Redundant and Distinct Roles of Two 14-3-3 Proteins in Fusarium sacchari, Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions.

A chromosomal-scale genome assembly of modern cultivated hybrid sugarcane provides insights into origination and evolution.

Sugarcane is a vital crop with significant economic and industrial value. However, the cultivated sugarcane's ultra-complex genome still needs to be resolved due to its high ploidy and extensive recombination between the two subgenomes. Here, we generate a chromosomal-scale, haplotype-resolved genome assembly for a hybrid sugarcane cultivar ZZ1. This assembly contains 10.4 Gb genomic sequences and 68,509 annotated genes with defined alleles in two sub-genomes distributed in 99 original and 15 recombined chromosomes. RNA-seq data analysis shows that sugar accumulation-associated gene families have been primarily expanded from the ZZSO subgenome. However, genes responding to pokkah boeng disease susceptibility have been derived dominantly from the ZZSS subgenome. The region harboring the possible smut resistance genes has expanded significantly. Among them, the expansion of WAK and FLS2 families is proposed to have occurred during the breeding of ZZ1. Our findings provide insights into the complex genome of hybrid sugarcane cultivars and pave the way for future genomics and molecular breeding studies in sugarcane.

The Interaction between Hypovirulence-Associated Chrysoviruses and Their Host Fusarium Species.

Chrysoviruses are isometric virus particles (35-50 nm in diameter) with a genome composed of double-stranded RNAs (dsRNA). These viruses belonged to the Chrysoviridae family, named after the first member isolated from Penicillium chrysogenum. Phylogenetic classification has divided the chrysoviruses into Alphachrysovirus and Betachrysovirus genera. Currently, these chrysoviruses have been found to infect many fungi, including Fusarium species, and cause changes in the phenotype and decline in the pathogenicity of the host. Thus, it is a microbial resource with great biocontrol potential against Fusarium species, causing destructive plant diseases and substantial economic losses. This review provides a comprehensive overview of three chrysovirus isolates (Fusarium graminearum virus 2 (FgV2), Fusarium graminearum virus-ch9 (FgV-ch9), and Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1)) reported to decline the pathogenicity of Fusarium hosts. It also summarizes the recent studies on host response regulation, host RNA interference, and chrysovirus transmission. The information provided in the review will be a reference for analyzing the interaction of Fusarium species with chrysovirus and proposing opportunities for research on the biocontrol of Fusarium diseases. Finally, we present reasons for conducting further studies on exploring the interaction between chrysoviruses and Fusarium and improving the accumulation and transmission efficiency of these chrysoviruses.

New approach for sugarcane disease recognition through visible and near-infrared spectroscopy and a modified wavelength selection method using machine learning models.

The proliferation of pathogenic fungi in sugarcane crops poses a significant threat to agricultural productivity and economic sustainability. Early identification and management of sugarcane diseases are therefore crucial to mitigate the adverse impacts of these pathogens. In this study, visible and near-infrared spectroscopy (380-1400 nm) combined with a novel wavelength selection method, referred to as modified flower pollination algorithm (MFPA), was utilized for sugarcane disease recognition. The selected wavelengths were incorporated into machine learning models, including Naïve Bayes, random forest, and support vector machine (SVM). The developed simplified SVM model, which utilized the MFPA wavelength selection method yielded the best performances, achieving a precision value of 0.9753, a sensitivity value of 0.9259, a specificity value of 0.9524, and an accuracy of 0.9487. These results outperformed those obtained by other wavelength selection approaches, including the selectivity ratio, variable importance in projection, and the baseline method of the flower pollination algorithm.

Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Genetic Diversity of "Candidatus Liberibacter asiaticus" Based on Four Hypervariable Genomic Regions in China.

Huanglongbing (HLB; greening disease), caused by Candidatus Liberibacter asiaticus (CLas), is the most damaging citrus disease worldwide. The disease has spread throughout the citrus-producing regions of Guangxi, Guangdong, Fujian, and others in China. A total of 1,788 HLB-like symptomatic or asymptomatic samples were collected from the Guangxi and Fujian provinces of China to decipher the genetic diversity of CLas and its correlation with geographic region and host plant. The disease was the most severe in orange and the least in pomelo. CLas bacteria associated with the specific geographical and citrus variety infected more than 50% of the HLB-like symptomatic samples. We identified 6,286 minor variations by comparing 35 published CLas genomes and observed a highly heterogeneous variation distribution across the genome, including four highly diverse nonprophages and three prophage segments. Four hypervariable genomic regions (HGRs) were identified to determine the genetic diversity among the CLas isolates collected from Guangxi and Fujian, China. A phylogenetic tree constructed from four HGRs showed that 100 CLas strains could be separated into four distinct clades. Ten new strains with high variations of prophage regions were identified in the mandarin and tangerine grown in new plantation areas of Guangxi. Characterizing these HGR variations in the CLas bacteria may provide insight into their evolution and adaptation to host plants and insects. IMPORTANCE The hypervariable genomic regions derived from 35 published CLas genomes were used to decipher the genetic diversity of CLas strains and identify 10 new strains with high variations in prophage regions. Characterizing these variations in the CLas bacteria might provide insight into their evolution and adaptation to host plants and insects in China.

Dynamic DNA methylation changes reveal tissue-specific gene expression in sugarcane.

DNA methylation is an important mechanism for the dynamic regulation of gene expression and silencing of transposons during plant developmental processes. Here, we analyzed genome-wide methylation patterns in sugarcane (Saccharum officinarum) leaves, roots, rinds, and piths at single-base resolution. DNA methylation patterns were similar among the different sugarcane tissues, whereas DNA methylation levels differed. We also found that DNA methylation in different genic regions or sequence contexts plays different roles in gene expression. Differences in methylation among tissues resulted in many differentially methylated regions (DMRs) between tissues, particularly CHH DMRs. Genes overlapping with DMRs tended to be differentially expressed (DEGs) between tissues, and these DMR-associated DEGs were enriched in biological pathways related to tissue function, such as photosynthesis, sucrose synthesis, stress response, transport, and metabolism. Moreover, we observed many DNA methylation valleys (DMVs), which always overlapped with transcription factors (TFs) and sucrose-related genes, such as WRKY, bZIP, WOX, SPS, and FBPase. Collectively, these findings provide significant insights into the complicated interplay between DNA methylation and gene expression and shed light on the epigenetic regulation of sucrose-related genes in sugarcane.

Complete genome sequence of biocontrol strain Paenibacillus peoriae HJ-2 and further analysis of its biocontrol mechanism.

BACKGROUND: Paris polyphylla is a herb widely used in traditional Chinese medicine to treat various diseases. Stem rot diseases seriously affected the yield of P. polyphylla in subtropical areas of China. Therefore, cost-effective, chemical-free, eco-friendly strategies to control stem rot on P. polyphylla are valuable and urgently needed. RESULTS: In this paper, we reported the biocontrol efficiency of Paenibacillus peoriae HJ-2 and its complete genome sequence. Strain HJ-2 could serve as a potential biocontrol agent against stem rot on P. polyphylla in the greenhouse and field. The genome of HJ-2 consists of a single 6,001,192 bp chromosome with an average GC content of 45% and 5,237 predicted protein coding genes, 39 rRNAs and 108 tRNAs. The phylogenetic tree indicated that HJ-2 is most closely related to P. peoriae IBSD35. Functional analysis of genome revealed numerous genes/gene clusters involved in plant colonization, biofilm formation, plant growth promotion, antibiotic and resistance inducers synthesis. Moreover, metabolic pathways that potentially contribute to biocontrol mechanisms were identified. CONCLUSIONS: This study revealed that P. peoriae HJ-2 could serve as a potential BCA against stem rot on P. polyphylla. Based on genome analysis, the genome of HJ-2 contains more than 70 genes and 12 putative gene clusters related to secondary metabolites, which have previously been described as being involved in chemotaxis motility, biofilm formation, growth promotion, antifungal activity and resistance inducers biosynthesis. Compared with other strains, variation in the genes/gene clusters may lead to different antimicrobial spectra and biocontrol efficacies.

First report of stalk bacterial soft rot of sugarcane caused by Dickeya zeae in China.

In late September 2019, seven stalks of about 1400 stalks of sugarcane cultivar Zhongzhe 1 exhibited soft rot symptoms in a trial plot in Beihai city, Guangxi province of China. Symptoms included scorched and collapsed leaves, maceration of stalks, and sour smelling exudates from the stalks (Supplementary Fig. S1). Severely diseased stalks had collapsed and were dead. Internal stalk fragments of 5 × 5 mm were collected at the junction of healthy and diseased tissue after surface-sterilization of stalks with 70% ethanol for one minute, and three times rinsing with sterile distilled water. Stalk fragments were placed on Luria-Bertani agar medium (1 % w/v tryptone, 0.5 % w/v yeast extract, 1 % w/v NaCl, 1 % w/v agar, pH7.0) and plates were put in an incubator at 30°C for 48h. Four types of bacterial colonies were obtained, and small and white colonies with irregular margins were the most dominant. A single colony of each type was diluted in sterile distilled water and aliquots of each suspension were streaked on fresh medium plates to obtain pure cultures. Ten eight-week-old stalks (11 th leaf stage) of sugarcane plants, which derived from cuttings of symptomless cultivar Zhongzhe 1, were inoculated by injection of 300 µl of bacterial suspension (3.5x108 CFU/ml) into the stalks. Another 10 stalks were injected with pure water and served as control. The inoculated plants were kept in a greenhouse at 25-37℃.Among the four types of bacteria, only strain BH9 induced symptoms that were identical to those of diseased canes observed in the field (Supplementary Fig. S1). Elongated water-soaked lesions were observed around the inoculation sites three days post inoculation. Five of the 10 BH9-inoculated plants had collapsed two days later. Water-soaked stalks had a sour smell similar to the filed diseased plants. Eight days post inoculation, all BH9-inoculated plants exhibited symptoms but control plants remained symptomless up to 30 days after inoculation. Uniform white colonies with irregular margins were isolated from the inoculated stalks that developed soft rot symptom, and these bacteria caused again stalk soft rot symptoms when inoculated to a new batch of 10 healthy plants. The 16S rRNA gene of strain BH9 was amplified by PCR with primer pair fD2/rP1 and the PCR amplicons from three independent colonies were sequenced. The sequences of the three amplicons were identical (Accession No. MT723897). BLAST alignments of the 16S rDNA sequence from BH9 strain with the GenBank database revealed that BH9 belonged to the genus Dickeya (98.5% identity between D. zeae BH9 and D. zeae EC1). Further PCR assays and sequencing of three genes, DNA polymerase III gamma subunit gene dnaX with primers dnaXf/dnaXr, DNA gyrase gene gyrB with primers gyrBf1/gyrBr1, and recombinase A gene recA with primers recAf/recAr, were performed to identify the species within the genus Dickeya (Zhang et al., 2014). BH9 sequences of these genes (Accession No. MT723898 to MT723900) had highest identity (97.5%, 97.6%, and 97.7%, respectively) with those from D. zeae EC1 (GenBank accession No. CP006929.1). To determine the evolutionary relationship of BH9 to other Dickeya species and strains, a phylogenetic analysis was performed using dnaX, gyrB, and recA sequences. As shown in Supplementary Fig. S2, BH9 clustered with D. zeae strains and formed a lineage distinguishable from other Dickeya species. Among the closest strains, D. zeae NCPPB3531 (Accession No. CM001980.1) was isolated from potato and D. zeae CSL RW192 (Accession No. CM001972.1) from river water (Pritchard et al., 2013). Consequently, strain BH9 was identified as D. zeae. This bacterial species has been reported to cause soft rot in rice (Pu et al., 2012), banana (Zhang et al., 2014), maize (Martinez-Cisneros et al., 2014), and clivia (Hu et al., 2018). To the best of our knowledge, this is the first report of a bacterial stalk rot caused by D. Zeae in sugarcane. In fact, low incidence of D. zeae-caused stalk soft rot was recently found in sugarcane fields in Fusui County, about 150 km north to Beihai. Given the potential threat of this disease to the local sugarcane industry, the mode of transmission, cultivar resistance, and measures to control the disease should be investigated.

Virome Identification and Characterization of Fusarium sacchari and F. andiyazi: Causative Agents of Pokkah Boeng Disease in Sugarcane.

Fusarium sacchari and Fusarium andiyazi are two devastating sugarcane pathogens that cause pokkah boeng disease (PBD) in China. RNA_Seq was conducted to identify mycoviruses in F. sacchari and F. andiyazi isolates collected from PBD symptom-showing sugarcane plants across China. Fifteen isolates with a normal, debilitated, or abnormal phenotype in colony morphology were screened out for the existence of dsRNA from 104 Fusarium isolates. By sequencing the mixed pool of dsRNA from these Fusarium isolates, a total of 26 contigs representing complete or partial genome sequences of ten mycoviruses and their strains were identified, including one virus belonging to Hypoviridae, two mitoviruses with seven strains belonging to Narnaviridae, one virus of Chrysoviridae, and one alphavirus-like virus. RT-PCR amplification with primers specific to individual mycoviruses revealed that mitoviruses were the most prevalent and the alphavirus-like virus and chrysovirus were the least prevalent. In terms of host preference, more mitoviruses were found in F. andiyazi than in F. sacchari. Fusarium sacchari hypovirus 1 with a 13.9 kb genome and a defective genome of 12.2 kb, shares 54% identity at the amino acid level to the Wuhan insect virus 14, which is an unclassified hypovirus identified from insect meta-transcriptomics. The alphavirus-like virus, Fusarium sacchari alphavirus-like virus 1 (FsALV1), seemed to hold a distinct status amid fungal alphavirus-like viruses, with the highest identity of 27% at the amino acid level to Sclerotium rolfsii alphavirus-like virus 3 and 29% to a hepevirus, Ferret hepatitis E virus. While six of the seven mitoviruses shared 72-94% identities to known mitoviruses, Fusarium andiyazi mitovirus 2 was most similar to Alternaria brassicicola mitovirus with an identity of only 49% between the two viruses. Transmission of FsALV1 and Fusarium sacchari chrysovirus 1 (FsCV1) from F. sacharri to F. commune was observed and the characterization of the four-segment dsRNA chrysovirus was performed with aid of electron microscopy and analysis of the encapsidated RNAs. These findings provide insight into the diversity and spectrum of mycoviruses in PBD pathogens and should be useful for exploring agents to control the disease.

Identification and Functional Analysis of the Pheromone Response Factor Gene of Sporisorium scitamineum.

The sugarcane smut fungus Sporisorium scitamineum is bipolar and produces sporidia of two different mating types. During infection, haploid cells of opposite mating types can fuse to form dikaryotic hyphae that can colonize plant tissue. Mating and filamentation are therefore essential for S. scitamineum pathogenesis. In this study, we obtained one T-DNA insertion mutant disrupted in the gene encoding the pheromone response factor (Prf1), hereinafter named SsPRF1, of S. scitamineum, via Agrobacterium tumefaciens-mediated transformation (ATMT) mutagenesis. Targeted deletion of SsPRF1 resulted in mutants with phenotypes similar to the T-DNA insertion mutant, including failure to mate with a compatible wild-type partner strain and being non-pathogenic on its host sugarcane. qRT-PCR analyses showed that SsPRF1 was essential for the transcription of pheromone-responsive mating type genes of the a1 locus. These results show that SsPRF1 is involved in mating and pathogenicity and plays a key role in pheromone signaling and filamentous growth in S. scitamineum.

Draft Genome Sequence of Ralstonia solanacearum Strain Rs-T02, Which Represents the Most Prevalent Phylotype in Guangxi, China.

Ralstonia solanacearumstrain Rs-T02 was originally isolated from a bacterial wilt of tomato plant in Nanning City of Guangxi Province, China. It represents the most prevalent phylotype in Guangxi. Here, we present the draft genome sequence of this strain, which comprises 5,225 genes and 5,976,011 nucleotides with an average G+C content of 66.79%. There are 968 different genes between this isolate and the previously reported genome sequence ofRalstonia solanacearumGMl l000 (race l, biovar 3, phylotype I), and the genome sequence information of this isolate may be useful for comparative genomic studies to determine the genetic diversity in this species.

Complete Genome Sequence of Mulberry Vein Banding Associated Virus, a New Tospovirus Infecting Mulberry.

Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5'-/3'-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.

Δ(1)-pyrroline-5-carboxylate/glutamate biogenesis is required for fungal virulence and sporulation.

Proline dehydrogenase (Prodh) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5Cdh) are two key enzymes in the cellular biogenesis of glutamate. Recombinant Prodh and P5Cdh proteins of the chestnut blight fungus Cryphonectria parasitica were investigated and showed activity in in vitro assays. Additionally, the C. parasitica Prodh and P5Cdh genes were able to complement the Saccharomyces cerevisiae put1 and put2 null mutants, respectively, to allow these proline auxotrophic yeast mutants to grow on media with proline as the sole source of nitrogen. Deletion of the Prodh gene in C. parasitica resulted in hypovirulence and a lower level of sporulation, whereas deletion of P5Cdh resulted in hypovirulence though no effect on sporulation; both Δprodh and Δp5cdh mutants were unable to grow on minimal medium with proline as the sole nitrogen source. In a wild-type strain, the intracellular level of proline and the activity of Prodh and P5Cdh increased after supplementation of exogenous proline, though the intracellular Δ(1)-pyrroline-5-carboxylate (P5C) content remained unchanged. Prodh and P5Cdh were both transcriptionally down-regulated in cells infected with hypovirus. The disruption of other genes with products involved in the conversion of arginine to ornithine, ornithine and glutamate to P5C, and P5C to proline in the cytosol did not appear to affect virulence; however, asexual sporulation was reduced in the Δpro1 and Δpro2 mutants. Taken together, our results showed that Prodh, P5Cdh and related mitochondrial functions are essential for virulence and that proline/glutamate pathway components may represent down-stream targets of hypovirus regulation in C. parasitica.