caused leaf spot disease in Bayberry in Guizhou, China.

Bayberry (Myrica rubra) is one of the most important fruit in China. In July 2023, circular leaf spots were noted on spring bayberry (cv. Dongkui Arbutus) tree planted in Guanshanhu, Guiyang, Guizhou, China (26°37'3"N，106°39'59"E). The disease incidence (diseased plants/total plants × 100) varied from 55 to 60% with 15 to 20% disease severity (diseased leaf area/total leaf area × 100). Disease incidence and severity were calculated from five infected fields of bayberry using by SPSS Statistics 22.0 (IBM Corp., NY, USA). The abundance of leaf spots on the trees was predominantly observed on the young leaves. Symptoms included round or irregular spots on leaves with grayish-red during early infection and later transferred to brown dark spots on the edge of the leaves, accompanied by a pale yellow aperture on the periphery. Sixty diseased leaves (three leaves from each diseased plant) from twenty plants (four plants from each infected field) in the same growth period were collected randomly and cut into small pieces (2 mm × 2 mm), disinfected with 75% alcohol for 30 s and with 1% NaClO for 60 s and rinsed 3 times with ddH2O. The diseased tissues were plated on PDA plate having Streptomycin and then incubated at 25 ˚C for 2 d under 16: 8 h light and dark regime. The single hypha was picked from plates and transferred to a new PDA plate having Streptomycin for purification. Sixty five pure cultures were obtained through single spore isolation and purification. After ITS-BLAST, the results indicated that among the cultures, sixty were Nigrospora sp. (92.31%), two were Flavodon sp. (3.07%) and three were Aureobasidium sp. (4.62%). Thus, the detection rate of Nigrospora sp. was the highest. From sixty obtained isolates with same morphological characters, six isolates (GUCC 23-0016, GUCC23-0017, GUCC23-0018, GUCC24-0244, GUCC24-0245 and GUCC 24-0246) were selected for pathogenicity and identification. Conidia (n = 40) were black, oval or round, smooth and size range was 11-16 × 12-14.5 µm in diameter. Based on disease symptoms and morphological characters, the pathogen was primarily recognized as a Nigrospora sp. (Wang et al. 2017). PCR was performed for each of the six isolates to amplify the internal transcribed spacer (ITS), the large subunit rDNA (LSU), β-tubulin 2 (tub2) and translation elongation factor 1-alpha (tef1) genes with primers sets of ITS4/ITS5 (Innis et al. 1990), LR0R/LR5 (Vilgalys & Hester 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF-986R (Carbone and Kohn 1999), respectively and then PCR products were sequenced. GenBank accession numbers are OR647487, OR647489, OR647490, PQ803982, PQ803983 and PQ804003 for ITS, PQ351182, PQ351183, PQ351185, PQ805435, PQ804004 and PQ805436 for LSU, OR670516, OR670517 OR670518, PQ807000, PQ807001 and PQ807002 for tub2 and OR725093, OR725094 , OR725095, PQ807003, PQ807004 and PQ807005 for tef1. The BLAST results showed that DNA sequences of the present isolates were 100% identical to the type strain of N. aurantiaca (CGMCC 3.18130: KX986064.1, NG_069394, KY019295.1, KY019465.1) and the phylogenetic tree with four gene loci also indicated that our isolates belonged to N. aurantiaca. The pathogenicity test was conducted following Koch's postulates. Ten bayberry trees of approximate ten years old were inoculated with six above isolates (GUCC 23-0016, GUCC23-0017, GUCC23-0018, GUCC24-0244, GUCC24-0245 and GUCC 24-0246) and two healthy trees were used as control. Twenty leaves from each tree were disinfected with 75% ethanol prior to pathogen inoculation. The leaves were inoculated by spraying a conidial suspension (1 × 105 spores/mL) of each isolate, while ddH2O was sprayed on forty control leaves from two trees. Pathogen inoculation was performed under natural field conditions. After 7 d, similar symptoms to those observed in the field were noted on inoculated leaves, while the control leaves were still healthy. We also inoculated bayberry detached leaves in vitro by spraying with conidia suspension (1 × 105 spores/mL) of each isolate, while ddH2O as control. Inoculated leaves were maintained in a greenhouse with temperature ranging from 23-25 ˚C at 75% relative humidity. After 7 d, similar symptoms were observed on the inoculated leaves, while the control remained healthy. Both experiments were repeated three times. The pathogen was re-isolated from both in vivo and in vitro inoculated leaves and were re-identified as N. aurantiaca through morphological identification and molecular identification by code amplification of ITS, LSU, tub2 and tef1. Thus, we proposed that N. aurantiaca caused the leaf spot of bayberry. Leaf spot diseases caused by N. aurantiaca on tobacco, Chinese chestnut and olive were reported in Guizhou (Huang et al. 2021), in Yanyuan (Luo et al. 2020) and Croatia (Petrović et al. 2023), respectively. However, to our knowledge, this is the first report of N. aurantiaca causing leaf spot diseases on bayberry in China. The identification of this disease offered a basis for further research on its proper management.