1 J. Hortl. Sci. Vol. 17(2) : 00-00, 2022 This is an open access article d istributed under the terms of Creative Commons Attribution-NonCommer cial-ShareAl ike 4.0 International License, which permits unrestricted non-commercial use, d istribution, and reproduction in any med ium, provide d the original author and source are credited. Original Research Paper INTRODUCTION Ya r d long bea n (Vigna unguiculata subsp. sesquipedalis (L.) Verdcourt), a trailing type of vegetable cowpea (2n= 24) is one of the most popular and remunerative vegetable crops traditionally grown in Kerala. Due to the favourable a gro climatic conditions, the crop has gained much importance and has come to occupy a prime position among the vegetable crops raised in the state. But the production of vegetable cowpea is hindered by an array of diseases that cause growth suppression or death of plants, leading to reduction in yield and productivity. Sreeja (2014) conducted periodical survey in the potentia l cowpea gr owing a r ea s of Thiruvananthapuram district and reported that among the six major fungal diseases, anthracnose was found to be the most predominant one (0-55%). Disease index ranged between 0 - 33.30 and considerable yield losses were attributable to the disease. Anthracnose is a destructive fungal disease caused by Colletotrichum spp. In India, the incidence of a nthr a cnose disea se wa s fir st r epor ted fr om Maharashtra (Rao, 1966). According to Emechebe and Lagoke (2002), all stages of the crop and its parts like hypocotyls, stem, peduncle, flowers, leaves and pods were seriously affected. Previous attempts to identify r elia ble sour ces of r esista nce ha ve led to the identification of few bush type resistant cowpea (Kumar, 1999), as most of the trailing cultivars are susceptible to anthracnose, but high yielding compared to the bushy or semi-trailing types. The disease is pan-tropical in distribution and is being widely recorded in regions where conditions are wet and humid. Once the infection is incited under favourable condition, its management using fungicides is difficult. Breeding resistant varieties is suggested as the only practical strategy, especially under hot and humid condition. The present study identified the resistant varieties of vegetable cowpea through artificial inoculation followed by detached leaf assay. MATERIALS AND METHODS Fifty yard-long bean genotypes belonging to bush, semi erect and pole types were screened against anthracnose disease through artificial inoculation under pot cultur e. Seeds of vegeta ble cowpea genotypes were collected from different parts of India, including the released varieties of SAUs and ICAR institutes and stored at refrigerated conditions for the Screening of yard long bean (Vigna unguiculata subsp. sesquipedalis (L.) Verdcourt) genotypes for resistance to Colletotrichum gloeosporoides Merin E.G.*, Sarada S. and Joy M. College of Agriculture, Kerala Agricultural University, Trivandrum, Kerala, India *Corresponding author Email : merinelzageorge5010@gmail.com ABSTRACT Anthracnose is one of the most destructive fungal diseases caused by Colletotrichum gloeosporoides in yard long bean, leading to complete crop loss at all stages and its parts like hypocotyls, stem, peduncle, flowers, leaves and pods were seriously affected. Few bush type cowpea cultivars have been earlier identified as reliable sources of resistance while trailing types are susceptible, but high yielding. Breeding resistant varieties is suggested as the only practical strategy, especially under hot and humid condition. Fifty-yard-long bean genotypes belonging to bush, semi erect and pole types were screened against anthracnose disease through artificial inoculation under pot culture. The present study identified the resistant varieties of vegetable cowpea through artificial inoculation followed by detached leaf assay. Among the 50 varieties of yard long bean observed, Kanakamony, dual purpose yard long bean was found highly resistant with disease severity of 3.67% followed by Arimbra local. Keywords: Anthracnose, Colletotrichum gloeosporoides and yard long bean 2 Merin E.G., et al J. Hortl. Sci. Vol. 17(2) : 00-00, 2022 study. The most virulent isolate of C. gloeosporoides was used for artificial inoculation (Fig. 1 1). To isolate the fungal spores for artificial inoculation, infected plant parts were collected from field, washed in tap water, and surface sterilised using 0.1% mercuric chloride followed by washing thrice with autoclaved distilled water. The fungus was cultured in potato dextrose agar medium and mycelial growth was observed through microscope. All Petri dishes were incubated at room temperature (25±20C). Petri dishes were examined for the growth of the pathogen and the morphological characteristics were observed. Inoculum was prepared from secondary culture of 7 day- old culture, by scrapping off the mycelium and spores and suspended in 100 ml sterile distilled water. Seven seeds each of fifty genotypes were sown in two pots. After the germination, five healthy seedlings were retained and excess thinned out. Artificial inoculation was done on 20 days old seedlings kept in pots inside the greenhouse. Each plant was sprayed with 20 ml of spor e suspension of vir ulent str a in of C. gloeosporoides having a concentration of 106 spores ml-1 by following serial dilution method. The plants were covered with moistened polythene covers to maintain high humidity (Fig. 2). Inoculated plants were observed daily for disease incidence. Observations on disease incidence were taken on three, five, seven, nine, fifteen, twenty, twenty-five and thirty days. Final observation was taken when the disease was well expressed. Disease severity was assessed using the scale 0-5 reported by Latunde and Dada (1990). 0. No infection 0.5. Hypersensitive spots on main stem only 1: Trace of infection – Small anthracnose lesions on main stem, petioles of lower leaf only 2. Slight infection – Lesions on stem, petioles and branches 3. Moderate infection – Advanced anthracnose lesions on stem, petioles, branches, veins on the abaxial surface of leaves 4. Severe – Advanced anthracnose lesions on stem, petioles, branches, leaves, veins and peduncles 5. Very Severe – Advanced anthracnose lesions on stem, petioles, branches, leaf veins, spreading lesions on peduncle and pods Based on the percentage of plant area infected, disease severity/ intensity was calculated using the following formula (Wheeler, 1969). Per cent disease severity = Sum of all numerical ratings x 100 Total no. of plants taken for Maximum observation Disease category Per cent disease incidence was calculated by using the following formula. Per cent disease incidence = No. of plants infected x 100 Total no. of plants observed Based on the per cent disease severity, the genotypes wer e gr ouped into 5 categories a s a dopted by Rajkumar et al. (1995). Disease severity Category 0 Immune 1 - 10 Highly resistant 10.1 - 25 Moderately resistant 25.1 - 50 Moderately susceptible Above 50 Highly susceptible Five plants of the resistant genotype identified through artificial inoculation were grown in the field and detached leaf assay was done just before flowering to confirm resistance. RESULTS Observations on disease incidence were recorded at 10 days after infection. The infection was first observed in leaves, reddish brown streaks were very prominent on veins and veinlets. Prominent mildew symptoms were also observed on the leaf lamina (Fig. 3). Finally, leaves became chlorotic and detached from the infected plant. Reddish brown lesions were also developed on the stem. These individual lesions coalesced to form large sunken lesions and covered the whole stem causing drying up of the veins which is known as vine blackening. The pods were rottened, grey in colour, cover ed with bla ck fr uiting bodies of fungus. According to the visible symptoms, the plants were awarded disease scores. Among the 50 genotypes tested, Kanakamony, a dual purpose yard long bean variety was found to be highly resistant with disease severity of 3.67%, followed by Arimbra local with 3 Screening of yard long bean Sl.No. Treatments Source Percent Disease Severity Disease Reaction 1 Anaswara KAU 22.15 Moderately resistant 2 Bagyalakshmi KAU 13.90 Moderately resistant 3 Kanakamony KAU 3.67 Highly resistant 4 Arimbra local KAU 9.58 Highly resistant 5 Vyjyanthi KAU 56.84 Highly susceptible 6 Mithra KAU 46.90 Moderately susceptible 7 Githika KAU 56.70 Highly susceptible 8 Lola KAU 54.90 Highly susceptible 9 Manjari KAU 38.80 Moderately susceptible 10 Sharika KAU 63.80 Highly susceptible 11 Vellayani Jyothika KAU 55.90 Highly susceptible 12 KAU Deepika KAU 50.90 Highly susceptible 13 CO6 TNAU 24.70 Moderately resistant 14 Pusa Beej IARI 66.45 Highly susceptible 15 Kashi Kanchan IIVR 47.64 Moderately resistant 16 Arka Garima IIHR 23.90 Moderately resistant 17 Arka Mangala IIHR 24.30 Moderately resistant 18 Arka Samradhi IIHR 21.60 Moderately resistant 19 FH 31 Farm House, Trivandrum 56.50 Highly susceptible 20 FH 55 Farm House, Trivandrum 53.50 Highly susceptible 21 FH 7 Farm House, Trivandrum 70.78 Highly susceptible 22 VS 38 KAU 64.99 Highly susceptible 23 VS 53 KAU 50.12 Highly susceptible 24 VS 58 KAU 44.64 Moderately susceptible 25 VS 16 KAU 70.00 Highly susceptible 26 TCR 17 NBPGR 69.80 Highly susceptible 27 TCR 18 NBPGR 41.50 Moderately susceptible 28 TCR 19 NBPGR 56.00 Highly susceptible 29 TCR 50 NBPGR 47.25 Moderately susceptible 30 TCR 53 NBPGR 26.80 Moderately susceptible 31 TCR 54 NBPGR 36.00 Moderately susceptible 32 TCR 55 NBPGR 78.91 Highly susceptible 33 TCR 56 NBPGR 36.00 Moderately susceptible 34 TCR 60 NBPGR 59.49 Highly susceptible 35 TCR 61 NBPGR 54.00 Highly susceptible 36 TCR 63 NBPGR 59.45 Highly susceptible Table 1 : Percent disease severity and disease reaction of yard long bean genotypes for anthracnose incidence 4 37 TCR 68 NBPGR 31.70 Moderately susceptible 38 TCR 69 NBPGR 69.22 Highly susceptible 39 TCR 70 NBPGR 36.23 Moderately susceptible 40 TCR 74 NBPGR 26.89 Moderately susceptible 41 TCR 75 NBPGR 44.47 Moderately susceptible 42 TCR 77 NBPGR 35.50 Moderately susceptible 43 TCR 80 NBPGR 37.95 Moderately susceptible 44 TCR 84 NBPGR 54.25 Highly susceptible 45 TCR 86 NBPGR 33.63 Moderately susceptible 46 TCR 91 NBPGR 55.20 Highly susceptible 47 TCR 92 NBPGR 33.30 Moderately susceptible 48 TCR 93 NBPGR 65.33 Highly susceptible 49 TCR 96 NBPGR 39.82 Moderately susceptible 50 TCR 125 NBPGR 37.49 Moderately susceptible SE (m)± - 1.13 - C.D. (0.05) - 3.22 - 9.58% disease severity. TCR 55(78.91 %) was found to be highly susceptible followed by FH-7 (70.78 %) (Table 1). Among the genotypes evaluated, eight showed moderately resistant reaction from 13.90 to 23.40%. In the eighteen moderately susceptible genotypes, much difference could not be noticed in disease severity values and most of them were pole types. In deta ched lea f a ssa y, the lea ves of Kanakamony was symptomless, and the resistance was confirmed in vitro (Fig. 4). DISCUSSION Anthracnose is one of the most destructive fungal diseases caused by Colletotrichum gloeosporoides in yard long bean, leading to complete crop loss in all stages and its parts like hypocotyls, stem, peduncle, flowers, leaves and pods were seriously affected. C. gloeosporioides has a wide host range including Brassica campestris, legumes, pigeon pea, soybean, brinjal, pumpkin, cucumber, tomato, spinach, mung bea n, br oa d bea n, cowpea , etc. (Sha r ma a nd Kulshrestha, 2015). Symptomatology studies on anthracnose of cowpea was conducted by Sreeja (2014). The initial symptoms appeared as minute, circular to irregular spots on the leaves which later increased in size and turned light to dark brown in colour and coalesced together to form large, necrotic spots on the leaves with shot holes. On the stem and vines, symptoms appeared as spindle shaped lesions with light grey centre and reddish-brown margin which Merin E.G., et al Fig. 1 Fig. 2 Fig. 3 Fig. 4 5 enlarge upto 10-12 mm in length. In the later stages, small, irregular deep-seated reddish-brown spots appear on the pods also. Isolation, characterization and identifica tion of the pa thogen r evea led tha t Colletotrichum gloeosporioides was associated with anthracnose. Adebitan and Olufajo (1998) reported that grain types exhibited better resistance to anthracnose. Artificial inoculation of fifty genotypes confirmed the resistance in Kanakamony, followed by Arimbra local, hence these varieties are valuable sources for anthracnose resistance breeding programme. Kanakamony is a dual-purpose cowpea (grain cum vegetable) belonging to the sub-species cylindrica. Shiny et al. (2015) reported that susceptible bush-type cultivar Pusa Komal and pole type cultivar Lola showed 100 and 68.80 % disease severity while the immune bush type cultivar Kanakamony was free from the symptoms and pole type Arimbra Local showed 8.80 % disease severity using SDS-PAGE. These results further support that Kanakamony is highly resistant to C. gloeosporoides causing anthracnose. Since fungicides and chemicals are not fully effective, the transfer of resistance from the cultivars to trailing types can only be a durable solution. Hence the results revealed that the highly resistant genotypes Kanakamony and Ar imbr a local could be recommended for cr op improvement programmes in trailing type vegetable cowpea for enhancing the resistance to anthracnose. ACKNOWLEDGMENTS Merin Elza George expresses her sincere gratitude to Kerala Agricultural University, India for the complete funding of this research work, which forms part of her Ph.D program in Vegetable Science. REFERENCE Adebitan S.A. and Olufajo O. 1998. Field evaluation of cowpea (Vigna unguiculata) varieties for grain and fodder production and for multiple disease resistance in Nigeria. Ind. J. Agric. Sci., 68:152–154. Emechebe, A.M. and Lagoke, S.T.O. 2002. Recent advances in research on cowpea diseases. In: Challenges and opportunities for enhancing sustainable cowpea production. IITA, Nigeria pp. 94-123. Kumar, M. P. 1999. Anthracnose disease of vegetable cowpea [Vigna unguiculata ssp. sesquipedalis (L. ) Ver dcour t]. M. Sc. 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