Fungal communities are more sensitive to mildew than bacterial communities during tobacco storage processes.

Mildew poses a significant threat to tobacco production; however, there is limited information on the structure of the abundant and rare microbial subcommunities in moldy tobacco leaves. In this study, we employed high-throughput sequencing technology to discern the disparities in the composition, diversity, and co-occurrence patterns of abundant and rare fungal and bacterial subcommunities between moldy and normal tobacco leaves collected from Guizhou, Shanghai, and Jilin provinces, China. Furthermore, we explored the correlation between microorganisms and metabolites by integrating the metabolic profiles of moldy and normal tobacco leaves. The results showed that the fungi are more sensitive to mildew than bacteria, and that the fungal abundant taxa exhibit greater resistance and environmental adaptability than the rare taxa. The loss of rare taxa results in irreversible changes in the diversity, richness, and composition of the fungal community. Moreover, rare fungal taxa and abundant bacterial taxa played crucial roles in maintaining the stability and functionality of the tobacco microecosystem. In moldy tobacco, however, the disappearance of rare taxa as key nodes resulted in reduced connectivity and stability within the fungal network. In addition, metabolomic analysis showed that the contents of indoles, pyridines, polyketones, phenols, and peptides were significantly enriched in the moldy tobacco leaves, while the contents of amino acids, carbohydrates, lipids, and other compounds were significantly reduced in these leaves. Most metabolites showed negative correlations with Dothideomycetes, Alphaproteobacteria, and Gammaproteobacteria, but showed positive correlations with Eurotiales and Bacilli. This study has demonstrated that abundant fungal taxa are the predominant biological agents responsible for tobacco mildew, while bacteria may indirectly contribute to this process through the production and degradation of metabolites. KEY POINTS: • Fungi exhibited greater sensitivity to mildew of tobacco leaf compared to bacteria • Rare fungal taxa underwent significant damage during the mildew process • Mildew may damage the defense system of the tobacco leaf microecosystem.

Plant volatiles mediated the orientation preference of slugs to different plant species.

BACKGROUND: Slugs mechanically damage plant leaves, resulting in significant economic losses. However, there are limited cost-efficient strategies available in slug management. By studying how slugs utilize plant volatiles to locate host plants, we can gain insights into the design of attractants and repellents. RESULTS: Bioassay results suggest slugs (Agriolimax agrestis) prefer to orientate to lettuce (Lactuca sativa), cabbage (Brassica oleracea L.), and young tobacco seedlings, compared with old tobacco seedlings. We analyzed the volatomics of lettuce, cabbage, young and old tobacco seedlings. 2-(2-butoxyethoxy)-ethanol acetate (2EA) had high abundance while nonanal, decanal, and ß-cylocitral had relatively low content in volatiles. Old tobacco seedlings released significantly more hexanal but fewer 1,4-dihydro-4-oxopyridazine (DO). In olfactory tests, hexanal, nonanal, decanal, and ß-cylocitral showed strong repellency to slugs, while DO at a dose of 500 ng/µL and 2EA at a dose of 1% were attractive to slugs. The two alkanes, hexadecane and heptadecane, had no effect on slug orientating to host plants. DO and 2EA can thus alleviate the repellency of hexanal, nonanal, decanal and ß-cylocitral. CONCLUSION: The high emission of hexanal in old tobacco seedlings helps repel slugs, while 2EA and DO attract slugs to lettuce and cabbage. These findings suggest that these chemicals can be utilized in the design of repellents and attractants, and contribute to constructing a push-pull system for slug control. © 2023 Society of Chemical Industry.

Biocontrol potential of Bacillus velezensis EM-1 associated with suppressive rhizosphere soil microbes against tobacco bacterial wilt.

Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most devastating diseases. Microbial keystone taxa were proposed as promising targets in plant disease control. In this study, we obtained an antagonistic Bacillus isolate EM-1 from bacterial wilt-suppressive soil, and it was considered rhizosphere-resident bacteria based on high (100%) 16S rRNA gene similarity to sequences derived from high-throughput amplicon sequencing. According to 16S rRNA gene sequencing and MLSA, strain EM-1 was identified as Bacillus velezensis. This strain could inhibit the growth of R. solanacearum, reduce the colonization of R. solanacearum in tobacco roots, and decrease the incidence of bacterial wilt disease. In addition, strain EM-1 also showed a strong inhibitory effect on other phytopathogens, such as Alternaria alternata and Phytophthora nicotianae, indicating a wide antagonistic spectrum. The antimicrobial ability of EM-1 can be attributed to its volatile, lipopeptide and polyketide metabolites. Iturin A (C14, C15, and C16) was the main lipopeptide, and macrolactin A and macrolactin W were the main polyketides in the fermentation broth of EM-1, while heptanone and its derivatives were dominant among the volatile organic compounds. Among them, heptanones and macrolactins, but not iturins, might be the main potential antibacterial substances. Complete genome sequencing was performed, and the biosynthetic gene clusters responsible for iturin A and macrolactin were identified. Moreover, strain EM-1 can also induce plant resistance by increasing the activity of CAT and PPO in tobacco. These results indicated that EM-1 can serve as a biocontrol Bacillus strain for tobacco bacterial wilt control. This study provides a better insight into the strategy of exploring biocontrol agent based on rhizosphere microbiome.

Potassium accumulation characteristics and expression of related genes involved in potassium metabolism in a high-potassium variety: tobacco (Nicotiana tabacum) as a model.

We investigated potassium (K) accumulation characteristics and expression of K metabolism related genes in one high-K variety (ND202) and a common variety (NC89) of tobacco (Nicotiana tabacum L.). Results showed that K accumulation and leaf K content in ND202 were higher than those in NC89. The distribution rate and K accumulation in the leaves of ND202 increased significantly, while the distribution rate in the roots and stems had lower values. In addition, the maximum K accumulation rate and high-speed K accumulation duration in ND202 were found to be better than those in NC89. The expression of NKT1 in the upper and middle leaves of ND202 had an advantage, and the relative expression of NtKC1 and NtTPK1 in both the upper and middle leaves, as well as the roots, was also significantly upregulated. Conversely, the expression of NTRK1 in the lower leaves and roots of ND202 was weaker. ND202 had significantly greater expression levels of NtHAK1 than NC89 in the upper and middle leaves and roots; moreover, the expression of NtKT12 in the upper leaves and roots of ND202 was also higher. In comparison with common varieties, high-K varieties had a stronger ability to absorb and accumulate K. They also possessed higher expression of K+ channel- and transporter-related genes and showed a superior K accumulation rate and longer duration of high-speed K accumulation. Furthermore, K accumulation rate at 40-60days can be suggested as an important reference for the selection of high-K tobacco varieties.

Molecular characterization of a putative alphapartitivirus from Impatiens balsamina L.

Two double stranded RNAs (dsRNAs) that likely represent the genome of an alphapartitivirus, tentatively named "impatiens cryptic virus 1" (ICV1), were recovered from Impatiens balsamina L. RNA1 (2008 bp) codes for the RNA-dependent RNA polymerase (RdRp) of ICV1, which shares <83% amino acid sequence identity with the RdRps of other alphapartitiviruses. RNA2 (1906 bp) codes for the coat protein (CP) of ICV1, which shares <60% amino acid sequence identity with the CPs of other alphapartitiviruses. Phylogenetic analysis suggested that ICV1 is closely related to plant alphapartitiviruses, including vicia cryptic virus, beet cryptic virus 1, carrot cryptic virus, and white clover cryptic virus 1. Using primers specific for RNA1 or RNA2, ICV1 could be detected in I. balsamina from various parts of China.

Isolation and Genome Sequence of a Novel Phosphate-Solubilizing Rhizobacterium Bacillus altitudinis GQYP101 and Its Effects on Rhizosphere Microbial Community Structure and Functional Traits of Corn Seedling.

Bacillus altitudinis is a widely distributed soil bacterium that has various functional activities, including remediation of contaminated soil, degradation of herbicides, and enhancement of plant growth. B. altitudinis GQYP101 was isolated from the rhizosphere soil of Lycium barbarum L. and demonstrated potential as a plant growth-promoting bacterium. In this work, strain GQYP101 could solubilize phosphorus, and increased the stem diameter, maximum leaf area, and fresh weight of corn in a pot experiment. Nitrogen and phosphorus contents of corn seedlings (aerial part) increased by 100% and 47.9%, respectively, after application of strain GQYP101. Concurrently, nitrogen and phosphorus contents of corn root also increased, by 55.40% and 20.3%, respectively. Furthermore, rhizosphere soil nutrients were altered and the content of available phosphorus increased by 73.2% after application of strain GQYP101. The mechanism by which strain GQYP101 improved plant growth was further investigated by whole genome sequence analysis. Strain GQYP101 comprises a circular chromosome and a linear plasmid. Some key genes of strain GQYP101 were identified that were related to phosphate solubilization, alkaline phosphatase, chemotaxis, and motility. The findings of this study may provide a theoretical basis for strain GQYP101 to enhance crop yield as microbial fertilizer.

The relationship between material transformation, microbial community and amino acids and alkaloid metabolites in the mushroom residue-prickly ash seed oil meal composting with biocontrol agent addition.

This study investigated the effects of adding biocontrol microbes on metabolites and pathogenic microorganisms during mushroom residue composting and the relationships of metabolite changes with microbes and material transformation. The results showed that the addition of Bacillus subtilis (BS) and Trichoderma harzianum (TH) with mushroom residue promoted the conversion of organic carbon and nitrogen. The abundance of pathogenic microbes was increased in biocontrol microbial treatments. BS or TH treatments increased the levels of amino acids, carbohydrates, and bacteriostatic alkaloid metabolites. Network analysis revealed that the main microorganisms significantly related to alkaloid metabolites were Rhabdanaerobium, Atopostipes, Planifilum and Ureibacillus. The increased bacterial abundance and decreased NO3--N and TOC were closely related to the increases in amino acid and alkaloid metabolites after biocontrol agent treatments. Generally, adding biocontrol microbes is an effective way to increase the levels of antibacterial metabolites, but there is a risk of increasing the abundance of pathogenic microbes.

Effects of biocontrol Bacillus and fermentation bacteria additions on the microbial community, functions and antibiotic resistance genes of prickly ash seed oil meal-biochar compost.

This study evaluated the effects of biocontrol Bacillus and fermenting bacteria addition on the microbial community, metabolic functions and antibiotic resistance genes (ARGs) of new prickly ash seed oil meal (PSOM)-biochar composting. The results showed that the addition of Bacillus subtilis and fermentation bacteria significantly increased the NH4+-N, bacterial abundance and fungal diversity of compost while decreasing the relative abundances (RAs) of carbon metabolism genes in mature compost. NH4+-N was significantly correlated with microbial abundance and diversity, and its increase was closely related to microbial amino acid metabolism. The addition of biocontrol and fermenting bacteria changed the RAs of ARGs, which was caused by changes in the potential hosts Proteobacteria, Bacteroidota and Firmicutes in the compost. Consequently, adding Bacillus and fermenting bacteria into PSOM to make composting was suggested as an effective method to promote nutrient transformation, regulate microbial activity and decrease RAs of tetracycline and vancomycin ARGs.

Overexpression of NtSnRK2.2 enhances salt tolerance in Nicotiana tabacum by regulating carbohydrate metabolism and lateral root development.

SnRK2 is a plant-specific protein kinase family implicated in environmental stress tolerance. Individual SnRK2 genes have acquired distinct regulatory properties in response to various environmental stresses. In this study, NtSnRK2.2, a SnRK2 subclass II member in Nicotiana tabacum L., was cloned and characterised. Sequence alignment analysis showed that SnRK2.2 exhibits widespread sequence differences across Nicotiana species. The tissue expression pattern of NtSnRK2.2 showed a root-predominant expression. To investigate its biological function, NtSnRK2.2 was overexpressed in tobacco, which subsequently resulted in increased soluble sugars and more lateral roots under a normal condition. A salt-stress tolerance assay showed that NtSnRK2.2-overexpressing plants exhibited enhanced salt tolerance, which was further confirmed based on its better root architecture and increase in soluble sugars, thereby implying that NtSnRK2.2 is a multifunctional regulatory factor in plants. Together, our results indicated the possible role played by NtSnRK2.2 in maintaining metabolic homeostasis via the regulation of carbohydrate metabolism in response to environmental stress.

The Antifungal Effect of Garlic Essential Oil on Phytophthora nicotianae and the Inhibitory Component Involved.

This study explored the chemical compositions of garlic essential oil, the inhibitory activity of garlic essential oil and diallyl disulfide (DADS) against Phytophthora nicotianae, and the effects on mycelial plasma membrane permeability and P. nicotianae inhibition. In total, 29 compounds were detected in garlic essential oil, of which 26 were detected by gas chromatography‒mass spectrometry (GC-MS) and 21 by headspace solid-phase microextraction (HS-SPME) GC-MS. DADS (60.12% and 19.09%) and trisulfide di-2-propenyl (14.18% and 17.98%) were the major components identified by HS-SPME GC-MS and GC-MS analysis, respectively. Half-inhibitory concentration (Ec50, antagonism) and minimum inhibitory concentration (MIC, fumigation) of DADS against P. nicotianae were 150.83 µL/L and 20 µL/L, respectively, while Ec50 of garlic essential oil was 1108.25 µL/L. Mycelial membrane permeability gradually increased in a concentration-dependent manner, and cell death increased at 450 µL/L DADS. Furthermore, DADS treatment significantly reduced the incidence of tobacco black shank and the number of P. nicotianae pathogens in rhizosphere soil. DADS also promoted root development of tobacco seedlings at low concentrations, which was inhibited at high concentrations. Therefore, DADS may play an important role in the antifungal effect against P. nicotianae by destroying mycelial cell membrane integrity, causing an increase in cell membrane permeability, and leading to cell death.

Bioactive 3-Decalinoyltetramic Acids Derivatives From a Marine-Derived Strain of the Fungus Fusarium equiseti D39.

Two novel 3-decalinoyltetramic acid (3DTA) derivatives, namely fusarisetins C and D (1 and 2), and four known derivatives (3-6) were isolated from the marine-derived fungus Fusarium equiseti D39. Their structures were determined by spectroscopic data, vibrational circular dichroism (VCD) calculations, and X-ray crystallography. Compound 2 was identified as the first fusarisetin to possess an unprecedented carbon skeleton with a tetracyclic ring system comprised of a decalin moiety (6/6) and a tetramic acid moiety. A plausible biosynthetic pathway for the isolated compounds was proposed. All 3DTAs derivatives exhibited a potent phytotoxicity, and 5 also displayed a remarkable anti-phytopathogenic activity superior to the positive control resulting in damage of the cell membrane of Pseudomonas syringae and ensuing leakage of the intracellular components. Here, the phytotoxicity of fusarisetins has been reported for the first time. The OSMAC fermentation optimization approach to give 5 was performed by varying the culture media and salinities. The results showed that potato liquid medium with 1% salinity is the most favorable condition for the production of 5.

Phytotoxicity and anti-phytopathogenic activities of marine-derived fungi and their secondary metabolites.

To find new pesticides for agricultural use, 133 fungal strains were isolated from coastal marine habitats, from which 37 independent isolates were identified, belonging to 20 genera in nine orders, and the diversity of the isolated fungi were discussed. The phytotoxicity and anti-phytopathogenic fungal and bacterial activities of these 37 extracts, and two previously isolated fungal extracts were evaluated, displaying different levels of bioactivity. Based on the bioactive and chemical screening, an Alternaria sp. (P8) strain, which showed prominent bioactivity and contained abundant secondary metabolites was selected for further chemical investigation; one new compound, a benzopyranone (1), and seven known compounds (2-8) were obtained. Their structures were determined by analysing extensive NMR spectroscopic data and ECD comparisons. Compounds 1, 2, and 6-8 showed obvious phytotoxicity, especially against amaranth, and compound 1 also showed potent antifungal activity toward Alternaria brassicicola. To the best of our knowledge, this is the first report of the phytotoxicity of marine-derived fungi and their secondary metabolites. These studies should provide the foundation for future research into the use of such fungal extracts to combat weeds and diseases in agriculture.

In vitro and in vivo activities of eugenol against tobacco black shank caused by Phytophthora nicotianae.

Phytophthora nicotianae causes serious black shank disease in tobacco. Syringa oblata essential oil and its main components were evaluated to develop an effective and environmentally friendly biocontrol agent. Eugenol, which exhibited the strongest activity, was intensively investigated in vitro and in vivo. The mycelial growth of P. nicotianae was inhibited by eugenol at a minimum inhibitory concentration of 200µgmL-1, and inhibition occurred in a dose-dependent manner. Extracellular pH and extracellular conductivity results indicated that eugenol increased membrane permeability. Flow cytometry and fluorescent staining results further showed that eugenol disrupted mycelial membranes but did not affect spore membrane integrity. The in vivo results confirmed that treatment of tobacco with various concentrations of eugenol formulations reduced disease incidence and better controlled against the disease. Our results suggested that the ability of eugenol to control tobacco black shank depended on its ability to damage mycelial membranes and that eugenol formulations have potential as an eco-friendly antifungal agent for controlling tobacco blank shank.

Complete Genome Sequence of Paenibacillus polymyxa YC0136, a Plant Growth-Promoting Rhizobacterium Isolated from Tobacco Rhizosphere.

Paenibacillus polymyxa strain YC0136 is a plant growth-promoting rhizobacterium with antimicrobial activity, which was isolated from tobacco rhizosphere. Here, we report the complete genome sequence of P. polymyxa YC0136. Several genes with antifungal and antibacterial activity were discovered.

Complete Genome Sequence of Paenibacillus polymyxa YC0573, a Plant Growth-Promoting Rhizobacterium with Antimicrobial Activity.

Paenibacillus polymyxa strain YC0573 is a plant growth-promoting rhizobacterium with antimicrobial activity, which was isolated from tobacco rhizosphere. Here, we report the complete genome sequence of P. polymyxa YC0573. Antifungal and antibacterial genes were discovered.

[Influence of tobacco-Chuanminshen violaceum rotation on microbe community in soil].

Soil microbes are the important indicator of soil quality. For exploring Chuanminshen violaceum planting to microbial effects in tobacco soil, this paper adopted Illumina MiSeq high-throughput sequencing to research the change of bacteria and fungi at the phylum and genus in the soil. The results showed that the Ch. violaceum planting increased the biodiversity of bacteria and fungi. The influence on fungi was greater than that on bacteria. It greatly increased the sequence of fungi, it obtained 32 978 16S rDNA and 32 229 18S rDNA sequence number. There was no change of the top three phylums in bacteria, but the content changed, Proteobacteria and Acidobacteria reduced by 1.73% and 1.4% respectively, and Actinobacteria increased by 0.65%. The advantage phylum Ascomycete in tobacco reduced by 27.99% to be second advantage phylum after Ch. violaceum planting, and the second advantage phylum Basidiomycete increased by 23.69% to become the first dominant fungi. At the genus, Ch. violaceum planting changed the order of dominant genus and the abundance was also changed. Some changed largely such as uncultured Acidobacteriaceae Subgroup-1, Gemmatimonas, Subgroup-2,uncultured Nitrosomonadaceae for bacteria, norank Sordariales, norank Agaricomycetes, Phialophora for fungi. Especially the rotation increased antagonistic microbes and physiological microbes and decreased pathogenic microbes. So the Ch. violaceum planting can improve the microbe community in tobacco soil.