First Report of Cladosporium tenuissimum causing Postharvest rot of Elaeagnus conferta Fruit in Kunming, China.

Abstract

Elaeagnus conferta Roxb. is a perennial evergreen climbing shrub and is mainly native to India, Vietnam, Malaysia, and South China (Gupta & Singh, 2021). Various parts of this plant are used to treat multiple diseases(Gupta et al., 2021). Between during the months of March and April of 2021, in Kunming city of grower fields, Yunnan Province (N 25°02'; E 102°42'), southwest China. Some postharvest E. conferta fruits showed brown spots of decay with a greyish mycelium, which symptom only appears on fruit, and did not find it on this plant. The incidence of this disease in postharvest E. conferta fruits ranges from 45 % to 65 % in natural conditions. This pathogen is harmful and causes many plant diseases. Such as rice, oriental persimmon, pear, panicles of mango, and so on (Cho & Shin, 2004; Guillén-Sánchez et al., 2007; Lee et al., 2009). The infected fruit samples surface was disinfected with 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, then aseptic water washing three times. The fruit tissue is rich in carbohydrates and water content, which aid the growth of fungal species. Putting these diseased tissues on a potato dextrose agar (PDA) medium, cultured at 25 ± 1 ℃ for 7 days. The colonies grow on the PDA medium, then separated and puried again. Three pure cultures (YNGH01, YNGH03, YNGH05) were obtained, which were stored in 15 % glycerol at -80 ℃ refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Agricultural University. After 7 days of cultivation, the colonies were round and the diameter attained up to 38 mm, the surface of the colony showed tiled, fluffy, with a velvet-like texture, greyish-green to smoke-gray, slightly raised in the middle, the edges were radial hollow and wrinkle (Fig. 1A). Conidiophores were solitary, erect, unbranched or rarely branched, slightly flexuous at the apex, septate, dark brown, 254 to 680 µm long, 3.6 to 4.5 µm wide, top of the conidiophores or the rostral were slightly swollen (Fig. 1B). Conidia were light gray or grey, solitary or bispora, irregular in shape and size (Fig. 1C), nearly circular (3.21 × 3.31 µm), oval to lemon-shaped (6.59 × 3.21 µm) or elliptical (8.35 × 4.65 µm). The CTAB method extracts 3 isolates (YNGH01, YNGH03, YNGH05) genomic DNA (Aboul-Maaty & Oraby, 2019). To confirm identity with molecular identification, performed by three different genomic DNA regions, fragments of internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (TEF-1α), and actin (ACT) genomic regions. These genomic DNA were amplified with primers ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Carbone & Kohn, 1999). The sequences of these isolates were uploaded to GenBank (YNGH01: ON753810, ON868696, ON912090 YNGH03: ON753812, ON868698, ON912092, and YNGH05: ON753814, ON868700, ON912094). NCBI's BLASTn search of those ITS sequences showed 99.81% similar to C. tenuissimum (MG873077.1), and sequences TEF-1α and ACT were 100% identical to several isolates of C. tenuissimum (OM256526.1 and MT154171.1). Combined the ITS region, TEF-1α, and actin (ACT) genomic regions of isolates YNGH01, YNGH03 and YNGH05 to construct a phylogenetic tree with MEGA11. Maximum likelihood phylogenetic analyses further confirmed the results (Fig. 2)(Santos et al., 2020). Healthy and mature E. conferta fruits were used for pathogenicity test. Pathogens were washed with sterilized water at a final concentration of 2× 106 spores/mL (Jo et al., 2018). The test was divided into A and B groups (A: The surface of fruits was pierced with a sterilized needle that carried pathogenic fungus of final concentration at 2×106 spores/mL B: Sprayed at the concentration of 2×106 spores/mL on fruits). The control fruits were treated with sterilized water and stored at 25 ± 1 ℃ with a relative humidity of 80 %, average group with 10 fruits in this test, which was repeated three times. After 7 days, the fruits of group A were initially sesame seed size of the disease spots, nearly round, irregular, with grayish-brown spots, and slightly depressed. Later, the lesion gradually turns dark brown (Fig. 1D). And group B began with small patches of brown fungal growth on the pericarp, with the development of the disease, the necrotic spots enlarged and developed irregular and coalesced, the color of spots became gray or black gradually (Fig. 1E). The symptoms were similar to previously observed and the pathogen was reisolated and identified as C. tenuissimum. Control fruits were healthy (Fig. 1F). The pathogens test fulfilled Koch's postulates. According to morphology (Bensch et al., 2012), rDNA-ITS, TEF-1α, and ACT sequence analysis, phylogenetic analysis, and pathogenicity test, the pathogen was identified as C. tenuissimum. To our knowledge, this is the first report of C. tenuissimum occurring on E. conferta fruits in China.