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September 22, 2019  |  

Comparative genomic analyses reveal the features for adaptation to nematodes in fungi.

Nematophagous (NP) fungi are ecologically important components of the soil microbiome in natural ecosystems. Esteya vermicola (Ev) has been reported as a NP fungus with a poorly understood evolutionary history and mechanism of adaptation to parasitism. Furthermore, NP fungal genomic basis of lifestyle was still unclear. We sequenced and annotated the Ev genome (34.2 Mbp) and integrated genetic makeup and evolution of pathogenic genes to investigate NP fungi. The results revealed that NP fungi had some abundant pathogenic genes corresponding to their niche. A number of gene families involved in pathogenicity were expanded, and some pathogenic orthologous genes underwent positive selection. NP fungi with diverse morphological features exhibit similarities of evolutionary convergence in attacking nematodes, but their genetic makeup and microscopic mechanism are different. Endoparasitic NP fungi showed similarity in large number of transporters and secondary metabolite coding genes. Noteworthy, expanded families of transporters and endo-beta-glucanase implied great genetic potential of Ev in quickly perturbing nematode metabolism and parasitic behavior. These results facilitate our understanding of NP fungal genomic features for adaptation to nematodes and lay a solid theoretical foundation for further research and application.© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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September 22, 2019  |  

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019  |  

Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome.

Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence.We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes.The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

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September 22, 2019  |  

Nuclear and mitochondrial genomes of the hybrid fungal plant pathogen Verticillium longisporum display a mosaic structure

Allopolyploidization, genome duplication through interspecific hybridization, is an important evolutionary mechanism that can enable organisms to adapt to environmental changes or stresses. This increased adaptive potential of allopolyploids can be particularly relevant for plant pathogens in their quest for host immune response evasion. Allodiploidization likely caused the shift in host range of the fungal pathogen plant Verticillium longisporum, as V. longisporum mainly infects Brassicaceae plants in contrast to haploid Verticillium spp. In this study, we investigated the allodiploid genome structure of V. longisporum and its evolution in the hybridization aftermath. The nuclear genome of V. longisporum displays a mosaic structure, as numerous contigs consists of sections of both parental origins. V. longisporum encountered extensive genome rearrangements, whereas the contribution of gene conversion is negligible. Thus, the mosaic genome structure mainly resulted from genomic rearrangements between parental chromosome sets. Furthermore, a mosaic structure was also found in the mitochondrial genome, demonstrating its bi-parental inheritance. In conclusion, the nuclear and mitochondrial genomes of V. longisporum parents interacted dynamically in the hybridization aftermath. Conceivably, novel combinations of DNA sequence of different parental origin facilitated genome stability after hybridization and consecutive niche adaptation of V. longisporum.

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September 22, 2019  |  

Identification of candidate genes at the Dp-fl locus conferring resistance against the rosy apple aphid Dysaphis plantaginea

The cultivated apple is susceptible to several pests including the rosy apple aphid (RAA; Dysaphis plantaginea Passerini), control of which is mainly based on chemical treatments. A few cases of resistance to aphids have been described in apple germplasm resources, laying the basis for the development of new resistant cultivars by breeding. The cultivar ‘Florina’ is resistant to RAA, and recently, the Dp-fl locus responsible for its resistance was mapped on linkage group 8 of the apple genome. In this paper, a chromosome walking approach was performed by using a ‘Florina’ bacterial artificial chromosome (BAC) library. The walking started from the available tightly linked molecular markers flanking the resistance region. Various walking steps were performed in order to identify the minimum tiling path of BAC clones covering the Dp-fl region from both the “resistant” and “susceptible” chromosomes of ‘Florina’. A genomic region of about 279 Kb encompassing the Dp-fl resistance locus was fully sequenced by the PacBio technology. Through the development of new polymorphic markers, the mapping interval around the resistance locus was narrowed down to a physical region of 95 Kb. The annotation of this sequence resulted in the identification of four candidate genes putatively involved in the RAA resistance response.

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September 22, 2019  |  

Complete genome sequence of Pseudomonas Parafulva PRS09-11288, a biocontrol strain produces the antibiotic phenazine-1-carboxylic acid.

Rhizoctonia solani is a plant pathogenic fungus, which can infect a wide range of economic crops including rice. In this case, biological control of this pathogen is one of the fundmental way to effectively control this pathogen. The Pseudomonas parafulva strain PRS09-11288 was isolated from rice rhizosphere and shows biocontrol ability against R. solani. Here, we analyzed the P. parafulva genome, which is ~?4.7 Mb, with 4310 coding sequences, 76 tRNAs, and 7 rRNAs. Genome analysis identified a phenazine biosynthetic pathway, which can produce antibiotic phenazine-1-carboxylic acid (PCA). This compound is responsible for biocontrol ability against R. solani Kühn, which is one of the most serious fungus disease on rice. Analysis of the phenazine biosynthesis gene mutant, ?phzF, which is very important in this pathway, confirmed the relationship between the pathway and PCA production using LC-MS profiles. The annotated full genome sequence of this strain sheds light on the role of P. parafulva PRS09-11288 as a biocontrol bacterium.

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September 22, 2019  |  

Microbiome and infectivity studies reveal complex polyspecies tree disease in Acute Oak Decline.

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch’s postulates to address the role of microbial communities in disease.

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September 22, 2019  |  

Molecular characterization of NBS-LRR genes in the soybean Rsv3 locus reveals several divergent alleles that likely confer resistance to the soybean mosaic virus.

The divergence patterns of NBS – LRR genes in soybean Rsv3 locus were deciphered and several divergent alleles ( NBS_C, NBS_D and Columbia NBS_E ) were identified as the likely functional candidates of Rsv3. The soybean Rsv3 locus, which confers resistance to the soybean mosaic virus (SMV), has been previously mapped to a region containing five nucleotide binding site-leucine-rich repeats (NBS-LRR) genes (referred to as nbs_A-E) in Williams 82. In resistant cultivars, however, the number of NBS-LRR genes in this region and their divergence from susceptible alleles remain unclear. In the present study, we constructed and screened a bacterial artificial chromosome (BAC) library for an Rsv3-possessing cultivar, Zaoshu 18. Sequencing two positive BAC inserts on the Rsv3 locus revealed that Zaoshu 18 possesses the same gene content and order as Williams 82, but two of the NBS-LRR genes, NBS_C and NBS_D, exhibit distinct features that were not observed in the Williams 82 alleles. Obtaining these NBS-LRR genes from eight additional cultivars demonstrated that the NBS_A-D genes diverged into two different alleles: the nbs_A-D alleles were associated with the rsv3-type cultivars, whereas the NBS_A-D alleles were associated with the Rsv3-possessing cultivars. For the NBS_E gene, the cultivar Columbia possesses an allele (NBS_E) that differed from that in Zaoshu 18 and rsv3-type cultivars (nbs_E). Exchanged fragments were further detected on alleles of the NBS_C-E genes, suggesting that recombination is a major force responsible for allele divergence. Also, the LRR domains of the NBS_C-E genes exhibited extremely strong signals of positive selection. Overall, the divergence patterns of the NBS-LRR genes in Rsv3 locus elucidated by this study indicate that not only NBS_C but also NBS_D and Columbia NBS_E are likely functional alleles that confer resistance to SMV.

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September 22, 2019  |  

Complete genome sequence of Bacillus velezensis 157 isolated from Eucommia ulmoides with pathogenic bacteria inhibiting and lignocellulolytic enzymes production by SSF.

Bacillus velezensis 157 was isolated from the bark of Eucommia ulmoides, and exhibited antagonistic activity against a broad spectrum of pathogenic bacteria and fungi. Moreover, B. velezensis 157 also showed various lignocellulolytic activities including cellulase, xylanase, a-amylase, and pectinase, which had the ability of using the agro-industrial waste (soybean meal, wheat bran, sugarcane bagasse, wheat straw, rice husk, maize flour and maize straw) under solid-state fermentation and obtained several industrially valuable enzymes. Soybean meal appeared to be the most efficient substrate for the single fermentation of B. velezensis 157. Highest yield of pectinase (19.15 ± 2.66 U g-1), cellulase (46.69 ± 1.19 U g-1) and amylase (2097.18 ± 15.28 U g-1) was achieved on untreated soybean meal. Highest yield of xylanase (22.35 ± 2.24 U g-1) was obtained on untreated wheat bran. Here, we report the complete genome sequence of the B. velezensis 157, composed of a circular 4,013,317 bp chromosome with 3789 coding genes and a G + C content of 46.41%, one circular 8439 bp plasmid and a G + C content of 40.32%. The genome contained a total of 8 candidate gene clusters (bacillaene, difficidin, macrolactin, butirosin, bacillibactin, bacilysin, fengycin and surfactin), and dedicates over 15.8% of the whole genome to synthesize secondary metabolite biosynthesis. In addition, the genes encoding enzymes involved in degradation of cellulose, xylan, lignin, starch, mannan, galactoside and arabinan were found in the B. velezensis 157 genome. Thus, the study of B. velezensis 157 broadened that B. velezensis can not only be used as biocontrol agents, but also has potentially a wide range of applications in lignocellulosic biomass conversion.

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September 22, 2019  |  

Insights on a founder effect: the case of Xylella fastidiosa in the Salento area of Apulia, Italy

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa, causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca. In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of 14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity.

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September 22, 2019  |  

De novo assembly and phasing of dikaryotic genomes from two isolates of Puccinia coronata f. sp. avenae, the causal agent of oat crown rust.

Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae, is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp; approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenaeIMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae, which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae, resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of P. coronata f. sp. avenae. Copyright © 2018 Miller et al.

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September 22, 2019  |  

Pseudomonas orientalis F9: A potent antagonist against phytopathogens with phytotoxic effect in the apple flower.

In light of public concerns over the use of pesticides and antibiotics in plant protection and the subsequent selection for spread of resistant bacteria in the environment, it is inevitable to broaden our knowledge about viable alternatives, such as natural antagonists and their mode of action. The genus Pseudomonas is known for its metabolic versatility and genetic plasticity, encompassing pathogens as well as antagonists. We characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, and determined its antagonistic activity against several phytopathogenic bacteria, in particular Erwinia amylovora, the causal agent of fire blight. P. orientalis F9 displayed antagonistic activity against a broad suite of phytopathogenic bacteria in the in vitro tests. The promising results from this analysis led to an ex vivo assay with E. amylovora CFBP1430Rif and P. orientalis F9 infected detached apple flowers. F9 diminished the fire blight pathogen in the flowers but also revealed phytotoxic traits. The experimental results were discussed in light of the complete genome sequence of F9, which revealed the strain to carry phenazine genes. Phenazines are known to contribute to antagonistic activity of bacterial strains against soil pathogens. When tested in the cress assay with Pythium ultimum as pathogen, F9 showed results comparable to the known antagonist P. protegens CHA0.

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September 22, 2019  |  

Pantoea ananatis genetic diversity analysis reveals limited genomic diversity as well as accessory genes correlated with onion pathogenicity.

Pantoea ananatis is a member of the family Enterobacteriaceae and an enigmatic plant pathogen with a broad host range. Although P. ananatis strains can be aggressive on onion causing foliar necrosis and onion center rot, previous genomic analysis has shown that P. ananatis lacks the primary virulence secretion systems associated with other plant pathogens. We assessed a collection of fifty P. ananatis strains collected from Georgia over three decades to determine genetic factors that correlated with onion pathogenic potential. Previous genetic analysis studies have compared strains isolated from different hosts with varying diseases potential and isolation sources. Strains varied greatly in their pathogenic potential and aggressiveness on different cultivated Allium species like onion, leek, shallot, and chive. Using multi-locus sequence analysis (MLSA) and repetitive extragenic palindrome repeat (rep)-PCR techniques, we did not observe any correlation between onion pathogenic potential and genetic diversity among strains. Whole genome sequencing and pan-genomic analysis of a sub-set of 10 strains aided in the identification of a novel series of genetic regions, likely plasmid borne, and correlating with onion pathogenicity observed on single contigs of the genetic assemblies. We named these loci Onion Virulence Regions (OVR) A-D. The OVR loci contain genes involved in redox regulation as well as pectate lyase and rhamnogalacturonase genes. Previous studies have not identified distinct genetic loci or plasmids correlating with onion foliar pathogenicity or pathogenicity on a single host pathosystem. The lack of focus on a single host system for this phytopathgenic disease necessitates the pan-genomic analysis performed in this study.

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September 22, 2019  |  

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry.

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.

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September 22, 2019  |  

Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes.

Xanthomonas virus (phage) XacN1 is a novel jumbo myovirus infecting Xanthomonas citri, the causative agent of Asian citrus canker. Its linear 384,670?bp double-stranded DNA genome encodes 592 proteins and presents the longest (66?kbp) direct terminal repeats (DTRs) among sequenced viral genomes. The DTRs harbor 56 tRNA genes, which correspond to all 20 amino acids and represent the largest number of tRNA genes reported in a viral genome. Codon usage analysis revealed a propensity for the phage encoded tRNAs to target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes and seven additional translation-related genes as well as a chaperonin gene found in the XacN1 genome suggests a relative independence of phage replication on host molecular machinery, leading to a prediction of a wide host range for this jumbo phage. We confirmed the prediction by showing a wider host range of XacN1 than other X. citri phages in an infection test against a panel of host strains. Phylogenetic analyses revealed a clade of phages composed of XacN1 and ten other jumbo phages, indicating an evolutionary stable large genome size for this group of phages.

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