Using fungal-bacterial community analysis to explore potential microbiomes to manage Meloidogyne incongnita.

Rhizosphere microbial communities strongly affect outbreaks of root-knot nematode (RKN) disease. However, little is known about the interactions among fungi, bacteria and RKN. The bacterial and fungal community compositions in the rhizospheres of four representative tobacco varieties, both resistant and susceptible to RKN, were characterized using 16S rRNA gene sequencing for bacteria and internal transcribed spacer gene sequencing for fungi. Our findings revealed that the fungi played crucial roles in facilitating the cross-kingdom and symbiotic fungal-bacterial interactions to suppress RKN. Moreover, our investigation suggested Microbacterium as a potential microbial antagonist against RKN based on its enhanced presence in RKN-resistant tobacco genotypes, and the relative abundance of Microbacterium was 34.49% greater in the rhizosphere of resistant tobacco than that of susceptible tobacco significantly. Notably, the richness of fungal community enhanced tobacco's microbe-associated resistance to RKN through the positive regulation of the richness and diversity of bacterial community and the relative abundance of Microbacterium. This study underscores the critical role of the fungus-dominated fungal-bacterial community in bolstering tobacco resistance against RKN. The potential antagonistic role of Microbacterium presents promising avenues for innovative RKN management strategies.

Overexpression of NtEXPA7 promotes seedling growth and resistance to root-knot nematode in tobacco.

Root-knot nematode (RKN) is one of the most damaging plant pathogen in the world. They exhibit a wide host range and cause serious crop losses. The cell wall, encasing every plant cell, plays a crucial role in defending of RKN invasion. Expansins are a group of cell wall proteins inducing cell wall loosening and extensibility. They are widely involved in the regulation of plant growth and the response to biotic and abiotic stresses. In this study, we have characterized the biological function of tobacco (Nicotiana tabacum) NtEXPA7, the homologue of Solyc08g080060.2 (SlEXPA18), of which the transcription level was significantly reduced in susceptible tomato upon RKN infection. The expression of NtEXPA7 was up-regulated after inoculation of RKNs. The NtEXPA7 protein resided in the cell wall. Overexpression of NtEXPA7 promoted the seedling growth of transgenic tobacco. Meanwhile the increased expression of NtEXPA7 was beneficial to enhance the resistance against RKNs. This study expands the understanding of biological role of expansin in coordinate plant growth and disease resistance.

Transcriptome analysis and functional verification reveal the roles of copper in resistance to potato virus Y infection in tobacco.

Potato virus Y (PVY) is one of the most important pathogens in the genus Potyvirus that seriously harms agricultural production. Copper (Cu), as a micronutrient, is closely related to plant immune response. In this study, we found that foliar application of Cu could inhibit PVY infection to some extent, especially at 7 days post inoculation (dpi). To explore the effect of Cu on PVY infection, transcriptome sequencing analysis was performed on PVY-infected tobacco with or without Cu application. Several key pathways regulated by Cu were identified, including plant-pathogen interaction, inorganic ion transport and metabolism, and photosynthesis. Moreover, the results of virus-induced gene silencing (VIGS) assays revealed that NbMLP423, NbPIP2, NbFd and NbEXPA played positive roles in resistance to PVY infection in Nicotiana benthamiana. In addition, transgenic tobacco plants overexpressing NtEXPA11 showed increased resistance to PVY infection. These results contribute to clarify the role and regulatory mechanism of Cu against PVY infection, and provide candidate genes for disease resistance breeding.

Molecular characterization and pathogenicity of a novel monopartite geminivirus infecting tobacco in China.

The occurrence of geminiviruses causes significant economic losses in many economically important crops. In this study, a novel geminivirus isolated from tobacco in Sichuan province of China, named tomato leaf curl Chuxiong virus (TLCCxV), was characterized by small RNA-based deep sequencing. The full-length of TLCCxV genome was determined to be 2744 nucleotides (nt) encoding six open reading frames. Phylogenetic and genome-wide pairwise identity analysis revealed that TLCCxV shared less than 91% identities with reported geminiviruses. A TLCCxV infectious clone was constructed and successfully infected Nicotiana benthamiana, N. tabacum, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants. Furthermore, expression of the V2, C1 and C4 proteins through a potato virus X vector caused severe chlorosis or necrosis symptom in N. benthamiana. Taken together, we identified a new geminivirus in tobacco plants, and found that V2, C1 and C4 contribute to symptom development.

RNA interference-based exogenous double-stranded RNAs confer resistance to Rhizoctonia solani AG-3 on Nicotiana tabacum.

BACKGROUND: Rhizoctonia solani Kühn is a pathogenic fungus causing tobacco target spot disease, and leads to great losses worldwide. At present, resistant varieties and effective control strategy on tobacco target spot disease are very limited. Host-induced gene silencing (HIGS) as well as the exogenous dsRNA can be used to suppress disease progression, and reveal the function of crucial genes involved in the growth and pathogenesis of the fungus. RESULTS: The silencing of endoPGs or RPMK1 in host plants by TRV-based HIGS resulted in a significant reduction in disease development in Nicotiana benthamiana. In vitro analysis validated that red fluorescence signals were consistently observed in the hyphae treated with Cy3-fluorescein-labeled dsRNA at 12, 24, 48 and 72 h postinoculation (hpi). Additionally, application of dsRNA-endoPGs, dsRNA-RPMK1 and dsRNA-PGMK (fusion of partial endoPGs and RPMK1 sequences) effectively inhibited the hyphal growth of R. solani YC-9 in vitro and suppressed disease progression in the leaves, and quantitative real-time PCR confirmed that the application of dsRNAs significantly reduced the expression levels of endoPGs and RPMK1. CONCLUSION: These results provide theoretical basis and new direction for RNAi approaches on the prevention and control of disease caused by R. solani. © 2024 Society of Chemical Industry.

Insights into the genetic variability and evolutionary dynamics of tomato spotted wilt orthotospovirus in China.

BACKGROUND: Viral diseases are posing threat to annual production and quality of tobacco in China. Recently, tomato spotted wilt orthotospovirus (TSWV) has been reported to infect three major crops including tobacco. Current study was aimed to investigate the population dynamics and molecular diversity of the TSWV. In the current study, to assess and identify the prevalence and evolutionary history of TSWV in tobacco crops in China, full-length genome sequences of TSWV isolates from tobacco, were identified and analyzed. METHODS: After trimming and validation, sequences of new isolates were submitted to GenBank. We identified the full-length genomes of ten TSWV isolates, infecting tobacco plants from various regions of China. Besides these, six isolates were partially sequenced. Phylogenetic analysis was performed to assess the relativeness of newly identified sequences and corresponding sequences from GenBank. Recombination and population dynamics analysis was performed using RDP4, RAT, and statistical estimation. Reassortment analysis was performed using MegaX software. RESULTS: Phylogenetic analysis of 41 newly identified sequences, depicted that the majority of the Chinese isolates have separate placement in the tree. RDP4 software predicted that RNA M of newly reported isolate YNKM-2 had a recombinant region spanning from 3111 to 3811 bp. The indication of parental sequences (YNKMXD and YNHHKY) from newly identified isolates, revealed the conservation of local TSWV population. Genetic diversity and population dynamics analysis also support the same trend. RNA M was highlighted to be more capable of mutating or evolving as revealed by data obtained from RDP4, RAT, population dynamics, and phylogenetic analyses. Reassortment analysis revealed that it might have happened in L segment of TSWV isolate YNKMXD (reported herein). CONCLUSION: Taken together, this is the first detailed study revealing the pattern of TWSV genetic diversity, and population dynamics helping to better understand the ability of this pathogen to drastically reduce the tobacco production in China. Also, this is a valuable addition to the existing worldwide profile of TSWV, especially in China, where a few studies related to TSWV have been reported including only one complete genome of this virus isolated from tobacco plants.

Identification and physiological activity of (methoxymethyl)triphenylphosphonium chloride as a new phytotoxin isolated from Rhizoctonia solani AG-3 TB.

Rhizoctonia solani as a cosmopolitan fungus is the causative agent of many crop diseases and leads to significant economic losses in crop production. To explore the toxin structure and physiological activity of R. solani AG-3 TB, high-performance liquid chromatography (HPLC), infrared absorption spectrum (IR), and nuclear magnetic resonance spectrum (NMR) were required. Here, the compound (methoxymethyl)triphenylphosphonium chloride (MMC) with the molecular formula C20H20ClOP was purified and identified from R. solani AG-3 TB. The pure compound MMC treated at 20 µg/mL, 50 µg/mL, and 100 µg/mL can cause obvious necrosis on leaves, increase active oxygen species (AOS), decrease chlorophyll content, and damage cellular structure. The results enrich the understanding of toxin compounds for R. solani and provide valuable insights into the toxicology of R. solani AG-3 TB.

Classification models for Tobacco Mosaic Virus and Potato Virus Y using hyperspectral and machine learning techniques.

Tobacco Mosaic Virus (TMV) and Potato Virus Y (PVY) pose significant threats to crop production. Non-destructive and accurate surveillance is crucial to effective disease control. In this study, we propose the adoption of hyperspectral and machine learning technologies to discern the type and severity of tobacco leaves affected by PVY and TMV infection. Initially, we applied three preprocessing methods - Multivariate Scattering Correction (MSC), Standard Normal Variate (SNV), and Savitzky-Golay smoothing filter (SavGol) - to corrected the leaf full-length spectral sheet data (350-2500nm). Subsequently, we employed two classifiers, support vector machine (SVM) and random forest (RF), to establish supervised classification models, including binary classification models (healthy/diseased leaves or PVY/TMV infected leaves) and six-class classification models (healthy and various severity levels of diseased leaves). Based on the core evaluation index, our models achieved accuracies in the range of 91-100% in the binary classification. In general, SVM demonstrated superior performance compared to RF in distinguishing leaves infected with PVY and TMV. Different combinations of preprocessing methods and classifiers have distinct capabilities in the six-class classification. Notably, SavGol united with SVM gave an excellent performance in the identification of different PVY severity levels with 98.1% average precision, and also achieved a high recognition rate (96.2%) in the different TMV severity level classifications. The results further highlighted that the effective wavelengths captured by SVM, 700nm and 1800nm, would be valuable for estimating disease severity levels. Our study underscores the efficacy of integrating hyperspectral technology and machine learning, showcasing their potential for accurate and non-destructive monitoring of plant viral diseases.

Transcriptome sequencing and functional verification revealed the roles of exogenous magnesium in tobacco anti-PVY infection.

Potato virus Y (PVY) infection causes necrosis and curling of leaves, which seriously affect the yield and quality of Solanaceous crops. The roles of nutrient elements in the regulation of plant resistance to virus infection has been widely reported, while the mechanisms are poorly studied. Previous studies in our laboratory have demonstrated that foliar spraying of MgSO4 could induce Nicotiana tabacum resistance to PVY by increasing the activity of defense-related enzymes. Consistent with the results, we found that exogenous magnesium (Mg) had a certain effect on N. tabacum anti-PVY infection. Meanwhile, Illumina RNA sequencing revealed that Mg induced resistance to PVY infection was mainly by regulating carbohydrate metabolism and transportation, nitrogen metabolism, Ca2+ signal transduction and oxidative phosphorylation. Moreover, we used virus-induced gene silencing assays to verify the function of homologs of five N. tabacum genes involved in above pathways in N. benthamiana. The results showed that NbTPS and NbGBE were conducive to PVY infection, while NbPPases and NbNR were related to resistance to PVY infection. These results suggested a novel strategy for resistance to PVY infection and provided a theoretical basis for virus-resistance breeding.

Effects of different botanical oil meal mixed with cow manure organic fertilizers on soil microbial community and function and tobacco yield and quality.

Introduction: The continuous application of cow manure in soil for many years leads to the accumulation of heavy metals, pathogenic microorganisms, and antibiotic resistance genes. Therefore, in recent years, cow manure has often been mixed with botanical oil meal as organic fertilizer applied to farmland to improve soil and crop quality. However, the effects of various botanical oil meal and cow manure mixed organic fertilizers on soil microbial composition, community structure, and function, tobacco yield, and quality remain unclear. Methods: Therefore, we prepared organic manure via solid fermentation by mixing cow manure with different oil meals (soybean meal, rape meal, peanut bran, sesame meal). Then, we studied its effects on soil microbial community structure and function, physicochemical properties, enzyme activities, tobacco yield and quality; then we analyzed the correlations between these factors. Results and discussion: Compared with cow manure alone, the four kinds of mixed botanical oil meal and cow manure improved the yield and quality of flue-cured tobacco to different degrees. Peanut bran, which significantly improved the soil available phosphorus, available potassium, and NO3--N, was the best addition. Compared with cow manure alone, soil fungal diversity was significantly decreased when rape meal or peanut bran was combined with cow manure, while soil bacterial and fungal abundance was significantly increased when rape meal was added compared with soybean meal or peanut bran. The addition of different botanical oil meals significantly enriched the subgroup_7 and Spingomonas bacteria and Chaetomium and Penicillium fungi in the soil. The relative abundances of functional genes of xenobiotics biodegradation and metabolism, soil endophytic fungi, and wood saprotroph functional groups increased. In addition, alkaline phosphatase had the greatest effect on soil microorganisms, while NO3--N had the least effect on soil microorganisms. In conclusion, the mixed application of cow manure and botanical oil meal increased the available phosphorus and potassium contents in soil; enriched beneficial microorganisms; promoted the metabolic function of soil microorganisms; increased the yield and quality of tobacco; and improved the soil microecology.

Analysis of lysine acetylation in tomato spot wilt virus infection in Nicotiana benthamiana.

Introduction: Kac is a model for all acylation modification studies. Kac plays a critical role in eukaryotes and prokaryotes. It is mainly involved in six major biological functions: gene expression, signal transduction, cell development, protein conversion, metabolism, and metabolite transport. Method: We investigated and compared the acetylation modification of proteins in healthy and tomato spot wilt virus (TSWV)-infected Nicotiana benthamiana leaves. Result: We identified 3,418 acetylated lysine sites on 1962 proteins acetylation of proteins in the TSWV-infected and control groups were compared; it was observed that 408 sites on 294 proteins were upregulated and 284 sites on 219 proteins (involved in pentose phosphate, photosynthesis, and carbon fixation in photosynthesis) were downregulated after the infection. Overall, 35 conserved motifs were identified, of which xxxkxxxxx_K_ Rxxxxxxxxx represented 1,334 (31.63%) enrichment motifs and was the most common combination. Bioinformatic analysis revealed that most of the proteins with Kac sites were located in the chloroplast and cytoplasm. They were involved in biological processes, such as cellular and metabolic processes. Discussion: In conclusion, our results revealed that Kac may participate in the regulation of TSWV infection in N. benthamiana.

Comprehensive analysis of lysine lactylation in Frankliniella occidentalis.

Western flower thrips (Frankliniella occidentalis) are among the most important pests globally that transmit destructive plant viruses and infest multiple commercial crops. Lysine lactylation (Klac) is a recently discovered novel post-translational modification (PTM). We used liquid chromatography-mass spectrometry to identify the global lactylated proteome of F. occidentalis, and further enriched the identified lactylated proteins using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). In the present study, we identified 1,458 Klac sites in 469 proteins from F. occidentalis. Bioinformatics analysis showed that Klac was widely distributed in F. occidentalis proteins, and these Klac modified proteins participated in multiple biological processes. GO and KEGG enrichment analysis revealed that Klac proteins were significantly enriched in multiple cellular compartments and metabolic pathways, such as the ribosome and carbon metabolism pathways. Two Klac proteins were found to be involved in the regulation of the TSWV (Tomato spotted wilt virus) transmission in F. occidentalis. This study provides a systematic report and a rich dataset of lactylation in F. occidentalis proteome for potential studies on the Klac protein of this notorious pest.

Integrative transcriptome analysis revealed the pathogenic molecular basis of Rhizoctonia solani AG-3 TB at three progressive stages of infection.

Rhizoctonia solani has a broad host range and results in significant losses in agricultural production. Here, an integrated transcriptomic analysis was performed to reveal the critical genes responsible for the pathogenesis of R. solani AG-3 TB on Nicotiana tabacum at different infection stages. The results showed that various differential expressed genes (DEGs) were enriched in fatty acid metabolism, amino sugar, carbon metabolism, and cellular carbohydrate biosynthetic process at the early (6-12 hpi), middle (24-36 hpi), and late stage (48-72 hpi) of infection. Specifically, several critical genes such as shikimate kinase that were involved in the biosynthesis of an important fungal toxin, phenylacetic acid (PAA) showed markedly increase at 24 hpi. Additionally, the genes expression levels of carbohydrate-active enzymes (CAZymes) and cell wall degrading enzymes (CWDEs) were significantly increased at the late infection stage. Furthermore, we identified 807 potential secreted proteins and 78 small cysteine-rich proteins, which may function as fungal effectors and involved in the pathogenicity. These results provide valuable insights into critical and potential genes as well as the pathways involved in the pathogenesis of R. solani AG-3 TB.

Bacillus velezensis SYL-3 suppresses Alternaria alternata and tobacco mosaic virus infecting Nicotiana tabacum by regulating the phyllosphere microbial community.

The occurrence of plant diseases is closely associated with the imbalance of plant tissue microecological environment. The regulation of the phyllosphere microbial communities has become a new and alternative approach to the biological control of foliar diseases. In this study, Bacillus velezensis SYL-3 isolated from Luzhou exhibited an effective inhibitory effect against Alternaria alternata and tobacco mosaic virus (TMV). The analysis of phyllosphere microbiome by PacBio sequencing indicated that SYL-3 treatment significantly altered fungal and bacterial communities on the leaves of Nicotiana tabacum plants and reduced the disease index caused by A. alternata and TMV. Specifically, the abundance of P. seudomo, Sphingomonas, Massilia, and Cladosporium in the SYL-3 treatment group increased by 19.00, 9.49, 3.34, and 12.29%, respectively, while the abundances of Pantoea, Enterobacter, Sampaiozyma, and Rachicladosporium were reduced. Moreover, the abundance of beneficial bacteria, such as Pseudomonas and Sphingomonas, was negatively correlated with the disease indexes of A. alternata and TMV. The PICRUSt data also predicted the composition of functional genes, with significant differences being apparent between SYL-3 and the control treatment group. Further functional analysis of the microbiome also showed that SYL-3 may induce host disease resistance by motivating host defense-related pathways. These results collectively indicate that SYL-3 may suppress disease progression caused by A. alternata or TMV by improving the microbial community composition on tobacco leaves.

Application of microbial organic fertilizers promotes the utilization of nutrients and restoration of microbial community structure and function in rhizosphere soils after dazomet fumigation.

Introduction: Soil fumigant dazomet is a broad-spectrum nematicide and fungicide that can kill non-target microbes. Fungicides or organic fertilizers are often added after fumigation to improve the recovery of soil microbes. However, the effect of adding microbial organic fertilizers (MOF) after fumigation on the structure and function of rhizosphere soil microbial communities of crops is unclear. Methods: Therefore, we investigated the effects of adding Junweinong and Junlisu MOFs after dazomet fumigation on the structure and function of rhizosphere microbial communities and its relationship with soil properties and enzyme activities. Results and discussion: The results showed that the addition of these two MOFs after dazomet fumigation significantly reduced the rhizosphere soil available phosphorus, available potassium, organic matter content, and urease, alkaline phosphatase, and catalase activities, but increased the soil pH compared with the fumigation treatment. The application of MOFs after fumigation resulted in significant enrichment of bacteria such as Gaiella, norank_f_Vicinamibacteraceae, and Flavisolibacter and fungi such as Peroneutypa, Olpidium, and Microascus in the rhizosphere soil of the crop and increased the relative abundance of functional genes of 13 kinds of amino acids metabolism, pyruvate metabolism, TCA cycle, and pentose phosphate pathway as well as endophytic and epiphytic functional groups in the rhizosphere soil. In particular, NH4 +-N, pH, and AK had the greatest effect on rhizosphere microorganisms. Overall, the addition of MOFs after fumigation promoted crop root nutrient uptake, enhanced rhizosphere soil microbial metabolism, allowed more beneficial communities to colonize the roots, and promoted soil microbiological health.

Sequencing and phylogenetic characterization of a novel RNA virus in Arma chinensis.

The complete genome of a novel virus from Arma chinensis was determined by RNA sequencing and rapid amplification of cDNA ends. This virus has a single-stranded RNA genome of 10,540 nucleotides (nt) excluding the poly(A) tail. Two non-overlapping open reading frames (ORFs) in the sense direction were predicted: one long ORF at the 5' end of the genome (6,219 nt) that encodes a polypeptide of 2,072 amino acids (aa), and one short ORF at the 3' end of the genome (3,033 nt) that encodes a polypeptide of 1,010 aa. Phylogenetic analysis indicated that the virus clusters within a large cluster of currently unidentified picorna-like viruses with a high bootstrap value. We named the virus isolate Arma chinensis picorna-like virus 1 (AcPV-1). The prevalence of AcPV-1 infection in samples of Arma chinensis from the wild was at a low level (5.48%, 8 positives in 146 samples). Keywords: Arma chinensis; genomic characterization; phylogenetic analysis; Arma chinensis picorna-like virus 1; prevalence.

Sequencing and phylogenetic characterization of a novel RNA virus genome from Harmonia axyridis.

As a natural predator of many insect pests on its native Asian range, Harmonia axyridis remains amongst the insects whose pathogenic or beneficial microorganisms are yet to be studied. The genome nucleotide (nt) and amino acid sequences of open reading frames (ORFs) of the novel RNA virus were identified. Neighbor-joining (NJ) were constructed using MEGA7 software packages with nt sequences and conserved amino acid sequences of predicted RNA-dependent RNA polymerase (RdRp).The complete genome of a novel virus named Harmonia axyridis virus 1 was determined by RNA-seq and rapid amplification of cDNA ends from H. axyridis, which had a single-stranded RNA genome of 8868 nts in length and contains two putative ORFs. ORF1 encodes a polypeptide of 2182 amino acids, which contained conserved domains for 2 picornavirus-like capsid proteins and one RNA helicase. ORF2 encodes a polypeptide of 655 amino acids, which contained 1 RdRp domain. Phylogenetic analysis of whole genome nt sequences and RdRp deduced amino acid sequences suggested that the virus clustered with several unclassified Hubei picorna-like virus. To our knowledge, this is the first full annotated genome of a novel member of the unclassified group of RNA viruses, infecting H. axyridis in natural field conditions.