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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 .

Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (Fon), is a soilborne disease that significantly limits yield in watermelon ( Citrullus lanatus) and occasionally causes the loss of an entire year's harvest. Reference-quality de novo genomic assemblies of pathogenic and nonpathogenic strains were generated using a combination of next-generation and third-generation sequencing technologies. Chromosomal-level genomes were produced with representatives from three Fon races, facilitating comparative genomic analysis and the identification of chromosomal structural variation. Syntenic analysis between isolates allowed for differentiation of the core and lineage-specific portions of their genomes. This research will support future efforts to refine the scientific understanding of the molecular and genetic factors underpinning the Fon host range, develop diagnostic assays for each of the four races, and decipher the evolutionary history of race 3. [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 .

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.

The authors of Cardenas et al. 111:1893-1896 (2021) retracted this article because posterior analyses have identified that the assembled genomes contained a substantial proportion of other fungi beyond the orange rust Puccinia kuehnii, mainly Ascomycetes for one isolate and Ustilaginales for the other (metagenomes). This article was retracted on 11 March 2022. 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.

Phytophthora parasitica is a devastating plant pathogen that has a wide host range. As a new approach, silver nanoparticles (AgNPs) were assessed to control it. Previously AgNPs were shown to inhibit mycelial growth, zoospore production and germination, and germ tube elongation. However, the mechanism(s) of bioactivity of AgNPs on changes in metabolic patterns in P. parasitica has not been completely resolved. P. parasitica was exposed to AgNPs at several time points, RNA-Sequences were extracted, and then selected key genes in several of the pathways were verified for the level of expression using qRT-PCR. For RNA-Seq, the reads were mapped to the P. parasitica genome INRA 310.3. Principle component analysis illustrated low variation among biological replicates and emphasized the reproducibility of results. In this paper, seven candidate genes identified by RNA-Seq were evaluated for their expression using qRT-PCR. Interestingly, qRT-PCR confirmed that the genes that were significantly altered were found to be involved in major cellular pathways such as glutathione metabolism and ribosome biogenesis. qRT-PCR also revealed heat shock protein, ABC transporter, and Glutathione-S-Transferase (GST) genes were significantly affected. These genes are related to oxidative stress. Expression of GST and heat-shock protein significantly increased at 1 hr post-exposure to AgNPs, indicating a rapid response. The analysis based on qRT-PCR makes it evident that key genes involved in many major pathways were significantly altered on exposure to AgNPs.

Some fungal epithiodiketopiperazine alkaloids display α,β-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.

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. Fil: Wang, Fuqian. Wuhan Nº 1 Hospital; China. University of Hawaii at Hilo; Estados Unidos Fil: Sarotti, Ariel Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina Fil: Jiang, Guangde. University of Florida; Estados Unidos Fil: Huguet Tapia, José C.. University of Florida; Estados Unidos Fil: Zheng, Shao Liang. Harvard University; Estados Unidos Fil: Wu, Xiaohua. University of Hawaii at Hilo; Estados Unidos Fil: Li, Chunshun. University of Hawaii at Hilo; Estados Unidos Fil: Ding, Yousong. University of Florida; Estados Unidos Fil: Cao, Shugeng. University Hawaii Cancer Research Center; Estados Unidos. University of Hawaii at Hilo; Estados Unidos

Significance Plants 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.

Pitch canker, caused by the fungal pathogen

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.

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.

Significance Pathogenic bacteria acquire new virulence strategies for exploiting their hosts. This work reveals that the bacterial wheat pathogen Xanthomonas translucens uses a transcription activation-like (TAL) effector to promote virulence by directly activating the host gene 9-cis-epoxycarotenoid dioxygenase, the rate-limiting enzyme in biosynthesis of abscisic acid that is normally involved in water management within the host plant. Evolutionarily, TAL effectors are a relatively new class of virulence factors limited to a few species of pathogenic bacteria, and this work adds to the diversity of host susceptibility genes that can be exploited by pathogens through TAL effector gene function., Plants are vulnerable to disease through pathogen manipulation of phytohormone levels, which otherwise regulate development, abiotic, and biotic responses. Here, we show that the wheat pathogen Xanthomonas translucens pv. undulosa elevates expression of the host gene encoding 9-cis-epoxycarotenoid dioxygenase (TaNCED-5BS), which catalyzes the rate-limiting step in the biosynthesis of the phytohormone abscisic acid and a component of a major abiotic stress-response pathway, to promote disease susceptibility. Gene induction is mediated by a type III transcription activator-like effector. The induction of TaNCED-5BS results in elevated abscisic acid levels, reduced host transpiration and water loss, enhanced spread of bacteria in infected leaves, and decreased expression of the central defense gene TaNPR1. The results represent an appropriation of host physiology by a bacterial virulence effector.

Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae (Xoo), secretes one or more of six known transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11, SWEET13 and SWEET14, the expression of which is required for disease susceptibility. We used CRISPR–Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14, which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance., Genome editing of three SWEET gene promoters endows rice with resistance to all Xanthomonas bacterial blight strains tested.

Summary Transcription Activator‐Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time‐consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102‐nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single‐strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

The crystallization of silicon for photovoltaic applications is currently performed by directional solidification in amorphous silica crucibles. In order to avoid sticking, silica crucibles are coated with a layer of silicon nitride which acts as an interface releasing agent between the silicon and the crucible. Due to silica softening and subsequent transformations during the melting-solidification cycle, Si3N4-coated silica crucibles can be used only one time. A more interesting solution would be to have a graphite crucible which could be used several times in view of its high mechanical performances at elevated temperatures. The goal of this study is to determine in which way the Si3N4 coating can also be used for graphite crucibles. The study is conducted by means of the sessile drop technique and microstructure characterizations carried out by optical microscopy. For comparison purposes, experiments are also performed for the standard configuration of Si3N4-coated silica.

Plants are in constant association with a variety of microbes, and although much is known about how symbiotic and pathogenic microbes interact with plants, less is known about the population dynamics, adaptive traits, and transcriptional features of endophytic commensal microbes that live inside leaves. In this study, we evaluated the long-term population and transcriptional dynamics of two bacterial microbiota endophytes and compared them to those of a commensal-simulating non-pathogenic mutant of the bacterial pathogen Pseudomonas syringae. We found that population densities of all three endophytic phyllosphere bacteria remained static over a long period of time, which was caused by a continual equilibrium between bacterial multiplication and death, as evidenced by treatment of plants with antibiotics that only targeted dividing bacteria or by in planta visualization of bacteria carrying a fluorescent division reporter. Population stasis could not be explained by a lack of resources, as Arabidopsis leaves could support population densities up to 100 times higher than the normal microbiota populations, nor was population stasis reversed by significantly quenching PAMP-triggered immunity. Long-term temporal in planta transcriptomic analysis of these three bacterial endophytes revealed up-regulation of protein translation, the generation of energy, and the response to stress, and interestingly, for the microbiota strains, the longer the bacteria remained inside plants, the greater the up-regulation of some of these processes. Further transcriptomic analysis of in planta populations of commensal-simulating Pseudomonas syringae revealed a remarkable resemblance to those of in vitro bacteria in stationary phase, a metabolically active physiological state in which the production of secondary metabolites and stress responses are induced. This study provides novel insight into how endophytic bacteria survive and thrive inside plant leaves, and reshapes our current understanding of what it means to be part of the endophytic microbiota in the phyllosphere.

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., Bacterial TAL effectors hijack host gene expression to mediate disease susceptibility, and host plants counteract TAL effectors with resistance genes to thwart pathogen infection. This study reports the cloning of Xa7, demonstrates its induction by the essential virulence TAL effectors AvrXa7 and PthXo3, and documents its prevalence in indica rice cultivars.

Background Haplaxius crudus (the American palm cixiid) is a major insect pest of various economically important palms. H. crudus persists in tropical and subtropical regions where it is known to transmit the lethal yellowing (LY) phytoplasma. It has been implicated as the putative vector of Lethal bronzing (LB), a destructive phytoplasma-induced palm disease affecting over 16 species of ornamental and agricultural palms. To date, no mitochondrial genomes for species in the family Cixiidae are sequenced. Analysis of mitochondrial DNA sequences of H. crudus has proven useful for proper species diagnosis and population studies which could benefit management programs aimed at moving infective insects. These analyses describe the first mitochondrial genome from the American palm cixiid, Haplaxius crudus and an insect in the family Cixiidae. Results In this study, the complete mitochondrial genome of H. crudus was assembled and characterized from PacBio Sequel II long sequencing reads using the University of Florida’s HiPerGator supercomputer. The circular mitogenome of H. crudus is 15,845 bp long and encodes 37 mitochondrial genes (including 13 protein coding genes (PCGs), 22 tRNAs and 2 rRNAs) in addition to a putative non-coding internal control region. The nucleotide composition of H. crudus is asymmetric with a bias toward A and T (44.8 %A, 13.4 %C, 8.5 %G and 33.3 %T). Protein-coding genes (PCGs) possess the standard invertebrate mitochondrial start codons with few exceptions while the gene content and order of the H. crudus mitogenome is identical to most completely sequenced insect mitochondrial genomes. Phylogenetic analysis indicated that H. crudus is closely related to the planthopper in the family Delphacidae: N. lugens, which is the established sister group to Cixiidae. Conclusions Our studies have elucidated the first reference mitochondrial genome of Haplaxius crudus, providing structural analysis of the circular genome and encoded gene regions. The present results provide future opportunities to assess the diversity and origin of H. crudus. This study demonstrates the significance of understanding the structure and function of the mitochondrial genome to inform effective diagnostic and management strategies for insect pests.

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.

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 (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 IC(50) values in the range of 0.56 to 1.86 μM. The gene clusters mined from the sequenced genome support their putative biosynthetic pathways.

Phytophthora parasitica is one of the most widespread Phytophthora species, which is known to cause root rot, foot rot/gummosis and brown rot of fruits in citrus. In this study, we have analyzed the transcriptome of a commonly used citrus rootstock Carrizo citrange in response to P. parasitica infection using the RNA-seq technology. In total, we have identified 6692 differentially expressed transcripts (DETs) among P. parasitica-inoculated and mock-treated roots. Of these, 3960 genes were differentially expressed at 24 h post inoculation and 5521 genes were differentially expressed at 48 h post inoculation. Gene ontology analysis of DETs suggested substantial transcriptional reprogramming of diverse cellular processes particularly the biotic stress response pathways in Carrizo citrange roots. Many R genes, transcription factors, and several other genes putatively involved in plant immunity were differentially modulated in citrus roots in response to P. parasitica infection. Analysis reported here lays out a strong foundation for future studies aimed at improving resistance of citrus rootstocks to P. parasitica.

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.

Phytophthora capsici is an oomycete plant pathogen with a wide host range. Worldwide, P. capsici is known for causing the principal disease of chili pepper crops. Our goal was to expand the available genome resources for this diverse pathogen by generating whole-genome sequences for six isolates of P. capsici from Mexico.

TALes (Transcription Activator-Like effectors) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains ofXanthomonas oryzaepv. oryzae (Xoo) and some strains of otherXanthomonaspathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively throughin vitrohomologous recombination and single strand annealing and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately one month compared to three months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

Phytophthora parasiticais one of the most widespreadPhytophthoraspecies, which is known to cause root rot, foot rot/gummosis and brown rot of fruits in citrus. In this study, we have analyzed the transcriptome of a commonly used citrus rootstock Carrizo citrange in response toP. parasiticainfection using the RNA-seq technology. In total, we have identified 6692 differentially expressed transcripts (DETs) amongP. parasitica-inoculated and mock-treated roots. Of these, 3960 genes were differentially expressed at 24 hours post inoculation and 5521 genes were differentially expressed at 48 hours post inoculation. Gene ontology analysis of DETs suggested substantial transcriptional reprogramming of diverse cellular processes particularly the biotic stress response pathways in Carrizo citrange roots. ManyRgenes, transcription factors, and several other genes putatively involved in plant immunity were differentially modulated in citrus roots in response toP. parasiticainfection. Analysis reported here lays out a strong foundation for future studies aimed at improving resistance of citrus rootstocks toP. parasitica.

Pseudomonas syringae sensu strict , (phylogroup 2; referred to as P. syringae) consists of an environmentally ubiquitous bacterial population associated with diseases of numerous plant species. Recent studies using multilocus sequence analysis have indicated the clonal expansion of several P. syringae lineages, located in phylogroups 2a and 2b, in association with outbreaks of bacterial spot disease of watermelon, cantaloupe, and squash in the United States. To investigate the evolutionary processes that led to the emergence of these epidemic lineages, we sequenced the genomes of six P. syringae strains that were isolated from cucurbits grown in the United States, Europe, and China over a period of more than a decade, as well as eight strains that were isolated from watermelon and squash grown in six different Florida counties during the 2013 and 2014 seasons. These data were subjected to comparative analyses along with 42 previously sequenced genomes of P. syringae stains collected from diverse plant species and environments available from GenBank. Maximum likelihood reconstruction of the P. syringae core genome revealed the presence of a hybrid phylogenetic group, comprised of cucurbit strains collected in Florida, Italy, Serbia, and France, which emerged through genome-wide homologous recombination between phylogroups 2a and 2b. Functional analysis of the recombinant core genome showed that pathways involved in the ATP-dependent transport and metabolism of amino acids, bacterial motility, and secretion systems were enriched for recombination. A survey of described virulence factors indicated the convergent acquisition of several accessory type 3 secreted effectors (T3SEs) among phylogenetically distinct lineages through integrative and conjugative element and plasmid loci. Finally, pathogenicity assays on watermelon and squash showed qualitative differences in virulence between strains of the same clonal lineage, which correlated with T3SEs acquired through various mechanisms of horizontal gene transfer (HGT). This study provides novel insights into the interplay of homologous recombination and HGT toward pathogen emergence and highlights the dynamic nature of P. syringae sensu lato genomes.

Approximately 10 Streptomyces species cause disease on underground plant structures. The most economically important of these is potato scab, and the most studied of these pathogens is Streptomyces scabiei (syn. S. scabies). The main pathogenicity determinant of scab-causing Streptomyces species is a nitrated diketopiperazine, known as thaxtomin A (ThxA). In the pathogenic species Streptomyces turgidiscabies, ThxA biosynthetic genes reside on a mobile pathogenicity island (PAI). However, the mobilization of PAIs in other Streptomyces species remains uncharacterized. Here, we investigated the mobilization of the PAI of S. scabiei 87-22. Based on whole genome sequences, we inferred the evolutionary relationships of pathogenic Streptomyces species and discovered that Streptomyces sp. strain 96-12, a novel pathogenic species isolated from potatoes in Egypt, was phylogenetically grouped with nonpathogenic species rather than with known pathogenic species. We also found that Streptomyces sp. strain 96-12 contains a PAI that is almost identical to the PAI in S. scabiei 87-22, despite significant differences in their genome sequences. This suggested direct or indirect in vivo mobilization of the PAI between S. scabiei and nonpathogenic Streptomyces species. To test whether the S. scabiei 87-22 PAI could, indeed, be mobilized, S. scabiei 87-22 deletion mutants containing antibiotic resistance markers in the PAI were mated with Streptomyces diastatochromogenes, a nonpathogenic species. The PAI of S. scabiei was site-specifically inserted into the aviX1 gene of S. diastatochromogenes and conferred pathogenicity in radish seedling assays. Our results demonstrated that S. scabiei, the earliest described Streptomyces pathogen, could be the source of a PAI responsible for the emergence of novel pathogenic species.

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.

Isolation and characterization of common scab (CS) pathogen Streptomyces spp. from Uruguayan potato tubers and soil samples were done in response to significant economic losses due to CS on potato in autumn 2010. Seventy of the 331 isolates were classified as pathogenic owing to their ability to induce necrosis on tuber disks and stunting of radish seedling. Streptomyces spp. causing CS on potato in Uruguay were found to represent a range of different species by virtue of their diverse morphological and physiological traits as well as rep-PCR, rpoB phylogenetic analysis, and multi-locus sequences analysis. We identified isolates primarily as Streptomyces scabiei, S. acidiscabies, and S. europaeiscabiei. However, some of the pathogenic isolates still remain to be identified at the species level. This highlights the need for improved methods for discrimination among pathogenic Streptomyces species. The presence of Streptomyces pathogenicity island (PAI) genes was analyzed, including genes encoding for thaxtomin synthetase (txtA, txtB), tomatinase (tomA), and a necrosis protein (nec1). Among the isolates that were pathogenic, 50% contained the four pathogenicity genes, 33% had an atypical composition of PAI marker genes, and 17% did not contain any genes. The absence of the genes reported to be involved in thaxtomin biosynthesis (txtA, txtB) was confirmed by whole-genome sequencing of two representative strains of this group. This finding suggests the participation of other virulence factors in plant pathogenicity.

Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT–PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR–Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant ‘mega’ rice lines that will empower farmers to plant lines that are most likely to resist rice blight., Strategic deployment of blight-resistant rice lines is enabled by a molecular diagnostic kit.

Parasites and other symbionts are crucial components of ecosystems, regulating host populations and supporting food webs. However, most symbiont systems, especially those involving commensals and mutualists, are relatively poorly understood. In this study, we have investigated the nature of the symbiotic relationship between birds and their most abundant and diverse ectosymbionts: the vane-dwelling feather mites. For this purpose, we studied the diet of feather mites using two complementary methods. First, we used light microscopy to examine the gut contents of 1,300 individual feather mites representing 100 mite genera (18 families) from 190 bird species belonging to 72 families and 19 orders. Second, we used high-throughput sequencing (HTS) and DNA metabarcoding to determine gut contents from 1,833 individual mites of 18 species inhabiting 18 bird species. Results showed fungi and potentially bacteria as the main food resources for feather mites (apart from potential bird uropygial gland oil). Diatoms and plant matter appeared as rare food resources for feather mites. Importantly, we did not find any evidence of feather mites feeding upon bird resources (e.g., blood, skin) other than potentially uropygial gland oil. In addition, we found a high prevalence of both keratinophilic and pathogenic fungal taxa in the feather mite species examined. Altogether, our results shed light on the long-standing question of the nature of the relationship between birds and their vane-dwelling feather mites, supporting previous evidence for a commensalistic–mutualistic role of feather mites, which are revealed as likely fungivore–microbivore–detritivore symbionts of bird feathers.

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), an uncultured α-proteobacterium, is the most destructive disease of citrus trees worldwide. In previous studies, trunk injections of penicillin reduced CLas titers and HLB symptoms in citrus. However, antibiotic effects on the whole plant microbial community, which include effects on taxa that interact with CLas, have not yet been addressed. In this study, we investigated the effects of penicillin injection (0, 1000, and 6000 mg L−1) on rhizospheric and endophytic bacterial communities of grapefruit trees in field and greenhouse experiments through culture-independent high-throughput sequencing. DNA extractions from petioles and roots were subjected to 16S rRNA high-throughput sequencing, and reads were clustered by sequence similarity into operational taxonomic units (OTUs). Principal coordinates analysis based on weighted-UniFrac distances did not reveal differences in bacterial communities among treatments in any of the sample sources. However, pairwise linear discriminant analysis indicated significant differences in relative abundance of some taxa (including CLas) among treatments. Network analysis showed that penicillin produced major changes in root bacterial community structure by affecting interspecific microbial associations. This study provides new knowledge of the effect of antimicrobial treatments on interspecific relationships in citrus microbial communities.

Here, we report the draft genome sequence of Methylobacterium sp. strain V23, a bacterium isolated from accretion ice of the subglacial Lake Vostok (3,592 meters below the surface). This genome makes possible the study of ancient and psychrophilic genes and proteins from a subglacial environment isolated from the surface for at least 15 million years. Fil: Sapp, Amanda. University of Florida; Estados Unidos Fil: Huguet-Tapia, José C.. University of Florida; Estados Unidos Fil: Sanchez Lamas, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Antelo, Giuliano Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Primo, Emiliano David. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Bonomi, Hernán Ruy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Université de Montpellier; Francia Fil: Christner, Brent C.. University of Florida; Estados Unidos Fil: Oteroc, Lisandro H.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Two Streptomyces spp. strains responsible for potato common scab infections in Uruguay which do not produce diketopiperazines were identified through whole-genome sequencing, and the virulence factor produced by one of them was isolated and characterized. Phylogenetic analysis showed that both pathogenic strains can be identified as S. niveiscabiei, and the structure of the phytotoxin was elucidated as that of the polyketide desmethylmensacarcin using MS and NMR methods. The metabolite is produced in yields of ∼200 mg/L of culture media, induces deep necrotic lesions on potato tubers, stuns root and shoot growth in radish seedlings, and is comparatively more aggressive than thaxtomin A. This is the first time that desmethylmensacarcin, a member of a class of compounds known for their antitumor and antibiotic activity, is associated with phytotoxicity. More importantly, it represents the discovery of a new virulence factor related to potato common scab, an economically-important disease affecting potato production worldwide.

Background: Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains. Results: We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains. Conclusions: We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods. EEA Bella Vista Fil: Gochez, Alberto Martin. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; Argentina Fil: Huguet-Tapia, Jose Carlos. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Minsavage, Gerald V. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Shantharaj, Deepak. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Jalan, Neha. University of Florida. Department of Microbiology and Cell Science. Citrus Research and Education Center; Estados Unidos Fil: Strauß, Annett. University of Tübingen; Alemania Fil: Lahaye, Thomas. University of Tübingen; Alemania Fil: Wang, Nian. University of Florida. Department of Microbiology and Cell Science. Citrus Research and Education Center; Estados Unidos Fil: Canteros, Blanca Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bella Vista; Argentina Fil: Jones, Jeffrey B. University of Florida. Department of Plant Pathology; Estados Unidos Fil: Potnis, Neha. Auburn University. Department of Entomology and Plant Pathology; Estados Unidos

Strains of the Ralstonia solanacearum species complex in the phylotype IIB group are capable of causing Bacterial Wilt disease in potato and tomato at temperatures lower than 24°C. The capability of these strains to survive and to incite infection at temperatures colder than their normally tropical boundaries represents a threat to United States agriculture in temperate regions. In this work, we used a comparative genomics approach to identify orthologous genes linked to the lower temperature virulence phenotype. Six R. solanacearum cool virulent (CV) strains were compared to six strains non-pathogenic at low temperature (NPLT). CV strains can cause Bacterial Wilt symptoms at temperatures below 24°C, while NPLT cannot. Four R. solanacearum strains were sequenced for this work in order to complete the comparison. An orthologous genes comparison identified 44 genes present only in CV strains and 19 genes present only in NPLT strains. Gene annotation revealed a high percentage of genes compared with whole genomes in the transcriptional regulator and transport categories. A single nucleotide polymorphism (SNP) analysis identified 265 genes containing conserved non-synonymous SNPs in CV strains. Ten genes in the pathogenicity category were identified in this group. Comparisons of type 3 secretion system, type 6 secretion system (T6SS) clusters, and associated effectors did not indicate a correlation with the CV phenotype except for one T6SS VGR effector potentially associated with the CV phenotype. This is the first R. solanacearum genomic comparative analysis of multiple strains with different temperature related virulence. The candidate genes identified by this comparison are potential factors involved in virulence at low temperatures that need to be investigated. The high percentage of transcriptional regulators among the genes present only in CV strains supports the hypothesis that temperature dependent regulation of virulence genes explains the differential virulence phenotype at low temperatures. This comparison contributes to find new possible connections of temperature dependent virulence to the previously described complex regulatory system involving quorum-sensing, phenotype conversion (phcA), acyl-HSL production and responses to SA. It also added novel candidate T6SS effectors and useful detailed information about the T6SS in R. solanacearum.

Strains of the Ralstonia solanacearum species complex in the phylotype IIB group are capable of causing Bacterial Wilt disease in potato and tomato at temperatures lower than 24°C. The capability of these strains to survive and to incite infection at temperatures colder than their normally tropical boundaries represents a threat to United States agriculture in temperate regions. In this work, we used a comparative genomics approach to identify orthologous genes linked to the lower temperature virulence phenotype. Six R. solanacearum cool virulent (CV) strains were compared to six strains non-pathogenic at low temperature (NPLT). CV strains can cause Bacterial Wilt symptoms at temperatures below 24°C, while NPLT cannot. Four R. solanacearum strains were sequenced for this work in order to complete the comparison. An orthologous genes comparison identified 44 genes present only in CV strains and 19 genes present only in NPLT strains. Gene annotation revealed a high percentage of genes compared with whole genomes in the transcriptional regulator and transport categories. A single nucleotide polymorphism (SNP) analysis identified 265 genes containing conserved non-synonymous SNPs in CV strains. Ten genes in the pathogenicity category were identified in this group. Comparisons of type 3 secretion system, type 6 secretion system (T6SS) clusters, and associated effectors did not indicate a correlation with the CV phenotype except for one T6SS VGR effector potentially associated with the CV phenotype. This is the first R. solanacearum genomic comparative analysis of multiple strains with different temperature related virulence. The candidate genes identified by this comparison are potential factors involved in virulence at low temperatures that need to be investigated. The high percentage of transcriptional regulators among the genes present only in CV strains supports the hypothesis that temperature dependent regulation of virulence genes explains the differential virulence phenotype at low temperatures. This comparison contributes to find new possible connections of temperature dependent virulence to the previously described complex regulatory system involving quorum-sensing, phenotype conversion (phcA), acyl-HSL production and responses to SA. It also added novel candidate T6SS effectors and useful detailed information about the T6SS in R. solanacearum.

During infection, Citrus tristeza virus (CTV) produces a non-coding subgenomic RNA referred to as low-molecular-weight tristeza 1 (LMT1), which for a long time has been considered as a by-product of the complex CTV replication machinery. In this study, we investigated the role of LMT1 in the virus infection cycle using a CTV variant that does not produce LMT1 (CTV-LMT1d). We showed that lack of LMT1 did not halt virus ability to replicate or form proper virions. However, the mutant virus demonstrated significantly reduced invasiveness and systemic spread in Nicotiana benthamiana as well as an inability to establish infection in citrus. Introduction of CTV-LMT1d into the herbaceous host resulted in elevation of the levels of salicylic acid (SA) and SA-responsive pathogenesis-related genes beyond those upon inoculation with wild-type (WT) virus (CTV-WT). Further analysis showed that the LMT1 RNA produced by CTV-WT or via ectopic expression in the N. benthamiana leaves suppressed SA accumulation and up-regulated an alternative oxidase gene, which appeared to mitigate the accumulation of reactive oxygen species. To the best of our knowledge, this is the first report of a plant viral long non-coding RNA being involved in counter-acting host response by subverting the SA-mediated plant defense.

The study of biological diversification of oomycetes has been a difficult task for more than a century. Pioneer researchers used morphological characters to describe this heterogeneous group, and physiological and genetic tools expanded knowledge of these microorganisms. However, research on oomycete diversification is limited by conflicting phylogenies. Using whole genomic data from 17 oomycete taxa, we obtained a dataset of 277 core orthologous genes shared among these genomes. Analyses of this dataset resulted in highly congruent and strongly supported estimates of oomycete phylogeny when we used concatenated maximum likelihood and coalescent-based methods; the one important exception was the position of Albugo. Our results supported the position of Phytopythium vexans (formerly in Pythium clade K) as a sister clade to the Phytophthora-Hyaloperonospora clade. The remaining clades comprising Pythium sensu lato formed two monophyletic groups. One group was composed of three taxa that correspond to Pythium clades A, B and C, and the other group contained taxa representing clades F, G and I, in agreement with previous Pythium phylogenies. However, the group containing Pythium clades F, G and I was placed as sister to the Phytophthora-Hyaloperonospora-Phytopythium clade, thus confirming the lack of monophyly of Pythium sensu lato. Multispecies coalescent methods revealed that the white blister rust, Albugo laibachii, could not be placed with a high degree of confidence. Our analyses show that genomic data can resolve the oomycete phylogeny and provide a phylogenetic framework to study the evolution of oomycete lifestyles.

Phyllosticta citricarpa, the citrus black spot pathogen, was first identified in Florida in March 2010. Subsequently, this pathogen has become established in Florida but can be easily confused with the endemic nonpathogenic citrus endophyte P. capitalensis. In this study, the mating-type (MAT) loci of P. citricarpa and P. capitalensis were identified via draft genome sequencing and were characterized at the structural and sequence levels. P. citricarpa was determined to have an idiomorphic, heterothallic MAT locus structure, whereas P. capitalensis was found to have a single MAT locus consistent with a homothallic mating system. A survey of P. citricarpa isolates from Florida revealed that only the MAT1-2 idiomorph existed in the Floridian population. In contrast, isolates collected from Australia exhibited a 1:1 ratio of MAT1-1 and MAT1-2 isolates. Development and analysis of simple sequence repeat markers revealed a single multilocus genotype (MLG) in the Floridian population (n = 70) and 11 MLG within the Australian population (n = 24). These results indicate that isolates of P. citricarpa from Florida are likely descendent from a single clonal lineage and are reproducing asexually. The disease management focus in Florida will need to be concentrated on the production and dispersal of pycnidiospores.