First report of brown rot of nectarine caused by Monilia yunnanensis in Tibet.

Brown rot caused by Monilinia spp. is one of the most important diseases of stone fruits. To date, three species of Monilinia have been found to occur on Prunus species worldwide: Monilinia fructicola (G. Winter) Honey, Monilinia fructigena (Aderhold & Ruhland) Honey, and Monilinia laxa (Aderhold & Ruhland) Honey (Zhu et al. 2005; Hu et al. 2011a). While M. fructicola is widespread in the Americas, and parts of Europe and Asia (CABI, 2010), M. laxa and M. fructigena are the primary species causing brown rot of peach in Europe (Bryde et al. 1977). In China, a new species Monilia yunnanensis was identified in 2011 (Hu et al. 2011b; Zhao et al. 2013; Yin et al. 2015; Yin et al. 2017). However, the species causing brown rot of nectarine (Prunus persica var. nectarina) in Tibet have not been undertaken. In the summer of 2017-2018, brown rot disease of nectarine was observed in Nyingchi, Tibet, and approximately 30% of nectarines were affected annually. Therefore, the brown rot disease of nectarine is one of the main factors that restrict the yield and quality of nectarine fruit production, and causes severe economic losses in Tibet. Thirty-six nectarine fruit with typical brown rot symptoms were collected from Tibet during the summer of 2017-2018. In order to isolate the causal agent, small pieces of pericarp were disinfected with 75% ethanol for 1 min, and then for 1 min in 1% NaOCl, rinsed in sterile distilled water for three times, dried on sterile paper and placed on potato dextrose agar (PDA). Thirty-six single-spore isolates were obtained and all morphologically similar, and three representative isolates 2-1, 2-16 and 2-31 which were from different period and years in 2017-2018 were characterized phylogenetically and morphologically to identify them to species level. Pathogenicity of each representative isolate was confirmed by inoculating five surface-disinfected mature nectarines with mycelial plugs in the wound of the fruit. Nectarine fruit inoculated with sterile PDA plugs served as the negative control. The inoculated nectarines developed brown lesions after 6 days incubation at 22°C, and the pathogen was successfully re-isolated. There were no symptoms on the control nectarine fruit. The isolates 2-1, 2-16 and 2-31 produced gray-green colonies with even margins and concentric rings of sporogenous mycelium after 3 days incubation, and abundant black-colored stromata on the media after 16 days of incubation at 22°C, resembling those described for M. yunnanensis (Hu et al. 2011b). Conidia were one-celled, hyaline, ellipsoid to lemon shape (9.24 to 15.58 μm), and borne in branched monilioid chains. The average daily growth of mycelium on PDA at 22°C was 11.56 mm. Therefore, the isolates 2-1, 2-16 and 2-31 were preliminarily identified as M. yunnanensis based on the morphological investigations (Hu et al. 2011b). Morphological identification was confirmed by phylogenetic analysis based on sequences of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and β-tubulin (TUB2) genes of 2-1, 2-16 and 2-31 which were amplified using primers Mon-G3pdhF/Mon-G3pdhR and Mon-TubF1/Mon-TubR1 (Hu et al. 2011b). In both G3PDH and TUB2 phylogenetic trees, the isolates 2-1, 2-16 and 2-31 formed monophyletic clades within a derived clade with the M. yunnanensis isolates. Additionally, the three isolates were more closely related to M. yunnanensis (HQ908782.1 and HQ908783.1) than to other Monilinia species. Based on morphological and molecular identification, the isolates 2-1, 2-16 and 2-31 were identified as M. yunnanensis. Previously, M. yunnanensis has been reported as a new species causing brown rot of peach in China (Hu et al, 2011b). To our knowledge, this is the first report of M. yunnanensis causing nectarine fruit brown rot in Tibet. These findings suggest that M. yunnanensis is spreading on its principal host plants and causing substantial economic losses in the Tibet fruit production.