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Cigar tobacco (Nicotiana tabacum L.) is widely planted in Yunnan, which is becoming an important economic crop in China. In March 2023, root rot of cigar tobacco (cv. Yunxue 38) was observed in Baoshan (98°51'E, 24°58'N), and in July 2022 root rot of tobacco (cv. Yunyan 87) was observed in Dali (99°54'E, 26°30'N), Yunnan Province, China. The average disease incidences surveyed in the fields reached 10%. At the early stage, the bottom leaves showed wilting and turned yellow, and the roots became brown. Following the disease development, the color of roots turned to dark brown and ultimately necrosis. To isolate the causal agent, small pieces (5×5 mm) of diseased root from 6 symptomatic plant samples (three samples of cv. Yunxue 38 and three samples of cv. Yunyan 87) were cut. Pieces were surface-sterilized by dipping in 75% ethanol for 30 s, rinsed three times with sterile distilled water, then transferred to potato dextrose agar (PDA) medium and incubated at 28°C in the dark. Six fungal isolates cultured for 14 days were obtained. They were morphologically similar, so a representative isolate was selected for the following experiment. The colonies grew slowly on PDA, and their color were light pink initially, then changed to amaranth. Hyphae were hyaline and septate. Microconidia were hardly produced on PDA plates. After 14 days of culture on V8 juice agar, the colonies showed white aerial mycelia, and ellipsoidal and transparent conidia were observed, which measured 6.5 to 8.3 × 3.4 to 5.0 µm (n=20). Also, the pycnidia were measured 150 to 220 µm, that were subglobose in dark brown with brown setae. These morphological characteristics of 22DL91 were identical to S. terrestris (Boerema et al. 2004). For molecular identification, DNA was extracted and the PCR products of ITS region and polymerase II second largest subunit (RPB2), amplified with the primers ITS1/ITS4 and RPB2-5F/RPB2-7cR, were sequenced. By BLASTn analysis, the obtained ITS sequences showed 100% homology and the RPB2 sequences showed 95% homology with S. terrestris strains in GenBank (accession ON006851 and OM417590). The sequences were deposited in NCBI with accession numbers OR539491 (ITS) and OR554276 (RPB2), respectively. Based on the morphology and phylogenetic analysis, the isolate was 22DL91 identified as S. terrestris. Pathogenicity was evaluated on 50-day-old cigar tobacco seedlings (cv. Yunxue 38) and tobacco seedlings (cv. Yunyan 87). Ten plants were inoculated with 20 mL of conidial suspension of 105 conidia/mL poured onto the roots and ten control seedlings dipped in sterile water as controls (Luo et al. 2023). After 14 days, all inoculated seedlings showed the symptoms with leaves yellowing and root rot, whereas the control seedlings had no symptoms. Moreover, the fungus S. terrestris was reisolated from the infected roots, fulfilling Koch's postulates. This fungus was previously known to cause pink root on garlic in China (Zhang et al. 2019). To our knowledge, this is the first report of S. terrestris causing root rot of Nicotiana tabacum in China. Therefore, this finding will provide valuable information for prevention and management of root rot on tobacco.
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Tobacco (Nicotiana tabacum L.) was an important economic crop in China. A survey in Yunnan Province in the last several years showed that the incidence of tobacco root rot was 3 to 30%. In July 2021, root rot symptoms were observed with an average incidence of 5% on tobacco (cultivar Yunyan 87) in Dali (25.61° N, 100.27° E). Typical disease symptoms included plants stunted at early stages, brown-colored withering lower leaves and roots that became brown. Under high humidity conditions, symptoms of rot expanded in the roots, also the whole plant became wilted and stunted, and some plants ultimately died. Infected pieces of stem tissues and root were dissected and then sterilized with 2% NaOCl for 30 s, rinsed three times with sterile distilled water, and dried with sterilized filter paper. Three pieces were plated onto potato dextrose agar (PDA) for 3 days at 25°C with a 12-h light period. Colonies on PDA were characterized by white to pale yellow flocculent aerial mycelium, and a pink to red pigment in the agar. To induce sporulation, mycelium on PDA was transferred to carnation leaf agar (CLA) medium. After incubation for 7 days, a single spore was isolated from representative isolate 21DL16 for morphological and molecular analyses. Macroconidia observed on CLA were falcate, slightly curved, three to five septate, measured 33.1 to 53.7 × 3.2 to 4.6 µm (n=50), with a typical foot shaped basal cell. Morphological characteristics of the fungus were in agreement with the description of Fusarium graminearum (Leslie and Summerell 2006). For further identification, the internal transcribed spacer (ITS) region rDNA, translation elongation factor 1É (EF-1α) and RNA polymerase II second largest subunit (RPB2) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell et al. 2015) and RPB2-5F/RPB2-7cR (Reeb et al. 2004), respectively. Although the ITS sequence (GenBank accession no. OM392025) cannot distinguish F. meridionale from F. graminearum, combined phylogenetic analysis of the sequence of TEF1 (ON062055) and RPB2 (ON211932) clearly showed that the pathogen is F. meridionale that the sequences were 100% similarity, 0.0e-value and 100% query coverage to F. meridionale. Pathogenicity studies were conducted on six-leaf-stage tobacco seedlings cultivar Yunyan 87. A conidial suspension (1×105 spores/mL) was poured over the roots of tobacco seedlings. Three seedlings were treated with sterile water that served as controls. All 10 seedlings were maintained at 25°C at 70% relative humidity. After 5 days, the lower leaves showed symptoms of wilting and the roots of all inoculated seedlings become discolored, that were similar with the original symptoms, whereas the control seedlings did not develop symptoms. The fungus reisolated from the inoculated seedlings was identical to F. meridionale using the EF-1α gene sequence. To date, Fusarium root rot on tobacco in China was caused by F. oxysporium (Chen 2013). However, to the best of our knowledge, this is the first report of F. meridionale causing root rot on tobacco in China. Identification of F. meridionale as a root rot agent might provide important insight for disease management practices on tobacco caused by Fusarium species.
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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 C16ââ:ââ1 ω7c (summed feature 3), C16ââ:ââ0 and C18ââ:ââ1 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 (98.36â%) and R. pickettii K-288T (98.22â%). The 16S rRNA gene sequence identities between strain 56D2T and other members of the genus Ralstonia were below 98.00â%. 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 95â%, and the in silico DNA-DNA hybridization values (yielded by formula 2) were less than 70â%. 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).
Asunto(s)
Ácidos Grasos , Fosfolípidos , Ácidos Grasos/química , Nicotiana , Ralstonia/genética , ARN Ribosómico 16S/genética , China , Análisis de Secuencia de ADN , Composición de Base , Filogenia , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Bacterias/genéticaRESUMEN
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 (99.92â%), P. capsici Pc19-1T (99.67â%) and P. ovata F51T (98.94â%) . 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 95â%, and the in silico DNA-DNA hybridization (isDDH) values (yielded by formula 2) were less than 70â%. The major fatty acids were C16ââ:ââ1ω7c and/or C16ââ:ââ1ω6c (summed feature 3), C16ââ:ââ0 and C18ââ:ââ1ω7c and/or C18ââ:ââ1ω6c (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).
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Fosfatidiletanolaminas , Pseudomonas , ARN Ribosómico 16S/genética , Filogenia , Composición de Base , Nicotiana , ADN Bacteriano/genética , Cardiolipinas , Técnicas de Tipificación Bacteriana , Ácidos Grasos/química , Genes Bacterianos , Análisis de Secuencia de ADN , China , Fosfolípidos , Fosfatidilcolinas , Glucolípidos , Quinonas , NucleótidosRESUMEN
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°3'26â³, E 98°25'6â³) 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.
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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%.
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Nicotiana , Ralstonia solanacearum , China , Filogenia , Enfermedades de las Plantas , RalstoniaRESUMEN
Many Gram-negative bacteria employ N-acylhomoserine lactones (AHLs) as quorum-sensing (QS) signal molecules to regulate virulence expression in a density-dependent manner. Quorum quenching (QQ) via enzymatic inactivation of AHLs is a promising strategy to reduce bacterial infections and drug resistance. Herein, a thermostable AHL lactonase (AidB), which could degrade different AHLs, with or without a substitution of carbonyl or hydroxyl at the C-3 position, was identified from the soil bacterium Bosea sp. strain F3-2. Ultrahigh-performance liquid chromatography analysis demonstrated that AidB is an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone (HSL) ring. AidB was thermostable in the range 30 to 80°C and showed maximum activity after preincubation at 60°C for 30 min. The optimum temperature of AidB was 60°C, and the enzyme could be stably stored in double-distilled water (ddH2O) at 4°C or room temperature. AidB homologs were found only in Rhizobiales and Rhodospirillales of the Alphaproteobacteria AidB from Agrobacterium tumefaciens and AidB from Rhizobium multihospitium (with amino acid identities of 50.6% and 52.8% to AidB, respectively) also showed thermostable AHL degradation activity. When introduced into bacteria, plasmid-expressed AidB attenuated pyocyanin production by Pseudomonas aeruginosa PAO1 and the pathogenicity of Pectobacterium carotovorum subsp. carotovorum Z3-3, suggesting that AidB is a potential therapeutic agent by degrading AHLs.IMPORTANCE A quorum-sensing system using AHLs as the signal in many bacterial pathogens is a critical virulence regulator and an attractive target for anti-infective drugs. In this work, we identified a novel AHL lactonase, AidB, from a soil bacterial strain, Bosea sp. F3-2. The expression of aidB reduced the production of AHL signals and QS-dependent virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum The homologs of AidB with AHL-degrading activities were found only in several genera belonging to the Alphaproteobacteria Remarkably, AidB is a thermostable enzyme that retained its catalytic activity after treatment at 80°C for 30 min and exhibits reliable storage stability at both 4°C and room temperature. These properties might make it more suitable for practical application.
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Bradyrhizobiaceae/enzimología , Bradyrhizobiaceae/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , 4-Butirolactona/análogos & derivados , Acil-Butirolactonas/metabolismo , Agrobacterium tumefaciens/metabolismo , Secuencia de Aminoácidos , Bacterias/metabolismo , Proteínas Bacterianas , Bradyrhizobiaceae/genética , Estabilidad de Enzimas , Pectobacterium carotovorum/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Percepción de Quorum , Virulencia , Factores de Virulencia/metabolismo , Secuenciación Completa del GenomaRESUMEN
A Gram-negative, aerobic, rod-shaped, motile bacterium with multi-flagella, strain RST, was isolated from bacterial wilt of tobacco in Yuxi city of Yunnan province, China. The strain contains the major fatty acids of C16:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The polar lipid profile of strain RST consists of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified aminophospholipid. Strain RST contains ubiquinones Q-7 and Q-8. 16S rRNA gene sequence (1,407 bp) analysis showed that strain RST is closely related to members of the genus Ralstonia and shares the highest sequence identities with R. pseudosolanacearum LMG 9673T (99.50%), R. syzygii subsp. indonesiensis LMG 27703T (99.50%), R. solanacearum LMG 2299T (99.28%), and R. syzygii subsp. celebesensis LMG 27706T (99.21%). The 16S rRNA gene sequence identities between strain RST and other members of the genus Ralstonia were below 98.00%. Genome sequencing yielded a genome size of 5.61 Mbp and a G + C content of 67.1 mol%. The genomic comparison showed average nucleotide identity (ANIb) values between strain RST and R. pseudosolanacearum LMG 9673T, R. solanacearum LMG 2299T, and R. syzygii subsp. indonesiensis UQRS 627T of 95.23, 89.43, and 91.41%, respectively, and the corresponding digital DNA-DNA hybridization (dDDH) values (yielded by formula 2) were 66.20, 44.80, and 47.50%, respectively. In addition, strains belonging to R. solanacearum phylotype I shared both ANIb and dDDH with strain RST above the species cut-off values of 96 and 70%, respectively. The ANIb and dDDH values between the genome sequences from 12 strains of R. solanacearum phylotype III (Current R. pseudosolanacearum) and those of strain RST were below the species cut-off values. Based on these data, we concluded that strains of phylotype I, including RST, represent a novel species of the genus Ralstonia, for which the name Ralstonia nicotianae sp. nov. is proposed. The type strain of Ralstonia nicotianae sp. nov. is RST (=GDMCC 1.3533T = JCM 35814T).
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IMPORTANCE: DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.
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Ralstonia solanacearum , Ralstonia solanacearum/genética , ADN Bacteriano/genética , ADN Bacteriano/análisis , Reacción en Cadena de la Polimerasa/métodos , Enfermedades de las Plantas/microbiologíaRESUMEN
The Fic (filamentation induced by cyclic AMP) domain is a widely distributed motif with a conserved sequence of HPFx[D/E]GN[G/K]R, some of which regulate cellular activity by catalyzing the transfer of the AMP moiety from ATP to protein substrates. Some Fic proteins, including Fic-1 from the soil bacterium Pseudomonas fluorescens strain 2P24, have been shown to inhibit bacterial DNA replication by AMPylating the subunit B of DNA gyrase (GyrB), but the biochemical activity and cellular target of most Fic proteins remain unknown. Here, we report that Fic-2, which is another Fic protein from strain 2P24 and Fic-1 AMPylate the topoisomerase IV ParE at Tyr109. We also examined Fic proteins from several phylogenetically diverse bacteria and found that those from Yersinia pseudotuberculosis and Staphylococcus aureus AMPylate ParE and GrlB, the counterpart of ParE in Gram-positive bacteria, respectively. Modification by Fic-1 of P. fluorescens and FicY of Y. pseudotuberculosis inhibits the relaxation activity of topoisomerase IV. Consistent with the inhibition of ParE activity, ectopic expression of these Fic proteins causes cell filamentation akin to the canonical par phenotype in which nucleoids are assembled in the center of elongated cells, a process accompanied by the induction of the SOS response. Our results establish that Fic proteins from diverse bacterial species regulate chromosome division and cell separation in bacteria by targeting ParE.
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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.