First Report of Charcoal Rot on Tobacco Caused by Macrophomina phaseolina in Hunan Province, China.

Flue-cured tobacco (Nicotiana tabacum L.) is a significant cash crop globally. In August 2022, necrotic lesions on stem associated with root rot and wilting were observed on flue-cured tobacco (Cv. Yunyan 87) in fields located in Banxin village (27.95N,109.60E) of Fenghuang county in Xiangxi Autonomous Prefecture, Hunan Province, China. The affected and damaged area of tobacco is approximately 10 hectares, with adisease incidence of 60%. Lots of small black speckling within the lower stem of the affected plant, vascular tissue changed to black, dry rot, and looked like charcoal breezes. Small pieces were cut from healthy and diseased tissues, surface sterilized with 5% NaClO for 3 min and 75% ethanol for 1 min, rinsed with sterile distilled water and air-drying, incubated on oat medium incubated at 28℃ for five days. These isolates grew fast and produced typical black microsclerotia. The morphological were septate hyphae and microsclerotia. The microsclerotia were black and regularly round, with a 42.5 - 92.9 µm diameter. These morphological features were consistent with Macrophomina phaseolina (Smith and Wyllie 1999). The internal transcribed spacer (ITS) rDNA and translation elongation factor 1-α (TEF1-α) genes of three representative isolates were amplified and sequenced using the primers ITS1/ITS4 and EF1-728F/EF2R (Machado et al. 2019). Our resulting sequences (GenBank accessions OR435093, OR435101, OR435102 for ITS; OR891780, OR891781 and OR891782 for EF1-α) showed 99-100% similarity with M. phaseolina by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software with the NJ method. The combined sequences grouped with isolates to M. phaseolina with 100% bootstrap support. The strain XF22 has been sent to the China General Microbiological Culture Collection Center (CGMCC3.25349). Pathogenicity tests were conducted by inoculating potted plants (six plants per isolate, three times) from 45 day-old tobacco seedlings cv. Yunyan 87. Stems were randomly gently scratched with sterile needles, and a 5 mm agar disc with mycelium of the pathogen was attached to the surface of each wound, with a sterilized agar disc as control. Inoculated seedlings were incubated in growth chambers at 26℃ and 60% RH with a 12 h photoperiod/day. After ten days, symptoms that brown or black lesions on the inoculated lesions were dotted with numerous black, hard microsclerotia similar to those naturally occurring on the diseased plants, but not on the control plants. The same pathogen was re-isolated consistently, fulfilling Koch's postulates. Based on morphological, molecular, and pathogenicity test results, these isolates were identified as M. phaseolina. Charcoal rot of tobacco, caused by M. phaseolina was previously found in Guangxi in 1989 (Zhu et al. 2002), while this is the first report of M. phaseolina causing charcoal rot on flue-cured tobacco in Hunan, China. We speculate that the planting area is influenced by the preceding crop sesame. The soil carries M. phaseolina, which can cause stem rot of sesame, leading to the occurrence of tobacco charcoal rot. Our results indicated that charcoal rot caused by M. phaseolina is a new threat to flue-cured tobacco production and lue-cured tobacco might be acting as a reservoir and spreading this pathogen to other economically crops in China.

First Report of Black Rot on Persimmon Fruit Caused by Diaporthe eres in China.

The fruit of persimmon (Diospyros kaki L.) is popular with consumers for its delicious taste and is widely grown in China. In September 2022, fruit rot symptoms were found on persimmons grown at the Tobacco Research Institute of Chinese Academy of Agricultural Sciences (120°26'43.879″E, 36°7'59.794″N) in Qingdao City, Shandong Province, China. Diseases incidence on fruit is 70%. Typical symptoms were small, black spots, which gradually expanded, and became rot lesions, causing fruit rot and serious losses. To isolate the pathogen, small sections (approximately 2 × 2 mm) of symptomatic tissues from five persimmon fruits were surface-sterilized in 5% NaClO, followed by 75% ethanol, rinsed with sterile distilled water, and then incubated on potato dextrose agar (PDA) plates incubated at 28℃ for 5 days. By single spore isolation method, seven dominant fungal isolates developed with similar colonies. They were initially white and gradually turned to gray after 7 d. The alpha conidia were unicellular, colorless, elliptic or fusiform, and 5.0 to 8.0 µm × 2.5 to 3.5 µm in size. Beta conidia were unicellular, colorless, filiform, often hooked at one end and 18 to 28 µm × 1.3 to 1.5 µm in size. These morphological features were consistent with Diaporthe spp (Udayanga et al. 2014). To confirm identity of the causal fungus, the internal transcribed spacer (ITS) rDNA, translation elongation factor 1-a (TEF1-a), b-tubulin (TUB), and histone H3 (HIS) genes of three representative isolates (De2, De3 and DeR2) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b and CYLH3F/H3-1b as described by Chaisiri et al. (2021). The resulting sequences were deposited in GenBank (accession nos. OP963195, OP963386 and OP963389 for ITS; OP968958, OP968959 and OP9689600 for EF1-α; OP978141, OP978142 and OP978143 for TUB; OP968955, OP96896 and OP968957 for HIS), and showed 99-100% similarity with D. eres by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software, with the NJ method. In the phylogenetic tree, the combined sequences attributed the three isolates to the D. eres. On the basis of morphological and molecular characteristics, the pathogen was identified as D. eres. The strain De2 was preserved in the Agricultural Culture Collection of China (preservation number: ACCC 35482). Pathogenicity tests were performed by wound inoculation on healthy persimmon fruit. These were surface sterilized with 75% ethanol for 1 min, rinsed in sterile water three times, and allowed to dry. A 5-mm agar disc with mycelium of the causal pathogen was placed on the surface of fruit, which was wounded with a sterile needle in advance, with the sterile PDA plugs used as control. Three persimmons were used for each treatment, repeat three times. All the treated fruits were incubated in the sterilized transparent plastic cans at 28°C and 90% RH. After 3 days inoculation, the first symptom of rot lesion appeared on the fruit with mycelial plugs, whereas the control fruit remained symptomless. The same causal pathogens were re-isolated from the lesions and identified by morphologyand confirmed to fulfill Koch's postulates. This is the first report of D. eres causing fruit rot on persimmon. This finding showed that Diaporthe eres is a causal agent of persimmon fruit rot in China.

Identification, genetic diversity, and pathogenicity of Ralstonia pseudosolanacearum causing cigar tobacco bacterial wilt in China.

Ralstonia pseudosolanacearum, previously known as R. solanacearum species complex (RSSC) phylotypes I and III, is a plant pathogenic bacterium causing significant yield losses in economical crops. In the May of 2020 and 2021, cigar tobacco bacterial wilt was first observed in fields in Danzhou, Hainan Province, China. A total of eight bacterial isolates were isolated and identified as R. pseudosolanacearum with race 1, biovar III by 16S rRNA gene sequencing, Biolog, and host identification. The amino acid sequence showed that Hainan strains and 15 R. pseudosolanacearum reference strains from flue-cured tobacco in Shandong and Guizhou Provinces, all belonged to RS1000 type containing the avrA gene, only Guizhou strains also had the popP1 gene. On the basis of phylotype-specific multiplex PCR amplification, mismatch repair gene and endoglucanase gene-base tree, Hainan strains were identified as phylotype I sequevar 70, and showed stronger pathogenic capabilities on three different varieties than those reference strains. This is the first report of cigar tobacco bacterial wilt caused by R. pseudosolanacearum sequevar 70. The results revealed the diversity of RSSC in Nicotiana tabacum in China and provided useful information regarding the epidemiology of cigar tobacco wilt disease, as well as the breeding for disease resistance in local cigar tobacco.