First Record of Rhizoctonia solani AG 1-IB on Mucuna pruriens in India.

Mucuna is the source of L-Dopa (L 3,4 dihydroxyphenylalanine), a precursor of a dopamine used to treat Parkinson's disease. Leaf blight symptoms were observed on Mucuna pruriens plants in October to November 2010 in a field at the Indian Council of Agricultural Research Complex for the Northeastern Hill Region, Umiam, Meghalaya, India. Symptoms included black necrotic areas on leaves, collapsed leaf tissue and, occasionally, fungal growth visible on the leaf. In advanced infections, dead leaves were attached to the stem, followed by defoliation with only infected pods still attached. Approximately 10% of plants were infected in ~0.5 ha surveyed. Symptomatic leaf pieces were washed with sterile water, surface-sterilized using 4% NaOCl for 30 s, washed again, blotted dry, and plated on PDA amended with streptomycin (100 ppm). Characteristics of three fungal isolates were typical of Rhizoctonia solani J.G. Kühn [teleomorph = Thanatephorus cucumeris (A. B. Frank) Donk], i.e., hyphal branching at 90° angles, basal constriction at the hyphal branching point with a septum close to the lateral hyphum (3), and presence of multinucleate hyphal cells confirmed using DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine) staining (1). A culture was deposited at the Agharkar Research Institute, Pune, India (NFCCI No. 2602). The ITS1-5.8S-ITS2 region of nuclear rDNA of one isolate was sequenced after amplification with primers ITS1 and ITS4 (4), (GenBank Accession No. JQ675536). A BLAST search revealed 99% similarity of the sequence with that of 2 R. solani AG 1-IB isolates (AB122137 and AB000039). Sequences were aligned using MAFT Version 6. Maximum parsimony analysis using MEGA 5 placed the test isolate in AG 1-IB with 99% bootstrap support. PCR assays with primers for R. solani AG 1-IB produced a DNA band of ~300 bp (2). Koch's postulates were completed by inoculating 5-mm colonized plugs of PDA at the soil line of each of 5 potted, 40-day-old plants of M. pruriens, and covering the base of each plant with moistened cheesecloth. In addition, 3 plants were inoculated with colonized plugs at the junction of the lamina and petiole of 9 leaves/plant, spraying the plants with sterilized water, and covering the plants with polythene for 3 days. In addition, 10 detached leaves were inoculated with colonized PDA plugs and incubated in a moist chamber. Three non-inoculated plants served as a control treatment for the first 2 methods, and 10 leaves as a control treatment for the third method with sterilized PDA plugs. Symptoms of leaf blight (necrosis from base to leaf tip, with abundant fungal growth) developed in 6 to 7 days on plants inoculated at the soil line, 4 days on leaves inoculated at the junction of the lamina and petiole, and 2 to 3 days on detached leaves. Control plants and leaves remained asymptomatic for all 3 methods. R. solani was reisolated from inoculated plants as described above, and confirmed to be AG 1-IB. The fungus was not reisolated from control plants or leaves. To our knowledge, this is the first record of R. solani AG 1-IB causing leaf blight on M. pruriens in India. References: (1) M. M. Kulik and P. D. Dery. Biotech. Histochem. 70:95, 1995. (2) M. Matsumoto. Mycoscience 43:185, 2002. (3) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society Press, St Paul, MN, 1991. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Leaf Blight of Basella alba Caused by Rhizoctonia solani AG 1-IB in India.

Thick succulent leaves of Malabar spinach (Basella alba L.) are used for human consumption in India. Symptoms of leaf blight were observed on this plant in October 2010 at Umiam, Meghalaya, India. Symptoms started from the lower leaves and spread to the upper part. Water-soaked lesions covered the whole leaf and gradually the leaves shredded and got detached from the plants. Whole plants were seen defoliated due to severe infection. Lesions were visible on the stem also. The pathogen was isolated on potato dextrose agar amended with streptomycin (100 ppm). Fungus isolated from infected plants had typical characters associated with Rhizoctonia solani J. G. Kühn [teleomorph Thanatephorus cucumeris (A.B. Frank) Donk], i.e., hyphal ramification angles of ~90°, basal constriction, and a septum next to the lateral hyphae (2). Nuclear staining with DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine) confirmed that hyphal cells were multinucleate (1). Molecular analysis was conducted using sequence data (JQ675535) containing ITS1 and 5.8S and ITS 2 of nrDNA, which was obtained after amplification using universal primers ITS1 and 4. BLAST search revealed 99 to 100% similarity with AG 1-IB (GU585667, GU270581). Living culture has been deposited in Agharkar Research Institute, Pune, India (NFCCI No. 2601). Phylogenetic analysis was also conducted using MEGA 5. It also placed the isolate in AG 1-IB clade with 99% bootstrap support in MP (maximum parsimony) analysis. Three healthy plants were inoculated using colonized PDA bits from actively growing culture. Sterilized PDA bits were kept on control plants. Plants were sprayed with water and covered with cheesecloth for 3 days. Inoculated plants developed symptoms after 5 days whereas control plants remained healthy. Inoculations were also done on detached leaves kept in a moisture chamber using colonized PDA bits. In this case, symptoms developed within 3 days. Detached leaves with sterilized PDA bits remained healthy. R. solani was also reisolated from inoculated plants. To our knowledge, this is the first record of AG 1-IB based on molecular evidence on B. alba from India. This new host may accelerate the spread of this pathogen to other crops. References: (1) M. M. Kulik and P. D. Dery. Biotech. Histochem. 70:95, 1995. (2) B. Sneh et al. Identification of Rhizoctonia species. APS Press, St Paul, MN, 1991.

First Record of Rhizoctonia solani AG 2-1 on Ctenanthe oppenheimiana in India.

Ctenanthe oppenheimiana (= Maranta oppenheimiana) is a common foliage plant also known as "never never" plant. Plants grow best in high-humidity conditions. Potted plants in Barapani, Meghalaya, India were found to be infected with a foliar disease. Initial symptoms were leaf margin necrosis that expanded to cover large areas of the leaf blade during March and April of 2007. Leaf portions from the margin of infected and healthy tissue were washed with sterile water and then surface sterilized with 4% sodium hypochlorite for 30 s. These bits were again washed twice with sterile water, blotted dry, plated on water agar, and maintained at 25°C. After 24 h, mycelial growth was transferred to potato dextrose agar. Fungal colonies were brown with fluffy, aerial mycelium Hypha was branched at 90° with lateral branches having constriction at the point of origin. Hyphal cells were determined to be multinucleate when stained with 4',6-diamidino-2-phenylindole. Sclerotia were 3 to 5.6 mm in diameter (average 4.1 mm) and were not differentiated into cortex and medulla. The above characteristics were consistent with a Rhizoctonia sp. (2). Anastomosis group (AG) was determined by pairing isolates with tester isolates of R. solani and its subgroups (AG 1-1A, AG 2-1, AG 2-2 IIIB, AG 3, AG 4HG-II, AG 5, AG 6, AG 7, AG 8, AG 9, AG 10, AG 11, AG 12, AG 13, and AG BI) and staining hyphae with Safranin-O 0.03% and KOH 3% solutions. Anastomosis was positive only between the Ctenanthe isolate and AG 2-1 tester strain. (1). The Ctenanthe isolate was grown on potato dextrose broth for 8 days. The broth culture was blended in a Waring blender for 2 min and 10 ml was mixed with 90 ml of sterile water. The mycelium suspension was sprayed on five healthy plants of C. oppenheimiana Noninoculated plants served as a control. Inoculated leaves developed initial symptoms of marginal leaf necrosis after 6 to 8 days and expanded leaf necrosis, similar to the original symptoms, after 12 to 15 days. No symptoms developed on noninoculated plants. R. solani was isolated from leaf lesions of the inoculated plants, confirming Koch's postulate To our knowledge, this is a new report from India. References: (1) G. C. MacNish et al. Phytopathology 83:922, 1993. (2) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St Paul, MN, 1991.

First Report of Leaf Blight of Chinese Ground Orchid (Phaius tankervilliae) Caused by Botrytis cinerea in India.

Chinese ground orchid (Phaius tankervilliae Banks:Blume) is a beautiful, terrestrial orchid, which belongs to the family Orchidaceae. It is used as a cut flower, which lasts for 4 to 5 weeks. This species is considered endangered and rare in nature. In June of 2007, potted plants of P. tankervilliae in Shillong, Meghalaya (northeast India; maximum temperature 24°C, minimum temperature 18°C, and 83.5% relative humidity) exhibited leaf blight. Symptoms included water-soaked lesions and dense, gray mold growing on infected tissues. Thirty-six percent of the plants surveyed were found to have this disease. For isolation, diseased tissue was surface disinfested by soaking it in 0.5% sodium hypochlorite for 1 min, air dried, plated on potato dextrose agar, and incubated at 20°C. Mycelia were initially white but later turned gray. Mature, unicellular, ellipsoid, hyaline conidia (6.3 to 8.2 × 9.6 to 11.4 µm) were formed in botryose heads. Hard, black, irregular-shaped sclerotia (average size 1.8 × 2.3 mm) were formed after 15 days. On the basis of these morphological characters, the pathogen was identified as Botrytis cinerea Pers.:Fr. (1). Pathogenicity was confirmed by spraying plants with a spore suspension (106 spores per ml), which were then maintained under high humidity for 48 h at 20 to 22°C by covering with cheesecloth. Five potted plants were inoculated and five were sprayed with sterile water. Lesions and spore masses that were identical to those observed appeared 5 to 6 days after inoculation. Water-treated control plants remained asymptomatic. B. cinerea was reisolated from inoculated plants. A literature search revealed no previous record of this disease in India. So, to our knowledge, this is the first record of B. cinerea on P. tankervilliae in India. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, England, 1971.