Peanut (Arachis hypogaea L.), one of the most important oilseed crops in tropical and subtropical regions of the world (Kumar and Kirti 2011), is widely cultivated for its high protein and oil content in seeds. In August 2019, about 30% of A. hypogaea plants were found infected by leaf spot in the peanut-growing regions of Shandong Province, China. Disease symptoms appeared as the irregular and brown necrotic lesions on leaves that were 0.5 to 5.0 mm in diam. Twenty symptomatic plants were randomly sampled from peanut planting areas in Weihai and Yantai City. Small pieces (3 mm2) were cut from lesions, dipped in a 0.5% NaClO for 10 min, rinsed three times with sterilized distilled water, dried, placed onto potato-dextrose agar (PDA), and incubated in the dark at 25°C for 10 days. Three typical Cladosporium-like strains were isolated from diseased leaves of peanut. The colonies were grey to olivaceous green, reverse olivaceous black and woolly. The conidiophores were solitary, macronematous, unbranched or branched, straight or flexuous, cylindrical, slightly swollen at the apex, smooth. Conidiogenous cells were integrated, terminal and intercalary, with numerous loci on nodulose swelling. Ramoconidia were cylindrical, oblong, fusiform, 8.0 to 19.5×2.0 to 4.5 µm, aseptate or 1 septum, pale brown. Conidia were catenate, in densely branched chains, ellipsoid, ovoid, limoniform, aseptate, 4.0 to 11.5×2.5 to 5.5 µm, smooth, with conspicuous hila. The conidia easily break off from the chains. The morphological characteristics of these isolates matched the descriptions of Cladosporium tenuissimum (Bensch et al. 2010). For the molecular identification, the partial actin (act) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the respective primers ACT-512F/ACT-783R and EF1-728F/EF1-986R (Carbone and Kohn 1999). The representative sequences, deposited in GenBank (act: OL332701, OL332702 and OL332703; tef1: OL322090, OL322091 and OL322092), exhibited 99.6% and 100% identical to C. tenuissimum ex-type isolate CBS 125995 (HM148687 and HM148442). Phylogenetic analysis was done by Neighbor-Joining (NJ) analysis based on act+tef1 sequences. These three isolates were identified as C. tenuissimum by morphological and molecular characteristics. Pathogenicity of each C. tenuissimum isolate was tested on peanut in the greenhouse at 28°C with 75% relative humidity. Twenty plants of A. hypogaea were inoculated with the conidial suspension (1.0 × 105 conidia/ml) on the leaf surface. Ten plants were mock inoculated with sterile water as controls. Within 2 weeks, inoculated plants exhibited dark necrotic lesions on leaves which were similar to the symptoms observed in the field, while the mock inoculated plants remained symptomless. The fungal pathogen which was reisolated from inoculated rather than mock inoculated leaf tissues was identical to the original pathogen on the basis of morphological and molecular analysis, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. tenuissimum on peanut in China. The C. tenuissimum infection poses a serious threat by reducing the yield and quality of peanut in Shandong Province. This research is especially valuable to enhance epidemiological studies and implement effective control strategies.
Keywords: