Genome sequencing and comparative genomics reveal insights into pathogenicity and evolution of Fusarium zanthoxyli, the causal agent of stem canker in prickly ash.

BACKGROUND: Fusarium zanthoxyli is a destructive pathogen causing stem canker in prickly ash, an ecologically and economically important forest tree. However, the genome lack of F. zanthoxyli has hindered research on its interaction with prickly ash and the development of precise control strategies for stem canker. RESULTS: In this study, we sequenced and annotated a relatively high-quality genome of F. zanthoxyli with a size of 43.39 Mb, encoding 11,316 putative genes. Pathogenicity-related factors are predicted, comprising 495 CAZymes, 217 effectors, 156 CYP450s, and 202 enzymes associated with secondary metabolism. Besides, a comparative genomics analysis revealed Fusarium and Colletotrichum diverged from a shared ancestor approximately 141.1 ~ 88.4 million years ago (MYA). Additionally, a phylogenomic investigation of 12 different phytopathogens within Fusarium indicated that F. zanthoxyli originated approximately 34.6 ~ 26.9 MYA, and events of gene expansion and contraction within them were also unveiled. Finally, utilizing conserved domain prediction, the results revealed that among the 59 unique genes, the most enriched domains were PnbA and ULP1. Among the 783 expanded genes, the most enriched domains were PKc_like kinases and those belonging to the APH_ChoK_Like family. CONCLUSION: This study sheds light on the genetic basis of F. zanthoxyli's pathogenicity and evolution which provides valuable information for future research on its molecular interactions with prickly ash and the development of effective strategies to combat stem canker.

Exogenous priming of chitosan induces resistance in Chinese prickly ash against stem canker caused by Fusarium zanthoxyli.

Stem canker is a highly destructive disease that threatens prickly ash plantations in China. This study demonstrated the effective control of stem canker in prickly ash using chitosan priming, reducing lesion areas by 46.77 % to 75.13 % across all chitosan treatments. The mechanisms underlying chitosan-induced systemic acquired resistance (SAR) in prickly ash were further investigated. Chitosan increased H2O2 levels and enhanced peroxidase and catalase enzyme activities. A well-constructed regulatory network depicting the genes involved in the SAR and their corresponding expression levels in prickly ash plants primed with chitosan was established based on transcriptomic analysis. Additionally, 224 ZbWRKYs were identified based on the whole genome of prickly ash, and their phylogenetic evolution, conserved motifs, domains and expression patterns of ZbWRKYs were comprehensively illustrated. The expression of 12 key genes related to the SAR was significantly increased by chitosan, as determined using reverse transcription-quantitative polymerase chain reaction. Furthermore, the activities of defensive enzymes and the accumulation of lignin and flavonoids in prickly ash were significantly enhanced by chitosan treatment. Taken together, this study provides valuable insights into the chitosan-mediated activation of the immune system in prickly ash, offering a promising eco-friendly approach for forest stem canker control.

Microbial cell wall sorption and Fe-Mn binding in rhizosphere contribute to the obstruction of cadmium from soil to rice.

Screening high-tolerant microorganisms to cadmium (Cd) and revealing their bio-obstruction mechanism could be significant for Cd regulation from farmland to the food chain. We examined the tolerance and bio-removal efficiency of Cd ions of two bacterial strains, Pseudomonas putida 23483 and Bacillus sp. GY16, and measured the accumulation of Cd ions in rice tissues and its different chemical forms in soil. The results showed that the two strains had high tolerance to Cd, but the removal efficiency was decreased successively with increasing Cd concentrations (0.05 to 5 mg kg-1). Cell-sorption accounted for the major proportion of Cd removal compared with excreta binding in both strains, which was conformed to the pseudo-second-order kinetics. At the subcellular level, Cd was mostly taken up by the cell mantle and cell wall, and only a small amount entered into the cytomembrane and cytoplasmic with time progressed (0 to 24 h) in each concentration. The cell mantle and cell wall sorption decreased with increasing Cd concentration, especially in the cytomembrane and cytoplasmic. The scanning electron microscope (SEM) and energy dispersive X-ray (EDS) analysis verified that Cd ions were attached to the cell surface, and the functional groups of C-H, C-N, C=O, N-H, and O-H in the cell surface may participate in cell-sorption process tested by the FTIR analysis. Furthermore, inoculation of the two strains significantly decreased Cd accumulation in rice straw and grain but increased in the root, increased Cd enrichment ratio in root from soil, decreased Cd translocation ratio from root to straw and grain, and increased the Cd concentrations of Fe-Mn binding form and residual form in rhizosphere soil. This study highlights that the two strains mainly removed Cd ions in solution through biosorption and passivated soil Cd as Fe-Mn combined form ascribe to its characteristics of manganese-oxidizing, eventually achieving bio-obstruction of Cd from soil to rice grain.

Population Density and Host Preference of the Japanese Pine Sawyer (Monochamus alternatus) in the Qinling-Daba Mountains of China.

Monochamus alternatus is a serious trunk-boring pest and is the most important and effective vector of the pine wood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. The pine wilt disease poses a serious threat to forest vegetation and ecological security in the Qinling-Daba Mountains and their surrounding areas. In order to clarify whether the population density of M. alternatus larvae is related to the host preference of M. alternatus adults, we investigated the population density of M. alternatus overwintering larvae and explored the host preference of M. alternatus adults on Pinus tabuliformis, P. armandii, and P. massoniana. The results show that the population density of M. alternatus larvae was significantly higher on P. armandii than those on P. massoniana and P. tabuliformis. The development of M. alternatus larvae was continuous according to the measurements of the head capsule width and the pronotum width. Adults of M. alternatus preferred to oviposit on P. armandii rather than on P. massoniana and P. tabuliformis. Our results indicate that the difference in the population density of M. alternatus larvae between different host plants was due to the oviposition preference of M. alternatus adults. In addition, the instars of M. alternatus larvae could not be accurately determined, because Dyar's law is not suitable for continuously developing individuals. This study could provide theoretical basis for the comprehensive prevention and control of the pine wilt disease in this region and adjacent areas.

Polycistronic Artificial microRNA-Mediated Resistance to Cucumber Green Mottle Mosaic Virus in Cucumber.

Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted viruses and cultivating completely resistant plants are needed. Here, we describe the design and application of RNA interference-based technologies, containing artificial microRNA (amiRNA) and synthetic trans-acting small interfering RNA (syn-tasiRNA), against conserved regions of different strains of the CGMMV genome. We used a rapid transient sensor system to identify effective anti-CGMMV amiRNAs. A virus seed transmission assay was developed, showing that the externally added polycistronic amiRNA and syn-tasiRNA can successfully block the accumulation of CGMMV in cucumber, but different virulent strains exhibited distinct influences on the expression of amiRNA due to the activity of the RNA-silencing suppressor. We also established stable transgenic cucumber plants expressing polycistronic amiRNA, which conferred disease resistance against CGMMV, and no sequence mutation was observed in CGMMV. This study demonstrates that RNA interference-based technologies can effectively prevent the occurrence and accumulation of CGMMV. The results provide a basis to establish and fine-tune approaches to prevent and treat seed-based transmission viral infections.

Artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in Nicotiana benthamiana.

MAIN CONCLUSION: We describe a Nicotiana benthamiana system for rapid identification of artificial microRNA (amiRNA) to control cucumber green mottle mosaic virus (CGMMV) disease. Although artificial miRNA technology has been used to control other viral diseases, it has not been applied to reduce severe cucumber green mottle mosaic virus (CGMMV) disease and crop loss in the economically important cucurbits. We used our system to identify three amiRNAs targeting CGMMV RNA (amiR1-CP, amiR4-MP and amiR6-Rep) and show that their expression reduces CGMMV replication and disease in virus-infected plants. This work streamlines the process of generating amiRNA virus-resistant crops and can be broadly applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control disease in diverse crops.

Expression profiling and regulatory network of cucumber microRNAs and their putative target genes in response to cucumber green mottle mosaic virus infection.

Cucumber green mottle mosaic virus (CGMMV) is an important pathogen of cucumber (Cucumis sativus). The molecular mechanisms mediating host-pathogen interactions are likely to be strongly influenced by microRNAs (miRNAs), which are known to regulate gene expression during the disease cycle. This study focused on 14 miRNAs (miR159, miR169, miR172, miR838, miR854, miR5658, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p, csa-miRn6-3p, csa-miRn7-5p and csa-miRn8-3p) and their target genes. The data collected was used to construct a regulatory network of miRNAs and target genes associated with cucumber-CGMMV interactions, which identified 608 potential target genes associated with all of the miRNAs except csa-miRn7-5p. Five of the miRNAs (miR159, miR838, miR854, miR5658 and csa-miRn6-3p) were found to be mutually linked by target genes, while another eight (miR169, miR172, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p and csa-miRn8-3p) formed subnetworks that did not display any connectivity with other miRNAs or their target genes. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to analyze the expression levels of the different miRNAs and their putative target genes in leaf, stem and root samples of cucumber over a 42-day period after inoculation with CGMMV. A positive correlation was found between some of the miRNAs and their respective target genes, although for most, the response varied greatly depending on the time point, indicating that additional factors are likely to be involved in the interaction between cucumber miRNAs and their target genes. Several miRNAs, including miR159 and csa-miRn6-3p, were linked to target genes that have been associated with plant responses to disease. A model linking miRNAs, their targets and downstream biological processes is proposed to indicate the roles of these miRNAs in the cucumber-CGMMV pathosystem.

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress.

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall.

Quantitative proteomics identifies 38 proteins that are differentially expressed in cucumber in response to cucumber green mottle mosaic virus infection.

BACKGROUND: Since it was first reported in 1935, Cucumber green mottle mosaic virus (CGMMV) has become a serious pathogen in a range of cucurbit crops. The virus is generally transmitted by propagation materials, and to date no effective chemical or cultural methods of control have been developed to combat its spread. The current study presents a preliminary analysis of the pathogenic mechanisms from the perspective of protein expression levels in an infected cucumber host, with the objective of elucidating the infection process and potential strategies to reduce both the economic and yield losses associated with CGMMV. METHODS: Isobaric tags for relative and absolute quantitation (iTRAQ) technology coupled with liquid chromatography-tandem mass spectrometric (LC-MS/MS) were used to identify the differentially expressed proteins in cucumber plants infected with CGMMV compared with mock-inoculated plants. The functions of the proteins were deduced by functional annotation and their involvement in metabolic processes explored by KEGG pathway analysis to identify their interactions during CGMMV infection, while their in vivo expression was further verified by qPCR. RESULTS: Infection by CGMMV altered both the expression level and absolute quantity of 38 proteins (fold change >0.6) in cucumber hosts. Of these, 23 were found to be up-regulated, while 15 were down-regulated. Gene ontology (GO) analysis revealed that 22 of the proteins had a combined function and were associated with molecular function (MF), biological process (BP) and cellular component (CC). Several other proteins had a dual function with 1, 7, and 2 proteins being associated with BP/CC, BP/MF, CC/MF, respectively. The remaining 3 proteins were only involved in MF. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 18 proteins that were involved in 13 separate metabolic pathways. These pathways were subsequently merged to generate three network diagrams illustrating the interactions between the different pathways, while qPCR was used to track the changes in expression levels of the proteins identified at 3 time points during CGMMV infection. Taken together these results greatly expand our understanding of the relationships between CGMMV and cucumber hosts. CONCLUSIONS: The results of the study indicate that CGMMV infection significantly changes the physiology of cucumbers, affecting the expression levels of individual proteins as well as entire metabolic pathways. The bioinformatic analysis also identified several pathogenesis-related (PR) proteins that could be useful in the development of disease-resistant plants.

[Phylogenetic and Bioinformatics Analysis of Replicase Gene Sequence of Cucumber Green Mottle Mosaic Virus].

The replicase genes of five isolates of Cucumber green mottle mosaic virus from Jiangsu, Zhejiang, Hunan and Beijing were amplificated, sequenced and analyzed. The similarities of nucleotide acid sequences indicated that 129 kD and 57 kD replicase genes of CGMMV-No. 1, CGMMV-No. 2, CGMMV-No. 3, CGMMV-No. 4 and CGMMV-No. 5 were 99.64% and 99.74%, respectively. The similarities of 129 kD and 57 kD replicase genes of CGMMV-No. 1, CGMMV-No. 3 and CGMMV-No. 4 were 99.95% and 99.94%, while they were lower between CGMMV-No. 2 and the rest of four reference sequences, just from 99.16% to 99.27% and from 99.04% to 99.18%. All reference sequences could be divided into six groups in neighbor-joining (NJ) phylogenetic trees based on the replicase gene sequences of 129 kD, 57 kD protein respectively. CGMMV-No. 1, CGMMV-No. 3 and CGMMV-No. 4 were clustered together with Shandong isolate (Accession No. KJ754195) in two NJ trees; CGMMV-No. 5 was clustered together with Liaoning isolate (Accession No. EF611826) in two NJ trees; CGMMV-No. 2 was clustered together with Korea watermelon isolate (Accession No. AF417242) in phylogenetic tree of 129 kD replicase gene of CGMMV; Interestingly, CGMMV-No. 2 was classified as a independent group in phylogenetic tree of 57 kD replicase gene of CGMMV. There were no significant hydrophobic and highly coiled coil regions on 129 kD and 57 kD proteins of tested CGMMV isolates. Except 129 kD protein of CGMMV-No. 4, the rest were unstable protein. The number of transmembrane helical segments (TMHs) of 129 kD protein of CGMMV-No. 1, CGMMV-No. 2, CGMMV-No. 3 and CGMMV-No. 5 were 6, 6, 2 and 4, respectively, which were 13, 13 and 5 on the 57 kD protein of CGMMV-No. 2, CGMMV-No. 4 and CGMMV-No. 5. The glycosylation site of 129 kD protein of tested CGMMV isolates were 2, 4, 4, 4 and 4, and that of 57 kD protein were 2, 5, 2, 5 and 2. There were difference between the disorders, globulins, phosphorylation sites and B cell antigen epitopes of 129 kD and 57 kD proteins of tested CGMMV isolates. The current results that there was no significant difference between the replicase gene sequences, it was stable and conservative for intra-species and clearly difference for inter-species. CGMMV-No. 1, CGMMV-No. 3, CGMMV-No. 4 and CGMMV-No. 5 had. a close genetic relationship with Shandong and Liangning isolates (Accession No. KJ754195 and EF611826), they are potentially originate from the same source. CGMMV-No. 2 was closer with Korea isolate. High sequence similarity of tested samples were gathered for a class in phylogenetic tree. It didn't show regularity of the bioinformatics analysis results of 129 kD and 57 kD proteins of tested CGMMV isolates. There was no corresponding relationship among the molecular phylogeny and the bioinformatics analysis of the tested CGMMV isolates.