Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani.

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved.IMPORTANCEThe genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored.

Genome sequencing and de novo assembly of Trichoderma longibrachiatum isolate collected from Florida agricultural soils.

We report a draft genome assembly of Trichoderma longibrachiatum isolate GEV 3550, obtained from Florida, United States of America.

Comparative genomics of the niche-specific plant pathogen Streptomyces ipomoeae reveal novel genome content and organization.

IMPORTANCE: While most plant-pathogenic Streptomyces species cause scab disease on a variety of plant hosts, Streptomyces ipomoeae is the sole causative agent of soil rot disease of sweet potato and closely related plant species. Here, genome sequencing of virulent and avirulent S. ipomoeae strains coupled with comparative genomic analyses has identified genome content and organization features unique to this streptomycete plant pathogen. The results here will enable future research into the mechanisms used by S. ipomoeae to cause disease and to persist in its niche environment.

Next-generation fungal identification using target enrichment and Nanopore sequencing.

BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

A snapshot of the transcriptome of Medicago truncatula (Fabales: Fabaceae) shoots and roots in response to an arbuscular mycorrhizal fungus and the pea aphid (Acyrthosiphon pisum) (Hemiptera: Aphididae).

Plants simultaneously interact with belowground symbionts such as arbuscular mycorrhizal (AM) fungi and aboveground antagonists such as aphids. Generally, plants gain access to valuable resources including nutrients and water through the AM symbiosis and are more resistant to pests. Nevertheless, aphids' performance improves on mycorrhizal plants, and it remains unclear whether a more nutritious food source and/or attenuated defenses are the contributing factors. This study examined the shoot and root transcriptome of barrel medic (Medicago truncatula Gaertn.) plants highly colonized by the AM fungus Rhizophagus irregularis (Blaszk., Wubet, Renker, and Buscot) C. Walker and A. Schüßler (Glomerales: Glomeraceae) and exposed to 7 days of mixed age pea aphid (Acyrthosiphon pisum (Harris)) herbivory. The RNA-seq samples chosen for this study showed that aphids were heavier when fed mycorrhizal plants compared to nonmycorrhizal plants. We hypothesized that (i) insect-related plant defense pathways will be downregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; (ii) pathways involved in nutrient acquisition, carbohydrate-related and amino acid transport will be upregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; and (iii) roots of mycorrhizal plants with aphids will exhibit mycorrhiza-induced resistance. The transcriptome data revealed that the gene repertoire related to defenses, nutrient transport, and carbohydrates differs between nonmycorrhizal and mycorrhizal plants with aphids, which could explain the weight gain in aphids. We also identified novel candidate genes that are differentially expressed in nonmycorrhizal plants with aphids, thus setting the stage for future functional studies.

Transcriptomic alterations in the sweet orange vasculature correlate with growth repression induced by a variant of citrus tristeza virus.

Citrus tristeza virus (CTV, family Closteroviridae) is an economically important pathogen of citrus. CTV resides in the phloem of the infected plants and induces a range of disease phenotypes, including stem pitting and quick decline as well as a number of other deleterious syndromes. To uncover the biological processes underlying the poorly understood damaging symptoms of CTV, we profiled the transcriptome of sweet orange (Citrus sinensis) phloem-rich bark tissues of non-infected, mock-inoculated trees and trees singly infected with two distinct variants of CTV, T36 or T68-1. The T36 and T68-1 variants accumulated in the infected plants at similar titers. With that, young trees infected with T68-1 were markedly repressed in growth, while the growth rate of the trees infected with T36 was comparable to the mock-inoculated trees. Only a small number of differentially expressed genes (DEGs) were identified in the nearly asymptomatic T36-infected trees, whereas almost fourfold the number of DEGs were identified with the growth-restricting T68-1 infection. DEGs were validated using quantitative reverse transcription-PCR. While T36 did not induce many noteworthy changes, T68-1 altered the expression of numerous host mRNAs encoding proteins within significant biological pathways, including immunity and stress response proteins, papain-like cysteine proteases (PLCPs), cell-wall modifying enzymes, vascular development proteins and others. The transcriptomic alterations in the T68-1-infected trees, in particular, the strong and persistent increase in the expression levels of PLCPs, appear to contribute to the observed stem growth repression. On the other hand, analysis of the viral small interfering RNAs revealed that the host RNA silencing-based response to the infection by T36 and that by T68-1 was comparable, and thus, the induction of this antiviral mechanism may not contribute to the difference in the observed symptoms. The DEGs identified in this study promote our understanding of the underlying mechanisms of the yet unexplained growth repression induced by severe CTV isolates in sweet orange trees.

Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria.

Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

Genome-Wide Identification, Characterization, and Comparative Analysis of NLR Resistance Genes in Coffea spp.

The largest family of disease resistance genes in plants are nucleotide-binding site leucine-rich repeat genes (NLRs). The products of these genes are responsible for recognizing avirulence proteins (Avr) of phytopathogens and triggering specific defense responses. Identifying NLRs in plant genomes with standard gene annotation software is challenging due to their multidomain nature, sequence diversity, and clustered genomic distribution. We present the results of a genome-wide scan and comparative analysis of NLR loci in three coffee species (Coffea canephora, Coffea eugenioides and their interspecific hybrid Coffea arabica). A total of 1311 non-redundant NLR loci were identified in C. arabica, 927 in C. canephora, and 1079 in C. eugenioides, of which 809, 562, and 695 are complete loci, respectively. The NLR-Annotator tool used in this study showed extremely high sensitivities and specificities (over 99%) and increased the detection of putative NLRs in the reference coffee genomes. The NLRs loci in coffee are distributed among all chromosomes and are organized mostly in clusters. The C. arabica genome presented a smaller number of NLR loci when compared to the sum of the parental genomes (C. canephora, and C. eugenioides). There are orthologous NLRs (orthogroups) shared between coffee, tomato, potato, and reference NLRs and those that are shared only among coffee species, which provides clues about the functionality and evolutionary history of these orthogroups. Phylogenetic analysis demonstrated orthologous NLRs shared between C. arabica and the parental genomes and those that were possibly lost. The NLR family members in coffee are subdivided into two main groups: TIR-NLR (TNL) and non-TNL. The non-TNLs seem to represent a repertoire of resistance genes that are important in coffee. These results will support functional studies and contribute to a more precise use of these genes for breeding disease-resistant coffee cultivars.

Identification of Fungi in the Botryosphaeriaceae Family Associated with Stem Blight of Vaccinium spp. in the Southeastern United States.

Stem blight is a major disease of blueberry caused by Botryosphaeriaceae fungi. Chemical and cultural management options are limited, putting emphasis on breeding efforts to identify sources of resistance. The efficacy and durability of host resistance could be impacted by the species composition of the pathogen population in a region and by the isolates employed in the screenings used to identify the resistance. Samples (365) were collected from southern highbush (SHB) and rabbiteye blueberry (REB) cultivars from 28 sites in the southeastern US (AL, FL, GA, NC, and SC). Colony morphology identified 86% of the isolates as Botryosphaeriaceae. Conidia morphology and Maximum Likelihood analysis of the Internal Transcribed Spacer rDNA regions (ITS), translation elongation factor one alpha (tef1-α), and ß-tubulin were used to identify isolates at genera or species level. A PCR-restriction fragment length polymorphism (PCR-RFLP) test was used to identify isolates to genus. Neofusicoccum and Lasiodiplodia were the predominant genera. N. kwambonambiense, N. ribis, L. theobromae and L. pseudotheobromae were the most common species isolated. Phylogenies conducted with a limited number of isolates indicated non-clonal and potentially diverse populations occur on blueberry that warrant additional study. Botryosphaeria corticis, B. dothidea, and Diplodia seriata were isolated infrequently.

A synthetic 'essentialome' for axenic culturing of 'Candidatus Liberibacter asiaticus'.

OBJECTIVE: 'Candidatus Liberibacter asiaticus' (CLas) is associated with the devastating citrus 'greening' disease. All attempts to achieve axenic growth and complete Koch's postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic 'Ca. Liberibacter' spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. RESULTS: Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of 'essential' Lcr genes to CLas.

Comparative Genomics of Fusarium circinatum Isolates Used to Screen Southern Pines for Pitch Canker Resistance.

Pitch canker, caused by the fungal pathogen Fusarium circinatum, is a global disease affecting many Pinus spp. Often fatal, this disease causes significant mortality in both commercially grown and natural pine forests and is an issue of current and growing concern. F. circinatum isolates collected from three locations in the U.S. state of Florida were shown to be virulent on both slash and loblolly pine, with two of the isolates causing equivalent and significantly larger lesions than those caused by the third isolate during pathogenicity trials. In addition, significant genetic variation in lesion length in the pedigreed slash pine population was evident and rankings of parents for lesion length were similar across isolates. Experimental data demonstrate that both host and pathogen genetics contribute to disease severity. High-quality genomic assemblies of all three isolates were created and compared for structural differences and gene content. No major structural differences were observed among the isolates; however, missing or altered genes do contribute to genomic variation in the pathogen population. This work evaluates in planta virulence among three isolates of F. circinatum, provides genomic resources to facilitate study of this organism, and details comparative genomic methods that may be used to explore the pathogen's contribution to disease development.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Shared in planta population and transcriptomic features of nonpathogenic members of endophytic phyllosphere microbiota.

SignificancePlants evolved in an environment colonized by a vast number of microbes, which collectively constitute the plant microbiota. The majority of microbiota taxa are nonpathogenic and may be beneficial to plants under certain ecological or environmental conditions. We conducted experiments to understand the features of long-term interactions of nonpathogenic microbiota members with plants. We found that a multiplication-death equilibrium explained the shared long-term static populations of nonpathogenic bacteria and that in planta bacterial transcriptomic signatures were characteristic of the stationary phase, a physiological state in which stress protection responses are induced. These results may have significant implications in understanding the bulk of "nonpathogenic" plant-microbiota interactions that occur in agricultural and natural ecosystems.

Draft Genome Sequence and De Novo Assembly of a Fusarium oxysporum f. sp. lycopersici Isolate Collected from the Andean Region in Colombia.

We report a draft genome assembly of the causal agent of tomato vascular wilt, Fusarium oxysporum f. sp. lycopersici isolate 59, obtained from the Andean region in Colombia.

The Xa7 resistance gene guards the rice susceptibility gene SWEET14 against exploitation by the bacterial blight pathogen.

Many plant disease resistance (R) genes function specifically in reaction to the presence of cognate effectors from a pathogen. Xanthomonas oryzae pathovar oryzae (Xoo) uses transcription activator-like effectors (TALes) to target specific rice genes for expression, thereby promoting host susceptibility to bacterial blight. Here, we report the molecular characterization of Xa7, the cognate R gene to the TALes AvrXa7 and PthXo3, which target the rice major susceptibility gene SWEET14. Xa7 was mapped to a unique 74-kb region. Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element (EBE) in its promoter and encoded a 113-amino-acid peptide of unknown function. Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains. Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana. The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility. Ectopic expression of Xa7 triggers cell death in N. benthamiana. Xa7 is prevalent in indica rice accessions from 3000 rice genomes. Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.

Draft Genome Sequence Resource for the Orange Rust Pathogen of Sugarcane Puccinia kuehnii.

Puccinia kuehnii is an obligate biotrophic fungal pathogen that causes orange rust of sugarcane, which is prevalent in many countries around the globe. In the United States, orange rust was first detected in sugarcane in Florida in 2007 and poses a persistent and economically damaging threat to the sugarcane industry in this region. Here, we generated the first genome assemblies for two isolates of P. kuehnii (1040 and 2143) collected in Florida in 2017 from two sugarcane cultivars, CL85-1040 and CP89-2143, respectively. These two rust genome resources will be of immense value for future genomic studies, particularly further exploration of the predicted secretomes that may help define key pathogenicity determinants for this economically important pathogen.

Fungal Epithiodiketopiperazines Carrying α,ß-Polysulfide Bridges from Penicillium steckii YE, and Their Chemical Interconversion.

Some fungal epithiodiketopiperazine alkaloids display α,ß-polysulfide bridges alongside diverse structural variations. However, the logic of their chemical diversity has rarely been explored. Here, we report the identification of three new (2, 3, 8) and five known (1, 4-7) epithiodiketopiperazines of this subtype from a marine-derived Penicillium sp. The structure elucidation was supported by multiple spectroscopic analyses. Importantly, we observed multiple nonenzymatic interconversions of these analogues in aqueous solutions and organic solvents. Furthermore, the same biosynthetic origin of these compounds was supported by one mined gene cluster. The dominant analogue (1) demonstrated selective cytotoxicity to androgen-sensitive prostate cancer cells and HIF-depleted colorectal cells and mild antiaging activities, linking the bioactivity to oxidative stress. These results provide crucial insight into the formation of fungal epithiodiketopiperazines through chemical interconversions.

Big Genes, Small Effectors: Pea Aphid Cassette Effector Families Composed From Miniature Exons.

Aphids secrete proteins from their stylets that evidence indicates function similar to pathogen effectors for virulence. Here, we describe two small candidate effector gene families of the pea aphid, Acyrthosiphon pisum, that share highly conserved secretory signal peptide coding regions and divergent non-secretory coding sequences derived from miniature exons. The KQY candidate effector family contains eleven members with additional isoforms, generated by alternative splicing. Pairwise comparisons indicate possible four unique KQY families based on coding regions without the secretory signal region. KQY1a, a representative of the family, is encoded by a 968 bp mRNA and a gene that spans 45.7 kbp of the genome. The locus consists of 37 exons, 33 of which are 15 bp or smaller. Additional KQY members, as well as members of the KHI family, share similar features. Differential expression analyses indicate that the genes are expressed preferentially in salivary glands. Proteomic analysis on salivary glands and saliva revealed 11 KQY members in salivary proteins, and KQY1a was detected in an artificial diet solution after aphid feeding. A single KQY locus and two KHI loci were identified in Myzus persicae, the peach aphid. Of the genes that can be anchored to chromosomes, loci are mostly scattered throughout the genome, except a two-gene region (KQY4/KQY6). We propose that the KQY family expanded in A. pisum through combinatorial assemblies of a common secretory signal cassette and novel coding regions, followed by classical gene duplication and divergence.

A draft genome of Steinernema diaprepesi.

Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.

Draft Genome Sequences of Three Fusarium circinatum Isolates Used To Inoculate a Pedigreed Population of Pinus elliottii Seedlings.

Here, we announce the draft genome sequences of three Fusarium circinatum isolates that were used to inoculate slash pines (Pinus elliottii) at the U.S. Forest Service Resistance Screening Center in Asheville, North Carolina. The genomes of these isolates were similar to other publicly available genomes, with average nucleotide identity values of >0.98.

Waikikiamides A-C: Complex Diketopiperazine Dimer and Diketopiperazine-Polyketide Hybrids from a Hawaiian Marine Fungal Strain Aspergillus sp. FM242.

Waikikiamides A-C (1-3), structurally complex diketopiperazine derivatives, and putative biogenic precursors, (+)-semivioxanthin (4), notoamide F (5), and (-)-notoamide A (6), were isolated from Aspergillus sp. FM242. 1 and 2, bearing a hendecacyclic ring system, represent a novel skeleton. 3 features the first unique heterodimer of two notoamide analogs with an N-O-C bridge. Compounds 1 and 3 exhibit antiproliferative activity with IC50 values in the range of 0.56 to 1.86 µM. The gene clusters mined from the sequenced genome support their putative biosynthetic pathways.