First report of leaf spot on Elaeagnus pungens caused by Neopestalotiopsis clavispora in China.

Abstract

Elaeagnus pungens Thunb. is a common traditional Chinese herbal medicine. It has high medicinal, edible, and ornamental value. In Sep. 2020, a leaf spot disease was found on E. pungens in the campus of Nanjing Forestry University, China (31°36'51"N, 119°11'8"E). The incidence rate was ca. 77%. The disease primarily appeared as small brown spots on the leaves. Then, the spots enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. At the late stage of symptom development, black spots (acervulus) appeared on the necrotic lesions (Fig. S2A-C). Eight symptomatic leaves were collected and surface-sterilized using 75% ethanol for 30 s followed by 1% NaClO for 1 min, and then washed three times in sterile distilled water. Cuttings (ca. 5×5 mm) were made from the margins of the lesions and placed on 2% of potato dextrose agar (PDA) in Petri plates and incubated at 25 ± 2 °C for 5 days. The isolation frequency of pathogens from diseased tissues was ca. 100%. A total of four fungal isolates 3-3-1, 3-3-2, 3-3-3, and 3-3-4 were obtained using the monosporic isolation method and stored in the Forest Pathology Laboratory at Nanjing Forestry University. For molecular identification, the internal transcribed spacer (ITS), partial translation elongation factor 1-alpha (TEF1-α), and partial β-tubulin (TUB2) were amplified from the isolate 3-3-1, 3-3-2, 3-3-3 and 3-3-4, with the corresponding primer sets published in Maharachchikumbura et al. (2014). The amplicons of ITS (ON510047, ON510048, ON510070, and ON510069), TEF1-α (ON808445, ON808446, ON808447, and ON808448), and TUB2 (ON808449, ON808450, ON808451, and ON808452) generated from the isolate 3-3-1, 3-3-2, 3-3-3, and 3-3-4 were sequenced and deposited in GenBank. The ITS, TEF1-α, and TUB2 of the isolate 3-3-1 shared the same nucleotide sequences with the corresponding sequences of the isolate 3-3-2, 3-3-3, and 3-3-4. The ITS, TEF1-α, and TUB2 sequences showed 100%, 97%, and 99% similarity to Neopestalotiopsis clavispora MFLUCC12-0281 (ex-type), respectively. Phylogenetic analysis using concatenated sequences of ITS, TEF1-α, and TUB2 also showed that isolate 3-3-1, 3-3-2, 3-3-3, and 3-3-4 clustered monophyletically with N. clavispora, and supported with a high bootstrap value (80%) (Fig. S1). Since these four isolates were same species based on phylogenetic analysis, isolate 3-3-4 was randomly chosen for the pathogenicity test and morphological analysis. Colonies of the isolate 3-3-4 grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface, and averaged 12.2 mm d-1 growth at 25 °C (Fig. S2F). Black conidiomata formed superficially, scattered over the PDA at two weeks post incubation, 170.15-1820.32 × 90.33-1230.12 µm (n = 109), and contained slimy black conidial mass (Fig. S2G). Conidiogenous cells were pear-shaped to cylindrical, transparent, and colorless to pale yellow with smooth cell walls (Fig. S2H). Conidia were spindle shaped, five cells, four septa, 18.46-25.9 × 5.3-9.37 μm, (av ± SD = 23.31 ± 1.81 × 7.33 ± 1.07 μm, n = 34) (Fig. S2I). Apical and basal cells were lighter in color, mostly hyaline, and the middle three cells were darker in color, mostly brown. The apical cell showed two to three colorless, transparent unbranched accessory filaments, 9.68-30.59 μm in length, (av ± SD = 20.57 ± 4.52 μm, n = 95), whereas the basal cell only a single appendage, 3.52-9.4 μm in length, (av ± SD = 5.32 ± 1.29 μm, n = 34) (Fig. S2I). These morphological characteristics were similar to N. clavispora described by Daengsuwan et al. (2021). Based on phylogenetic analysis and morphological characteristics, isolate 3-3-1, 3-3-2, 3-3-3, and 3-3-4 were identified as N. clavispora. Healthy potted seedlings of E. pungens (63-85 cm in height, 0.7-1.6 cm in diameter) were selected for the pathogenicity test in vivo. The surface-sterilized leaves were wounded with sterilized needles (1 mm in dia.) and inoculated with mycelial plugs and conidial suspensions, respectively. One part of the leaves were inoculated with mycelial plugs (5 mm in dia.) of isolate 3-3-4. The other part of the leaves were inoculated with 10 μL of conidial suspensions (1×106 spores mL-1). The inoculated plants were kept in a growth chamber at ca. 25 ± 2 °C and ca. 90% RH under a 12-h photoperiod. PDA discs without fungi and sterilized dH2O were used as controls, respectively. All experiments were repeated twice, and each treatment had six replicates at least. After 10 and 12 days post-inoculation, the necrotic lesions appeared on the leaves inoculated with the mycelial plugs and conidial suspensions of the isolate 3-3-4, respectively (Fig. S2D and E). However, no lesions were found on the plants inoculated with PDA discs and dH2O (Fig. S2D and E). Fungal isolates were re-isolated from the infected leaves and shared similar morphological characteristics of colonies and conidia with the original one. Thus, Koch's postulates were fulfilled. Neopestalotiopsis clavispora was determined as the pathogens of a variety of plant diseases such as leaf spot on Taxus chinensis, gray blight on Camellia sinensis, and root and crown rot on strawberry (Kirschbaum et al., 2018; Wang et al., 2019a, b). To our knowledge, this is the first report of leaf spot caused by N. clavispora on E. pungens worldwide. The discovery will be helpful for monitoring and control of this disease in the future.