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The genomes of eighteen Fusarium isolates cultured from diseased and healthy citrus trees were sequenced, assembled, and annotated. Isolate species identification was confirmed using single marker (TEF1-alpha) phylogenetic assessment. Studies of the traits and genotypes of plant-associated isolates are important to understanding the fungal contribution to phytobiomes of citrus.

The citrus root and rhizosphere microbiomes have been relatively well described in the literature, especially in the context of Huanglonbing disease. Yet questions addressing the assembly of root microbial endophytes have remained unanswered. In the above ground tree tissues, leaves and stems have been the research focus point, while flush and flower microbiomes, two important tissues in the vegetative and reproductive cycles of the tree, are not well described. In this study, the fungal and bacterial taxa in five biocompartments (bulk soil, rhizosphere, root endosphere, flower and flush) of citrus trees grown in a single California orchard were profiled using an amplicon-based metagenomic Illumina sequencing approach. Trees with no observable signs of abiotic or biotic stresses were sampled for two consecutive years during the floral development phase. The rhizosphere was the most biodiverse compartment compared to bulk soil, root endosphere, flower and flush microbiomes. In addition, the belowground bacteriome was more diverse than the mycobiome. Microbial richness decreased significantly from the root exosphere to the endosphere and was overall low in the above ground tissues. Root endophytic microbial community composition shared strong similarities to the rhizosphere but also contained few taxa from above ground tissues. Our data indicated compartmentalization of the microbiome with distinct profiles between above and below ground microbial communities. However, several taxa were present across all compartments suggesting the existence of a core citrus microbiota. These findings highlight key microbial taxa that could be engineered as biopesticides and biofertilizers for citriculture.

The Botryosphaeriaceae is a fungal family that includes many destructive vascular pathogens of woody plants (e.g., Botryosphaeria dieback of grape, Panicle blight of pistachio). Species in the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum, and Neoscytalidium attack a range of horticultural crops, but they vary in virulence and their abilities to infect their hosts via different infection courts (flowers, green shoots, woody twigs). Isolates of seventeen species, originating from symptomatic apricot, grape, pistachio, and walnut were tested for pathogenicity on grapevine wood after 4 months of incubation in potted plants in the greenhouse. Results revealed significant variation in virulence in terms of the length of the internal wood lesions caused by these seventeen species. Phylogenomic comparisons of the seventeen species of wood-colonizing fungi revealed clade-specific expansion of gene families representing putative virulence factors involved in toxin production and mobilization, wood degradation, and nutrient uptake. Statistical analyses of the evolution of the size of gene families revealed expansions of secondary metabolism and transporter gene families in Lasiodiplodia and of secreted cell wall degrading enzymes (CAZymes) in Botryosphaeria and Neofusicoccum genomes. In contrast, Diplodia, Dothiorella, and Neoscytalidium generally showed a contraction in the number of members of these gene families. Overall, species with expansions of gene families, such as secreted CAZymes, secondary metabolism, and transporters, were the most virulent (i.e., were associated with the largest lesions), based on our pathogenicity tests and published reports. This study represents the first comparative phylogenomic investigation into the evolution of possible virulence factors from diverse, cosmopolitan members of the Botryosphaeriaceae.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.

Plant microbiomes are critical components to plant health and can influence disease outcomes. We provide empirical data describing disease-induced shifts within the citrus microbiome at different levels of huanglongbing (HLB) disease severity. HLB is associated with an invasive phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’, that is introduced into the aerial portions of the tree by an insect vector. Disease manifests as aboveground foliar and fruit symptoms and significant root decline belowground. During the early phase of disease, there were depletions of putative keystone taxa in leaves and roots, followed by enrichments of putative beneficial taxa, suggesting a microbially derived immune response involved in plant protection that is ancillary to immune components encoded in the plant’s genome. In the late phase of disease, we observed enrichments of parasitic and saprophytic microorganisms, particularly in the roots. The community shifts within the root compartment are emblematic of a disease-induced dysbiosis where pathogens other than ‘Ca. L. asiaticus’ begin to dominate the community. Furthermore, we define key taxa enriched in trees with a slower rate of disease development, referred to as survivor trees, that are hallmarks of those found in trees in the early phase of disease that may be drivers of the survivor tree phenotype. We propose a disease ecology model that illustrates the relationship between the pathogen, the microbiome, and the host plant that highlights microorganisms that may serve as disease facilitators or antagonists.

BACKGROUND:DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. RESULTS:We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers. CONCLUSIONS:Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.

Huanglongbing (HLB), associated with the psyllid-vectored phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), is a disease threat to all citrus production worldwide. Currently, there are no sustainable curative or prophylactic treatments available. In this study, we utilized mass spectrometry (MS)-based metabolomics in combination with 3D molecular mapping to visualize complex chemistries within plant tissues to explore how these chemistries change in vivo in HLB-infected trees. We demonstrate how spatial information from molecular maps of branches and single leaves yields insight into the biology not accessible otherwise. In particular, we found evidence that flavonoid biosynthesis is disrupted in HLB-infected trees, and an increase in the polyamine, feruloylputrescine, is highly correlated with an increase in disease severity. Based on mechanistic details revealed by these molecular maps, followed by metabolic modeling, we formulated and tested the hypothesis that CLas infection either directly or indirectly converts the precursor compound, ferulic acid, to feruloylputrescine to suppress the antimicrobial effects of ferulic acid and biosynthetically downstream flavonoids. Using in vitro bioassays, we demonstrated that ferulic acid and bioflavonoids are indeed highly bactericidal to CLas, with the activity on par with a reference antibiotic, oxytetracycline, recently approved for HLB management. We propose these compounds should be evaluated as therapeutics alternatives to the antibiotics for HLB treatment. Overall, the utilized 3D metabolic mapping approach provides a promising methodological framework to identify pathogen-specific inhibitory compounds in planta for potential prophylactic or therapeutic applications. [ABSTRACT FROM AUTHOR]

Grapevines, like other perennial crops, are affected by so-called 'trunk diseases', which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development, especially under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e. metatranscriptomics) approach that could monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co-infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi-species reference for the mapping and quantification of DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition, as well as genome-wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting GTPs.

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

BackgroundTrunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development.ResultsThe integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution.ConclusionsThis study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management.

The citrus root and rhizosphere microbiomes have been relatively well described in the literature, especially in the context of Huanglonbing disease. Yet questions addressing the assembly of root microbial endophytes have remained unanswered. In the above ground tree tissues, leaves and stems have been the research focus point, while flush and flower microbiomes, two important tissues in the vegetative and reproductive cycles of the tree, are not well described. In this study, the fungal and bacterial taxa in five biocompartments (bulk soil, rhizosphere, root endosphere, flower and flush) of citrus trees grown in a single California orchard were profiled using an amplicon-based metagenomic Illumina sequencing approach. Trees with no observable signs of abiotic or biotic stresses were sampled for two consecutive years during the floral development phase. The rhizosphere was the most biodiverse compartment compared to bulk soil, root endosphere, flower and flush microbiomes. In addition, the belowground bacteriome was more diverse than the mycobiome. Microbial richness decreased significantly from the root exosphere to the endosphere and was overall low in the above ground tissues. Root endophytic microbial community composition shared strong similarities to the rhizosphere but also contained few taxa from above ground tissues. Our data indicated compartmentalization of the microbiome with distinct profiles between above and below ground microbial communities. However, several taxa were present across all compartments suggesting the existence of a core citrus microbiota. These findings highlight key microbial taxa that could be engineered as biopesticides and biofertilizers for citriculture. [ABSTRACT FROM AUTHOR]

English walnut (Juglans regia) has become an important crop in Chile, representing 11.5% of the total area of fruit trees, surpassed only by grapevine. As the Chilean walnut industry rapidly expands, young orchards are at risk from the emergence of new fungal diseases. Botryosphaeriaceae and Diaporthaceae fungi have been recognized as main causes of wood diseases in walnut, with symptoms of dieback, canker, and blight. In winter 2017, samples were collected from different orchards in Valparaíso and Maule regions. Fungal isolates recovered were cultured, characterized morphologically, and identified using DNA sequence analyses. Three species (Neofusicoccum parvum, Diplodia mutila, Diplodia seriata) were characterized in Botryosphaeriaceae and two (Diaporthe cynaroidis, Diaporthe australafricana) in Diaporthaceae. Pathogenicity tests showed that N. parvum was the most aggressive species to walnut. This study confirmed the presence of pathogenic Botryosphaeriaceae and Diaporthaceae in J. regia that should be considered an increasing risk for the growing Chilean walnut industry. [ABSTRACT FROM AUTHOR]

Summary: Grapevines, like other perennial crops, are affected by so‐called ‘trunk diseases’, which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co‐existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development, especially under vineyard conditions. The objective of this study was to develop and optimize a community‐level transcriptomics (i.e. metatranscriptomics) approach that could monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co‐infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi‐species reference for the mapping and quantification of DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition, as well as genome‐wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co‐infecting GTPs. [ABSTRACT FROM AUTHOR]

The number of reports associated with wood dieback caused by fungi in the Botryosphaeriaceae in numerous perennial crops worldwide has significantly increased in the past years. In this study, we investigated the interactions between the canker pathogen Neofusicoccum parvum and the almond tree host (Prunus dulcis), with an emphasis on varietal resistance and host response at the cell wall biochemical and histological levels. Plant bioassays in a shaded house showed that among the four commonly planted commercial almond cultivars ('Butte', 'Carmel', 'Monterey', and 'Nonpareil'), there was no significant varietal difference with respect to resistance to the pathogen. Gummosis was triggered only by fungal infection, not by wounding. A two-dimensional nuclear magnetic resonance and liquid chromatography determination of cell wall polymers showed that infected almond trees differed significantly in their glycosyl and lignin composition compared with healthy, noninfected trees. Response to fungal infection involved a significant increase in lignin, a decrease in glucans, and an overall enrichment in other carbohydrates with a profile similar to those observed in gums. Histological observations revealed the presence of guaiacyl-rich cell wall reinforcements. Confocal microscopy suggested that N. parvum colonized mainly the lumina of xylem vessels and parenchyma cells, and to a lesser extent the gum ducts. We discuss the relevance of these findings in the context of the compartmentalization of decay in trees model in almond and its potential involvement in the vulnerability of the host toward fungal wood canker diseases. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems and leaves using traditional microbial culturing integrated with culture- independent m ethods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar-diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the non-culturable Candidatus Liberibacter asiaticus bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacteria Pantoea, Bacillus and Curtobacterium. Purified bioactive natural products with anti-CLas activity, were identified from the fungus, C. cladosporiodes. Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C and D as the active agents against L. crescens. This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand functional roles of members of the microbiome with the long-term goal of developing anti-CLas bioinoculants that thrive in the citrus holosystem. [ABSTRACT FROM AUTHOR]