RESUMEN
Garden pea (Pisum sativum L.) is an important vegetable and pulse crop grown worldwide. Leaf blight symptoms with chlorotic lesions were observed on the pea genotypes, 2006-2008A and Arkel in field conditions at Asian Vegetable Research and Development Centre (AVRDC), Hyderabad (17°23' N; 78°29' E) and greenhouse conditions at University of Hyderabad, Hyderabad, India (17° 27' N; 78° 19' E) since 2010. The blight symptoms showed black lesions coated by a fine velvety layer of olive-grey conidia. The disease incidence on field and greenhouse pea plants ranged from 3-6%. The Cladosporium isolates, Gp01 and Gp02 were isolated from pea genotypes, Arkel and 2006-2008A in greenhouse and field conditions respectively. One fungus was predominantly isolated from infected leaf tissue plated onto potato dextrose agar (PDA), and it was identified as Cladosporium sp. based on colony morphology and conidial appearance. Single-spored cultures grown at 26±2 °C on PDA showed olive green mycelia on the obverse-side and dark green on reverse-side (Fig. 1 a,b). The conidia were limoniform with dimensions ranging from 5.91-7.33 x 3.19-4.58 µm (Table 1) and were produced on solitary conidiophores containing 1-2 septa, with no constriction whereas the aerial hyphae were septate and unbranched or rarely branched (Fig.1 c-d). To verify the pathogenicity of the fungus, detached leaf assay was carried on 30-day old Arkel leaves by point inoculation with 20 µL (106 spores/ml) spore suspension using a needle-less syringe to infiltrate the spores into the abaxial surface and the inoculated leaves were incubated at 26±2 °C for 3 days in petri plates containing moistened autoclaved absorbent cotton. Pea leaves inoculated with water served as controls. The experiment was repeated with a minimum of three times with five leaves per replication and three replications per experiment for each of the isolates. The appressorial germ tube formation was observed by scanning electron microscopy (SEM) at 12 hours post inoculation (hpi) (Fig.1 e-f). Thirty-day old pea plants were spray-inoculated with a spore suspension of 1-2 ml per plant with a concentration of 106 spores/ ml while the control plants were mock-inoculated (sprayed) with water. 5 plants per replicate per isolate with three replications per experiment and a minimum of three experiments were carried. The inoculated plants were covered with polythene zip-lock bags for two days post inoculation (dpi) and maintained in greenhouse at 26±2 °C with natural photoperiod of 14 hours. Wilting and necrosis symptoms accompanied by chlorotic lesions were observed on pea plants at 7 days post inoculation (dpi) (Fig. 2). Complying with Koch's postulates, the pathogen, which was re-isolated from the first appearance at 3 dpi from infected pea showed similar morphological characteristics as those used for inoculation. Sequencing analysis of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers (White et al. 1990), D1-D2 region sequencing (Kwiatkowski et al. 2012), Translation elongation factor 1-alpha (TEF) and Actin (ACT) gene sequencing (Nam et al. 2015) of Gp01 and Gp02 showed significant similarity with C. cladosporioides (Table S1). Further, concatenated phylogenetic trees constructed by maximum likelihood method using all the four (ITS, D1-D2, TEF and ACTIN) gene sequences of these isolates along with other reported Cladosporium species (Fig. S1a) and specific isolates of C. cladosporiodes which are reported as "sensu stricto" (Bensch et al. 2010) collected from different substrata (Fig. S1b) confirmed them as C. cladosporioides. Gp01 and Gp02 were also confirmed by Microbial Type Culture Collection (MTCC, India) as C. cladosporioides with Acc. No. MTCC 9994 and MTCC 9995. C. cladosporioides has been previously reported on Dalbergia sp. and Eucalyptus sp in India and on garden pea in other countries (Bensch et al. 2010). However, owing to the impact of this pathogen on growth and productivity of pea crop, further in-depth study needs to be carried. To our understanding this is the first report of Cladosporium cladosporioides infecting garden pea in India.
