RESISTANCE OF RUBBER CLONES RECOMMENDED IN INDONESIA TO CORYNESPORA AND COLLETOTRICHUM LEAF FALL DISEASES

RESISTANCE OF RUBBER CLONES RECOMMENDED IN INDONESIA TO CORYNESPORA AND COLLETOTRICHUM LEAF FALL DISEASES Alchemi Putri Juliantika Kusdiana, Afdholiatus Syafaah, and Tri Rappani Febbiyanti Sembawa Research Centre, Indonesian Rubber Research Institute Abstract Leaf fall disease caused by Corynespora cassiicola and Colletotrichum sp. can causes reduction of latex production in rubber plantations. Hence screening for disease development in each rubber clone needs to be carried out. Resistance level of 25 rubber clones recommended in Indonesia to leaf fall diseases were examined on rubber plants grown in polybags placed in a greenhouse using three isolates each of C. cassiicola and Colletotrichum sp. isolated from the rubber clones RRIM 600, GT 1, and PR 303. The results showed that 9 rubber clones i.e AVROS 2037, BPM 1, BPM 109, IRR 42, IRR 107, IRR 220, PB 260, PB 330, and RRIC 100 had a high resistance to both the leaf fall diseases while 4 clones i.e BPM 24, PR 261, PR 300, and PR 303 had a low resistance. The other clones had a high resistance against only one of the diseases. Keywords: Colletotrichum sp., Corynespora cassiicola, leaf fall disease, rubber clones. INTRODUCTION Rubber (Hevea brasiliensis Muell. Arg) is one of the important plantation crops in several Asian and African countries. attack can be a limiting factor in rubber production. Corynespora leaf fall caused by Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei and Colletotrichum leaf fall caused by Colletotrichum sp. are important leaf diseases. Leaf diseases attack plants of all age levels, both in nurseries or in gardens (Suryaningtyas, 2012; Jayashinge, 2000). Leaf fall disease intensity is affected by the condition of plants, weather, and the clones (Pawirosoemardjo, 2004). Corynespora leaf fall disease occurs during the dry months after the refoliation (Manju et al, 2001) and Colletotrichum leaf fall is more prominent during the rainy and humid months (Manju et al., 2014). Epidemics and recurrent attack of this disease can cause economic losses at all ages of the plant. C. cassiicola attack young copper brown and late copper brown leaf stages (Fernando et al., 2010a). The early symptoms is black patches on the leaf and extend into the smaller leaf veins and the spots will appear 811

Proceedings of International Rubber Conference 2017 like fish bones (Jinji et al., 2007). Later, the spots become more widespread, round or irregular with dried center and may fall off leaving a hole. This symptom is due the toxins accumulation at the site of infection. Furthermore, the lesion vicinity becomes chlorotic due to the breakdown of chloroplasts (Jayasinghe & Fernando, 2011). The affected leaves become yellow or brown gradually and then fall. In addition to attacking the leaves, pathogens also attack the petiole, shoots, stems, or branches (Suryaningtyas, 2012). Colletotrichum sp. in rubber can attack the young or old leaves, but usually the young rubber leaf is the main target showing higher disease intensity (Febbiyanti & Kusdiana, 2013). Young leaves when attacked limp and turn black, wrinkled, and finally fall. Infected mature leaves have raised brown or black spot, wrinkle, and rarely fall down (Situmorang & Budiman, 2003). Although cultivation of resistant clones is suggested for disease management, resistance can be broken by the emergence of new more virulent races of pathogen. Monitoring of disease in each clone needs to be done systematically (Situmorang et al., 2004). This study is an attempt to screen the rubber clones recommended in Indonesia, for disease resistance. MATERIALS AND METHODS The study was carried out in the Greenhouse of Sembawa Research Centre, South Sumatra from December 2015 to September 2016, on rubber plants grown in polybags using three replications. Twenty five rubber clones recommended in Indonesia used in this research were AVROS 2037, BPM 1, BPM 24, BPM 107, BPM 109, GT 1, IRR 5, IRR 32, IRR 39, IRR 42, IRR 104, IRR 107, IRR 112, IRR 118, IRR 220, PB 260, PB 330, PB 340, PR 255, PR 261, PR 300, PR 303, RRIC 100, RRIM 600, and RRIM 712. The pathogen isolates consisted of three isolates of C. cassiicola (CC-RRIM 600, CC-GT 1, and CC-PR 303) and three isolates of Colletotrichum sp. (CS-RRIM 600, CS-GT 1, and CS-PR 303) from RRIM 600, GT 1, and PR 303 rubber clones. Those isolates were high of virulence levels (Oktavia et al. 2016b). 812

Resistance of rubber clones recommended in Indonesia to corynespora and Preparation of Pathogen Isolate Leaves infected by C. cassiicola and Colletotrichum sp. were collected from three rubber clones RRIM 600, GT 1, and PR 303 from the field. Pathogens were isolated on potato dextrose agar (PDA) media purified and identified by microscopic. Production of C. cassiicola Inoculum C. cassiicola pathogen inoculum was prepared by Situmorang (2002) method. A total of 8-10 discs (Ø 5 mm) of isolate cultures were placed in reverse position on the abaxial surface of steriled young leaves in petri dishes, so the mycelia were in direct contact to the leaf surface. The leaf position was reversed in 3 to 4 days after incubation. The conidia were produced on the adaxial surface after 4-5 days. The leaves were dried for one day at room temperature and the conidia formed were removed using a brush, mixed with sterile water and filtered using gauze filter to separate the clumped conidia. Conidia concentrations were calculated and adjusted using a haemocytometer. Production of Colletotrichum sp. Inoculum Colletotrichum sp. inoculum was obtained from two weeks old cultures. The conidia were removed from the potato dextrose agar media using a brush, mixed with sterile water and filtered using gauze filter to separate the clumped conidia. Conidia concentrations were calculated and adjusted using a haemocytometer. Inoculation of Rubber Plants Inoculation was carried out by spraying one ml of inoculum with a concentration of 4x10 4 conidia/ml using a sprayer on the abaxial surface of the young brown leaves. The leaves were covered with transparent plastic bags to retain moisture during incubation. The plastic bags were removed after four days of incubation. Observation and Data Analysis Observation of disease intensity was carried out 12 days after inoculation. was graded using the scale as shown in Table 1. 813

Table 1. attack scale Proceedings of International Rubber Conference 2017 Scale 0 No symptoms 1 Few blackish brown spots 2 1-50% of the leaf area is brownish yellow 3 51-100% of the leaf area is brownish yellow or leaf fall The disease intensity (%) was calculated using the Towsendt & Hueberger formula, 1943 (Sinaga, 2006): n n. v DI i 1 Z. N x100% where: DI : disease intensity; n : number of plant scale v; v : scale to-i; Z : highest score; N : number of plants observed. Based on the percentage of disease intensity and virulence levels of pathogen isolates the clones were classified as follows (modified from Situmorang, 2002): Table 2. of virulence level Attacks scale 0-5% highly resistant 6-33% resistant 34-67% susceptible 68-100% highly susceptible RESULTS AND DISCUSSION The results of screening for resistance against C. cassiicola on the 25 rubber clones recommended in Indonesia showed the presence of different virulence levels of each isolate ranging from highly resistant to highly susceptible levels (Table 3). The results of previous studies suggest that each rubber clone has a different resistance level to C. cassiicola isolates (Situmorang et al., 2000; Hadi et al., 2004; Oktavia et al., 2016a). 814

Resistance of rubber clones recommended in Indonesia to corynespora and Table 3. Corynespora leaf fall disease intensity of recommended rubber clones to three of C. cassiicola isolates CC-RRIM 600, CC-GT 1, and CC-PR 303. CC-RRIM 600 CC-GT 1 CC-PR 303 Clone Intensity (%) AVROS 2037 55.56 susceptible 13.89 resistant 0.00 highly resistant BPM 1 25.00 resistant 2.78 highly resistant 0.00 highly resistant BPM 24 100.00 highly susceptible 69.44 highly susceptible 83.33 highly susceptible BPM 107 2.78 highly resistant 0.00 highly resistant 0.00 highly resistant BPM 109 16.67 resistant 0.00 highly resistant 8.33 resistant GT 1 66.67 susceptible 91.67 highly susceptible 75.00 highly susceptible IRR 5 69.44 highly susceptible 100.00 highly susceptible 38.89 susceptible IRR 32 97.22 highly susceptible 86.11 highly susceptible 61.11 susceptible IRR 39 75.00 highly susceptible 100.00 highly susceptible 100.00 highly susceptible IRR 42 19.44 resistant 16.67 resistant 19.44 resistant IRR 104 69.44 highly susceptible 2.28 highly resistant 13.89 resistant IRR 107 16.67 resistant 16.67 resistant 0.00 highly resistant IRR 112 86.11 highly susceptible 75.00 highly susceptible 33.33 resistant IRR 118 77.78 highly susceptible 75.00 highly susceptible 63.89 susceptible IRR 220 50.00 susceptible 5.56 highly resistant 44.44 susceptible PB 260 0.00 highly resistant 5.56 highly resistant 0.00 highly resistant PB 330 25.00 resistant 0.00 highly resistant 0.00 highly resistant PB 340 100.00 highly susceptible 100.00 highly susceptible 0.00 highly resistant PR 255 25.00 resistant 16.67 resistant 19.44 resistant PR 261 100.00 highly susceptible 100.00 highly susceptible 100.00 highly susceptible PR 300 100.00 highly susceptible 13.89 resistant 33.33 resistant PR 303 100.00 highly susceptible 91.67 highly susceptible 88.89 highly susceptible RRIC 100 13.89 resistant 13.89 resistant 0.00 highly resistant RRIM 600 66.67 susceptible 86.11 highly susceptible 16.67 resistant RRIM 712 0.00 highly resistant 5.56 highly resistant 13.89 resistant 815

Proceedings of International Rubber Conference 2017 C. cassiicola spores of CC-RRIM 600 had the greater effect of 54.33% compared to other spores i.e CC-GT 1 of 43.54% and CC-PR 303 of 32.55%. This can be due to higher virulence level. Situmorang et al. (2007) also reported that RRIM 600 rubber clone is an important source of infection in Indonesian rubber plantations. The testing of Colletotrichum sp. on the rubber clones showed only two different virulence levels, resistant and susceptible (Table 4). The effect of spore treatment on the disease intensity showed that the spores of Colletotrichum sp. CS-PR 303 had the greater effect of 32.94% compared to other spores CS-RRIM 600 of 31.37% and CS-GT 1 of 25.13 %. The testing of resistance of Corynespora against 25 recommended rubber clones showed different resistance responses. Two clones i.e BPM 107 and PB 260 with the disease intensity value range from 0.93% to 1.85% are highly resistant. 11 clones i.e AVROS 2037, BPM 1, BPM 109, IRR 42, IRR 104, IRR 107, IRR 220, PB 330, PR 255, RRIC 100 and RRIM 712 are found resistant with disease intensity values ranging from 6.48% to 30.56%. Seven clones i.e IRR 5, IRR 32, IRR 39, IRR 112, PB 340, PR 300, and RRIM 600 are found susceptible with disease intensity values ranging from 44.44% to 66.67%. Five clones i.e BPM 24, GT 1, IRR 118, PR 261, and PR 303 are found highly susceptible with disease intensity value ranging from 72.22% to 100.00% (Table 5). These results are similar to previous studies which stated that clones PB 260 and RRIC 100 are resistant to Corynespora leaf fall disease (Fernando et al., 2010b), GT 1 and RRIM 600 were included in highly susceptible group on the basic of laboratory and field evaluation (Narayanan & Mydin, 2012; Jacob, 2003; Jayasinghe, 2000). The testing of recommended rubber clone by Kusdiana & Syafaah (2015) on laboratory scale revealed nine resistant clones that have the same resistance level as observed now in the greenhouse test i.e AVROS 2037, BPM 1, BPM 107, BPM 109, IRR 42, IRR 104, IRR 107, PB 260, and PB 330. In addition, there were two clones that are reported as susceptible i.e PR 300 and RRIM 600. 816

Resistance of rubber clones recommended in Indonesia to corynespora and Table 4. Colletotrichum leaf fall disease intensity of recommended rubber clones to three of Colletotrichum sp. isolates CS-RRIM 600, CS- GT 1, and CS-PR 303. CS-RRIM 600 CS-GT 1 CS-PR 303 Clone AVROS 2037 24.07 resistant 20.37 resistant 25.00 resistant BPM 1 29.62 resistant 17.59 resistant 52.78 susceptible BPM 24 34.26 susceptible 36.11 susceptible 40.74 susceptible BPM 107 40.74 susceptible 39.81 susceptible 50.00 susceptible BPM 109 31.48 resistant 29.63 resistant 25.00 resistant GT 1 25.00 resistant 34.26 susceptible 39.81 susceptible IRR 5 17.59 resistant 10.19 resistant 18.52 resistant IRR 32 32.41 resistant 25.00 resistant 36.11 susceptible IRR 39 37.04 susceptible 18.52 resistant 20.37 resistant IRR 42 25.00 resistant 16.67 resistant 17.59 resistant IRR 104 43.52 susceptible 21.30 resistant 54.63 susceptible IRR 107 25.00 resistant 18.52 resistant 18.52 resistant IRR 112 26.85 resistant 28.70 resistant 21.30 resistant IRR 118 27.78 resistant 12.50 resistant 16.67 resistant IRR 220 22.22 resistant 28.70 resistant 37.96 susceptible PB 260 36.11 susceptible 25.93 resistant 19.44 resistant PB 330 20.37 resistant 15.74 resistant 30.56 resistant PB 340 34.26 susceptible 20.37 resistant 24.07 resistant PR 255 45.37 susceptible 37.04 susceptible 50.93 susceptible PR 261 43.52 susceptible 28.70 resistant 31.48 resistant PR 300 34.26 susceptible 16.67 resistant 52.78 susceptible PR 303 24.07 resistant 25.93 resistant 40.74 susceptible RRIC 100 35.19 susceptible 19.44 resistant 11.11 resistant RRIM 600 24.07 resistant 36.11 susceptible 27.78 resistant RRIM 712 44.44 susceptible 44.44 susceptible 59.72 susceptible 817

Testing to Colletotrichum leaf fall disease in greenhouses showed that 17 clones are resistant i.e AVROS 2037, BPM 1, BPM 109, GT1, IRR 5, IRR 32, IRR 39, IRR 42, IRR 107, IRR 112, IRR 118, IRR 220, PB 260, PB 330, PB 340, RRIC 100, and RRIM 600 with disease intensity values ranging from 15.43% to 33.03%. Eight clones were found susceptible i.e BPM 24, BPM 107, IRR 104, PR 255, PR 261, PR 300, PR 303, and RRIM 712 with disease intensity value ranging from 30.25% to 48, 26% (Table 5). Narayanan & Mydin (2012) reported that PB 260 and RRIM 600 are resistant to Colletotrichum leaf fall disease. Table 5. Corynespora and Colletotrichum leaf fall disease intensity of recommended rubber clones in greenhouse C. cassiicola C. gloeosporioides Clones Virulence Level Virulence Level AVROS 2037 23.15 resistant 23.15 resistant BPM 1 9.26 resistant 33.33 resistant BPM 24 84.26 highly susceptible 37.04 susceptible BPM 107 0.93 highly resistant 43.52 susceptible BPM 109 8.33 resistant 28.70 resistant GT 1 77.78 highly susceptible 33.03 resistant IRR 5 44.44 susceptible 15.43 resistant IRR 32 62.04 susceptible 31.17 resistant IRR 39 63.89 susceptible 25.31 resistant IRR 42 18.52 resistant 19.75 resistant IRR 104 28.70 resistant 39.81 susceptible IRR 107 11.11 resistant 20.68 resistant IRR 112 64.81 susceptible 25.62 resistant IRR 118 72.22 highly susceptible 19.79 resistant IRR 220 30.56 resistant 29.63 resistant PB 260 1.85 highly resistant 27.16 resistant PB 330 8.33 resistant 22.22 resistant PB 340 66.67 susceptible 26.23 resistant PR 255 20.37 resistant 44.44 susceptible PR 261 100.00 highly susceptible 34.57 susceptible PR 300 49.07 susceptible 34.57 susceptible PR 303 93.52 highly susceptible 30.25 susceptible RRIC 100 9.26 resistant 21.91 resistant RRIM 600 56.48 susceptible 29.32 resistant RRIM 712 6.48 resistant 48.26 susceptible CONCLUSION Testing of resistance of Corynespora and Colletotrichum leaf fall disease in greenhouse showed that nine clones are resistant to both the diseases i.e AVROS 2037, BPM 1, BPM 109, IRR 42, IRR 107, IRR 220, PB 260, PB 330, and RRIC 100. Four clones were susceptible i.e BPM 818

Physiological characters and production toward tapping panel dryness of PB 260 24, PR 261, PR 300, and PR 303. The other clones were only resistant to one of the diseases. REFERENCES Febbiyanti, T. R. & Kusdiana, A. P. J. (2013). Pengaruh infeksi jamur Colletotrichum gloeosporioides terhadap kerusakan daun tanaman karet. Prosiding Konferensi Nasional Karet (pp. 251-258). Bogor, Indonesia: Pusat Penelitian Karet. Fernando, T. H. P. S., Jayasinghe, C. K., Wijesundera, R. L. C, & Siriwardena, D. (2010a). Susceptibility of different leaf stages of Hevea to Corynespora cassiicola. Journal of the Rubber Research Institute of Sri Lanka, 90, 58-63. Fernando, T. H. P. S., Jayasinghe, C.K., Wijesundera, R.L.C., Silva, W.P.K., & Nishantha, E.A.D.N. (2010b). Evaluation of screening methods against Corynespora leaf fall disease of rubber (Hevea brasiliensis). J. Plant Dis Protec, 117(1), 24-29. Hadi, H., Hartana, A., & Sinaga, M. (2004). Analisis genetika pewarisan sifat ketahanan tanaman karet terhadap penyakit gugur daun corynespora. Hayati, 11(1), 1-5. Jacob, C. K. (2003). of potential threat to rubber in India. Planters Chronicle, 92(10), 451-461. Jayashinge, C. K. (2000). Corynespora leaf fall: The most challenging rubber disease in Asian and African Continents. Bull Rub Res Inst Sri Lanka, 42, 56-64. Jayasinghe, C. K., & Fernando, T. H. P. S. (2011). Corynespora Leaf Fall of Hevea Rubber The Most Threatening Leaf in Asia & African Continents. Common Fund for Commodities & International Rubber Research and Development Board. Jinji, P., Xin, Z., Yangxian, Q., Yixian, X., Huiqiang, Z., & He, Z. (2007). First record of Corynespora leaf fall disease of Hevea rubber tree in China. Australasian Plant Notes, 2, 35 36. Kusdiana, A. P. J., & Syafaah, A. (2015). Pengujian resistensi klon rekomendasi tanaman karet terhadap penyakit gugur daun Corynespora. Prosiding Dies Natalis Fakultas Pertanian UNSRI ke- 52 tahun 2015 (pp. 693-701). Palembang, Indonesia: Fakultas Pertanian, Universitas Sriwijaya. 819

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