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Among plant-associated bacteria, agrobacteria occupy a special place. These bacteria are feared in the field as agricultural pathogens. They cause abnormal growth deformations and significant economic damage to a broad range of plant species. However, these bacteria are revered in the laboratory as models and tools. They are studied to discover and understand basic biological phenomena and used in fundamental plant research and biotechnology. Agrobacterial pathogenicity and capability for transformation are one and the same and rely on functions encoded largely on their oncogenic plasmids. Here, we synthesize a substantial body of elegant work that elucidated agrobacterial virulence mechanisms and described their ecology. We review findings in the context of the natural diversity that has been recently unveiled for agrobacteria and emphasize their genomics and plasmids. We also identify areas of research that can capitalize on recent findings to further transform our understanding of agrobacterial virulence and ecology. Expected final online publication date for the Annual Review of Phytopathology, Volume 61 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Globalization has made agricultural commodities more accessible, available, and affordable. But their global movement increases the potential for invasion by pathogens and necessitates development and implementation of sensitive, rapid, and scalable surveillance methods. Here, we used 35 strains, isolated by multiple diagnostic laboratories, as a case study for using whole genome sequence data in a plant disease diagnostic setting. Twenty-seven of the strains were isolated in 2022 and identified as

Specialization in mutualisms is thought to be a major driver of diversification, but few studies have explored how novel specialization evolves, or its relation to the evolution of other niche axes. A fundamental question is whether generalist interactions evolve to become more specialized (i.e., oscillation hypothesis) or if partner switches evolve without any change in niche breadth (i.e., musical chairs hypothesis). We examined alternative models for the evolution of specialization by estimating the mutualistic, climatic, and edaphic niche breadths of sister plant species, combining phylogenetic, environmental, and experimental data on Acmispon strigosus and Acmispon wrangelianus genotypes across their overlapping ranges in California. We found that specialization along all three niche axes was asymmetric across species, such that the species with broader climatic and edaphic niches, Acmispon strigosus, was also able to gain benefit from and invest in associating with a broader set of microbial mutualists. Our data are consistent with the oscillation model of specialization, and a parallel narrowing of the edaphic, climatic, and mutualistic dimensions of the host species niche. Our findings provide novel evidence that the evolution of specialization in mutualism is accompanied by specialization in other niche dimensions.

The genus Streptomyces includes several phytopathogenic species that cause common scab, a devastating disease of tuber and root crops, in particular potato. The diversity of species that cause common scab is unknown. Likewise, the genomic context necessary for bacteria to incite common scab symptom development is not fully characterized. Here, we phenotyped and sequenced the genomes of five strains from a poorly studied Streptomyces lineage. These strains form a new species-level group. When genome sequences within just these five strains are compared, there are no polymorphisms of loci implicated in virulence. Each genome contains the pathogenicity island that encodes for the production of thaxtomin A, a phytotoxin necessary for common scab. Yet, not all sequenced strains produced thaxtomin A. Strains varied from nonpathogenic to highly virulent on two hosts. Unexpectedly, one strain that produced thaxtomin A and was pathogenic on radish was not aggressively pathogenic on potato. Therefore, while thaxtomin A biosynthetic genes and production of thaxtomin A are necessary, they are not sufficient for causing common scab of potato. Additionally, results show that even within a species-level group of Streptomyces strains, there can be aggressively pathogenic and nonpathogenic strains despite conservation of virulence genes.

NFAT-133 is a Streptomyces-derived aromatic polyketide compound with immunosuppressive, antidiabetic, and antitrypanosomal activities. It inhibits transcription mediated by nuclear factor of activated T cells (NFAT), leading to the suppression of interleukin-2 expression and T cell proliferation. It also activates the AMPK pathway in L6 myotubes and increases glucose uptake. In addition to NFAT-133, a number of its congeners, e.g., panowamycins and benwamycins, have been identified. However, little is known about their modes of formation in the producing organisms. Through genome sequencing of Streptomyces pactum ATCC 27456, gene inactivation, and genetic complementation experiments, the biosynthetic gene cluster of NFAT-133 and its congeners has been identified. The cluster contains a highly disordered genetic organization of type I modular polyketide synthase genes with several genes that are necessary for the formation of the aromatic core unit and tailoring processes. In addition, a number of new analogs of NFAT-133 were isolated and their chemical structures elucidated. It is suggested that the heptaketide NFAT-133 is derived from an octaketide intermediate, TM-123. The current study shows yet another unusual biosynthetic pathway involving a noncanonical polyketide synthase assembly line to produce a group of small molecules with valuable bioactivities.

Acquisition of mobile genetic elements can confer novel traits to bacteria. Some integrative and conjugative elements confer upon members of Bradyrhizobium the capacity to fix nitrogen in symbiosis with legumes. These so-called symbiosis integrative conjugative elements (symICEs) can be extremely large and vary as monopartite and polypartite configurations within chromosomes of related strains. These features are predicted to impose fitness costs and have defied explanation. Here, we show that chromosome architecture is largely conserved despite diversity in genome composition, variations in locations of attachment sites recognized by integrases of symICEs, and differences in large-scale chromosomal changes that occur upon integration. Conversely, many simulated nonnative chromosome–symICE combinations are predicted to result in lethal deletions or disruptions to architecture. Findings suggest that there is compatibility between chromosomes and symICEs. We hypothesize that the size and structural flexibility of symICEs are important for generating combinations that maintain chromosome architecture across a genus of nitrogen-fixing bacteria with diverse and dynamic genomes.

Bacterial canker of stone fruit is a destructive disease of Prunus species. Here we characterized five Pseudomonas syringae (Ps) strains isolated from bacterial cankers of stone fruits in East Azerbaijan province, Iran. Koch’s postulates were performed demonstrating pathogenicity on apricot cuttings. Ice-nucleation activity and syringomycin synthesis were determined phenotypically and confirmed molecularly in all five strains. Strains were variably resistant to copper. The strains were then compared to a sample of 227 strains of the Ps species complex using multilocus sequence analysis (MLSA) of the four housekeeping genes gap1, gltA, gyrB, and rpoD. The concatenated sequences of the four genes provided a deeply-resolved phylogeny. Iranian strains all belonged to Ps phylogroup 2 but clustered into three separate sub-groups. They had the greatest similarity to strains isolated from stone fruit trees in the USA, UK, and Lebanon but also to strains isolated from other plant species and the water cycle. We conclude that canker of stone fruit is caused by a diverse group of Ps phylogroup 2 strains with global distribution. Further sampling, host range testing, and genome sequencing will be needed to get further insight into the relative role of the water cycle and other modes of long distance movement in the dissemination of Ps strains that cause bacterial canker of stone fruit.

The type III secretion system (T3SS) of plant-pathogenic Pseudomonas syringae is essential for virulence. Genes encoding the T3SS are not constitutively expressed and must be induced upon infection. Plant-derived metabolites, including sugars such as fructose and sucrose, are inducers of T3SS-encoding genes, yet the molecular mechanisms underlying perception of these host signals by P. syringae are unknown. Here, we report that sugar-induced expression of type III secretion A (setA), predicted to encode a DeoR-type transcription factor, is required for maximal sugar-induced expression of T3SS-associated genes in P. syringae DC3000. From a Tn5 transposon mutagenesis screen, we identified two independent mutants with insertions in setA. When both setA::Tn5 mutants were cultured in minimal medium containing fructose, genes encoding the T3SS master regulator HrpL and effector AvrRpm1 were expressed at lower levels relative to that of a wild-type strain. Decreased hrpL and avrRpm1 expression also occurred in a setA::Tn5 mutant in response to glucose, sucrose, galactose, and mannitol, demonstrating that setA is genetically required for T3SS induction by many different sugars. Expression of upstream regulators hrpR/S and rpoN was not altered in setA::Tn5, indicating that SetA positively regulates hrpL expression independently of increased transcription of these genes. In addition to decreased response to defined sugar signals, a setA::Tn5 mutant had decreased T3SS deployment during infection and was compromised in its ability to grow in planta and cause disease. These data suggest that SetA is necessary for P. syringae to effectively respond to T3SS-inducing sugar signals encountered during infection.

Fourteen strains of Streptomyces isolated from scab lesions on potato are described as members of a novel species based on genetic distance, morphological observation and biochemical analyses. Morphological and biochemical characteristics of these strains are distinct from other described phytopathogenic species. Strain NE06-02DT has white aerial mycelium and grey, cylindrical, smooth spores on rectus-flexibilis spore chains. Members of this species group can utilize most of the International Streptomyces Project sugars, utilize melibiose and trehalose, produce melanin, grow on 6–7 % NaCl and pH 5–5.5 media, and are susceptible to oleandomycin (100 µg ml−1), streptomycin (20 µg ml−1) and penicillin G (30 µg ml−1). Though the 16S rRNA gene sequences from several members of this novel species are identical to the Streptomyces bottropensis 16S rRNA gene sequence, whole-genome average nucleotide identity and multi-locus sequence analysis confirm that the strains are members of a novel species. Strains belonging to this novel species have been isolated from the United States, Egypt and China with the earliest known members being isolated in 1961 from common scab lesions of potato in both California, USA, and Maine, USA. The name Streptomyces caniscabiei sp. nov. is proposed for strain NE06-02DT (=DSM111602T=ATCC TSD-236T) and the other members of this novel species group.

Background Many named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. Results We utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. Conclusions We demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

The effective elimination of Listeria monocytogenes through cleaning and sanitation is of great importance to the food processing industry. Specifically in fresh produce operations, the lack of a kill step requires effective cleaning and sanitation to mitigate the risk of cross-contamination from the environment. As facilities rely on sanitizers to control L. monocytogenes, reports of the development of tolerance to sanitizers and other antimicrobials through cross-resistance is of particular concern. We investigated the potential for six L. monocytogenes isolates from fresh produce handling and processing facilities and packinghouses to develop cross-resistance between a commercial sanitizer and antibiotics. Experimental adaptation of isolates belonging to hypervirulent clonal complexes (CC2, CC4, and CC6) to a commercial quaternary ammonium compound sanitizer (cQAC) resulted in elevated minimum inhibitory concentrations (2–3 ppm) and minimum bactericidal concentrations (3–4 ppm). Susceptibility to cQAC was restored for all adapted (qAD) isolates in the presence of reserpine, a known efflux pump inhibitor. Reduced sensitivity to 7/17 tested antibiotics (chloramphenicol, ciprofloxacin, clindamycin, kanamycin, novobiocin, penicillin, and streptomycin) was observed in all tested isolates. qAD isolates remained susceptible to antibiotics commonly used in the treatment of listeriosis (i.e., ampicillin and gentamicin). The whole genome sequencing of qAD strains, followed by comparative genomic analysis, revealed several mutations in fepR, the regulator for FepA fluoroquinolone efflux pump. The results suggest that mutations in fepR play a role in the reduction in antibiotic susceptibility following low level adaptation to cQAC. Further investigation into the cross-resistance mechanisms and pressures leading to the development of this phenomenon among L. monocytogenes isolates recovered from different sources is needed to better understand the likelihood of cross-resistance development in food chain isolates and the implications for the food industry.

Members of the agrobacteria–rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

The type VI secretion system (T6SS) is used by many Gram-negative bacteria to deploy toxic effectors for interbacterial competition. This system provides a competitive advantage in planta to agrobacteria, a diverse group with phytopathogenic members capable of genetically transforming plants. To inform on the ecology and evolution of agrobacteria, we revealed processes that diversify their effector gene collections. From genome sequences of diverse strains, we identified T6SS loci, functionally validated associated effector genes for toxicity, and predicted genes homologous to those that encode proteins known to interact with effectors. The gene loci were analyzed in a phylogenetic framework, and results show that strains of some species-level groups have different patterns of T6SS expression and are enriched in specific sets of T6SS loci. Findings also demonstrate that the modularity of T6SS loci and their associated genes engenders dynamicity, promoting reshuffling of entire loci, fragments therein, and domains to swap toxic effector genes across species. However, diversification is constrained by the need to maintain specific combinations of gene subtypes, congruent with observations that certain genes function together to regulate T6SS loading and activation. Data are consistent with a scenario where species can acquire unique T6SS loci that are then reshuffled across the genus in a restricted manner to generate new combinations of effector genes. IMPORTANCE The T6SS is used by several taxa of Gram-negative bacteria to secrete toxic effector proteins to attack others. Diversification of effector collections shapes bacterial interactions and impacts the health of hosts and ecosystems in which bacteria reside. We uncovered the diversity of T6SS loci across a genus of plant-associated bacteria and show that diversification is driven by the acquisition of new loci and reshuffling among species. However, linkages between specific subtypes of genes need to be maintained to ensure that proteins whose interactions are necessary to activate the T6SS remain together. Results reveal how organization of gene loci and domain structure of genes provides flexibility to diversify under the constraints imposed by the system. Findings inform on the evolution of a mechanism that influences bacterial communities.

Recent listeriosis outbreaks linked to fresh produce suggest the need to better understand and mitigate L. monocytogenes contamination in packing and processing environments. Using whole genome sequencing (WGS) and phenotype screening assays for sanitizer tolerance, we characterized 48 L. monocytogenes isolates previously recovered from environmental samples in five produce handling facilities. Within the studied population there were 10 sequence types (STs) and 16 cgMLST types (CTs). Pairwise single nucleotide polymorphisms (SNPs) ranged from 0 to 3047 SNPs within a CT, revealing closely and distantly related isolates indicative of both sporadic and continuous contamination events within the facility. Within Facility 1, we identified a closely related cluster (0–2 SNPs) of isolates belonging to clonal complex 37 (CC37; CT9492), with isolates recovered during sampling events 1-year apart and in various locations inside and outside the facility. The accessory genome of these CC37 isolates varied from 94 to 210 genes. Notable genetic elements and mutations amongst the isolates included the bcrABC cassette (2/48), associated with QAC tolerance; mutations in the actA gene on the Listeria pathogenicity island (LIPI) 1 (20/48); presence of LIPI-3 (21/48) and LIPI-4 (23/48). This work highlights the potential use of WGS in tracing the pathogen within a facility and understanding properties of L. monocytogenes in produce settings.

The effective elimination of Listeria monocytogenes through cleaning and sanitation is of great importance to the food processing industry. Specifically in fresh produce operations, the lack of a kill step requires effective cleaning and sanitation to mitigate the risk of cross-contamination from the environment. As facilities rely on sanitizers to control L. monocytogenes, reports of the development of tolerance to sanitizers and other antimicrobials through cross-resistance is of particular concern. We investigated the potential for six L. monocytogenes isolates from fresh produce handling and processing facilities and packinghouses to develop cross-resistance between a commercial sanitizer and antibiotics. Experimental adaptation of isolates belonging to hypervirulent clonal complexes (CC2, CC4, and CC6) to a commercial quaternary ammonium compound sanitizer (cQAC) resulted in elevated minimum inhibitory concentrations (2–3 ppm) and minimum bactericidal concentrations (3–4 ppm). Susceptibility to cQAC was restored for all adapted (qAD) isolates in the presence of reserpine, a known efflux pump inhibitor. Reduced sensitivity to 7/17 tested antibiotics (chloramphenicol, ciprofloxacin, clindamycin, kanamycin, novobiocin, penicillin, and streptomycin) was observed in all tested isolates. qAD isolates remained susceptible to antibiotics commonly used in the treatment of listeriosis (i.e., ampicillin and gentamicin). The whole genome sequencing of qAD strains, followed by comparative genomic analysis, revealed several mutations in fepR, the regulator for FepA fluoroquinolone efflux pump. The results suggest that mutations in fepR play a role in the reduction in antibiotic susceptibility following low level adaptation to cQAC. Further investigation into the cross-resistance mechanisms and pressures leading to the development of this phenomenon among L. monocytogenes isolates recovered from different sources is needed to better understand the likelihood of cross-resistance development in food chain isolates and the implications for the food industry.

Recent listeriosis outbreaks linked to fresh produce suggest the need to better understand and mitigate L. monocytogenes contamination in packing and processing environments. Using whole genome sequencing (WGS) and phenotype screening assays for sanitizer tolerance, we characterized 48 L. monocytogenes isolates previously recovered from environmental samples in five produce handling facilities. Within the studied population there were 10 sequence types (STs) and 16 cgMLST types (CTs). Pairwise single nucleotide polymorphisms (SNPs) ranged from 0 to 3047 SNPs within a CT, revealing closely and distantly related isolates indicative of both sporadic and continuous contamination events within the facility. Within Facility 1, we identified a closely related cluster (0–2 SNPs) of isolates belonging to clonal complex 37 (CC37; CT9492), with isolates recovered during sampling events 1-year apart and in various locations inside and outside the facility. The accessory genome of these CC37 isolates varied from 94 to 210 genes. Notable genetic elements and mutations amongst the isolates included the bcrABC cassette (2/48), associated with QAC tolerance; mutations in the actA gene on the Listeria pathogenicity island (LIPI) 1 (20/48); presence of LIPI-3 (21/48) and LIPI-4 (23/48). This work highlights the potential use of WGS in tracing the pathogen within a facility and understanding properties of L. monocytogenes in produce settings.

Gram-positive bacteria are prominent members of plant-associated microbial communities. Although many are hypothesized to be beneficial, some are causative agents of economically important diseases of crop plants. Because the features of Gram-positive bacteria are fundamentally different relative to those of Gram-negative bacteria, the evolution and ecology as well as the mechanisms used to colonize and infect plants also differ. Here, we discuss recent advances in our understanding of Gram-positive, plant-associated bacteria and provide a framework for future research directions on these important plant symbionts.

Diseases have a significant cost to agriculture. Findings from analyses of whole-genome sequences show great promise for informing strategies to mitigate risks from diseases caused by phytopathogens. Genomic approaches can be used to dramatically shorten response times to outbreaks and inform disease management in novel ways. However, the use of these approaches requires expertise in working with big, complex data sets and an understanding of their pitfalls and limitations to infer well-supported conclusions. We suggest using an evolutionary framework to guide the use of genomic approaches in epidemiology and diagnostics of plant pathogens. We also describe steps that are necessary for realizing these as standard approaches in disease surveillance.

BackgroundMany bacterial taxa are species complexes and uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilizedAgrobacterium tumefaciens, a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes.ResultsWe utilized 35 genome assemblies to achieve a comprehensive and balanced sampling ofA. tumefaciens. Our confident inference of gene content and core-genome phylogeny supported a quantitative guideline for delineating 12 species and allowed for robust investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins.ConclusionsWe demonstrated that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. Our strategies for multi-level investigations at scales that range from whole-genomes to intragenic domains and phylogenetic depths of between- and within-species are applicable to other bacteria. Finally, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

SummaryAgrobacterium tumefaciens, is a member of the Alphaproteobacteria that pathogenizes plants, and associates with biotic and abiotic surfaces via a single cellular pole.A. tumefaciensproduces the unipolar polysaccharide (UPP) at the site of surface contact. UPP production is normally surface-contact inducible, but elevated levels of the second messenger cyclic diguanylate monophosphate (cdGMP) bypass this requirement. Multiple lines of evidence suggest that the UPP has a central polysaccharide component. Using anA. tumefaciensderivative with elevated cdGMP and mutationally disabled for other dispensable polysaccharides, a series of related genetic screens have identified a large number of genes involved in UPP biosynthesis, most of which are Wzx-Wzy-type polysaccharide biosynthetic components. Extensive analyses of UPP production in these mutants have revealed that the UPP is comprised of two genetically, chemically and spatially discrete forms of polysaccharide, and that each requires a specific Wzy-type polymerase. Other important biosynthetic, processing and regulatory functions for UPP production are also revealed, some of which are common to both polysaccharides, and a subset of which are specific to each species. Many of the UPP genes identified are conserved among diverse rhizobia, whereas others are more lineage specific.Plain language summaryBacteria attach to a wide variety of surfaces including host tissues, via externalized structures described as adhesins. We define a large set of genes involved in synthesis of a complex unipolar adhesin comprised of two distinct polysaccharides, that is required for surface attachment by the plant-associated pathogenAgrobacterium tumefaciens.

The genus

Agrobacteria virulence writ large Plasmids are widespread among bacteria and are important because they spread virulence and antibiotic resistance traits, among others. They are horizontally transferred between strains and species, so it is difficult to work out their evolution and epidemiology. Agrobacteria, a diverse grouping of species that infect plants, inject oncogenic Ti and Ri plasmids, which cause crown galls and hairy root diseases, respectively. The upside is that these plasmids have become valuable biotechnological tools. Weisberg et al. combed through an 80-year-old collection of Agrobacterium strains but found a surprisingly low diversity of plasmids. It is puzzling how limited the number of plasmid lineages is despite reported high levels of plasmid recombination, but what is clear is how plant production systems have influenced plasmid spread into various genomic backbones. Science , this issue p. eaba5256

Rhodococcus is a genus of Gram-positive bacteria with species that can cause growth deformations to a large number of plant species. This ability to cause disease is hypothesized to be dependent on a cluster of three gene loci on an almost 200 kb-sized linear plasmid. To reevaluate the roles of some of the genes in pathogenicity, we constructed and characterized deletion mutants of fasR and four fas genes. Findings confirmed that fasR, which encodes a putative transcriptional regulator, is necessary for pathogenesis. However, three of the fas genes, implicated in the metabolism of plant growth promoting cytokinins, are dispensable for the ability of the pathogen to cause disease. We also used long-read sequencing technology to generate high quality genome sequences for two phytopathogenic strains in which virulence genes are diverged in sequence and/or hypothesized to have recombined into the chromosome. Surprisingly, findings showed that the two strains carry extremely diverse virulence plasmids. Ortholog clustering identified only 12 genes present on all three virulence plasmids. Rhodococcus requires a small number of horizontally acquired traits to be pathogenic and the transmission of the corresponding genes, via recombination and conjugation, has the potential to rapidly diversify plasmids and bacterial populations.

Rhodococcus is a genus of Gram-positive bacteria with species that can cause growth deformations to a large number of plant species. This ability to cause disease is hypothesized to be dependent on a cluster of three gene loci on an almost 200 kb-sized linear plasmid. To reevaluate the roles of some of the genes in pathogenicity, we constructed and characterized deletion mutants of fasR and four fas genes. Findings confirmed that fasR, which encodes a putative transcriptional regulator, is necessary for pathogenesis. However, three of the fas genes, implicated in the metabolism of plant growth promoting cytokinins, are dispensable for the ability of the pathogen to cause disease. We also used long-read sequencing technology to generate high quality genome sequences for two phytopathogenic strains in which virulence genes are diverged in sequence and/or hypothesized to have recombined into the chromosome. Surprisingly, findings showed that the two strains carry extremely diverse virulence plasmids. Ortholog clustering identified only 12 genes present on all three virulence plasmids. Rhodococcus requires a small number of horizontally acquired traits to be pathogenic and the transmission of the corresponding genes, via recombination and conjugation, has the potential to rapidly diversify plasmids and bacterial populations.

Indonesian http://mts.hindawi.com/update/) in our Manuscript Tracking System and after you have logged in click on the ORCID link at the top of the page. This link will take you to the ORCID website where you will be able to create an account for yourself. Once you have done so, your new ORCID will be saved in our Manuscript Tracking System automatically."?>marine environments are known to house diverse organisms. However, the potential for bacteria from these environments as a source of antibacterial agents has not been widely studied. This study aims to explore the antibacterial potential of secondary metabolites produced by bacterial symbionts from sponges and corals collected in the Indonesian waters. Extracts of 12 bacterial isolates from sponges or corals were prepared by cultivating the bacteria under a number of different media conditions and using agar well diffusion assays to test for antibacterial activity. In addition, the morphology, physiology, and biochemical characteristics and 16S rRNA sequence of each isolate were used to determine their taxonomic classification. All tested bacterial isolates were able to produce secondary metabolites with various levels of antibacterial activity depending on medium composition and culture conditions. Two of the bacteria (RS3 and RC4) showed strong antibacterial activities against both Gram-negative and Gram-positive bacteria. A number of isolates (RS1, RS3, and RC2) were co-cultured with mycolic acid-containing bacteria, Mycobacterium smegmatis or Rhodococcus sp. However, no improvements in their antibacterial activity were observed. All of the 12 bacteria tested were identified as Streptomyces spp. LC-MS analysis of EtOAc extracts from the most active strains RS3 and RC4 revealed the presence of a number of dactinomycin analogues and potentially new secondary metabolites. Symbiotic Streptomyces spp. from sponges and corals of the Indonesian marine environments have great potential as a source of broad-spectrum antibacterial agents.

The type III secretion system (T3SS) of plant-pathogenic

The detection of pathogen-associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is an essential part of plant immunity. Until recently, elf18, an epitope of elongation factor-Tu (EF-Tu), was the sole confirmed PAMP of Ralstonia solanacearum, the causal agent of bacterial wilt disease, limiting our understanding of R. solanacearum-plant interactions. Therefore, we set out to identify additional R. solanacearum PAMPs based on the hypothesis that genes encoding PAMPs are under selection to avoid recognition by plant PRRs. We calculated Tajima's D, a population genetic test statistic which identifies genes that do not evolve neutrally, for 3003 genes conserved in 37 R. solanacearum genomes. The screen flagged 49 non-neutrally evolving genes, including not only EF-Tu but also the gene for Cold Shock Protein C, which encodes the PAMP csp22. Importantly, an R. solanacearum allele of this PAMP was recently identified in a parallel independent study. Genes coding for efflux pumps, some with known roles in virulence, were also flagged by Tajima's D. We conclude that Tajima's D is a straightforward test to identify genes encoding PAMPs and other virulence-related genes in plant pathogen genomes.

Poison ivy [Toxicodendron radicans (L.) Kuntz] is a widely recognized native plant species because of its production of urushiol, which is responsible for delayed contact dermatitis symptoms in humans. Poison ivy is predicted to become both more prevalent and more noxious in response to projected patterns of climate change. Future studies on poison ivy chemical ecology will require reverse genetics to investigate urushiol metabolism. A prerequisite for reverse genetic procedures is the introduction and expression of recombinant DNA into poison ivy tissues. Poison ivy leaves and cotyledons were marginally susceptible to vacuum- and syringe-agroinfiltration and expression of two firefly luciferase (LUC)–based reporter genes. The efficacy of agroinfiltration and transient LUC expression was dependent on leaf age and plant growth environmental conditions, with young leaves grown in magenta boxes showing highest transient LUC expression levels. Agroinfiltrated leaves showed an Agrobacterium-dependent accumulation of brown–colored pigments. Biolistic transformation of a LUC reporter gene did not show brown pigment accumulation and readily displayed transient LUC bioluminescence in both leaves and cotyledon tissues. These studies establish best practices for introducing and transiently expressing recombinant DNA into poison ivy leaf and cotyledon tissues, on which future reverse genetic procedures can be developed.

Fluorescent Pseudomonas spp. are widely studied for their beneficial activities to plants. To explore the genetic diversity of Pseudomonas spp. in tropical regions, we collected 76 isolates from a Brazilian soil. Genomes were sequenced and compared to known strains, mostly collected from temperate regions. Phylogenetic analyses classified the isolates in the P. fluorescens (57) and P. putida (19) groups. Among the isolates in the P. fluorescens group, most (37) were classified in the P. koreensis subgroup and two in the P. jessenii subgroup. The remaining 18 isolates fell into two phylogenetic subclades distinct from currently recognized P. fluorescens subgroups, and probably represent new subgroups. Consistent with their phylogenetic distance from described subgroups, the genome sequences of strains in these subclades are asyntenous to the genome sequences of members of their neighbour subgroups. The tropical isolates have several functional genes also present in known fluorescent Pseudomonas spp. strains. However, members of the new subclades share exclusive genes not detected in other subgroups, pointing to the potential for novel functions. Additionally, we identified 12 potential new species among the 76 isolates from the tropical soil. The unexplored diversity found in the tropical soil is possibly related to biogeographical patterns.

Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.

Pseudomonas syringaeis a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requiresP. syringaeto rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria includingP. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacAmutant ofP. syringae pv.tomatoDC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation inanmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele ofanmKpartially increased growth of AC811 in Arabidopsis leaves. In addition to the defectiveanmKallele, we also show that the Tn5insertion ingacAexerts a polar effect onuvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-typeanmKallele together with increased expression ofuvrCfully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects inanmKanduvrCare together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of thegacA-uvrClocus.

Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.

Pseudomonas syringae is a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requires P. syringae to rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria including P. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacA mutant of P. syringae pv. tomato DC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation in anmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele of anmK partially increased growth of AC811 in Arabidopsis leaves. In addition to the defective anmK allele, we also show that the Tn5 insertion in gacA exerts a polar effect on uvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-type anmK allele together with increased expression of uvrC fully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects in anmK and uvrC are together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of the gacA-uvrC locus.

Legumes preferentially associate with and reward beneficial rhizobia in root nodules, but the processes by which rhizobia evolve to provide benefits to novel hosts remain poorly understood. Using cycles of in planta and in vitro evolution, we experimentally simulated lifestyles where rhizobia repeatedly interact with novel plant genotypes with which they initially provide negligible benefits. Using a full-factorial replicated design, we independently evolved two rhizobia strains in associations with each of two Lotus japonicus genotypes that vary in regulation of nodule formation. We evaluated phenotypic evolution of rhizobia by quantifying fitness, growth effects and histological features on hosts, and molecular evolution via genome resequencing. Rhizobia evolved enhanced host benefits and caused changes in nodule development in one of the four host–symbiont combinations, that appeared to be driven by reduced costs during symbiosis, rather than increased nitrogen fixation. Descendant populations included genetic changes that could alter rhizobial infection or proliferation in host tissues, but lack of evidence for fixation of these mutations weakens the results. Evolution of enhanced rhizobial benefits occurred only in a subset of experiments, suggesting a role for host–symbiont genotype interactions in mediating the evolution of enhanced benefits from symbionts.

Bacterial mutualists generate major fitness benefits for eukaryotes, reshaping the host phenotype and its interactions with the environment. Yet, microbial mutualist populations are predicted to generate mutants that defect from providing costly services to hosts while maintaining the capacity to exploit host resources. Here, we examined the mutualist service of symbiotic nitrogen fixation in a metapopulation of root-nodulatingBradyrhizobiumspp.that associate with the native legumeAcmispon strigosus. We quantified mutualism traits of 85Bradyrhizobiumisolates gathered from a 700 km transect in California spanning 10 sampledA. strigosuspopulations. We clonally inoculated eachBradyrhizobiumisolate ontoA. strigosushosts and quantified nodulation capacity and net effects of infection, including host growth and isotopic nitrogen concentration. SixBradyrhizobiumisolates from five populations were categorized as ineffective because they formed nodules but did not enhance host growth via nitrogen fixation. Six additional isolates from three populations failed to form root nodules. Phylogenetic reconstruction inferred two types of mutualism breakdown, including three to four independent losses of effectiveness and five losses of nodulation capacity onA. strigosus. The evolutionary and genomic drivers of these mutualism breakdown events remain poorly understood.

Significance International trade has resulted in the introduction of plant diseases into natural ecosystems around the world. These introductions have potentially catastrophic impacts on ecosystem structure and function. Leveraging genomic tools, natural variation within a tree species, and a high-throughput phenotyping platform, we present a framework that can be broadly applied to rapidly identify candidate genes associated with resistance and susceptibility to introduced plant diseases. The unprecedented speed and accuracy with which the candidate genes can be identified in woody trees demonstrates the potential of genomics to mitigate the impacts of invasive diseases on forest health., Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca. 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose–binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.

Rathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicus. R. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria., Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.

Randall et al., 2018 and Vereecke, 2018 have raised concerns about a paper we published (Savory et al., 2017). Here, we respond to those concerns.

Bulk soil and rhizosphere are soil compartments selecting different microbial communities. However, it is unknown whether this selection also can change the genome content of specific bacterial taxa, splitting a population in distinct ecotypes. To answer this question we compared the genome sequences of 53 isolates obtained from sugarcane rhizosphere (28) and bulk soil (25). These isolates were previously classified in the Pseudomonas koreensis subgroup of the P. fluorescens complex. Phylogenomics showed a trend of separation between bulk soil and rhizosphere isolates. Discriminant analysis of principal components (DAPC) identified differences in the accessory genome of rhizosphere and bulk soil sub-populations. We found significant changes in gene frequencies distinguishing rhizosphere from bulk soil ecotypes, for example, enrichment of phosphatases and xylose utilization (xut) genes, respectively. Phenotypic assays and deletion of xutA gene indicated that accumulation of xut genes in the bulk soil sub-population provided a higher growth capacity in a d-xylose medium, supporting the corresponding genomic differences. Despite the clear differences distinguishing the two ecotypes, all 53 isolates were classified in a single 16S rRNA gene OTU. Collectively, our results revealed that the gene pool and ecological behavior of a bacterial population can be different for ecotypes living in neighbouring soil habitats.

Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses., eLife digest All organisms live in a world teeming with bacteria. Some bacteria are beneficial and, for example, provide their hosts with nutrients. Others cause harm, for example, by stealing nutrients and causing disease. Many bacteria can also gain DNA from other bacteria, and the genes encoded within the new DNA can help them to live with other organisms. This can start the bacteria on an evolutionary path to becoming beneficial or harmful. Rhodococcus are bacteria that live in association with many species of plants, including trees. Most are harmless but some cause disease. Plants infected with harmful Rhodococcus can show deformed growth, which causes major losses to the nursery industry. Savory, Fuller, Weisberg et al. set out to understand how disease-causing Rhodococcus are introduced into nurseries, if they are transferred between nurseries, whether they persist in nurseries, and how to limit their spread. It turns out that harmless Rhodococcus are beneficial to plants. However, if these harmless bacteria gain a certain DNA molecule – called a virulence plasmid – they can convert into harmful bacteria. Further analysis showed that some nurseries repeatedly acquired the harmful bacteria. The pattern of affected nurseries suggested that some might have purchased diseased plants from a common provider. In other cases, the sources remained a mystery. Savory et al. also report that, contrary to previous findings, there is no evidence to support the diagnosis that Rhodococcus without a virulence plasmid are responsible for an unusual growth problem that has plagued the pistachio industry. In recent years, this incorrect diagnosis led to trees being unnecessarily destroyed, worsening the economic losses. These findings suggest that genes moving between bacteria can dramatically change how those bacteria interact with the organisms in which they live. It needs to be shown whether this is an exceptional process, unique to only certain groups of bacteria, or if it is more widespread in nature. These findings could inform future disease management strategies to better protect agricultural systems.