Morphological and cultural characterization of colletotrichum capsici, incitant of blight of chickpea in Andhra Pradesh, India

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1 Legume Research, 40 (3) 2017 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Morphological and cultural characterization of colletotrichum capsici, incitant of blight of chickpea in Andhra Pradesh, India M.Sunil Kumar*, R. Sarada Jayalakshmi Devi, B.V. Bhaskara Reddy 1 and L. Prasanthi 1 Department of Plant Pathology, S.V. Agricultural College, Tirupati , AndhraPradesh, India. Received: Accepted: DOI: /lr.v0iOF.9563 ABSTRACT In Andhra Pradesh, Chickpea is severely affected by wilt (Fusarium oxysporum f.sp. ciceri) and dry root rot (Rhizoctonia bataticola). In addition to these two diseases, Colletotrichum blight occurred in severe form during rabi 2009 and 2010 due to heavy unusual rains, which resulted in crop failure in many areas leading to re-sowing of the crops. Hence studies were conducted on Colletotrichum blight of chickpea, where a total of seven isolates were collected from major chickpea growing areas of Kurnool, Anantapur, Prakasam and Kadapa districts of Andhra Pradesh. The pathogen was isolated from infected plants and various morphological and cultural characteristics like size and shape of conidia, size of setae, chlamydospores, colony color, colony diameter, sectoring in the culture, mycelia dry weight and sporulation were studied and a great variability was observed among the isolates. Key words : Chickpea, Colletotrichum blight, Cultural characteristics, Morphological. INTRODUCTION Chickpea, cool season legume crop, is grown in several countries across the world as a food source. Seed is the main edible part of the plant and is a rich source of protein, carbohydrates and minerals especially for the vegetarian population. As other legume crops, chickpea can also fix atmospheric nitrogen through its symbiotic association with Rhizobium sp.; thus helping in enhancement of soil quality for subsequent cereal crop cultivation. Pulses contain 20 to 40 per cent protein which is two to three times more than that of cereals (Arora, 1988). Hence, they have been named as poor man s meat and rich man s vegetable. Chickpea is the most important pulse crop of India both in terms of area and production. India is the largest producer of chickpea in the world with a share of and per cent of the total area (11.97 m ha) and production (10.89 mt), respectively. Major constraints for potential chickpea production are diseases, insect pests and poor management practices. Chickpea is infected by 67 fungi, 3 bacteria, 22 viruses, mycoplasma and 80 nematodes (Nene et al., 1996). Dry root rot and Fusarium wilt are the major diseases of chickpea prevailing in Andhra Pradesh. In addition to these two diseases, incidence of Colletotrichum blight was observed in severe form in Kurnool, Prakasam and Anantapur districts of Andhra Pradesh during rabi 2009 and 2010 due to heavy unusual rains, which resulted in crop failure in many areas leading to the re-sowing of the crop. The pathogen associated was confirmed to be Colletotrichum capsici based on symptomatology and study of the causal agent. (Uday Krishna, 2012). MATERIALS AND METHODS A total of seven isolates were collected from seven different locations in four districts of Andhra Pradesh. These isolates were designated as Cb 1 (Cb = Colletotrichum blight) from Prakasam district, Cb 2 from Kurnool district, Cb 3, Cb 4, Cb 5 from Ananthpur district,cb 6 and Cb 7 from Kadapa district and morphological and cultural variability were studied among the isolates. Isolation and purification of the pathogen: Pathogen was isolated from the leaves and pods showing typical symptoms of Colletotrichum blight disease by tissue segmentation method (Rangaswamy and Mahadevan, 1999) on Potato Dextrose Agar (PDA) medium. All the isolates of the pathogen were purified by single spore isolation method (Rangaswamy and Mahadevan, 1999). A diluted spore suspension (approximately 3000 conidia/ml) of each isolate was prepared in sterile distilled water. One ml of spore suspension was poured in each water agar plate using sterilized pipette and the plate was tilted slowly for uniform spread of the spore suspension and kept in an incubator at 25 o C. Individual spores along with the medium were cut with sterilized needle and then transferred to PDA slants. Morphological characteristics: To study the morphology of conidia, setae and acervuli fifteen days old culture of each isolate grown on PDA medium was taken and slides were prepared by using lactophenol cotton blue and observed under compound microscope. Observations were recorded for size and shape of conidia, size of setae and chlamydospores. The shape and size (length and width) of *Corresponding author s sunilkmr8617@gmail.com; 1 Institute of frontier technology, RARS, Tirupati

2 conidia produced by the isolates of pathogen were recorded in 100 randomly selected conidia.length and width of setae for 100 randomly selected setae were observed and recorded.presence or absence of chlamydospores was observed with respect to each isolate grown on PDA medium and recorded. Cultural characteristics: Colony growth of each isolate was recorded as aerial fluffy or submerged felty, while colour of the colony was recorded as light grey to dark grey or whitish.twenty ml of the PDA medium was poured in sterilized petriplates and allowed to solidify. Mycelial discs of five mm diameter were cut from the margin of the seven days old culture of C. capsici for each isolate and placed in the centre of the petriplate under aseptic conditions. The plates were incubated for ten days in an incubator at 27 ± 2 C and the diameter of the colony was measured and recorded. Three replications were maintained for each isolate in the study.to study the mycelial dry weight of each isolate, 100 ml of Potato Dextrose Broth (PDB) was poured in 250 ml conical flask, plugged and sterilized. Mycelial discs of 5 mm diameter were cut from the margin of the seven day old culture of each isolate of the pathogen and transferred to the conical flask containing the sterilized medium under aseptic conditions. The flasks were incubated at 27 ± 2 C in an incubator for 15 days. The mycelial mat was removed aseptically washed thoroughly with distilled water and dried in an oven at 50 o C for 12 hours. Dried mycelial weight of each isolate was recorded using an electronic balance. Three replications were maintained for each isolate in the study.presence or absence of distinct sectors in pure culture of each isolate grown on PDA medium was observed and recorded.five mm discs of the fungal culture grown on PDA medium were cut by a sterilized cork borer from the centre to the margin at three different equidistant places along the radius. The three discs were taken in a test tube containing 15 ml sterile water and crushed by a glass rod. The suspension was shaken by a vortex mixer to separate the conidia. Spore suspension (0.02 ml) was poured with a graduated pipette in one chamber of the haemocytometer. The number of spores in a small square at the centre were counted and calculated for the total number of spores in 1 ml of the spore suspension. Three observations were recorded for each isolate. The extent of spore production was assayed with respect to each isolate by counting the number of spores present in one milliliter of spore suspension. The sporulation of each isolate was designed as excellent, good, moderate and poor based on the relative number of spores counted. RESULTS AND DISCUSSION Isolation and identification of the pathogen isolates: C. capsici isolates were isolated from the bits of infected leaflets and pods on PDA medium. Growth of the fungus was observed 2-3 days after incubation at 27 ± 2 C in all the isolates. White mycelial growth was observed at four days Volume 40 Issue 3 (June 2017) 593 after incubation later, turned from light grey to dark grey. Acervuli and conidial mass appeared as pinkish creamy growth. Acervuli were distinct, abundant and sub-conical with stiff divergent setae. Setae were dark in color but paler at the apex, swollen at the base and tapering at the apex and more or less erect and septate. Conidia were one celled, hyaline, smooth walled, with a central oil globule, curved, sickle shaped, tapering gradually at both ends with acute apex. The maximum colony growth was obtained ten days after incubation. Thereafter the growth of the culture ceased. The culture was submerged felty or raised fluffy in its growth. On the basis of conidial morphology, their size, acervuli and setae, the isolates were identified as C. capsici and the morphological characteristics tallied with the CMI descriptions (conidia length of C. capsici fell within the range of µm) given by Mordue (1971). Morphological characteristics Studies on morphological characteristics of seven isolates of the pathogen grown on PDA medium were made by recording the shape and size of conidia, size of setae and formation of chlamydospores. Conidia: The perusal of the data presented in Table 1 indicates that there was significant difference in size and shape of conidia among the seven isolates of C. capsici. The conidial length of different isolates varied from µm to µm and width from 2.90 µm to 3.81 µm. The maximum size of conidia ( µm) was observed in isolate Cb 1, while isolate Cb 4 showed minimum size ( µm). All the isolates produced falcate conidia, while Cb 2 produced both falcate and fusiform conidia as shown in Fig c. However, developmental relationship between falcate and fusiform conidia may need further investigation. Conidial dimension is an important and stable character within the species of Colletotrichum. In the present investigation, significant differences were observed with respect to shape and size of conidia among the seven isolates of C. capsici, even when same medium was used for growth of the isolates. These variations in the conidial size indicated the existence of variability in the pathogen. Setae: It is evident from the data presented in Table 1 that the isolates differed significantly in length and width of setae. Isolate Cb 3 produced the longest ( µm) setae, while the isolate Cb 7 produced shortest (87.63 µm) setae as shown in Fig a & b. The width of setae is highest in isolate Cb 3 (4.72 µm) and least in isolate Cb 4 (3.76 µm). Chlamydospores: Among the seven isolates, only the isolate collected from Kurnool (Cb 2) produced chlamydospores as shown in Fig d & e. Cultural characteristics Studies on cultural characteristics of seven isolates of the pathogen grown on PDA medium were made by

3 594 LEGUME RESEARCH - An International Journal Table 1: Morphological characteristics of conidia of seven isolates collected from major chickpea growing areas of Andhra Pradesh Isolates Length (µm)* Width (µm)* Shape of conidia Setae Range Mean Range Mean Length (µm)* Width (µm)* Cb a a Falcate c 3.85 bc Cb b d Fusiform and falcate c 4.10 b Cb a c Falcate a 4.72 a Cb c d Falcate b 3.76 c Cb c b Falcate e 3.91 bc Cb b b Falcate d 4.49 a Cb c a Falcate e 3.96 bc SEm± CD (5%) *Mean of 100 conidia recording colony type and colony colour, maximum radial growth, sectoring in the culture, mycelial dry weight and sporulation. Colony Type and colour: Variation was observed with respect to colony type and colour among the C. capsici isolates. Most of the isolates produced felty growth on PDA medium while, the isolates Cb 5 and Cb 7 produced fluffy growth. Colour of the cultures were dark grey in isolates Cb 1, Cb 2, Cb 4 and Cb 6, whereas it was light grey in isolate Cb 3 and whitish in isolates Cb 5 and Cb 7 as shown in Table 2 and Fig f. Maximum radial growth of isolates: Maximum radial growth was obtained 10 days after incubation on PDA medium in some of the isolates. Among the isolates diameter of the colony ranged between mm to mm as shown in Table 2 and Fig f. The isolate Cb 4 showed maximum growth (82.33 mm) followed by isolates Cb 1 (81.00 mm), Cb 6 (80.97 mm), and Cb 2 (80.43 mm). The isolate Cb 5 showed least colony growth (65.13 mm) followed by Cb 7 (66.17 mm). Mycelial dry weight: A large variation was recorded in the mycelial dry weight among the isolates which varied from mg (Cb 5) to mg (Cb 1) as shown in Table 2. Sectoring in the culture: Of the seven isolates studied, only two isolates from Kurnool (Cb 2), and Anantapur (Cb 4) showed sectoring in culture within 7-8 days after incubation at 27 ± 2 o C on PDA medium as shown in Fig f. Sporulation: Excellent sporulation (>30 x 10 4 conidia ml -1 ) was observed in isolates Cb 1 and Cb 2 after 15 days of incubation on PDA medium. Good sporulation ( conidia ml -1 ) was observed in isolates Cb 4, Cb 6 and Cb 7 followed by moderate sporulation ( conidia ml -1 ) in Cb 5 and poor sporulation in Cb 3(< conidia ml -1 ) (Table 2). In the present study, no distinct variation was observed among the isolates with respect to colour of conidia as it was hyaline for all the isolates. Similar observations were made by Singh et al. (1973) among the isolates of C. capsici infecting brinjal. Lubna Masoodi et al. (2012) observed falcate and fusiform conidia in different isolates of C. capsici. Conidial dimorphism has been demonstrated by Hazra et al. (1999) in case of sorghum anthracnose pathogen, C. graminicola. Vinaya Hemannavar (2008) reported that setae are dark in colour but paler at apex, swollen at the base and tapering at the apex and more or less erect and upto 150 µm long. Though production of setae is a genetically controlled character (Sutton and Waterson, 1970), Table 2: Cultural characteristics of seven isolates of Colletotrichumcapsici on PDA medium at 27 ± 2 C Isolates Colony colour Colony diameter* (mm) Mycelial dry weight* (mg) Sectoring Sporulation Cb 1 Dark grey a a Absent ++++ Cb 2 Dark grey a b Present ++++ Cb 3 Light grey b c Absent + Cb 4 Dark grey a c Present +++ Cb 5 Whitish c e Absent ++ Cb 6 Dark grey a b Absent +++ Cb 7 Whitish c d Absent +++ SEm± CD (5%) *Mean of three replications Excellent (> ml -1 ) Good ( ml -1 ) ++ - Moderate ( ml -1 ) + - Poor (< ml -1 )

4 a Volume 40 Issue 3 (June 2017) 595 b c d e f Fig 1: showing (a)setae of Cb 3, (b) setae of Cb 7, (c) conidia of Cb 2 showing falcate and fusiform shaped conidia, (d,e) Chlamydospores produced in Cb 2, (f) Isolates showing colony color, colony diameter and sectoring in culture atmospheric humidity plays an important role for expression of genetic potential (Frost, 1964). Development of setae is also related to the type of primordial for acervulus production. Thind and Jhooty (1990) reported that the colony diameter of C. capsici ranged from to 90 mm and the growth rate ranged from 4.3 to 7.5 mm per day. Angadi (1999)found that C. capsici on chilli crop produced maximum mycelial weight after sixteen days of incubation. Gupta (1981) studied on variation among the twelve isolates of C. capsici and reported that isolate Cols. 4 produced maximum sporulation and least in isolates Cols. 2, 3, 5, 6 and 9. REFERENCES Angadi, H.D. (1999). Studies on anthracnose of chilli (Capsicum annum) and its management.m.sc. (Agri.) Thesis, Univ. Agric. Sci., Bangalore, Karnataka, India. Arora, P.P. (1988). Line x tester analysis for combining ability in bengalgram.himachal Journal of Agriculture Research. 14: Frost, R.R. (1964). Setae formation in Colletotrichum spp. Nature. 201: Gupta, B.D. (1981). Sporulation and relative virulence among isolates of Colletotrichum capsici causing anthracnose of betelvine. Indian Phytopathology. 34:

5 596 LEGUME RESEARCH - An International Journal Hazra, S., Thakur, R.P., Umadevi, G and Thakur, K. (1999). Pathogenic amd Molecular variability among twelve isolates of Colletotrichum graminicola from sorghum. Journal of Mycology and Plant Pathology. 29: Lubna Masoodi, Ali, Shahzad and Sofi, T.A. (2012). Cultural, Morphological and Pathogenic Variability in Colletotrichum capsici causing Die-back and Fruit Rot of chilli. Asian Journal of Plant Pathology. DOI: /ajppaj Mordue, J.E.M. (1971). Colletotrichum capsici in CMI descriptions of pathogenic fungi and bacteria No Commonwealth Mycological Institute, Kew, Surry, England. Nene, Y.L., Sheila, V.K and Sharma, S.B. (1996). A world list of Chickpea and Pigeon pea pathogens.5th edn, ICRISAT, Patancheru India, 27p. Rangaswamy, G and Mahadevan, A. (1999). Diseases of Crop Plants in India. Prentice Hall of India Pvt. Ltd. New Delhi. 607 p. Singh, S.A., Singh, H and Chohan, J.S. (1973). Fruit rot of brinjal by Colletotrichum capsici. Indian Journal of Mycology and Plant Pathology. 3: Sutton, B.C and Waterson, J.M. (1970). CMI Descriptions of pathogenic fungi and Bacteria No Commonwealth Mycological Institute, Kew, Surry, England. Thind, T.S and Jhooty, J.S. (1990). Studies on variability in two Colletotrichum species causing anthracnose and fruit rot of chillies in Punjab. Indian Phytopathology. 43: Uday Krishna. (2012). Studies on Colletotrichum blight of chickpea. M.Sc. (Agri.) Thesis, ANGRAU., Hyderabad, Andhra Pradesh, India. Vinaya Hemannavar. (2008). Studies on seed borne aspects of anthracnose of chilli and its management. M. Sc. (Agri.) Thesis, Univ. Agric. Sci., Dharwad, Karnataka, India.