Pakistan Journal of Phytopathology, Published by: Pakistan Phytopathological Society www.pakps.com www.pjp.pakps.com info@pakps.com Pak. J. Phytopathol., Vol 23(1) : 05-08, 2011. SCREENING OF CHICKPEA GERMPALSM AGAINST ASCOCHYTA BLIGHT Aftab Ali Bokhari, M. Ashraf, Abdul Rehman, Arbab Ahmad and M. Iqbal Department of Plant Pathology,University of Agriculture Faisalabad ABSTRACT Ten Chickpea varieties/lines (08-AG-004, K-CH-70/02, K-CH-76/02, K-NOOR-91, K-70005, K-70008, K70022, K-CM-2008, YN-08004 and K-70009) of Pulses Research Institute, Faisalabad (PRI, Faisalabad) evaluated for the source of resistance against A. rabiei but none was found highly resistant. However, four lines (08-AG-004, k-CH-70/02, K-CH-76/02 and K-NOOR-91) were moderately resistant and five lines (K-70005, K70008, k-70022, k-CM-2008 and YN-08004) exhibited moderately susceptible reaction. Among twenty varieties/lines of Department of Plant Breeding and Genetics, University of Agriculture Faisalabad (PBG-UAF) evaluated for the source of resistance but none was found highly resistant and two lines PB-101 and PB-620 were resistant. However four lines viz. PAIDAR-91, PB-2000, PB-818 and PB-870 were found to be moderately resistant. Nine lines viz. AUG-27, M-98, PB-114, PB-115, PB-117, PB-781, PB-1049, PB-4025, and PB-5006 exhibited moderately susceptible reaction and three lines PB-205, PB-1205, and PB-1288 exhibited susceptible reaction. Key words: Ascochyta rabiei, blight, Chickpea/Gram, germplasm, PRI Faisalabad, PBG-UAF the foliar application and seed dressing fungicides (Reddy and Singh, 1984; Rauf et al., 1996), use of disease free seed, destruction of plant disease debris (Chaube and Pandey, 1986) and host plant resistance (Iqbal et al., 2002; Ahmad et al., 2006). However, due to lack of durable resistance in commercial chickpea cultivars (Iqbal et al., 1989) because the virulences of the pathogen are constantly changing in nature, previously released resistant cultivars have become susceptible due to appearance of new virulent strains/races (Jamil et al., 1995; Armstrang et al., 2001). Thus there is a need to continuously explore and identify the sources of resistance in chickpea germplasm and its incorporation into high yielding quality commercial chickpea varieties (Bashir et al., 1997). In addition, there is a need to explore the efficacy of certain fungicides to manage the disease below economic threshold level in the absence of resistant germplasm. INTRODUCTION Chickpea (Cicer arietinum L.) is third grain legume in the world and first in South Asia for its area and production. It is the most important legume crop of dry land agriculture. It is a rich and cheap source of vegetable protein for human nutrition (Hulse, 1991). It is an important pulse crop of Pakistan. It is the only crop that can be successfully grown during winter in the vast barani area of the country. It is grown on an area of 1052.3 thousand hectare that produces 837.8 thousand tons with an average yield of 697 kg per hectare (Anonymous 2007a). Its production is mainly concentrated in rainfed areas of Punjab (910.7 thousand hectares) followed by Sindh (55.9 thousand hectares) and NWFP (49.0 thousand hectares), whereas in Balochistan it is cultivated on a very small scale (36.7 thousand hectares) (Anonymous 2007b). Gram blight (Ascochyta rabiei Pass. Lab.) is the most serious disease of gram, which causes considerable degradation in quality and yield of the crop stand. Ascochyta blight disease occurs in epidemic form during the year receiving more than 13 cm rain fall. It has been reported to cause 50 to70 percent crop losses (Malik and Bashir, 1984) with favourable atmospheric condition for the disease development. Sometimes it may cause failure of the whole gram crop. Disease epidemics in Pakistan as well as in different parts of the world have been reported (Kausar, 1965; Radulescu et al., 1971; Kaiser, 1973). The disease appears regularly in alarming epidemic form both in the barani and irrigated areas of Pakistan. The disease can effectively be managed by MATERIALS AND METHODS Twenty lines/entries of gram germplasm were obtained from Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad (PBG-UAF) and ten lines/entries were collected from Pulses Research Institute (PRI), Ayub Agricultural Research Institute (AARI), Faisalabad. Lines/entries obtained from PBG-UAF consisted of PB-698, PB820, PB-205, PB-1205, PB-1288, AUG-27, M-98, PB-114, PB-115, PB-117, PB-781, PB-1049, PB4025, PB-5006, PAIDAR-91, PB-2000, PB-818, PB870, PB-101, PB-620 whereas lines/entries collected 5 from PRI, Faisalabad were K-70009, K-70005, K70008, K-70022, K-CM-2008, YN-08004, 08-AG004, K-CH-70/02, K-CH-76/02, K-NOOR-91. The test entries were sown in a single row sub-plot of 4m length and 30cm row spacing in a augmented design. The highly susceptible variety Punjab-l, as a check was planted as a single row after every two test lines of the germplasm for disease spread in the field and its distribution to test line on each side. Diseased pods of gram showing characteristics symptoms of blight disease were obtained from PRI and were kept at 5-8 °C until used for the isolation of Ascochyta rabiei. The isolation was carried out by the procedure followed by the Ilyas and Iqbal (1986). IInfected pods, by holding them in a forceps were surface flamed in such a way that only charring of outer pod layer could occur but the inner pod layer remained intact. The pods were then passed open and infected seeds were taken out aseptically with the help of another flame sterilized forceps. The naturally A. rabiei infected seeds, thus obtained were planted on autoclaved gram seed meal agar (GSMA) medium in Petri plates and were incubated at 24 °C for more than two weeks. The colonies of A. rabiei coming out of blighted seeds were isolated and purified by spore streak method (Pathak, 1986). The purified culture was identified and grown on GSMA slants and maintained at 5 °C for further studies. The mass multiplication of A. rabiei inoculum was carried by the method of Ilyas and Khan (1986). When the entries were in early to mid pod stage, they were spray inoculated with spore suspension of A. rabiei (18,000-20,000 spores/ml). The inoculum of A. rabiei was prepared by the mass culturing technique described by Ilyas and Khan (1986). The inoculum spray was carried every day in the evening till the development of blight symptoms on susceptible variety. The development of disease was further aided by the continuous spray of tap water every day. The data of blight severity were continued to be recorded till check line (Punjab-91) were completely blighted and died to assess the level of resistance/susceptibility of each test line, using following 1-9 grades disease rating scale developed by Reddy and Nene (1979). RESULTS AND DISCUSSION Field screening of gram germplasm for the source of resistance against Ascochyta rabiei revealed that none of the test lines/cultivars possessed immunity against this pathogen. However, two lines of PBGUAF viz. PB-101 and PB-620 exhibited resistance. Four lines of PRI viz. 08-AG-004, K-CH-70/02, KCH-76/02 and K-NOOR-91; and four lines PBGUAF viz. PAIDAR-91, PB-2000, PB-818 and PB870 exhibited moderately resistant reaction against the disease. Five lines of PRI i.e. K-70005, K-70008, K-70022, K-CM-2008, YN-08004 and nine lines of PBG-UAF viz. AUG-27, M-98, PB-114, PB-115, PB-117, PB-781, PB-1049, PB-4025, PB-5006 revealed moderately susceptible reaction to the disease. One line i.e. K-70009 of PRI, Faisalabad and three lines viz. PB-205, PB-1205 and PB-1288 of PBG-UAF were found susceptible to the disease. Two lines of PBG-UAF Table 1: Disease development during four months on 10 chickpea varieties/lines of Pulses Research Institute (PRI) Faisalabad Disease Severity During Varieties Mean Response 1st Month 2nd Month 3rd Month 4th Month 08-AG-004 0.67 lm* 1.67 kl 2.67 jk 5.67 d-f 2.67 E MR K-70005 3.67 h-j 4.67 f-h 6.00 de 8.67 a 5.75 B MS K-70008 3.67 h-j 4.67 f-h 6.67 cd 8.67 a 5.91 B MS K-70009 5.00 e-g 7.33 bc 8.67 a 9.00 a 7.50 A S K-70022 1.67 kl 4.00 g-i 5.00 e-g 8.00 ab 4.67 C MS K-CH-70/02 0.33 m 1.67 kl 2.67 j-k 5.67 d-f 2.58 E MR K-CH-76/02 0.33 m 1.67 kl 2.67 j-k 5.67 d-f 2.58 E MR K-CM-2008 1.00 lm 3.33 ij 4.33 gi 7.33 bc 4.00 D MS K-NOOR-91 0.33 m 1.67 kl 2.67 jk 5.67 d-f 2.58 E MR YN-08004 1.00 lm 3.33 ij 4.33 g-i 7.33 bc 4.00 D MS MEAN 1.76 D 3.40 C 4.57 B 7.17 A LSD = 0.504 * Means sharing similar letters do not differ significantly at p ≤ 0.05 by Duncan’s multiple range test 6 Table 2: Disease development during four months on 20 chickpea varieties/lines of PBG-UAF Disease Severity During Varieties Mean 1st Month 2nd Month 3rd Month 4th Month Response AUG-27 1.00 m-o* 3.33 h-j 4.33 f-h 7.33 b 4.00 C MS M-98 1.00 m-o 3.67 g-i 4.67 e-g 7.33 b 4.17 C MS PAIDAR-91 0.67 m-o 1.67 k-m 2.67 i-k 5.67 c-e 2.67 D MR PB-2000 0.33 no 1.67 k-m 3.00 i-j 6.00 cd 2.75 D MR PB-101 0.00 o 1.33 l-n 2.33 j-l 2.33 j-l 1.50 E R PB-114 1.33 l-n 3.67 g-i 4.67 e-g 7.33 b 4.25 C MS PB-115 1.33 l-n 3.67 g-i 4.33 f-h 7.33 b 4.17 C MS PB-117 1.00 m-o 3.33 h-j 4.33 f-h 7.33 b 4.00 C MS PB-205 3.67 g-i 4.67 e-g 6.67 bc 8.67 a 5.92 B S PB-620 0.00 o 1.33 l-n 2.33 j-l 2.33 j-l 1.50 E R PB-698 5.00 d-f 7.33 b 8.67 a 9.00 a 7.50 A HS PB-781 1.33 l-n 3.33 h-j 4.33 f-h 7.33 b 4.08 C MS PB-818 0.67 m-o 1.67 k-m 2.67 i-k 5.67 c-e 2.67 D MR PB-820 5.00 d-f 7.33 b 8.67 a 9.00 a 7.50 A HS PB-870 0.33 no 1.67 k-m 2.67 i-k 5.67 c-e 2.58 D MR PB-1049 1.33 l-n 3.33 h-j 4.33 f-h 7.33 b 4.08 C HS PB-1205 3.67 g-i 5.00 d-f 6.67 bc 8.67 a 6.00 B S PB-1288 3.67 g-i 5.33 d-f 6.67 bc 8.67 a 6.08 B S PB-4025 1.67 k-m 3.33 h-j 4.33 f-h 7.33 b 4.17 C MS PB-5006 1.33 l-n 3.33 h-j 4.33 f-h 7.33 b 4.08 C MS MEAN 1.72 D 3.50 C 4.63 B 6.88 A LSD = 0.46 *Means sharing similar letters do not differ significantly at p ≤ 0.05 by Duncan’s multiple range test Ascochyta rabiei and concluded that out of 150 lines sown in field and green house for there reaction against Ascochyta rabiei, seven showed moderate resistance. viz. PB-698 and PB-820 were highly susceptible. This revealed that there are good source of resistance in existing gram germplasm lines/mutant/cultivars that can further be exploited and incorporated into commercial cultivars. Many other workers have also reported the occurrence of moderate resistance to Ascochyta blight. Many sources of resistance to Asochyta rabiei have been reported during the last 50 years and generally these reports were based either on field observation during natural epidemics or on artificial inoculation tests in the field or greenhouse (Bashir et al., 1985; Alam et al., 2003; Iqbal et al., 2004; Chaudhary et al., 2005; Bashir et al., 2006). While Haq et al. (1981) identified blight resistant gram mutants CM-72 and CM-68 from his irradiated material. Most of the previously reported resistant cultivars have lost their resistance, probably due to the appearance of new physiological races of A. rabiei and need replacement. Similarly, Jalali et al. (1983) tested various genotypes for resistance against REFERENCES Ahmad, H.U., K.F. Chang, S.F. Hwang, B.D. Gossen, R.J. Howard and T.D. Warkentin. 2006. Component of disease resistance in desi and kabuli chickpea varieties against Ascochyta blight. J. Pl. Path. 5(3): 336-342. Alam, S. S., M. Hassan, M. A. Haq, T. M. Shah, B. M. Atta, and H. Syed. 2003. Screening for Ascochyta blight resistance in chickpea. Mycopath. 1(2): 129-130. Anonymous. 2007a. Economic survey of Pakistan, Government of Pakistan, Ministry of Food, Agriculture and Livestock, Economics Wing, Islamabad. 7 Anonymous. 2007b. Agricultural Indicators Federal Bureau of Statistics, Statistic Division, Ministry of Economic Affairs and Statistics, Government of Pakistan. Armstrang, C.L., G. Chongo, B.D. Gossen and C.J. Duczek. 2001. Mating type distribution and incidence of the teleomorph of Ascochyta rabiei (Didymella rabiei) in Canada. Can. J. Pl. Path. 23:110-113. Bashir, A., M. Yaqoob, M. Rahim, R. Khalid, and Najibullah. 2006. Source of resistance against disease complex in chickpea. Indus J. Biol. Sci. 3 (1): 660-663. Bashir, M., S. S. Alam and S. H. Qureshi. 1985. Chickpea germplasm evaluation for resistance to Ascochyta blight under artificial conditions. Int. Chickpea Newsletter No. 12: 24-26. Bashir, N., M I. Hashmi, and F.F. Jamil. 1997. Induction of systemic acquired resistance by oxalic acid in chickpea (Cicer arientinum L.) against Ascochyta rabiei. Pakistan J. Phytopath. 9(1): 18-20. Chaube, H.S., and B.K. Pandey. 1986. Transmission of seed borne inoculum of Ascochyta rabiei (Pass.) Labr. In chickpea seedling. Bull. Pure and Appl. Sci. 5: 18. Chaudhry, M.A., M. Faquir and A. Muhmmad. 2005. Screening of chickpea germplasm for resisance to Ascochyta blight. J. Agri. Res. 43(3): 229233. Haq, M. A., A. Shakoor, M. Sadiq and M. Hussain. 1981. Induction of Ascochyta blight resistant mutants in chickpea. Mutation Breeding Newsletter No. 17: 5-6 FAO/IAEA. Hulse, J.H., 1991. Nature, composition and utilization of grain legumes. In: Uses Tropical Legumes: Proc. Consultants' Meeting, pp: 11– 27, March 27-30, 1989, ICRISAT Patancheru, AP 502 324, India. Ilyas, M.B. and A. Iqbal. 1986. Evaluation of fungicides for the eradication of seed borne Ascochyta rabiei fungus. Pakistan J. Agri. Sci. 23(3-4): 145-149. Ilyas, M.B. and I.U. Khan. 1986. A low cost easy technique for the culturing of Ascochyta rabiei fungus. Pakistan J. Agri. Sci. 23(1): 60. Iqbal, S. M., A. Bakhsh, M. A. Zahid and A. M. Haqqani. 2004. Screening of resistance against Ascochyta blight in chickpea. Mycopath. 2(1): 7-10. Iqbal, S. M., A. Bukhsh., A. Shabbir and M. Bashir. 1989. Field evaluation of fungicides for Ascochyta blight in lentil. Sar. J. Agri. 5(3): 307-308. Iqbal, S.M., A. Ghafoor, A. Bakhsh, M. Bashir. 2002. Disease resistance status of chickpea genotypes against Ascochyta blight. Pakistan J. Phytopath. 14(2): 135-139. Jamil, F.F., M. Sarwar, I. Haq, N. Bashir. 1995. Identification of pathotypes in Ascochyta rabiei (Pass.) Labr. The cause of chickpea blight in Pakistan. Pakistan J. Bot. 27: 193-199. Kaiser, W.J. 1973. Factors affecting growth, sporulation, pathogenicity and survival of Ascochyta rabiei. Mycologia 65: 444–457. Kausar, A.G. 1965. Epiphytology of recent epiphytotics of gram blight in West Pakistan. Pak. J. Agri. Sci. 2: 185-195. Malik, M. A. and M. Bashir. 1984. Strategies for controlling gram blight. Prog. Farm. 4(5): 2123. Pathak, M. V. 1986. Laboratory manual of plant pathology 2nd Edit. Oxford IBH Pub. Co. New Delhi, 23-25. Radulescu, E., E. Capetti, E. Schmidt and A. Cassian. 1971. Contributions to the study of anthracnose of chickpea (Mycosphaerella rabiei Kov.). Lucrari Stuntifice 14: 311–21. Rauf, C. A., M. R. Malik, S. M. Iqbal, S. Rahat and S. Hussain. 1996. Fungicides an economic tool to enhance productivity and net returns in chickpea crop. Sar. J. Agri. 12 (4): 445-448. Reddy, M. V. 1984. Laboratory techniques for isolation and multiplication of Ascochyta rabiei. p. 103-104. In: Proc. Symp. On the Role of Induced Mutations in Crop Improvement. Osmania Univ. Hyderabad, India. Reddy, M. V., and Y. L. Nene. 1979. A case of induced mutation in chickpea for Ascochyta blight resistance. p. 398-408. In: Proc. symp. On thr role of induced mutation in crop improvement. Osmania Univ. Hyderabad, India. Singh, K. B., M. V. Reddy and Y. L. Nene. 1981. Sources of resistance to Ascochyta blight of chickpea. Int. Chickpea Newsletter No. 4: 1617. Steel, R.G.D., J.H. Torrie and D.A. Deekey. 1997. Principles and procedures of statistics. A Biometrical Approach. 3rd Ed. McGraw Hill Book Co. Inc. N.Y. U.S.A. 8