The bacterial strain Chryseobacterium endophyticum Cas268 was isolated from tobacco rhizosphere soil in China. In this study, we report the draft genome sequence of this strain. The genome measures 4,299,272 bp in length and has a G + C content of 39.56%.

Ralstonia solanacearum is a destructive soil-borne pathogen that causes substantial yield losses in tobacco. Elucidating the structural basis of host resistance is therefore crucial for breeding durable resistance. In this study, we compared the bacterial wilt-susceptible variety Cuibi-1 (CB-1) and its near-isogenic line, the resistant tobacco mutant KCB-1 at the histological and biochemical levels to identify tissue-level determinants of resistance. Our results demonstrate that KCB-1 roots possessed constitutively higher levels of cellulose and pectin than CB-1, and maintained greater lignin stability upon R. solanacearum inoculation. These structural features collectively contribute to a reinforced physical barrier against pathogen ingress. However, neither the number nor the area of xylem conduits in roots correlated with resistance, suggesting that macroscopic anatomical features are not primary determinants. Notably, compared with CB-1, KCB-1 exhibited significantly lower bacterial colonization in both roots and stem xylem vessels, accompanied by higher stem polyphenol content, which likely inhibits pathogen-mediated cell wall degradation. Furthermore, the smaller xylem pit membrane apertures observed in KCB-1 may physically restrict the movement of R. solanacearum between adjacent xylem conduits. In addition, the expression levels of multiple defense-related genes were significantly upregulated in KCB-1 upon inoculation. Collectively, these findings reveal that resistance in KCB-1 is associated with a combination of cell wall reinforcement, chemical barriers, physical restrictions at the xylem level, and enhanced expression of defense-related genes. This study provides a theoretical basis and identifies candidate phenotypic traits for breeding bacterial wilt-resistant tobacco varieties.

We report here the draft genome sequence of Enterobacter ludwigii Cas398, a bacterial strain derived from the rhizosphere soil of tobacco in China. The genome is 4,626,841 bp in size, with a G + C content of 54.98%, and encodes 4,321 protein-coding genes.

Microplastics pose a serious threat to the stability of agricultural ecosystems by disrupting soil element cycling and plant growth. However, the extent and mechanisms of these impacts depend on microplastic biodegradability and remain poorly understood. Here, we conducted a mesocosm experiment to evaluate the influences of a biodegradable microplastic (poly(butylene adipate-co-butylene terephthalate), PBAT) and three conventional microplastics with distinct side-chain structures (polystyrene, PS; polyethylene, PE; polypropylene, PP) on soil-plant systems. We found that PBAT exerted more severe phytotoxic effects than conventional microplastics, leading to pronounced reductions in shoot height and root length, alongside the greatest declines in soil nitrate nitrogen. While conventional microplastics increased bacterial diversity and network robustness, PBAT fundamentally reshaped community assembly by reducing the importance of drift and homogeneous selection while intensifying dispersal limitation, thereby narrowing the ecological niche breadth. These assembly shifts were accompanied by distinct bacterial colonizers: PBAT enriched putative plastic-degrading taxa, whereas conventional microplastics favored oligotrophic groups with diminished element-cycling capacities. Structural equation modelling further confirmed that microplastic biodegradability exerted direct negative effects on soil nutrients and plant growth. Collectively, our findings uncover type-dependent microbial mechanisms linking microplastic properties to soil-plant ecosystem function, highlighting that biodegradable microplastics can exert stronger negative effects than conventional ones through distinct microbial assembly pathways.

Alkali/urea solvents enable rapid dissolution of chitin and chitosan, advancing homogeneous chemical modification and functional materials construction. Nevertheless, the pronounced tendency of these solutions to gel at elevated temperatures critically limits their utility in thermally driven reactions. We demonstrate that initially dissolved chitin chains retain residual inter-chain associations, facilitating hydrogen-bond-driven crosslinking and aggregation during heating. To circumvent this limitation, we introduced a post-dissolution low-temperature conditioning strategy (-20 °C for ≥24 h). This treatment profoundly enhances molecular dispersion, yielding solutions with exceptional thermal stability that retain fluidity for >24 h at 55 °C. Leveraging this stabilized system, we achieved efficient homogeneous alkylation, carboxylation, and quaternization of chitin at 55 °C. The reactions proceeded with high efficiency, affording derivatives with degrees of substitution (DS) exceeding 90%. Critically, quaternized chitin exhibited excellent water solubility. This work resolves a fundamental limitation in chitin processing and versatile and efficient route for thermally demanding homogeneous chemistry using alkali/urea solvents, broadening their applicability in biopolymer functionalization.

Sweet potato is a crop with high yield and adaptability that plays a crucial role in agricultural production and daily life. However, in recent years, the yield and quality of sweet potatoes have been increasingly affected by pathogens, such as fungi, bacteria, and viruses. Rapid and accurate identification and detection of diseases is critical for sustainable sweet potato production. This review first provides a systematic overview of the common diseases that affect sweet potato production, including their pathogens, symptoms, and geographical distribution. Subsequently, we discuss the advantages and challenges of traditional methods, molecular biology-based techniques, and emerging detection technologies for the identification of sweet potato diseases. We believe that the integration of new materials and technologies with artificial intelligence to develop rapid, sensitive, and accurate field-detection techniques or portable detection devices for sweet potato diseases represents a promising direction for future research.

Green manure rotation and alkaline amendments have been extensively utilized in soil remediation and disease control, with notable effects. Despite their effectiveness, the specific health-promoting mechanisms of these soil management practices remain unclear. This study investigated the assembly and function of root-associated microbial communities in a five-year field treated with green manure rotation and lime amendments. Compared to the control (CK), the disease incidence decreased by 20.48 % and 43.18 % in the green manure rotation (VV) and combined green manure rotation and lime amendment (VL) treatments, respectively. Meanwhile, the VL treatment notably reduced the soil density and instant nitrogen, while enhancing pH levels, effective phosphorus, exchangeable calcium content, ventilation porosity, and catalase activity. Additionally, VL treatment reduced the diversity and co-occurrence networks of root-associated bacterial communities. Crucially, functional genes related to the nitrogen (e.g., nitrogen fixation) and carbon (e.g., including carbon fixation and degradation) cycles were enriched in the VV and VL treatments. Notably, genera such as Chryseobacterium and Pseudomonas, were significantly enriched in the VL treatment. Experimental validation revealed that the strain Chryseobacterium sp. Cas268 was particularly abundant in the VL treatment, aiding host resistance bacterial wilt, which may adopt various defence mechanisms for protection, including inhibiting the expression of virulence genes, reducing biofilm formation of pathogens, and rapid growth rates. In summary, this study indicated that green manure rotation and lime amendments enhance plant disease resistance by modifying soil physiochemical properties and enriching beneficial bacteria and offers insights into environmentally friendly approaches to disease control in agricultural systems.

A bacterial strain Pseudomonas mediterranea Cas656 was isolated from the rhizosphere soils of tobacco in China. Here, we present the complete genome sequence of P. mediterranea Cas656. The genome comprises a 6,245,034 bp circular chromosome, with G + C contents of 60.92%.

Plant extracts and absolutes have high application value in several industries such as medicine, food, and fragrance. Especially in the field of fragrance, while there is expensive, they are prized by perfumers and provide a rich and lasting aroma. Owing to advancements in extraction technology, their yields have increased and their ingredients have become richer. However, no extraction technology is universal and each extraction technology has its own distinct advantages and disadvantages. Therefore, this review systematically characterizes the extraction technologies for plant extracts and absolutes, including traditional extraction technologies, such as maceration, percolation, reflux, and Soxhlet extraction, and green extraction technologies, such as microwave-assisted, ultrasonic-assisted, pressurized liquid, and supercritical fluid extractions. These extraction technologies are analyzed and compared in terms of their principles, advantages and disadvantages, improvement solutions, and applications. In addition, this review summarizes and compares new green extraction solvents and discusses the practical applications of these advanced extraction methods and solvents from different perspectives.

Ralstonia solanacearum is a soil-borne plant pathogenic bacterium that causes bacterial wilt disease, leading to substantial economic losses in over 250 crops, including tomatoes, tobacco, and potatoes. We developed a novel method for the specific detection of R. solanacearum by combining recombinase-aided amplification (RAA) with wild-type aerolysin nanopore. The probe Target-16 was hybridized to one strand of the RAA product, allowing the nicking endonuclease Nb.BsrDI to recognize specific cleavage sites and cleave the product into two short DNA strands. As these short DNA strands passed through the aerolysin nanopore, they produced distinct current-blockage signals that were markedly different from those generated by Target-16, thereby enabling on-site detection of R. solanacearum. This method revealed high sensitivity (10 pg/μL) and specificity and reproducibility. The strategy is less dependent on expensive instruments and pure DNA, and can be performed within 3.2 h at constant temperature, which can be utilized to detect R. solanacearum in tobacco samples, yielding results consistent with those obtained using PCR and DNA sequencing. This method provides a powerful tool for the rapid detection of R. solanacearum, especially suited for fieldwork or laboratories with limited resources.

A bacterial strain Bacillus velezensis Cas367 was isolated from the rhizosphere soils of tobacco in China. Here, we present the draft genome sequence of B. velezensis Cas367. The genome comprises a 3,889,832 bp, with G + C contents of 46.4%.

Cucumis melo L. is an important fruit with widespread consumption and commercial value. However, an undescribed disease affecting Hami melon (Cucumis melo L. var. Luhoutian) plants has consistently emerged in the Qihe region of Dezhou, Shandong Province of China since 2021. The disease can occur in both seedling and mature stages of Hami melon plants, and in severely diseased areas, the incidence rate was seen as 40 to 80%. During the seedling stage, the initial symptom is the appearance of water-soaked spots on the leaves. As the disease progresses, the leaves develop necrotic spots, and severely affected plants may exhibit stem rot and decay. In the mature stage, the disease primarily affects the leaves, causing necrotic spots and chlorosis. Under conditions of high humidity, black mold can be observed in the affected areas. Small pieces of symptomatic leaves from six different infected plants were collected and surface-sterilized with 5% NaClO for 3 min and 75% alcohol for 30 s for pathogen isolation (Wang et al., 2020). After rinsing with sterile water and blotted on sterile filter paper, the tissues were established on potato dextrose agar (PDA) media and incubated at 28℃ for 3-4 days. Pure isolates showed up at PDA were obtained through single-spore isolation. Colonies of all 16 isolates obtained by single-spore isolation had similar morphological characteristics on the PDA medium, the mycelium of the isolate appears dense and yellowish-brown on the PDA medium, and also secretes a brownish-red pigment on PDA. Under the opticalmicroscope, the perithecia from PDA media are subglobose spherical in shape, 80-100 μm in diameter, brownish by reflected light, wholly and densely hairy. Terminal hairs are very dense, greyish by reflected light, olive brown to reddish brown by transmitted light, thick-walled, arcuate, circinate, or spirally coiled at the apex. The ascospores within the perithecia are elliptical or droplet-shaped, initially colorless hyaline but later becoming subhyaline slightly gray, with dimensions of 7-9 μm × 4-5 μm. The morphological characteristics of the isolates were consistent with the description of Arcopilus aureus (Wang et.al. 2016). The internal transcribed spacer (ITS) region and β-tubulin genes of three randomly selected isolates were PCR amplified and sequenced using primers ITS4/ITS5 and Bt2a/Bt2b. The sequences of ITS and β-tubulin genes were submitted to NCBI with GenBank Accession No. OR539527 and OR640972, respectively. Based on morphological features and phylogenetic analysis, we concluded that the isolates belonged to A. aureus. Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of Hami melon seedlings (n=12) grown at 28°C with 60% humidity in a greenhouse, the assay was repeated three times. Symptoms appeared on the pathogen-inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as A. aureus based on morphological, and molecular phylogenetic analysis, while Koch'sostulate was used to confirm its life mode. To the best of our knowledge, this is the first report of leaf spot caused by A. aureus on Cucumis melo L. in China.

Background Viral diseases of sweet potatoes are causing severe crop losses worldwide. More than 30 viruses have been identified to infect sweet potatoes among which the sweet potato latent virus (SPLV), sweet potato mild speckling virus (SPMSV), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2) have been recognized as distinct species of the genus Potyvirus in the family Potyviridae. The sweet potato virus 2 (SPV2) is a primary pathogen affecting sweet potato crops. Methods In this study, we detected an SPV2 isolate (named SPV2-LN) in Ipomoea nil in China. The complete genomic sequence of SPV2-LN was obtained using sequencing of small RNAs, RT-PCR, and RACE amplification. The codon usage, phylogeny, recombination analysis and selective pressure analysis were assessed on the SPV2-LN genome. Results The complete genome of SPV2-LN consisted of 10,606 nt (GenBank No. OR842902), encoding 3425 amino acids. There were 28 codons in the SPV2-LN genome with a relative synonymous codon usage (RSCU) value greater than 1, of which 21 end in A/U. Among the 12 proteins of SPV2, P3 and P3N-PIPO exhibited the highest variability in their amino acid sequences, while P1 was the most conserved, with an amino acid sequence identity of 87-95.3%. The phylogenetic analysis showed that 21 SPV2 isolates were clustered into four groups, and SPV2-LN was clustered together with isolate yu-17-47 (MK778808) in group IV. Recombination analysis indicated no major recombination sites in SPV2-LN. Selective pressure analysis showed d(N)/d(S) of the 12 proteins of SPV2 were less than 1, indicating that all were undergoing negative selection, except for P1N-PISPO. Conclusion This study identified a sweet potato virus, SPV2-LN, in Ipomoea nil. Sequence identities and genome analysis showed high similarity between our isolate and a Chinese isolate, yu-17-47, isolated from sweet potato. These results will provide a theoretical basis for understanding the genetic evolution and viral spread of SPV2.

Background Beneficial root-associated microbiomes play crucial roles in enhancing plant growth and suppressing pathogenic threats, and their application for defending against pathogens has garnered increasing attention. Nonetheless, the dynamics of microbiome assembly and defense mechanisms during pathogen invasion remain largely unknown. In this study, we aimed to investigate the diversity and assembly of microbial communities within four niches (bulk soils, rhizosphere, rhizoplane, and endosphere) under the influence of the bacterial plant pathogen Ralstonia solanacearum . Results Our results revealed that healthy tobacco plants exhibited more diverse community compositions and more robust co-occurrence networks in root-associated niches compared to diseased tobacco plants. Stochastic processes (dispersal limitation and drift), rather than determinism, dominated the assembly processes, with a higher impact of drift observed in diseased plants than in healthy ones. Furthermore, during the invasion of R. solanacearum , the abundance of Fusarium genera, a known potential pathogen of Fusarium wilt, significantly increased in diseased plants. Moreover, the response strategies of the microbiomes to pathogens in diseased and healthy plants diverged. Diseased microbiomes recruited beneficial microbial taxa, such as Streptomyces and Bacilli , to mount defenses against pathogens, with an increased presence of microbial taxa negatively correlated with the pathogen. Conversely, the potential defense strategies varied across niches in healthy plants, with significant enrichments of functional genes related to biofilm formation in the rhizoplane and antibiotic biosynthesis in the endosphere. Conclusion Our study revealed the varied community composition and assembly mechanism of microbial communities between healthy and diseased tobacco plants along the soil-root continuum, providing new insights into niche-specific defense mechanisms against pathogen invasions. These findings may underscore the potential utilization of different functional prebiotics to enhance plants’ ability to fend off pathogens.

The soil microbial community plays a critical role in promoting robust plant growth and serves as an effective defence mechanism against root pathogens. Current research has focused on unravelling the compositions and functions of diverse microbial taxa in plant rhizospheres invaded by Ralstonia solanacearum, however, the specific mechanisms by which key microbial groups with distinct functions exert their effects remain unclear. In this study, we employed a combination of amplicon sequencing and metabolomics analysis to investigate the principal metabolic mechanisms of key microbial taxa in plant rhizosphere soil. Compared to the healthy tobacco rhizosphere samples, the bacterial diversity and co-occurrence network of the diseased tobacco rhizosphere soil were significantly reduced. Notably, certain genera, including Gaiella, Rhodoplanes, and MND1 (Nitrosomonadaceae), were found to be significantly more abundant in the rhizosphere of healthy plants than in that of diseased plants. Eight environmental factors, including exchangeable magnesium, available phosphorus, and pH, were found to be crucial factors influencing the composition of the microbial community. Ralstonia displayed negative correlations with pH, exchangeable magnesium, and cation exchange flux, but showed a positive correlation with available iron. Furthermore, metabolomic analysis revealed that the metabolic pathways related to the synthesis of various antibacterial compounds were significantly enriched in the healthy group. The correlation analysis results indicate that the bacterial genera Polycyclovorans, Lysobacter, Pseudomonas, and Nitrosospira may participate in the synthesis of antibacterial compounds. Collectively, our findings contribute to a more in-depth understanding of disease resistance mechanisms within healthy microbial communities and provide a theoretical foundation for the development of targeted strategies using beneficial microorganisms to suppress disease occurrence.

赤星病是烟草上的重要叶部病害,每年给烟草生产造成巨大损失,培育抗赤星病品种是防治烟草赤星病为害的根本措施,而对赤星病抗性关联遗传位点的发掘、鉴定和利用是培育抗赤星病烟草品种的关键基础.本研究利用抗赤星病的烟草材料'Beinhart 1000-1'和加工品质好但不抗赤星病的烟草品种'K326',通过种间杂交和F1代自交分离,创建了'Beinhart 1000-1'和'K326'的F2代杂交分离群体.随后,通过赤星病抗病性鉴定,本研究在F2代分离群体中选择了 19株抗赤星病材料和19株感赤星病材料,分别进行了基因组DNA提取,并通过BSA法进行了基因组重测序.在重测序数据分析基础上结合表型鉴定结果,本研究鉴定出差异SNP位点约170万个,进行烟草赤星病抗性关联SNP位点分析,找到赤星病抗性相关基因10个,促进了烟草抗赤星病分子育种的发展.

HomePlant DiseaseVol. 107, No. 3First Report of Bacterial Leaf Spot on Tobacco Caused by Pseudomonas psychrotolerans in China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Bacterial Leaf Spot on Tobacco Caused by Pseudomonas psychrotolerans in ChinaYichi Li, Dongkun Wang, Shoutao Cao, Xiaoqiang Wang, and Guangwei RenYichi LiChinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong 266100, China, Dongkun WangChinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong 266100, China, Shoutao CaoTechnology Center of China Tobacco Shandong Industrial Co., Ltd., Jinan 250100, China, Xiaoqiang Wang†Corresponding author: X. Wang; E-mail Address: [email protected]https://orcid.org/0000-0002-2173-6442Chinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong 266100, China, and Guangwei RenChinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong 266100, ChinaAffiliationsAuthors and Affiliations Yichi Li1 Dongkun Wang1 Shoutao Cao2 Xiaoqiang Wang1 † Guangwei Ren1 1Chinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong 266100, China 2Technology Center of China Tobacco Shandong Industrial Co., Ltd., Jinan 250100, China Published Online:14 Feb 2023https://doi.org/10.1094/PDIS-05-22-1069-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleTobacco (Nicotiana tabacum) is an economically important crop, and its productivity is challenged due to pathogen infection. In 2020 and 2021, a previously uncharacterized disease was observed on field-grown tobacco (var. NC102) in Zhucheng, Shandong Province, China (36°0′58″N, 119°7′14″E), where tobacco has been grown for decades. The disease was found throughout the growth period of tobacco and mainly occurred from the fast-growing period (about 13 to 16 leaves) to the leaf maturity stage. In severely diseased areas, the incidence rate reached 100%. Symptoms began as a chlorotic water stain like small spots, then the spots merged into larger irregular necrotic maculae around the chlorotic halos. Small pieces of symptomatic leaves from 10 different plants were collected for pathogen isolation. The pieces were surface sterilized with 75% ethanol for 40 s, washed with sterile water three times, and ground with a glass rod with 1 ml of sterile water, and 100 μl suspensions were spread on nutrient agar medium and incubated at 28°C for 48 h. Round yellow colonies with undulating edges showed up 48 h later. Three isolates were randomly picked up from each of the 10 plates for subsequent analysis. After purification and culture on a nutrient agar plate, the 16S rRNA gene of the 30 isolates was amplified with primers 27F and 1492R and the amplicons were sequenced and analyzed by sequence alignment. The results showed that the 16S rRNA nucleotide identity of the 30 isolates was 100%. The typical isolate ZC5 was selected for subsequent analysis, and the resulting 16S rRNA sequence was deposited in GenBank (accession OK092624). The 16S rRNA sequence identity with those of Pseudomonas psychrotolerans strain K3-2 (KY882083) and M3-1 (KY882120) was 100%. A phenotypic analysis by Biolog Gen III indicated that ZC5 showed highest similarity (98.3%) with strain P. oryzihabitans. P. oryzihabitans and P. psychrotolerans have a high degree of homology in the phylogenetic relationship based on a phylogenetic analysis of three concatenated sequences of gyrB, rpoB, and rpoD genes (Mulet et al. 2010). The gyrB (ON462356), rpoB (ON462355), and rpoD (ON462357) genes of ZC5 were also amplified and sequenced by primers gyrB-For/gyrB-Rev, rpoB-For/rpoB-Rev, and rpoD-For/rpoD-Rev (Hauser et al. 2004), respectively. While P. psychrotolerans and P. oryzihabitans form the same clade in phylogenies, strains of P. psychrotolerans form a unique subclade. ZC5 clustered more closely with the type strain of P. psychrotolerans LMG 21977 in the phylogenetic tree. Therefore, based on the concatenated sequences of gyrB, rpoB, and rpoD, ZC5 was confirmed as P. psychrotolerans. Based on morphology, biological characteristics, and phylogenetic analysis, the isolate was identified as P. psychrotolerans. Tobacco plants at the fast-growing stage were selected for pathogenicity tests. Tests were conducted by injecting 10 μl of bacterial suspension (108 CFU/ml) of ZC5 into leaves with a syringe. Sterile water was inoculated in the same way as a control. Six plants were selected for pathogenicity tests each time, five leaves of each tobacco plant were inoculated, and the tests were repeated three times. To simulate disease conditions in the natural environment, the inoculated plants were moved outdoors. The average temperature was 32°C during the day and 20°C at night. To maintain humidity, leaves were sprayed with water every 2 days. Symptoms appeared on the pathogen-inoculated leaves 7 days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as P. psychrotolerans based on morphological, molecular, and phylogenetic analysis, fulfilling Koch's postulates. To our knowledge, this is the first report of tobacco bacterial leaf spot caused by P. psychrotolerans.The author(s) declare no conflict of interest.References:Hauser, E., et al. 2004. Int. J. Syst. Evol. Microbiol. 54:1633. https://doi.org/10.1099/ijs.0.03024-0 Crossref, ISI, Google ScholarMulet, M., et al. 2010. Environ. Microbiol. 12:1513. https://doi.org/10.1111/j.1462-2920.2010.02181.x ISI, Google ScholarFunding: This work was supported by the National Natural Science Foundation of China (31901937), Research on Prevention and Control Technology of Tobacco Disease (110202101027[LS-02]), and Agricultural Science and Technology Innovation Program of China (ASTIP-TRIC04).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 107, No. 3 March 2023SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Article History Issue Date: 3 Apr 2023Published: 14 Feb 2023First Look: 19 Jul 2022Accepted: 11 Jul 2022 Page: 935 Information© 2023 The American Phytopathological SocietyFundingNational Natural Science Foundation of ChinaGrant/Award Number: 31901937Research on Prevention and Control Technology of Tobacco DiseaseGrant/Award Number: 110202101027(LS-02)Agricultural Science and Technology Innovation Program of ChinaGrant/Award Number: ASTIP-TRIC04Keywordsfield cropsNicotiana tabacumpathogen detectionprokaryotesPseudomonas psychrotoleransThe author(s) declare no conflict of interest.PDF download

Tobacco (Nicotiana tabacum) is an economically important crop, and its productivity is challenged due to pathogen infection. In 2020 and 2021, a previously uncharacterized disease was observed on field grown tobacco (Variety NC102) in Zhucheng City, Shandong Province, China (119°7'14" E, 36°0'58" N), where tobacco has been grown for decades. The disease can be found throughout the growth period of tobacco and mainly occurred from fast growing period (about 13-16 leaves) to leaf maturity stage. In severely diseased areas, the incidence rate can reach 100%. The symptoms first began as chlorotic water stain like small spots, then the spots merged into larger irregular necrotic maculae around the chlorotic halos. Small pieces of symptomatic leaves from 10 different infected plants were collected for pathogen isolation. The small pieces of discolored leaves were surface sterilized with 75% ethanol for 40s and washed with sterile water for three times. The sterilized leaves were ground with a glass rod with 1mL sterile water, and 100 μL suspensions were spread on nutrient agar medium then incubated at 28oC for 48 hours. Yellow round colonies with undulating edges were showed up on nutrient agar medium 48 hours later. Three isolates were randomly picked up from each of the 10 plates for subsequent analysis. After purification and culture on nutrient agar plate, the 16S rRNA gene of the 30 isolates were amplified with primers 27F and 1492R and the amplicons were sequenced and analyzed by sequence alignment. The sequence alignment results showed that the 16S rRNA nucleotide identity of the 30 isolates were 100%. One typical isolate named ZC5 was selected for subsequent analysis, and the resulting 16S rRNA sequence was deposited at GenBank, NCBI under accession OK092624. The 16S rRNA sequence identity with those of P. psychrotolerans strain K3-2 (KY882083) and M3-1 (KY882120) were 100%, respectively. The phenotypic analysis by Biolog Gen Ⅲ indicated that the bacterial isolate (ZC5) showed highest similarity (98.3%) with strain Pseudomonas oryzihabitans. P. oryzihabitans and P. psychrotolerans have a high degree of homology in the phylogenetic relationship based on the phylogenetic analysis of three concatenated sequences of gyrB, rpoB and rpoD genes (Mulet et al. 2010). The gyrB (ON462356), rpoB (ON462355), rpoD (ON462357) gene of isolate ZC5were also amplified and sequenced by using primers gyrB-For/gyrB-Rev, rpoB-For/rpoB-Rev and rpoD-For/rpoD-Rev (Hauser et al. 2004), respectively. While P. psychrotolerans and P. oryzihabitans form the same clade in phylogenies, strains of P. psychrotolerans do form a unique sub-clade. Isolate ZC5 clustered more closely with the type strain of P. psychrotolerans LMG 21977 in the phylogenetic tree. Therefore, based on the concatenated sequences of three genes (gyrB, rpoB and rpoD), the isolate ZC5 was confirmed as P. psychrotolerans. Based on morphological, Biolog characteristics and phylogenetic analysis, the isolate was identified as P. psychrotolerans. The tobacco plants at fast growing stage were selected for pathogenicity tests. Pathogenicity tests were conducted by injecting 10 μL bacterial suspension (108cfu/mL) of ZC5 into tobacco leaves with a syringe. Sterile water was inoculated into the tobacco leaves in the same way as the control. Six plants were selected for pathogenicity tests each time and five leaves of each tobacco plant were inoculated, and the tests were repeated three times. To simulate disease conditions in the natural environment, the inoculated plants were moved outdoors. The average temperature was 32°C during the day and 20°C at night. To maintain humidity, the tobacco leaves were sprayed with water every two days. Symptoms appeared on the pathogen inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as P. psychrotolerans based on morphological, molecular and phylogenetic analysis, which fulfilled Koch's postulates. To our knowledge, this is the first report of tobacco bacterial leaf spot caused by P. psychrotolerans.

A rapid, simple, and sensitive fluorescent detection method for brown spot of tobacco is established by lambda exonuclease-induced Mg2+-dependent DNAzyme amplification. It contains hybridization of the Alternaria alternata genome and HP1, digestion of the 5'-phosphorylated strand of the hybrid dsDNA by lambda exonuclease, acquisition of complete Mg2+-dependent DNAzyme, cleavage of the substrate modified with FAM and BHQ-1, and fluorescent detection. The proposed assay exhibits good sensitivity (10 pg L-1), selectivity and reproducibility. The method does not require pure DNA and expensive instruments, and can be performed within 2.5 hours. To the best of our knowledge, this is the first report of fluorescent detection of Alternaria alternata and its tobacco field samples. This method can be applied to the rapid and sensitive detection of Alternaria alternata in tobacco and its seedlings, and is particularly important for the green prevention and control of tobacco brown spot disease.

A combined chemical-bacterial process was developed to convert vegetable straw waste to high value antifungal iturins. Straws from three widely cultivated vegetable (cucumber, tomato and pepper) were evaluated as feedstocks for iturin production. Microwave assisted hydrolysis with very dilute acid (0.2% w/w H2SO4) achieved efficient reducing sugar recovery. The high glucose concentration in non-detoxified hydrolysate from pepper straw facilitated the optimal growth of Bacillus amyloliquefaciens strain Cas02 and stimulated the production of iturin. The fermentation parameters were optimised to enhance the iturin production efficiency. The obtained fermentation extract was further purified using macroporous adsorption resin, resulting in an iturin-rich extract that exhibited strong antifungal activity against Alternaria alternata with an IC50 of 176.44 mu g/mL. Each iturin homologue was identified using NMR. Overall, 1.58 g iturin-rich extract containing 164.06 mg/g iturins was obtained from 100 g pepper straw, illustrating the great potential of valorising pepper straw via this process.