Study on the effect of enzymatic treatment of tobacco on HnB cigarettes and microbial succession during fermentation.

Starch and cellulose are the fundamental components of tobacco, while their excessive content will affect the quality of tobacco. Enzymatic treatment with different enzymes is a promising method to modulate the chemical composition and improve the sensory quality of tobacco leaves. In this study, enzymatic treatments, such as amylase, cellulase, and their mixed enzymes, were used to improve tobacco quality, which could alter the content of total sugar, reducing sugar, starch, and cellulose in tobacco leaves. The amylase treatment changed surface structure of tobacco leaves, increased the content of neophytadiene in tobacco by 16.48%, and improved the total smoking score of heat-not-burn (HnB) cigarette products by 5.0 points compared with the control. The Bacillus, Rubrobacter, Brevundimonas, Methylobacterium, Stenotrophomonas, Acinetobacter, Pseudosagedia-chlorotica, and Sclerophora-peronella were found to be significant biomarkers in the fermentation process by LEfSe analysis. The Basidiomycota and Agaricomycetes were significantly correlated with aroma and flavor, taste, and total score of HnB. The results showed that microbial community succession occurred due to amylase treatment, which promoted the formation of aroma compounds, and regulated the chemical composition of tobacco, and improved tobacco quality during tobacco fermentation. This study provides a method for enzymatic treatment to upgrade the quality of tobacco raw materials, thereby improving the quality of HnB cigarettes, and the potential mechanism is also revealed by chemical composition and microbial community analysis. KEY POINTS: Enzymatic treatment can change the chemical composition of tobacco leaves. The microbial community was significantly affected by enzymatic treatment. The quality of HnB cigarettes was significantly improved by amylase treatment.

Ralstonia wenshanensis sp. nov., a novel bacterium isolated from a tobacco field in Yunnan, China.

A Gram-negative, aerobic, motile with paired polar flagella and rod-shaped bacterium strain (56D2T) was isolated from tobacco planting soil in Yunnan, PR China. Major fatty acids were C16  :  1 ω7c (summed feature 3), C16  :  0 and C18  :  1 ω7c (summed feature 8). The polar lipid profile of strain 56D2T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid and one unidentified glycolipid. Moreover, strain 56D2T contained ubiquinone Q-8 as the sole respiratory quinone. 16S rRNA gene sequence analysis showed that strain 56D2T was closely related to members of the genus Ralstonia and the two type strains with the highest sequence identities were R. mannitolilytica LMG 6866T (98.36 %) and R. pickettii K-288T (98.22 %). The 16S rRNA gene sequence identities between strain 56D2T and other members of the genus Ralstonia were below 98.00 %. Genome sequencing revealed a genome size of 5.87 Mb and a G+C content of 63.7 mol%. The average nucleotide identity values between strain 56D2T and R. pickettii K-288T, R. mannitolilytica LMG 6866 T and R. insidiosa CCUG 46789T were less than 95 %, and the in silico DNA-DNA hybridization values (yielded by formula 2) were less than 70 %. Based on these data, we conclude that strain 56D2T represents a novel species of the genus Ralstonia, for which the name Ralstonia wenshanensis sp. nov. is proposed. The type strain of Ralstonia wenshanensis sp. nov. is 56D2T (=CCTCC AB 2021466T=GDMCC 1.2886T=JCM 35178T).

Pseudomonas lijiangensis sp. nov., a novel phytopathogenic bacterium isolated from black spots of tobacco.

Three Gram-stain-negative, motile, with amphilophotrichous flagella, and rod-shaped bacteria (LJ1, LJ2T and LJ3) were isolated from lower leaves with black spots on flue-cured tobacco in Yunnan, PR China. The results of phylogenetic analysis based on 16S rRNA gene sequences indicate that all the strains from tobacco were closely related to the type strains of the Pseudomonas syringae group within the P. fluorescens lineage and LJ2T has the highest sequence identities with P. cichorii DSM 50259T (99.92 %), P. capsici Pc19-1T (99.67 %) and P. ovata F51T (98.94 %) . The 16S rRNA gene sequence identities between LJ2T and other members of the genus Pseudomonas were below 98.50%. The average nucleotide identity by blast (ANIb) values between LJ2T and P. cichorii DSM 50259T, P. capsici Pc19-1T and P. ovata F51T were less than 95 %, and the in silico DNA-DNA hybridization (isDDH) values (yielded by formula 2) were less than 70 %. The major fatty acids were C16  :  1ω7c and/or C16  :  1ω6c (summed feature 3), C16  :  0 and C18  :  1ω7c and/or C18  :  1ω6c (summed feature 8). The polar lipids profile of LJ2T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids and one unidentified glycolipid. The predominant respiratory quinone was Q-9. The DNA G+C content of LJ2T was 58.4 mol%. On the basis of these data, we concluded that LJ2T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas lijiangensis sp. nov. is proposed. The type strain of Pseudomonas lijiangensis sp. nov. is LJ2T (=CCTCC AB 2021465T=GDMCC 1.2884T=JCM 35177T).

First Report of Pectobacterium versatile Causing Stem Rot of Tobacco in China.

Tobacco is one of the most significant non-food cash crops (Lu et al. 2020). In March 2022, cigar tobacco plants showing characteristic symptoms of vascular discoloration, stem rotting, leaf wilting and rotting were observed in Tengchong city (N 25°3'26″, E 98°25'6″) of Yunnan province, China (Fig. S1). The disease incidence was about 5% on cultivar Yunxue 6 in a 33-ha field. Infected stems were collected from Tengchong for pathogen isolation and 16S rDNA sequence analysis was performed as previously described (Lu et al. 2021). Sequence analysis showed that tobacco isolates (GenBank accession numbers: ON795108, ON795107 and ON795106) had an identical sequence with that of the species type strain of Pectobacterium versatile CFBP 6051T and shared the sequence identities of 99.55% and 99.47% with P. carotovorum DSM 30168T and P. parvum s0421T, respectively. Furthermore, phylogenetic analysis showed that tobacco strains were clustered with Pectobacterium versatile CFBP 6051T (Fig. S2a). In API assays, strain 22TC1 was positive for ß-galactosidase activity, reduction of nitrates to nitrites, fermentation of glucose, hydrolysis of esculin and gelatin, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, malic acid and trisodium citrate; positive for the enzymatic substrates of alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, ß-galactosidase and α-glucosidase. Furthermore, the average nucleotide identity (ANI) analysis (Richter et al. 2015) showed that strain 22TC1 (GenBank accession number: JAMWYQ000000000) had the highest ANIb score of 96.76% and ANIm value of 97.19% with P. versatile CFBP 6051T. Similarly, in silico DNA-DNA hybridization (isDDH) value was 74.5% compared to P. versatile CFBP 6051T, isDDH values were 35.5-63.7% with the other Pectobacterium species, which below the 70% threshold value for species delineation (Meier-Kolthoff et al. 2021). The phylogenomic analysis also showed that strain 22TC1 was clustered with the species type strain of P. versatile CFBP 6051T. For pathogenicity tests, cell suspension with ten-fold dilution (approx. 1 x 108 CFU/ml) was injected into the leaf axils of two 2-month-old tobacco stems (cv. Yunyan 87). As a control, tobacco seedlings were inoculated with sterile distilled water. The plants were sealed in plastic bags and maintained in a growth chamber at 28°C for 2 d. The symptoms of water-soaked decay were observed within 24 h of inoculation. Whole-plant decay was at 2 days after injection. No symptoms were developed in the controls. Reisolation was performed on diseased stems and the identity of isolated bacteria was confirmed by PCR and sequencing of 16S rRNA. Similar results were obtained in two independent experiments. Based on the above-described data, the causal pathogen of stem rot on cigar tobacco in Tengchong was identified as P. versatile. To our knowledge, this is the first time that P. versatile is found to cause stem rot on tobacco. Pectobacterium species have been reported to cause seed-borne diseases on tobacco seedlings in the floating tray system and soil-borne diseases in tobacco fields (Wang et al. 2017; Xia and Mo 2007). Therefore, studying the possible transmission of the P. versatile to tobacco plants is necessary.

Complete Genome Sequence of Ralstonia syzygii subsp. indonesiensis Strain LLRS-1, Isolated from Wilted Tobacco in China.

Here, we present the complete genome sequence and annotation of Ralstonia syzygii subsp. indonesiensis strain LLRS-1, which caused bacterial wilt on flue-cured tobacco in Yunnan Province, southwest China. Strain LLRS-1 is the first R. syzygii strain identified to be pathogenic to tobacco in China. The completely assembled genome of strain LLRS-1 consists of a 3,648,314-bp circular chromosome and a 2,046,405-bp megaplasmid with 5,190 protein-coding genes, 55 transfer RNAs, 28 small RNAs, 3 structural RNAs (5S, 16S, and 23S), and a G+C content of 67.05%.

A novel putative betapartitivirus isolated from the plant-pathogenic fungus Rhizoctonia solani.

In this study, we describe the genome sequence of a novel double-stranded RNA (dsRNA) mycovirus, designated as "Rhizoctonia solani partitivirus 15" (RsPV15), from the phytopathogenic fungus Rhizoctonia solani. RsPV15 consists of two genomic double-stranded RNA segments, dsRNA-1 and dsRNA-2, which are 2433 bp and 2350 bp long, respectively. Each of the dsRNA segments contains a single open reading frame, encoding the putative RNA-dependent RNA polymerase and coat protein, respectively. Homology searches and phylogenetic analysis suggested that RsPV15 is a new member of the genus Betapartitivirus within the family Partitiviridae.

Ralstonia chuxiongensis sp. nov., Ralstonia mojiangensis sp. nov., and Ralstonia soli sp. nov., isolated from tobacco fields, are three novel species in the family Burkholderiaceae.

Eight Gram-negative, aerobic, motile with paired polar flagella and rod-shaped bacteria were isolated from six tobacco fields in Yunnan, PR China. 16S rRNA gene sequence analysis revealed that all the strains belonged to the genus Ralstonia. Among them, strain 22TCCZM03-6 had an identical 16S rRNA sequence to that of R. wenshanensis 56D2T, and the other strains were closely related to R. pickettii DSM 6297T (98.34­99.86%), R. wenshanensis 56D2T (98.70­99.64%), and R. insidiosa CCUG 46789T (97.34­98.56%). Genome sequencing yielded sizes ranging from 5.17 to 5.72 Mb, with overall G + C contents of 63.3­64.1%. Pairwise genome comparisons showed that strain 22TCCZM03-6 shared average nucleotide identity (ANI) and digital DNA­DNA hybridization (dDDH) values above the species cut-off with R. wenshanensis 56D2T, suggesting that strain 22TCCZM03-6 is a special strain of the R. wenshanensis. Five strains, including 21MJYT02-10T, 21LDWP02-16, 22TCJT01-1, 22TCCZM01-4, and 22TCJT01-2, had ANI values >95% and dDDH values >70% when compared with each other. These five strains had ANI values of 73.32­94.17% and dDDH of 22.0­55.20% with the type strains of the genus Ralstonia individually, supporting these five strains as a novel species in the genus Ralstonia. In addition, strains 21YRMH01-3T and 21MJYT02-11T represent two independent species. They both had ANI and dDDH values below the thresholds for species delineation when compared with the type species of the genus Ralstonia. In strains 21YRMH01-3T and 21MJYT02-10T, the main fatty acids were summed features 3, 8, and C16:0; however, strain 21MJYT02-11T contained C16:0, cyclo-C17:0, and summed features 3 as major fatty acids. The main polar lipids, including diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine, were identified from strains 21YRMH01-3T, 21MJYT02-10T, and 21MJYT02-11T. The ubiquinones Q-7 and Q-8 were also detected in these strains, with Q-8 being the predominant quinone. Based on the above data, we propose that the eight strains represent one known species and three novel species in the genus Ralstonia, for which the names Ralstonia chuxiongensis sp. nov., Ralstonia mojiangensis sp. nov., and Ralstonia soli sp. nov. are proposed. The type strains are 21YRMH01-3T (=GDMCC 1.3534T = JCM 35818T), 21MJYT02-10T (=GDMCC 1.3531T = JCM 35816T), and 21MJYT02-11T (=GDMCC 1.3532T = JCM 35817T), respectively.