First Report of Apple Brown Rot caused by Monilinia polystroma in Jilin Province in China.

Abstract

Brown rot caused by Monilinia spp. is an important postharvest disease. It affects fruit quality and can cause serious economic losses. In October 2021, typical brown rot symptoms on fruit were observed at an apple orchard in Xiaobaishan Township, Jilin Province, China (E126°39'10″, N43°44'21″). Over 1200 plants were surveyed in the orchard, and nearly 25% of the plants were infected. In this research, samples from ten different trees showing typical symptoms were isolated and identified. Freshly diseased fruits were surface sterilized with 75% ethanol for 15s, then fungal colonies were isolated from 3 mm diameter diseased tissue samples. The purified colonies were placed on potato dextrose agar (PDA), oatmeal agar (OA) and water agar (WA + Sterilized apple pulp) and incubated at 25 ℃ in a 12 h/12 h light-dark photoperiod for 5 days. The colonies became light to dark brown; they grew faster on PDA with a growth rate of 5.53 mm/d, most densely on OA and slowest on SA + sterilized apple pulp with thin mycelia. After 20 days, some transparent, oval, smooth conidia were observed on the SA + sterile apple culture medium. Conidia sizes were 8.27-16.54 (avg. 11.97) x 2.92-7.09 (avg. 4.52) um (n=50) (Hilber-Bodmer et al. 2012). Pure cultures of eight samples were isolated from single spores, and DNA was extracted with a commercial nucleic acid extraction kit (Omega, cat#D3390-01). Then, fragments of the internal transcribed spacer region (ITS), translation elongation factor 1 alpha (EF1-alpha), beta-tubulin (Bt), and glyceraldehyde-3-phosphate dehydrogenase genes(G3pdh) were PCR amplified using primes ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), BT-2a/BT-2b (Glass and Donaldson 1995), and G3pdhF/G3pdhR (Hu et al. 2011) respectively. The amplicons were sequenced by Takara Bio Inc. Sequences of all isolates were identical, thus one set of sequences were run with BLASTn against the NCBI GenBank nr database. The homology analysis showed that ITS (OQ170786), Bt (OQ179019), EF (OQ834046) and G3pdh (OQ834047) gene fragments were 100% (0/540 nt) , 100% (0/462 nt), 99.01% (3/304nt) and 99.61% (3/767 nt) similar to M. polystroma （NR154198.1, MT038414.1, LT632542.1, MT038415.1）respectively. Based on morphological characteristics and sequence analysis, the fungal isolate was identified as M. polystroma. Koch's postulates were conducted using ten healthy apple fruits that were surface disinfected with 0.2% sodium hypochlorite and repeatedly rinsed with sterile water. The test apples were wounded with a sterile needle, inoculated with mycelial agar plugs (3 mm diameter) on the wounds, and incubated at 25℃, 50% room humidity. The equivalent sterile PDA plugs were used as control and the experiment was repeated three times. After 5 days, brown rot symptoms appeared on the inoculated apples. 10 days later, nearly 1/3 of the inoculated apple surface appeared rotted, but the controls were symptomless. Subsequently, the same strain was re-isolated from the inoculated apples by pure culture and molecular identification. Therefore, M. polystroma (named JL-1) was confirmed as the causal agent of brown rot in Jilin Provincen China. M. polystroma is a typical pathogen of brown rot in the north of China, and only reported on apples in Shandong, apricots in Heilongjiang and pears in Hebei in China (Zhu et al. 2016) but never in Jilin. In addition, it was reported that the contribution of M. polystroma species to brown rot disease on apple and pear in China is 20% out of all the Monilinia spp. species that cause the disease, but M. polystroma virulence is not significantly different from other Monilinia species more widely distributed (Zhu et al, 2016). This is the first report of M. polystroma causing apple brown rot in Jilin Province of China. This finding will provide useful information for future diagnosis and management.