 1. Leaf spot a ELS, LLS Robut 33-1 x krapovickas 16 Non additive LLS governed by 5 recessive gene Nevill ( 1908, 1982) b ELS, LLS Crossed 6 resistant line in a diallel Non additive maternal effect in f 1 Best general combiners: ELS – NCGP- 343, NC-5, NC-3033; LLS- NCGP-343, NC 5; ELS+LLS NC3033, NCGP 343 Kornegay (1980) c LLS Crossed 5 resistant lines and 4 cultivars in an M X N mating design Additive and non additive gene action Male – NC 7, NC 6; female – NCAC 17090, 17132 was best combiner f 1 partially resistant to NC7 Walls and Wanye (1985) d ELS, LLS - Additive Analyzed f 2 best combiner GPNC 343, ELS and LLS inherited independently; heritability 0.4- 0.8 Anderson et al. ( 1986) 46 Table 7. Genetics of disease resistant in groundnut Conti…..

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SR.NO. DISEASES CROSSES GENETIC NATURE OF RESISTANT REMARKS REFERENCES 2. Rust a 3 susceptible and 3 resistant Digenic, trigenic F 2 ratios 15 S: 1R and 63 S: 1R Kishore (1981) b UF 49-16-10-3 X PI 314817 Duplicate recessive F 2 ratios 15 S: 1R and 63 S: 1R Knauft and Nordan (1983) C Crossed 2 susceptible and 2 2 resistant lines Digenic F 2 ratio 9S : 6I : 1R Tiwari et al. (1984) D Crossed 3 resistant ( EC 76446-292, NCAC 17090,PI 259747 AND 2 susceptible ( J 11 and Gangapuri) Digenic Additive x additive , additive x dominance Reddy et al. ( 1987 ) e Crossed 2 resistant ( PI 298115 X EC 76446-292 with 2 susceptible lines in a diallel recessive High heritability Liao et al. (1988) 47 Conti…..

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SR.NO. DISEASES CROSSES GENETIC NATURE OF RESISTANT REMARKS REFERENCES 3 Multiple disease 10- parent half diallel Additive resistant to ELS,LLS, rust , pstv , TSWV Studied F 1 , F 2 , F 3 ; parents and SCA significant for TSWV and PStv Anderson et al. (1990) 4 CBR Crossed NC 30339(R), Aregentine (R) and florigant (S) Additive NC 3033, Argentine and NC 2 showed high negative GCA in F 1 and f 2 Hadley et al. (1979) 5 Verticilium wilt Crossed P362 and P870-5 with P431 and P446-T Monogenic recessive Two tolerant lines Additive nature of gene action Khan et al. (1973) 6 Bacterial wilt Diallel of 3 resistant and 1 susceptible lines Partially dominant HR G1G1G2G2g3g3 MR G1G1g2g2g3g3 S g1g1g2g2G3G3 Liao et al. (1988 1990) 48

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Table 4. Resistance class distributions and probabilities for goodness-of-fit to designated ratios based on chi-square analysis for rust. Genotype Number of plants Ratio tested Probability Resistant Susceptible PI 314817 x UF 439-16-10-3 F 20 243 3:1 0.5 > P>0.20 PI 314817 x UF 439-16-10-3 F 34 268 11:1 0.10 >P> 0.05 PI 350680 x UF 439-16-10-3 F 22 284 3:1 P = 0.50 PI 350680 x UF 439-16-10-3 F 33 262 11:1 0.10> P>0.05 PI 315608 x UF 439-16-10-3 F 1 162 3:1 P < 0.01 PI 315608 x UF 439-16-10-3 F 9 176 11:1 0.10> P>0.05 Knauft (1987) ICRISAT, Patancheru 49

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Table 5. Segregation patterns of F 3 families derived from F 2 susceptible plants Cross F 3 family ratio 3:1 15:1 all susc Chi-square PI 314817 x UF 439-16-10-3 7 4 8 1.04 PI 350680 x UF 439-16-10-3 5 4 10 0.38 PI 315680 x UF 439-16-10-3 1 2 9 4.03 ICRISAT, Patancheru Knauft (1987) 50

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Table 8. Narrow-sense heritability for resistance to Late Leaf Spot Parameters Tainan 9 × RLRS 15 Lampang × RLRS 15 Khon Kaen 60-1 × RLRS 15 Disease resistance parameter Disease score at 70 days 1/ a a a Disease score at 80 days 1/ a a a Lesion number o.14 0.23 0.50 Lesion size (mm) a 0.98 0.91 Sporulation 2/ 0.29 0.99 a 51 1/ a scale of 1-9 where 1 = no disease and 9 = severe damage. 2/ a scale of 1-5 where 1 = very little sporulation and 5 = heavy sporulation. a = V P1F1 + V P2F2 > 2V F 2 expected h2 for these traits equal zero. Kormsa et al .(2002) Thailand

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Table 9. Estimates of different types of gene effect for peanut bud necrosis incidence at 60 days after planting in three crosses between resistant and susceptible peanut lines Gene effect IC 10 a x KK 60-1 b ICGV 86388 a x KK 60-1 b ICGV 86388 a x IC 10 a m 29.09 ± 0.77 c 34.35 ± 4.25 c 19.09 ± 2.81 c a 14.65 ± 1.08 -3.78 ± 7.25 NS d 16.99 ± 2.66 -11.10 ± 17.45 2.33 ± 7.74 aa NS -16.99 ± 17.39 -10.63 ± 15.87 ad NS 7.99 ± 8.01 NS dd NS NS NS m- mean; a - sum of additive effects; d- sum of dominance effects a -Resistant line. b- Susceptible line. aa- sum of additive x additive epistatic effects; ad- sum of additive x dominance epistatic effects; dd- sum of dominance x dominance epistatic effects. NS- non-significance at P ¼ 0.05. Pensuk et al. (2004) Thailand 52

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53 Table 10. Genetic analysis of six crosses of groundnut for Late Leaf Spot resistant Crosses Mean Rang Variance Coefficient of variation H 2 GAM G P G P Kadiri -3 x TCGS-150 7.3 5.0-9.0 1.3 2.2 15.6 20.3 59.1 24.7 Kadiri-3 X ICGV 88083 7.2 2.0-9.0 6.2 6.5 35.6 35.4 95.4 69.3 ICGV-91173 X TCGS-150 7.4 2.0-9.0 1.2 2.8 14.8 22.6 42.9 20.0 ICGV-91173 X ICGV -88083 8.1 1.0- 9.0 1.5 1.9 15.1 17.0 78.9 27.7 DRG- 102 X TCGS- 150 6.2 1.0- 9.0 2.3 6.3 24.5 40.5 36.5 12.3 DRG- 102 X ICGV- 88083 6.4 2.0-9.0 5.6 9.7 37.0 48.7 57.7 58.1 Jhon et al. (2008) Tirupati

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54 Table 11. Mean squares for general and specific combining abilities for parameters of resistance to Cercospora personata and Cercospora arachidicola for the F 1 detached leaf evaluation Effect df Lesion no. / 100 cm2 of leaf area Avg. lesion size Necrotic area Sporulation rating Latent period C. personatum GCA 6 0.13* 4.50** 0.39** 2.43** 9.93** SCA 9 0.08 0.61 0.12 0.18 3.28 Error 233 0.06 0.57 0.10 0 .24 2 .28 Anderson et al. (1986) North Carolina Conti… C. arachidicola GCA 6 0.50** 0.71 0.41** 0.35 6.20 S CA 9 0.17* 0.49 0.21* 0.39 7.56* Error 233 0.07 0.33 0.10 0 .22 3.25

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55 Table 12. Estimates of general combining ability effects for parameters of resistance to Cercosporidiurn perosonatum and Cercospora arachidicola for the F 1 detached leaf evaluation. Lesion no. / 100 cm2 of leaf area Avg. lesion size Necrotic area Sporulation rating Latent period C. personatum NC 17133(KF) 0.061 0.144 0.088 0.282 0.620 PI 350680 0.029 0.097 0.042 0.150 0.251 FESR 5-P2-B1 -0.071 -0.332 -0.127 -0.264 -0.467 NC 17090 -0.017 0.089 -0.001 -0.170 -0.405 PI 109839 0.001 -0.143 -0.015 0.056 -0.014 GP-SC 343 -0.036 -0.278 -0.074 -0.124 -0.077 NC 270806 0.000 0.394 0.064 0.087 -0.251 PI 269685 0.036 0.003 0.028 -0.022 -0.154 LSD (0.05) 0.130 0.376 0.194 0.238 0.770 Anderson et al. (1986) North Carolina Cont….

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56 Lesion no. / 100 cm2 of leaf area Avg. lesion size Necrotic area Sporulation rating Latent period C. arachidicola NC17133(KF) 0.088 -0.143 0.073 0.053 0.552 PI 350680 0.052 -0.086 0.038 -0.036 -0.071 FESR 5-P2-B1 -0.164 0.152 -0.101 0.001 -0.477 NC 17090 -0.008 0.069 0.019 -0.021 -0.009 PI 109839 0.011 0.012 0.012 0.038 0.102 GP-SC 343 -0.089 -0.07 -0.107 -0.112 0.098 NC 270806 0.042 0.059 0.07 0.107 -0.097 PI 269685 0.038 0 . ooo 0.037 -0.053 -0.104 LSD (0.05) 0.096 0.220 0.119 0.179 0.656

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57 Effect F 1 F 2 50 DAP 60 DAP 50 DAP 60 DAP GCA 66.3 ** 73.4 ** 73.3 ** 82.0 ** SCA 15.3 ** 11.5 ** 13.8 ** 9.6 ** Reciprocal 18.4 ** 15.1 ** 12.9 ** 8.3 *,** - significant at the 0.05 and 0.01% probability levels DAP - Days After Planting Table 13. Percentages of genotypes sum of squares attributed to GCA, SCA and reciprocal effects in the combining ability analysis for PBND incidence in the F 1 and F 2 generations of six diallel cross Pensuk et al. (2002) Thailand

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58 Breeding Methods

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59

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Table 12. Breeding strategies suggested for rapid and cost effective genetic enhancement in groundnut diseases. Trait Conventional breeding Wide crosses+ marker assisted backcross Genetic transformation Genetic basis Rust + oligogenic ELS + Polygenic LLS + Oligogenic and polygenic Bacterial wilt + oligogenic GRD + + Mono and digenic PBND + Not known ELS Early Leaf Spot, GRD Groundnut Rosette Disease LLS Late Leaf Spot PBND Groundnut Bud Necrosis Disease Dwivedi et al. (2003) (ICRISAT) Patancheru 60

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61 Conventional breeding

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62 Identification of resistant sources Crossing block Agronomic evaluation F1s; Space planted Screening in disease nursery F2 F3 F4 F5 Bulking of sister lines F6 and F7 Station trials Testing for other diseases and pests and for quality National and international programs National and international testing ICRISAT multilocational trials Release of resistant cultivars Fig 3. Basic scheme for development of rust resistant groundnut cultivars Reddy et al. (1987) (ICRISAT), Patancheru

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63 HYBRIDIZATION ICGV 87160 was bred and developed at ICRISAT Centre, Patancheru, India. It was derived by the bulk pedigree method from a cross of a Spanish variety, Ah 65, and a rust-resistant Valencia germplam line, NCAC-17090. ICGV 87160

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64 Its pedigree is (Ah 65 x NC Ac 17090) F2-B1-B1- B2-B1-B1-B1-B2 High-yielding variety resistant to rust and tolerant of late leaf spot. Tolerant of bud necrosis disease.

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65 ICGV 86699 originates from a single-plant selection made in CS 29, which in turn was developed through repeated selections From a cytologically unstable segregating population of ( Arachis batizocoi x A. duranensis) x A. hypogaea (CVNC 2) received from the North Carolin a State University, USA. ICGV 86699

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66 ICGV 86699 was developed using the bulk pedigree method in four subsequent generations of the single-plant selection in CS 29. Multiple resistance/tolerance to diseases Rust, Early and Late leaf spots, Peanut bud necrosis and Peanut mottle viruses, Stem and Pod rots ( Sclerotium rolfsii)

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67 ICGV 87157 was bred at ICRISAT Center, India It originated from a single plant selection made in F3 population A cross between a Spanish variety, argentine, and a rust and late leaf spot resistant parent, PI 259747. ICGV 87157

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68 Shows less bud necrosis disease incidence under field conditions than the popular Indian cultivars, JL 24 and Kadiri 3 Tolerant of stem and pod rot ( Sclerotium rolfsii) A high-yielding breeding line resistant to rust and tolerant of late leaf spot

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69 Mutation breeding

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Characters Foliar Disease Resistant Mutant VMG 1 Control (VRI 2) Days to maturity 114.9 ± 0.433 102.3 ± 0.472 Plant height (cm) 19.25 ± 2.70 41.45 ± 1.604 Number of primaries 6.5 ± 1.93 6.1 ± 0.585 Number of secondaries 11.45 ± 3.54 6.0 ± 1.619 Number of Flowers / inflorescence 2.5 ± 0.166 2.6 ± 0.163 Leaflet length (cm) 3.76 ± 0.151 4.562 ± 0.115 Leaflet width (cm) 2.34 ± 0.096 2.207 ± 0.055 Length/Width ratio 1.608 ± 0.024 2.089 ± 0.051 Number of seeds per pod 1.8 ± 0.133 1.8 ± 0.133 Pod length (cm) 1.55 ± 0.037 2.66 ± 0.039 VRI 2 was irradiated with 300 Gamma-rays during Rabi/summer 2007-08 Table 14 . Quantitative characters of groundnut foliar disease mutant VGM 1 Conti…. 70

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Characters Foliar Disease Resistant Mutant VMG 1 Control (VRI 2) Pod width (cm) 0.738 ± 0.021 1.15 ± 0.024 Seed length (cm) 0.662 ± 0.024 1.34 ± 0.023 Seed width (cm) 0.513 ± 0.016 0.735 ±0.0184 100 seed weight (g) 18.2 ± 0.207 47.44 ± 0.424 Shelling per cent 70.68 ± 0.168 72.43 ± 0.567 Pod yield (g) 7.437 ± 0.049 25.23±3.873 Oil content (%) 48.4 49.6 Reaction to rust 4.2 8.8 Reaction to late leaf spot 3.8 8.6 Mothilal et al., (2010) Tamil Nadu 71

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fig. Foliar Disease Resistant Mutant (left) with VRI 2 (right) 72

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73 TAG 24 was hybridized with V 9514 as the male parent in 2000. During 2001, F2 seeds from three F1 plants out of five were irradiated with 200 Gy of from a 60Co source. Both the treated and untreated F2 seeds were sown in the field. Selection was practiced in F3 and F3M2 and subsequent Generations for LLS and rust Diseases. At the end of F6 and F6M5 generations, five ideal recombinants named as Trombay foliar disease resistant groundnut (TFDRG) 1 to 5 were established. TFDRG 1 is the selection from the untreated F2 population while the rest are from gamma ray treated F2 populations. Trombay Groundnut Recombinants Resistant to Foliar Diseases Badigannavar et al. (2005) Trombay

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74 Table 15. Disease response of Trombay groundnut recombinants Genotypes Botanical variety Peanut bud Necrosis (%) Late leaf spot Rust TFDRG 1 vulgaris 10.0 2 1 TFDRG 2 vulgaris 12.2 3 5 TFDRG 3 hypogaea 4.9 2 3 TFDRG 4 vulgaris 4.9 3 2 TFDRG 5 vulgaris 10.0 1 3 TAG 24 vulgaris 9.9 7 7 VG 9514 hypogaea 6.2 1 1 * 1-9 scale, where 1= 0% and 9 = 81-100 % disease severity; % of infected plants Badigannavar et al. (2005) Trombay

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75 Table 16. Evaluation of some peanut mutant genotypes against pod rots disease complex under greenhouse conditions. Genotypes % Dry brown lesion % Pink discoloration % General breakdown %Apparently healthy pods RT-6 10.66 3.02 14.07 72.25 RT-7 8.31 2.27 8.89 80.53 RT-8 7.61 2.82 12.99 76.58 RT-9 12.32 3.22 16.54 67.92 RT-10 4.72 0.50 8.88 85.90 RT-11 9.90 1.31 9.82 78.97 RT-12 8.37 1.75 7.82 82.06 RT-13 7.92 1.59 13.13 77.37 RT-14 14.42 2.43 13.08 70.07 RT-15 14.36 2.54 20.60 62.50 Giza 5 16.18 5.69 23.75 54.39 L.S.D at 5% 2.39 2.37 2.41 2.31 Azzam et al. (2007) Egypt

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76 Biotechnology

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77 MW RP SP RB RB RB RB SB SB SB SB SB SB MW 300 bp 200 bp 100 bp Figure 6. SSR marker pPGPseq3A1 associated with resistance to rust in the F 2 population of the cross ICGV 99003 x TMV2 in groundnut MW - molecular weight, RP- resistant parent, S - susceptible parent RB- resistant bulk, SB - susceptible bulk Varma et al. (2005) Dharwad

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78 Filtrate (%) Raichur isolate Dharwad isolate KRG 1 TMV 2 KRG 1 TMV2 0 100 100 100 100 25 20 30 15 20 50 10 0 10 10 75 10 20 0 0 Table 17.Effect of S. rolfsii culture filtrate of Raichur and Dharwad isolates Effect of S. rolfssi culture filtrate on cell viability (TSS assay ) in calli of groundnut Filtrate (%) Raichur isolate Dharwad isolate 0 0.19 0.14 0.22 0.17 25 0.185 0.088 0.18 0.058 50 0.083 0.081 0.08 0.052 75 0.042 0.044 0.055 0.045 Ashok et al. (2004) Dharwad

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79 Genetic transformation and Southern blot analysis of peanut transgenic plants for late leaf spot resistant By employing pCAMBIA2300 harboring defensin BjD and the same Agrobacterium strain for transformation. They had generated transgenic peanut plants in the cultivar JL-24. Overall, 17 putative transformants were obtained from six independent transformation experiments using embryo axis explants. Out of these, 13 survived under the glass house conditions, 7 of which were found to be positive in a preliminary PCR analysis. They collected seeds from 11 T0 plants and the Southern analysis was done with PCR positive T1 plants. Cont….. Swathi et al. (2008) Hyderabad

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80 The genomic DNA of transgenic plants was digested with enzyme EcoR1, which has a single site on the T-DNA between the nptII and defensin expression cassette . The Southern blot was probed with the PCR amplified nptII fragment as probe. The varying length of hybridizing bands indicated that at least three of these primary transgenic plants represented single copy containing independent transformation events. Transgene integration was evident in the progeny of five plants out of eleven T0 plants that survived the transfer to the glass house.

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81 Fig 6. Southern blot analysis of peanut transgenic plants in T1 generation. DNA samples were digested with EcoRI and the blots probed with nptII. Please note single copy insertion in the transgenic plants 4, 5, 6, 7

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82 Fig 7. Defensin gene expression in peanut transgenic plants in T1 generation with defensin as a probe in the northern analysis and identification of plants for disease resistance analysis. Northern Hybridization Analysis

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83 Fig 8. Field analysis of late leaf spot infection (in the green house) after conidial spray. Note the development of leaf spots in the control plant. Transgenic plant 4-2 resisted the infection. Photograph taken after 28 days of treatment.

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84 Fig 9. Detached leaf assay for the leaf spot using conidial spray in Petri dishes. Please note the disease development on control and segregants in the progenies of transgenic plants that did not receive the transgene. Lesions were of a mixture of early and late leaf spots Evaluation of the transgenic peanut plants for resistance to tikka or late leaf spot disease-whole plant assay

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Disease resistance genes are often closely linked with loci conferring undesirable pod and seed characteristics. Disease resistant germplasm are late maturing types, have lower partitioning, and are sensitive to photoperiod than agronomically elite susceptible materials. Large genotype by- environment interactions for traits of economic importance. Limited gene introgression from wild Arachis species to cultivated groundnut . Major constraints to rapid genetic enhancement 85

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Achievements 86

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Diseases Genotypes Hot spot location Stem rot ISK - I- 2008- 7, IVK-I-2008-7, AVK-I-2008-5, ALSVT-I-2008-1 Less than 5% Kadiri IVK-I-08-18, LSVT-I-08-13, LSVT-II-07-8, IVT-VG-I-1 , Zero incidence is against 16.4% in GG 20 and highest 52.5% Junagadh ISK-I-2008-12,ISK-I-2008-20, <10% as against 39% in the check. JL-24,LGN-163, ICGV-99105, LGN-140,LGN-142,LGN-145 < 10% stem rot diseases as against 40% in check, JL 24 Latur Collar rot IVK-2008-7,and IVK-2008-15; IVK-II-2007-7 and ISK-I -2008-9 Zero incidence as against highest 23-27% Hanumangadh Early leaf spot ISK-I-2008-12 and 16, IVK-I-2008-1,12, 13, and 16; ISK-I-2007-12 Grade = 2 against highest 6 Kadiri IVK-I -2008-12, IVK-I-2008-13,IVK-II-2007-1,5,8. Grade =1 against highest grade 5-6 Hanumangadh Late leaf spot ISK-I-2008-1,2,12,16,17,18,20 and IVK-I-2008-1,2,3,7,11, Moderately resistant ( grade 3-4) Jalgaon Table 19. Disease resistant varieties DGR, annual report (2009) 87

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Diseases Genotypes Hot spot location Rust IVK-I-2007-06, 08 and ISK-I-2008-11, LSVT-I-08-6 Latur, Junagadh LLS + Rust LSVT-I-2008-05,7, 11; ISK-I-2008 12,20,23, Moderately resistant Latur, Junagadh PBND AVK-2008-10,ICG-1415,ICG- 5745 and NRCG CS-107; CS 241, 244,245 and 260 Less than 5% as against highest 30.8-64.30% Raichur PSND ISK-2007-10,IVK-I-2007-08 2.5 to 3.5 % as against highest 23.6% Kadiri 88

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Thank you 89