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Accelerating the pace of microbiome science to enhance crop productivity and agroecosystem health will require transdisciplinary studies, comparisons among datasets, and synthetic analyses of research from diverse crop management contexts. However, despite the widespread availability of crop-associated microbiome data, variation in field sampling and laboratory processing methodologies, as well as metadata collection and reporting, significantly constrains the potential for integrative and comparative analyses. Here we discuss the need for agriculture-specific metadata standards for microbiome research, and propose a list of “required” and “desirable” metadata categories and ontologies essential to be included in a future minimum information metadata standards checklist for describing agricultural microbiome studies. We begin by briefly reviewing existing metadata standards relevant to agricultural microbiome research, and describe ongoing efforts to enhance the potential for integration of data across research studies. Our goal is not to delineate a fixed list of metadata requirements. Instead, we hope to advance the field by providing a starting point for discussion, and inspire researchers to adopt standardized procedures for collecting and reporting consistent and well-annotated metadata for agricultural microbiome research.

Cucurbit yellow vine disease (CYVD), which is caused by the gram-negative bacterium Serratia marcescens and transmitted by squash bugs ( Anasa tristis DeGeer), is a devastating disease of cucurbit crops that is emerging rapidly in the eastern half of the U.S. The lack of a robust pathogenicity assay for CYVD in the laboratory has hampered functional tests using genomic sequences to investigate the biology of this phytopathogen. In this study we developed and validated a bioassay that yielded consistent and quantifiable CYVD symptoms on squash in the lab. We compared inoculation by wounding with a multipronged floral pin frog to inoculation by injection in which a needle was moved in and out of the stem multiple times in each of multiple piercings to mimic the feeding behavior of squash bugs. We found that inoculation by needle injection of ≥10 8 CFU/ml of S. marcescens into the stem of squash (Cucurbita pepo) plants at the cotyledon growth stage reproducibly induced CYVD symptoms, whereas injecting 10 6 or 10 7 CFU/ml did not. Additionally, we found that S. marcescens induced symptoms on all of the squash cultivars tested, and induced symptoms that have not been previously reported, including stem elongation and leaf cupping. In short, through our injection approach of mimicking the natural process of S. marcescens transmission by squash bug feeding, we obtained robust and quantifiable CYVD symptoms. This laboratory bioassay provides a crucial tool for investigating the biology and pathology of this emerging pathogen and for plant breeding screens aimed at combatting CYVD.

The phloem-limited bacterium ' Candidatus Liberibacter asiaticus' ( C Las) is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid Diaphorina citri . C Las severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M . exotica L.). In this study, 13 accessions of three Murraya species ( M. paniculata , M. sumatrana Roxb., and M. lucida [G.Forst.] Mabb.) and the Papuan form of a putative hybrid ( M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5'trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and quantitative real-time PCR (qPCR) on two to four occasions over 14 months. C Las was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M . paniculata and M. sumatrana , respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa . The species identification of the C Las-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded internal transcribed spacer (ITS) region. The results indicated that natural infection of M. paniculata and M. sumatrana with C Las can occur in Java. To our knowledge, this is the first demonstration of the natural infection of M. sumatrana with C Las. Further studies are required to determine whether infections persist in the absence of D. citri ., Competing Interests: The author(s) declare no conflict of interest.

Most Ralstonia solanacearum species complex strains cause bacterial wilts in tropical or subtropical zones, but the group known as Race 3 biovar 2 (R3bv2) is cool virulent and causes potato brown rot at lower temperatures. R3bv2 has invaded potato-growing regions around the world but is not established in the United States. Phylogenetically, R3bv2 corresponds to a subset of the R. solanacearum phylotype IIB clade, but little is known about the distribution of the cool virulence phenotype within phylotype IIB. Therefore, genomes of 76 potentially cool virulent phylotype IIB strains and 30 public genomes were phylogenetically analyzed. A single clonal lineage within the sequevar 1 subclade of phylotype IIB that originated in South America has caused nearly all brown rot outbreaks worldwide. To correlate genotypes with relevant phenotypes, we quantified virulence of ten Ralstonia strains on tomato and potato at both 22°C and 28°C. Cool virulence on tomato did not predict cool virulence on potato. We found that cool virulence is a quantitative trait. Strains in the sequevar 1 pandemic clonal lineage caused the most disease, while other R3bv2 strains were only moderately cool virulent. However, some non-R3bv2 strains were highly cool virulent and aggressively colonized potato tubers. Thus, cool virulence is not consistently correlated with strains historically classified as R3bv2 group. To aid detection of sequevar 1 strains, this group was genomically delimited in the LINbase web server and a sequevar 1 diagnostic primer pair was developed and validated. We discuss implications of these results for the R3bv2 definition.

The choline-glycine betaine pathway plays an important role in bacterial survival in hyperosmotic environments. Osmotic activation of the choline transporter BetT promotes the uptake of external choline for synthesizing the osmoprotective glycine betaine. Here, we report the cryo-electron microscopy structures of Pseudomonas syringae BetT in the apo and choline-bound states. Our structure shows that BetT forms a domain-swapped trimer with the C-terminal domain (CTD) of one protomer interacting with the transmembrane domain (TMD) of a neighboring protomer. The substrate choline is bound within a tryptophan prism at the central part of TMD. Together with functional characterization, our results suggest that in Pseudomonas species, including the plant pathogen P. syringae and the human pathogen Pseudomonas aeruginosa , BetT is locked at a low-activity state through CTD-mediated autoinhibition in the absence of osmotic stress, and its hyperosmotic activation involves the release of this autoinhibition.

Plant-microbe interaction research has had a transformative trajectory, from individual microbial isolate studies to comprehensive analyses of plant microbiomes within the broader phytobiome framework. Acknowledging the indispensable role of plant microbiomes in shaping plant health, agriculture, and ecosystem resilience, we underscore the urgent need for sustainable crop production strategies in the face of contemporary challenges. We discuss how the synergies between advancements in 'omics technologies and artificial intelligence can help advance the profound potential of plant microbiomes. Furthermore, we propose a multifaceted approach encompassing translational considerations, transdisciplinary research initiatives, public-private partnerships, regulatory policy development, and pragmatic expectations for the practical application of plant microbiome knowledge across diverse agricultural landscapes. We advocate for strategic collaboration and intentional transdisciplinary efforts to unlock the benefits offered by plant microbiomes and address pressing global issues in food security. By emphasizing a nuanced understanding of plant microbiome complexities and fostering realistic expectations, we encourage the scientific community to navigate the transformative journey from discoveries in the laboratory to field applications. As companies specializing in agricultural microbes and microbiomes undergo shifts, we highlight the necessity of understanding how to approach sustainable agriculture with site-specific management solutions. While cautioning against overpromising, we underscore the excitement of exploring the many impacts of microbiome-plant interactions. We emphasize the importance of collaborative endeavors with societal partners to accelerate our collective capacity to harness the diverse and yet-to-be-discovered beneficial activities of plant microbiomes.

The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C 4 -C 10 ) or acyl-ACPs (C 6 -C 10 ). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis.IMPORTANCEAcyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from β-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis., Competing Interests: The authors declare no conflict of interest.

Cucurbit yellow vine disease (CYVD) is caused by Serratia marcescens , vectored by squash bugs ( Anasa tristis ), and is an emerging disease in many parts of the U.S. CYVD can cause 100% yield losses in cucurbits (Bruton et al., 2003). In the summer of 2021, at the Iowa State University Horticultural Research Station (HRS) in Gilbert, Iowa, we observed leaf yellowing, vine decline, and honey-brown discoloration of the phloem of acorn squash ( Cucurbita pepo cv. Table Ace) plants in research fields that were infested with squash bugs. In 2022, we observed similar symptoms on pumpkin ( Cucurbita maxima cv. Howden) and muskmelon ( Cucumis melo cv. Athena) in different fields at the HRS and on giant pumpkins ( Cucurbita maxima cv. Prizewinner) in Jones and Ringgold counties. For up to 3 symptomatic plants of each cucurbit species per location, a 20-cm-long stem section immediately above the soil line was excised, surface sterilized by immersion in 10% sodium hypochlorite and 70% ethanol for 2 min each, then triple rinsed in sterile water. The interior of the cross-section tissue was blotted on Luria agar amended with cycloheximide (100 μg/ml) and tetracycline (20 μg/ml) (Stock et al. 2003). Whitish translucent colonies developed after incubation at 28°C for 48 h. The genomic DNA of three isolates from symptomatic plants of muskmelon (MK01), pumpkin (HFP01), and giant pumpkin (AP01), was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD). S. marcescens species-specific primers YV1 (5'-GGGACTTGCTCCCCGG-3') and YV4 (5'-AACGTCAATTGATGAACGTATTAAGT-3') (Bruton et al. 2003) were used to amplify part of the 16S rDNA gene, and the primers specific to S. marcescens CYVD strains A79F/A79R (Zhang et al., 2005) were used to amplify part of a major facilitatory superfamily (MFS) transporter gene strain. The sequences of the 16S rRNA PCR product for the three isolates were identical and were deposited in NCBI under Accession OR963533. They shared 100% (395/395 nt) identity with other CYVD strains (Rascoe et al. 2003) and those of other S. marcescens strains in NCBI. The sequences of the amplified region of the MFS transporter gene of the three isolates (NCBI Accession OR962261) were identical and showed a 98.8% (319/323 nt) identity to that of non-CYVD-causing S. marcescens strains, such as N10A28 (Accession CP033623.1). Koch's postulates were fulfilled by inoculating C. pepo cv. Zephyr plants with either strain HFP01 or phosphate buffer saline (PBS) (10 plants per treatment) 1 wk after seeding by injecting 300 µl of bacteria (~10 8 CFU/ml) or PBS using a syringe needle. Plants were incubated at 28°C in a growth room for 4 wks. CYVD symptoms similar to those observed in the field developed on 7 out of 10 plants inoculated with strain HFP01 in one study, and 9 out 10 plants in a replicate study, with none of the PBS-inoculated plants showing CYVD symptoms. Bacteria were isolated from the symptomatic plants with selection on tetracycline. The PCR fragments amplified with YV1/YV4 and A79F/A79R were the same size as those of the pre-inoculation strain HFP01. To our knowledge this is the first report of CYVD in Iowa and in the Upper Midwest of the U.S. CYVD is a devastating disease that poses a significant threat to cucurbit production. This report can serve as an alert for the region's growers and for the development of effective management practices.

The ecological significance of light perception in nonphotosynthetic bacteria remains largely elusive. In terrestrial environments, diurnal oscillations in light are often temporally coupled to other environmental changes, including increased temperature and evaporation. Here, we report that light functions as an anticipatory cue that triggers protective adaptations to tolerate a future rapid loss of environmental water. We demonstrate this photo-anticipatory stress tolerance in leaf-associated Pseudomonas syringae pv. syringae ( Pss ) and other plant- and soil-associated pseudomonads. We found that light influences the expression of 30% of the Pss genome, indicating that light is a global regulatory signal, and this signaling occurs almost entirely via a bacteriophytochrome photoreceptor that senses red, far-red, and blue wavelengths. Bacteriophytochrome-mediated light control disproportionally up-regulates water-stress adaptation functions and confers enhanced fitness when cells encounter light prior to water limitation. Given the rapid speed at which water can evaporate from leaf surfaces, such anticipatory activation of a protective response enhances fitness beyond that of a reactive stress response alone, with recurring diurnal wet-dry cycles likely further amplifying the fitness advantage over time. These findings demonstrate that nonphotosynthetic bacteria can use light as a cue to mount an adaptive anticipatory response against a physiologically unrelated but ecologically coupled stress.

Although floral nectar is a rich source of nutrients, it is rarely infected by microorganisms. Defense molecules such as proteins have been identified in this fluid, but defense peptides have been largely overlooked. Thus, the aim of this study was to perform an extensive peptidomic analysis of the ornamental tobacco floral nectar to seek peptides involved in nectar defense. Using LC-MS/MS, 793 peptides were sequenced and characterized. After extensive bioinformatics analysis, six peptides were selected for further characterization, synthesis, and evaluation of their antimicrobial properties against phytopathogenic fungi and bacteria. All six peptides had antimicrobial activity to some extent. However, the activity varied by peptide concentration and microorganism tested. An analysis of the action mechanism revealed damage in the cell membrane induced by peptides. The results show that floral nectar is rich in peptides and that, together with proteins and hydrogen peroxide, they contribute to plant defense against microorganisms during pollination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The predicted repertoire of type III secretion system effectors (T3SEs) in Erwinia tracheiphila , causal agent of cucurbit bacterial wilt, is much larger than in xylem pathogens in the closely related genera Erwinia and Pantoea . The genomes of strains BHKY and SCR3, which represent distinct E. tracheiphila clades, encode at least 6 clade-specific and 12 shared T3SEs. The strains expressed the majority of the T3SE genes examined in planta. Among the shared T3SE genes, eop1 was expressed most highly in both strains in squash ( Cucurbita pepo ) and muskmelon ( Cucumis melo ) but the clade-specific gene avrRpm2 was expressed 40- to 900-fold more than eop1 in BHKY. The T3SEs AvrRpm2, Eop1, SrfC, and DspE contributed to BHKY virulence on squash and muskmelon, as shown using combinatorial mutants involving six T3SEs, whereas OspG and AvrB4 contributed to BHKY virulence only on muskmelon, demonstrating host-specific virulence functions. Moreover, Eop1 was functionally redundant with AvrRpm2, SrfC, OspG, and AvrB4 in BHKY, and BHKY mutants lacking up to five effector genes showed similar virulence to mutants lacking only two genes. The T3SEs OspG, AvrB4, and DspE contributed additively to SCR3 virulence on muskmelon and were not functionally redundant with Eop1. Rather, loss of eop1 and avrB4 restored wild-type virulence to the avrB4 mutant, suggesting that Eop1 suppresses a functionally redundant effector in SCR3. These results highlight functional differences in effector inventories between two E. tracheiphila clades, provide the first evidence of OspG as a phytopathogen effector, and suggest that Eop1 may be a metaeffector influencing virulence. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Strains of Erwinia tracheiphila, causal agent of bacterial wilt of cucurbits, are divided into distinct clades. Et-melo clade strains wilt Cucumis spp. but not Cucurbita spp., thus exhibiting host specificity, whereas Et-C1 clade strains wilt Cucurbita spp. more rapidly than Cucumis melo , thus exhibiting a host preference. This study investigated the contribution of the effector proteins Eop1 and DspE to E. tracheiphila pathogenicity and host adaptation. Loss of eop1 did not enable Et-melo strains to infect squash ( Cucurbita pepo ) or an Et-C1 strain to induce a more rapid wilt of muskmelon ( Cucumis melo ), indicating that Eop1 did not function in host specificity or preference as in the related pathogen E. amylovora. However, overexpression of eop1 from Et-melo strain MDCuke but not from Et-C1 strain BHKY increased the virulence of a BHKY eop1 deletion mutant on muskmelon, demonstrating that the Eop1 variants in the two clades are distinct in their virulence functions. Loss of dspE from Et-melo strains reduced but did not eliminate virulence on hosts muskmelon and cucumber, whereas loss of dspE from an Et-C1 strain eliminated pathogenicity on hosts squash, muskmelon, and cucumber. Thus, the centrality of DspE to virulence differs in the two clades. Et-melo mutants lacking the chaperone DspF exhibited similar virulence to mutants lacking DspE, indicating that DspF is the sole chaperone for DspE in E. tracheiphila , unlike in E. amylovora . Collectively, these results provide the first functional evaluation of effectors in E. tracheiphila and demonstrate clade-specific differences in the roles of Eop1 and DspE.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Erwinia tracheiphila, the causal pathogen of bacterial wilt of cucurbit crops, is disseminated by cucumber beetles. A bacteriophage, designated FBB1 ( F u- B eattie- B eetle-1), was isolated from spotted cucumber beetles ( Diabrotica undecimpunctata ) that were collected from a field in which E. tracheiphila is endemic. FBB1 was classified into the Myoviridae family based on its morphology, which includes an elongated icosahedral head (106 × 82 nm) and a putatively contractile tail (120 nm). FBB1 infected all 62 E. tracheiphila strains examined and three Pantoea spp. strains. FBB1 virions were stable at 55°C for 1 h and tolerated a pH range from 3 to 12. FBB1 has a genome of 175,994 bp with 316 predicted coding sequences and a GC content of 36.5%. The genome contains genes for a major bacterial outer-membrane protein, a putative exopolysaccharide depolymerase, and 22 predicted transfer RNAs. The morphology and genome indicate that FBB1 is a T4-like virus and thus in the Tevenvirinae subfamily. FBB1 is the first virulent phage of E. tracheiphila to be reported and, to date, is one of only two bacteriophages to be isolated from insect vectors of phytopathogens. Collectively, the results support FBB1 as a promising candidate for biocontrol of E. tracheiphila based on its virulent (lytic) rather than lysogenic lifestyle, its infection of all E. tracheiphila strains examined to date, and its infection of a few nonpathogenic bacteria that could be used to support phage populations when pathogen numbers are low.

Xanthomonas citri pv. glycines (Xcg) is a major pathogen of soybean (Glycine max) in South Korea, despite the availability of soybean varieties with some resistance. We conducted a nationwide survey of the incidence and severity of bacterial pustule caused by Xcg. The percentage of infected fields was 7% to 17% between 2015 and 2017. We characterized the diversity of a nationwide collection of 106 Xcg isolates based on avrBs3 banding patterns. The isolates fell into 11 groups, each represented by a type strain; only two of these were similar to isolates collected from 1999 to 2002. The diversity of Xcg strains increased and the dominant strains changed between 1999 and 2017, with three new type strains comprising 44% of the isolates examined in 2012 to 2017. Pathogenicity tests did not show evidence for a shift in the races or aggressiveness of Xcg strains. Korean soybean cultivars, including the widely-grown Daewon cultivar, were susceptible to the 11 new type strains. The cultivar CNS, which carries the rxp resistance gene, was susceptible to most type strains, including two representing 83% of the Korean Xcg strains. In contrast, Williams 82, which also carries rxp, showed resistance to at least five type strains. Collectively, these results suggest that Williams 82 has resistance loci in addition to rxp. The widespread distribution of Xcg, the high virulence of the current endemic strains, and the low resistance of most Korean soybean cultivars collectively favor widespread disease in Korea in years that are favorable to pustule development.

Xanthomonas citri pv. glycines is a major pathogen of soybean in Korea. Here, we analyzed pathogenicity genes based on a comparative genome analysis of five Korean strains and one strain from the United States, 8ra. Whereas all six strains had nearly identical profiles of carbohydrate-active enzymes, they varied in diversity and number of candidate type III secretion system effector (T3SE) genes. The five Korean strains were similar in their effectors, but differed from the 8ra strain. Across the six strains, transcription activator-like effectors (TALEs) showed diverse repeat sizes and at least six forms of the repeat variable di-residue (RVD) sequences, with differences not correlated with the origin of the strains. However, a phylogenetic tree based on the alignment of RVD sequences showed two distinct clusters with 17.5 repeats, suggesting that two distinct 17.5 RVD clusters have evolved, potentially to adapt Xcg to growth on distinct soybean cultivars. The predicted effector binding elements of the TALEs fell into six groups and were strongly overlapping in sequence, suggesting evolving target specificity of the binding domains in soybean cultivars. Our findings reveal the variability and adaptability of T3SEs in the Xcg strains and enhance our understanding of Xcg pathogenicity in soybean.

Glutamicibacter sp. FBE-19 was isolated based on its strong antagonism to the cucurbit bacterial blight pathogen Erwinia tracheiphila on plates. Members of the Glutamicibacter genus can promote plant growth under saline conditions and antagonize fungi on plates via chitinolytic activity; however, their production of antibacterial compounds has not been examined. Here, we report the genome sequence of strain FBE-19. The genome is 3.85 Mbp with a G+C content of 60.1% and comprises 3,791 genes. Genes that may contribute to its antagonistic activity include genes for the secondary metabolites stenothricin, salinosporamide A, a second β-lactone compound, and a carotenoid. The Glutamicibacter sp. FBE-19 genome data may be a useful resource if this strain proves to be an effective biocontrol agent against E. tracheiphila .

Background: 'Candidatus Liberibacter asiaticus' is transmitted by Diaphorina citri, an insect with a wide range of hosts in Rutaceae. Species related to Citrus occur in Brazilian forests where they may serve as hosts for psyllids and infested citrus orchards., Results: The suitability of plants as hosts of D. citri was classified into four groups. Group I (high suitability): Citrus × aurantium 'Valencia', 'Citrus limonia', Murraya paniculata (syn. Murraya exotica L.) (Aurantioideae: Aurantieae) and Bergera koenigii (Aurantioideae: Clauseneae). Group II (intermediate to low suitability): Citrus (Poncirus) trifoliata 'Pomeroy', Citrus wintersii, Swinglea glutinosa (Aurantieae) and Clausena lansium (Clauseneae). Group III (not suitable): Aegle marmelos, Atalantia buxifolia, Citrus ('Microcitrus') sp. (Aurantieae) and Helietta apiculata (Amyridoideae). Group IV (non-hosts): Glycosmis pentaphylla (Clauseneae), Balfourodendron riedelianum, Casimiroa edulis, Esenbeckia febrifuga, Esenbeckia leiocarpa, Metrodorea stipularis, Zanthoxylum rhoifolium (Amyridoideae) and Dictyoloma vandellianum (Cneoroideae). Insects survived longer on newly differentiated leaves compared with fully expanded soft leaves. Psyllids either did not develop or did not survive for long on most Group IV species, all of which, with the exception of G. pentaphylla, occur naturally in Brazilian forests., Conclusion: Citrus relatives occurring in forests near citrus orchards are not suitable hosts of D. citri and, therefore, do not contribute to huanglongbing spread. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Current management of sudden death syndrome (SDS) of soybean, caused by Fusarium virguliforme, focuses on planting resistant varieties and improving soil drainage; however, these measures are not completely effective. A 6-year study evaluated the effects of cropping system diversification on SDS and soybean yield. SDS, root health, yield, and F. virguliforme density in soil were assessed in a naturally infested field trial comparing a 2-year cropping system consisting of a corn-soybean rotation and synthetic fertilizer applications with 3- and 4-year cropping systems consisting of corn-soybean-oat + red clover and corn-soybean-oat +alfalfa-alfalfa rotations, respectively, with both manure and low synthetic fertilizer rates. In 5 of 6 years, SDS incidence and severity were lower and yield higher in the 3- and 4-year systems than in the 2-year system. SDS severity and incidence were up to 17-fold lower in the diversified systems than in the 2-year system. Incidence and severity of SDS explained 45 to 87% of the variation in yield. Plants in the 2-year system generally showed more severe root rot and lower plant weights than plants in the diversified systems. F. virguliforme density in soil was up to fivefold greater in the 2-year system compared with the 4-year system. The processes responsible for the suppression of SDS and yield protection in the diversified cropping systems still need to be determined.

Plants collect, concentrate, and conduct light throughout their tissues, thus enhancing light availability to their resident microbes. This review explores the role of photosensing in the biology of plant-associated bacteria and fungi, including the molecular mechanisms of red-light sensing by phytochromes and blue-light sensing by LOV (light-oxygen-voltage) domain proteins in these microbes. Bacteriophytochromes function as major drivers of the bacterial transcriptome and mediate light-regulated suppression of virulence, motility, and conjugation in some phytopathogens and light-regulated induction of the photosynthetic apparatus in a stem-nodulating symbiont. Bacterial LOV proteins also influence light-mediated changes in both symbiotic and pathogenic phenotypes. Although red-light sensing by fungal phytopathogens is poorly understood, fungal LOV proteins contribute to blue-light regulation of traits, including asexual development and virulence. Collectively, these studies highlight that plant microbes have evolved to exploit light cues and that light sensing is often coupled with sensing other environmental signals.

Background: In insects, little is known about the co-evolution between their primary endosymbionts and hosts at the intraspecific level. This study examined co-diversification between the notorious agricultural pest Diaphorina citri and its primary endosymbionts (P-endosymbiont), 'Candidatus Carsonella ruddii' at the population level., Results: Maximum likelihood, haplotype network, principal components and Bayesian clustering identified three lineages for D. citri and its P-endosymbiont: a Western clade containing individuals from Pakistan, Bhutan (Phuentsholing), Vietnam (Son La), USA, Myanmar and China (Ruili, Yunnan); a Central clade, with accessions originating from Southwest China, Bhutan (Tsirang) and Bangladesh; and an Eastern clade containing individuals from Southeast Asia, and East and South China. A more diverse genetic structure was apparent in the host mitochondrial DNA than their P-endosymbionts; however, the two sets of data were strongly congruent., Conclusion: This study provides evidence for the co-diversification of D. citri and its P-endosymbiont during the migration from South Asia to East and Southeast Asia. We also suggest that the P-endosymbiont may facilitate investigations into the genealogy and migration history of the host. The biogeography of D. citri and its P-endosymbiont indicated that D. citri colonized and underwent a secondary dispersal from South Asia to East and Southeast Asia. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Salinity stress is one of the major abiotic stresses limiting crop production in arid and semi-arid regions. Interest is increasing in the application of PGPRs (plant growth promoting rhizobacteria) to ameliorate stresses such as salinity stress in crop production. The identification of salt-tolerant, or halophilic, PGPRs has the potential to promote saline soil-based agriculture. Halophytes are a useful reservoir of halotolerant bacteria with plant growth-promoting capabilities. Here, we review recent studies on the use of halophilic PGPRs to stimulate plant growth and increase the tolerance of non-halophytic crops to salinity. These studies illustrate that halophilic PGPRs from the rhizosphere of halophytic species can be effective bio-inoculants for promoting the production of non-halophytic species in saline soils. These studies support the viability of bioinoculation with halophilic PGPRs as a strategy for the sustainable enhancement of non-halophytic crop growth. The potential of this strategy is discussed within the context of ensuring sustainable food production for a world with an increasing population and continuing climate change. We also explore future research needs for using halotolerant PGPRs under salinity stress.

Light may be an important environmental signal for plant-associated bacteria, particularly those that live on leaves. An integrated network of red/far-red- and blue-light-responsive photosensory proteins is known to inhibit swarming motility in the foliar plant pathogen Pseudomonas syringae pv. syringae B728a. Here we elucidated factors in the red/far-red-light-sensing bacteriophytochrome BphP1 signal transduction pathway and report evidence for a role of BphP1 in multiple stages of the P syringae B728a life cycle. We report that BphP1 signaling involves the downstream regulator Bsi (bacteriophytochrome-regulated swarming inhibitor) and an acyl-homoserine lactone (AHL) signal. Loss of bphP1 or bsi resulted in the early initiation of swarm tendrils during swarming motility, a phenotype that was dependent on red/far-red light and reversed by exogenous AHL, illustrating that the BphP1-Bsi-AHL pathway inhibits the transition from a sessile state to a motile state. Loss of bphP1 or bsi resulted in larger water-soaked lesions induced on bean ( Phaseolus vulgaris ) pods and enhanced movement from soil and buried plant tissues to seeds, demonstrating that BphP1 and Bsi negatively regulate virulence and bacterial movement through soil to seeds. Moreover, BphP1, but not Bsi, contributed to leaf colonization; loss of bphP1 reduced survival on leaves immediately following inoculation but enhanced the size of the subsequently established populations. Neither Bsi nor Smp, a swarm motility-promoting regulator identified here, affected leaf colonization, indicating that BphP1-mediated contributions to leaf colonization are, at least in part, independent of swarming motility. These results demonstrate that P syringae B728a red-light sensing involves a multicomponent, branched regulatory pathway that affects several stages of its life cycle. IMPORTANCE Microbes on plants are particularly well positioned to exploit light cues based on the importance of light to plant growth. Photosensory proteins enable organisms to sense light and respond to light, but their roles in the life cycles of plant microbes are poorly understood. This study investigated the cellular components and ecological roles of red/far-red-light sensing in the foliar bacterial pathogen Pseudomonas syringae The study demonstrated that a bacteriophytochrome photosensory protein functions via a multicomponent, branched regulatory pathway that operates primarily through red/far-red-light-mediated inhibition. This pathway negatively regulates the transition from sessile to motile states under conditions conducive to swarming motility. It also negatively regulates virulence on bean pods, movement through soil to seeds, and survival following inoculation on leaves, but it positively contributes to the eventual establishment of leaf-borne populations. These results provide strong evidence that light sensing modulates behaviors at multiple stages in the life cycle of a nonphotosynthetic, plant microbe., (Copyright © 2017 McGrane and Beattie.)

We used morphological and molecular differences to confirm the identities of red scale (Aonidiella aurantii) and yellow scale (A. citrina), and their primary parasitoids, in Australia. An extension to the distribution of yellow scale was confirmed. Six primary parasitoids of red scale were identified: Aphytis chrysomphali, A. lingnanensis, A. melinus, Comperiella bifasciata, Encarsia citrina, and E. perniciosi. With the exception of A. lingnanensis, these parasitoids, and a species of Aphelinus, were detected in association with red scale during studies in citrus orchards in coastal New South Wales between 2009 and 2012. Two races of A. melinus were recorded: one from the Indian Subcontinent, the other previously only recorded in China. The studies, and reviews of historical records, led us to conclude that 4 parasitoids, A. lingnanensis, C. bifasciata, and both species of Encarsia, were present in Australia before successful or unsuccessful formal introductions between 1902 and 1970. The A. melinus race previously recorded in China may also have been present before the Indian Subcontinent race was formally introduced in 1961. We suggest the possibility that the natural distribution of some of the parasitoids may include East and Southeast Asia, and parts of Australasia. We found no reports of native armored scales being recorded on species and hybrids of Citrus introduced to Australia, and no reports of introduced armored scales being recorded on native Rutaceae, including 6 species of Citrus. However, we subsequently recorded yellow scale on Geijera parviflora, a native rutaceous tree., (© 2015 Institute of Zoology, Chinese Academy of Sciences.)

Modern industrial agriculture depends on high-density cultivation of genetically similar crop plants, creating favorable conditions for the emergence of novel pathogens with increased fitness in managed compared with ecologically intact settings. Here, we present the genome sequence of six strains of the cucurbit bacterial wilt pathogen Erwinia tracheiphila (Enterobacteriaceae) isolated from infected squash plants in New York, Pennsylvania, Kentucky, and Michigan. These genomes exhibit a high proportion of recent horizontal gene acquisitions, invasion and remarkable amplification of mobile genetic elements, and pseudogenization of approximately 20% of the coding sequences. These genome attributes indicate that E. tracheiphila recently emerged as a host-restricted pathogen. Furthermore, chromosomal rearrangements associated with phage and transposable element proliferation contribute to substantial differences in gene content and genetic architecture between the six E. tracheiphila strains and other Erwinia species. Together, these data lead us to hypothesize that E. tracheiphila has undergone recent evolution through both genome decay (pseudogenization) and genome expansion (horizontal gene transfer and mobile element amplification). Despite evidence of dramatic genomic changes, the six strains are genetically monomorphic, suggesting a recent population bottleneck and emergence into E. tracheiphila's current ecological niche., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Bacterial wilt threatens cucurbit crop production in the Midwestern and Northeastern United States. The pathogen, Erwinia tracheiphila, is a xylem-limited bacterium that affects most commercially important cucurbit species, including muskmelon, cucumber, and squash. Bacterial wilt is transmitted and overwintered by striped and spotted cucumber beetles. Since there are few commercially available resistant cultivars, disease management usually relies on use of insecticides to suppress vector populations. Although bacterial wilt was initially described more than 100 years ago, our knowledge of disease ecology and epidemiology advanced slowly for most of the 20th century. However, a recent wave of research has begun to fill in missing pieces of the bacterial wilt puzzle. This article-the first review of research toward understanding the cucurbit bacterial wilt pathosystem-recounts early findings and updates our understanding of the disease cycle, including pathogen and vector biology. We also highlight research areas that could lead to more efficient and ecologically based management of bacterial wilt.

The tomentose cochineal scale insect, Dactylopius tomentosus (Lamarck) (Hemiptera: Dactylopiidae), is an important biological control agent against invasive species of Cylindropuntia (Caryophyllales: Cactaceae). Recent studies have demonstrated that this scale is composed of host-affiliated biotypes with differential host specificity and fitness on particular host species. We investigated genetic variation and phylogenetic relationships among D. tomentosus biotypes and provenances to examine the possibility that genetic diversity may be related to their host-use pattern, and whether their phylogenetic relationships would give insights into taxonomic relatedness of their host plants. Nucleotide sequence comparison was accomplished using sequences of the mitochondrial cytochrome c oxidase I (COI) gene. Sequences of individuals from the same host plant within a region were identical and characterized by a unique haplotype. Individuals belonging to the same biotype but from different regions had similar haplotypes. However, haplotypes were not shared between different biotypes. Phylogenetic analysis grouped the monophyletic D. tomentosus into 3 well-resolved clades of biotypes. The phylogenetic relationships and clustering of biotypes corresponded with known taxonomic relatedness of their hosts. Two biotypes, Fulgida and Mamillata, tested positive for Wolbachia (α-Proteobacteria), a common endosymbiont of insects. The Wolbachia sequences were serendipitously detected by using insect-specific COI DNA barcoding primers and are most similar to Wolbachia Supergroup F strains. This study is the first molecular characterization of cochineal biotypes that, together with Wolbachia sequences, contribute to the better identification of the biotypes of cochineal insects and to the biological control of cacti using host-specific biotypes of the scale., (© 2014 Institute of Zoology, Chinese Academy of Sciences.)

Unlabelled: The plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean (Phaseolus vulgaris). To understand the contribution of distinct regulators to B728a fitness and pathogenicity, we performed a transcriptome analysis of strain B728a and nine regulatory mutants recovered from the surfaces and interior of leaves and exposed to environmental stresses in culture. The quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator, SalA, formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals and a plant signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. SalA functioned as a central regulator of iron status based on its reciprocal regulation of pyoverdine and achromobactin genes and also sulfur uptake, suggesting a role in the iron-sulfur balance. RetS functioned almost exclusively to repress secondary metabolite genes when the cells were not on leaves. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN, and AlgU in global regulation in B728a in planta and a high level of plasticity in these regulators' responses to distinct environmental signals., Importance: Leaves harbor abundant microorganisms, all of which must withstand challenges such as active plant defenses and a highly dynamic environment. Some of these microbes can influence plant health. Despite knowledge of individual regulators that affect the fitness or pathogenicity of foliar pathogens, our understanding of the relative importance of various global regulators to leaf colonization is limited. Pseudomonas syringae strain B728a is a plant pathogen and a good colonist of both the surfaces and interior of leaves. This study used global transcript profiles of strain B728a to investigate the complex regulatory network of putative quorum-sensing regulators, two-component regulators, and sigma factors in cells colonizing the leaf surface and leaf interior under stressful in vitro conditions. The results highlighted the value of evaluating these networks in planta due to the impact of leaf-specific environmental signals and suggested signal differences that may enable cells to differentiate surface versus interior leaf habitats., (Copyright © 2014 Yu et al.)

Mutualistic relationships between honeydew-producing insects and ants have been widely recognized for several decades. Iridomyrmex rufoniger (Lowne) is the commonest ant species associated with black scale, Saissetia oleae (Olivier), in the citrus orchards of the mid latitudes of coastal New South Wales. Citrus trees with high densities of both red and black scale and high ant activity were identified and the results of excluding ants from half of those trees (using a polybutene band on each trunk) were compared with the results of not excluding ants from the other half. Trees with a low incidence of black scale and ants were also studied. Exclusion of ants from trees was soon followed by collapse of black scale populations because most individuals were asphyxiated by their own honeydew. Also, parasitism of the red scale by Encarsia perniciosi (Tower) and Encarsia citrina Craw was significantly higher than in the control trees over the following year, as was the predation rate on red scale due to three coccinellid predators, Halmus chalybeus (Boisduval), Rhyzobius hirtellus Crotch and Rhyzobius lophanthae (Blaisdell). In contrast, another coccinellid, Orcus australasiae (Boisduval), and a noctuid moth larva, Mataeomera dubia Butler, were seen in low numbers on banded (ant exclusion) trees, probably because of the low availability of their black scale prey, but were significantly higher on control trees apparently because of their invulnerability to ants.

The foliar pathogen Pseudomonas syringae is a useful model for understanding the role of stress adaptation in leaf colonization. We investigated the mechanistic basis of differences in the osmotolerance of two P. syringae strains, B728a and DC3000. Consistent with its higher survival rates following inoculation onto leaves, B728a exhibited superior osmotolerance over DC3000 and higher rates of uptake of plant-derived osmoprotective compounds. A global transcriptome analysis of B728a and DC3000 following an osmotic upshift demonstrated markedly distinct responses between the strains; B728a showed primarily upregulation of genes, including components of the type VI secretion system (T6SS) and alginate biosynthetic pathways, whereas DC3000 showed no change or repression of orthologous genes, including downregulation of the T3SS. DC3000 uniquely exhibited improved growth upon deletion of the biosynthetic genes for the compatible solute N-acetylglutaminylglutamine amide (NAGGN) in a minimal medium, due possibly to NAGGN synthesis depleting the cellular glutamine pool. Both strains showed osmoreduction of glnA1 expression, suggesting that decreased glutamine synthetase activity contributes to glutamate accumulation as a compatible solute, and both strains showed osmoinduction of 5 of 12 predicted hydrophilins. Collectively, our results demonstrate that the superior epiphytic competence of B728a is consistent with its strong osmotolerance, a proactive response to an osmotic upshift, osmoinduction of alginate synthesis and the T6SS, and resiliency of the T3SS to water limitation, suggesting sustained T3SS expression under the water-limited conditions encountered during leaf colonization.

The quaternary ammonium compound (QAC) choline is a major component of membrane lipids in eukaryotes and, if available to microbial colonists of plants, could provide benefits for growth and protection from stress. Free choline is found in homogenized plant tissues, but its subcellular location and availability to plant microbes are not known. Whole-cell bacterial bioreporters of the phytopathogen Pseudomonas syringae were constructed that couple a QAC-responsive transcriptional fusion with well-characterized bacterial QAC transporters. These bioreporters demonstrated the presence of abundant free choline compounds released from germinating seeds and seedlings of the bean Phaseolus vulgaris, and a smaller but consistently detectable amount of QACs, probably choline, from leaves. The localization of P. syringae bioreporter cells to the surface and intercellular sites of plant tissues demonstrated the extracellular location of these QAC pools. Moreover, P. syringae mutants that were deficient in the uptake of choline compounds exhibited reduced fitness on leaves, highlighting the importance of extracellular choline to P. syringae on leaves. Our data support a model in which this choline pool is derived from the phospholipid phosphatidylcholine through plant-encoded phospholipases that release choline into the intercellular spaces of plant tissues, such as for membrane lipid recycling. The consequent extracellular release of choline compounds enables their interception and exploitation by plant-associated microbes, and thus provides a selective advantage for microbes such as P. syringae that are adapted to maximally exploit choline., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

The biological and regulatory roles of photosensory proteins are poorly understood for nonphotosynthetic bacteria. The foliar bacterial pathogen Pseudomonas syringae has three photosensory protein-encoding genes that are predicted to encode the blue-light-sensing LOV (light, oxygen, or voltage) histidine kinase (LOV-HK) and two red/far-red-light-sensing bacteriophytochromes, BphP1 and BphP2. We provide evidence that LOV-HK and BphP1 form an integrated network that regulates swarming motility in response to multiple light wavelengths. The swarming motility of P. syringae B728a deletion mutants indicated that LOV-HK positively regulates swarming motility in response to blue light and BphP1 negatively regulates swarming motility in response to red and far-red light. BphP2 does not detectably regulate swarming motility. The histidine kinase activity of each LOV-HK and BphP1 is required for this regulation based on the loss of complementation upon mutation of residues key to their kinase activity. Surprisingly, mutants lacking both lov and bphP1 were similar in motility to a bphP1 single mutant in blue light, indicating that the loss of bphP1 is epistatic to the loss of lov and also that BphP1 unexpectedly responds to blue light. Moreover, whereas expression of bphP1 did not alter motility under blue light in a bphP1 mutant, it reduced motility in a mutant lacking lov and bphP1, demonstrating that LOV-HK positively regulates motility by suppressing negative regulation by BphP1. These results are the first to show cross talk between the LOV protein and phytochrome signaling pathways in bacteria, and the similarity of this regulatory network to that of photoreceptors in plants suggests a possible common ancestry. IMPORTANCE Photosensory proteins enable organisms to perceive and respond to light. The biological and ecological roles of these proteins in nonphotosynthetic bacteria are largely unknown. This study discovered that a blue-light-sensing LOV (light, oxygen, or voltage) protein and a red/far-red-light-sensing bacteriophytochrome both regulate swarming motility in the foliar pathogen Pseudomonas syringae. These proteins form an integrated signaling network in which the bacteriophytochrome represses swarming motility in response to red, far-red, and blue light, and LOV positively regulates swarming motility by suppressing bacteriophytochrome-mediated blue-light signaling. This is the first example of cross talk between LOV and phytochrome signaling pathways in bacteria, which shows unexpected similarity to photoreceptor signaling in plants.

Many bacteria can accumulate glycine betaine for osmoprotection and catabolize it as a growth substrate, but how they regulate these opposing roles is poorly understood. In Pseudomonas syringae B728a, expression of the betaine catabolism genes was reduced by an osmotic upshift to an intermediate stress level, consistent with betaine accumulation, but was increased by an upshift to a high stress level, as confirmed by an accompanying increase in degradation of radiolabeled betaine. Deletion of the gbcAB betaine catabolism genes reduced osmotolerance at a high osmolarity, and this reduction was due to the relief of betaine-mediated suppression of compatible solute synthesis. This conclusion was supported by the findings that, at high osmolarity, the ΔgbcAB mutant accumulated high betaine levels and low endogenous solutes and exhibited reduced expression of the solute synthesis genes. Moreover, the ΔgbcAB mutant and a mutant deficient in the synthesis of the compatible solutes NAGGN and trehalose exhibited similar reductions in osmotolerance and also in fitness on bean leaves. Activation of betaine catabolism at high osmotic stress resulted, in part, from induction of gbdR, which encodes the transcriptional activator GbdR. Betaine catabolism was subject to partial repression by succinate under hyperosmotic stress conditions, in contrast to strong repression in the absence of stress, suggesting that betaine functions both in nutrition and as an intracellular signal modulating solute synthesis under hyperosmotic stress conditions. Collectively, these results begin to provide a detailed mechanistic understanding of how P. syringae transitions from reliance on exogenously derived betaine to the use of endogenous solutes during adaptation to hyperosmotic conditions.

Some strains of the foliar pathogen Pseudomonas syringae are adapted for growth and survival on leaf surfaces and in the leaf interior. Global transcriptome profiling was used to evaluate if these two habitats offer distinct environments for bacteria and thus present distinct driving forces for adaptation. The transcript profiles of Pseudomonas syringae pv. syringae B728a support a model in which leaf surface, or epiphytic, sites specifically favor flagellar motility, swarming motility based on 3-(3-hydroxyalkanoyloxy) alkanoic acid surfactant production, chemosensing, and chemotaxis,indicating active relocation primarily on the leaf surface. Epiphytic sites also promote high transcript levels for phenylalanine degradation, which may help counteract phenylpropanoid-based defenses before leaf entry. In contrast, intercellular, or apoplastic,sites favor the high-level expression of genes for GABA metabolism (degradation of these genes would attenuate GABA repression of virulence) and the synthesis of phytotoxins, two additional secondary metabolites, and syringolin A. These findings support roles for these compounds in virulence, including a role for syringolin A in suppressing defense responses beyond stomatal closure. A comparison of the transcriptomes from in planta cells and from cells exposed to osmotic stress, oxidative stress, and iron and nitrogen limitation indicated that water availability, in particular,was limited in both leaf habitats but was more severely limited in the apoplast than on the leaf surface under the conditions tested. These findings contribute to a coherent model of the adaptations of this widespread bacterial phytopathogen to distinct habitats within its host.

Pseudomonas aeruginosa uses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline in P. aeruginosa physiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, in P. aeruginosa growth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ΔbetT1 and ΔbetT1 ΔbetT3 mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdR-mediated cbcXWV induction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.

This review examines the many ways in which water influences the relations between foliar bacterial pathogens and plants. As a limited resource in aerial plant tissues, water is subject to manipulation by both plants and pathogens. A model is emerging that suggests that plants actively promote localized desiccation at the infection site and thus restrict pathogen growth as one component of defense. Similarly, many foliar pathogens manipulate water relations as one component of pathogenesis. Nonvascular pathogens do this using effectors and other molecules to alter hormonal responses and enhance intercellular watersoaking, whereas vascular pathogens use many mechanisms to cause wilt. Because of water limitations on phyllosphere surfaces, bacterial colonists, including pathogens, benefit from the protective effects of cellular aggregation, synthesis of hygroscopic polymers, and uptake and production of osmoprotective compounds. Moreover, these bacteria employ tactics for scavenging and distributing water to overcome water-driven barriers to nutrient acquisition, movement, and signal exchange on plant surfaces., (Copyright © 2011 by Annual Reviews. All rights reserved.)

Host specialization to form biotypes is common among phytophagous insects, and it has been hypothesised that biotypes of Dactylopius tomentosus L. (Hemiptera: Dactylopiidae) occur. D. tomentosus is an important biological control agent for Cylindropuntia cacti when they occur as weeds. Additionally, there is uncertainty surrounding the taxonomic status of some species of Cylindropuntia. This study aimed to confirm the existence of D. tomentosus biotypes and to assess whether host specialization can help to resolve this systematic uncertainty. For this study, the host specificity and performance of ten provenances of D. tomentosus collected from C. cholla, C. fulgida var. fulgida, C. imbricata, C. f. var. mamillata, C. rosea and C. tunicata and reared on C. cholla, C. fulgida var. fulgida, C. imbricata and C. rosea were investigated. Five life-history parameters were measured including: crawler development time and survival, female development time, and the weight and number of eggs produced by females. Results revealed significant variation in host specificity with provenances either thriving, surviving or dying on the different hosts, thus demonstrating the existence of biotypes. Also, host specificity was related to host species and not to the geographic locality from which either the host or provenance was sourced. These findings suggest that the characteristics of Cylindropuntia species may differ sufficiently, there by presenting different selection pressures that induce and sustain distinct biotypes of D. tomentosus. The observed host use patterns of the biotypes separated the plant species into two groups that accorded with known phylogenetic relationships among Cylindropuntia species, suggesting that biotypes can be used to elucidate their taxonomic relatedness. Besides advancing our knowledge of the ecology and evolution of D. tomentosus, these novel findings have important implications for the biological control of Cylindropuntia species.

Dactylopius tomentosus is composed of biotypes adapted to different Cylindropuntia species. One biotype is an important biological control agent of C. imbricata in South Africa while another has the potential for the control of C. fulgida var. fulgida. These two weed species occur in sympatry in some areas of South Africa, so the introduction of the second biotype could result in hybridization, which, in turn, could impact on the biological control programs through altered host specificity and fitness of the hybrids. To anticipate what might happen, reciprocal crosses were made between the two biotypes, and the biological performance of the resultant hybrids was compared with that of each parental lineage on C. imbricata and C. f. var. fulgida. The biotypes interbred freely and reciprocally in the laboratory. Comparisons of crawler and adult female traits showed differences in performance that were dependent on the origin of the maternal and paternal genomes. However, when all traits were combined into a 'fitness index', both hybrids clearly outperformed the parental lineages. The increase in fitness shown by the hybrids over their maternal lineage was greater on the alternative host of the maternal parent than on the natural host of the maternal parent. Therefore, in areas where the two cacti occur in sympatry, hybridization between the biotypes is not expected to be detrimental to the biological control of either weed.

Surprisingly little is known of the trehalose biosynthetic pathways in pseudomonads, despite the importance of trehalose to protecting cells from environmental stresses such as low water availability. The genome of the foliar pathogen Pseudomonas syringae pv. tomato strain DC3000 contains genes for two trehalose biosynthetic pathways, TreS and TreYZ, and lacks genes for the more common OtsAB pathway. Deletion of either the treS (PSPTO_2760-2762) or treY/treZ (PSPTO_3125-3134) locus eliminated trehalose accumulation and reduced bacterial growth under hyperosmotic conditions. In evaluating the role of trehalose in P. syringae fitness on leaves, we found that a double deletion mutant lacking these loci exhibited poorer survival than the wild type on tomato leaves over a 2-week period in a growth chamber. Similarly, this mutant exhibited reduced survival on leaves of susceptible and resistant cultivars of the host plant tomato and of the non-host plant soybean over a 10-day period in field plots. Thus, the trehalose biosynthetic loci in P. syringae, which are highly conserved among pseudomonads, contributed to DC3000 fitness on leaves, supporting a role for trehalose in P. syringae survival and population maintenance in the phyllosphere.

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Cylindropuntia fulgida (Engelmann) F.M. Knuth var. fulgida (Engelmann) F.M. Knuth (Cff) (Caryophyllales: Cactaceae) is native to Mexico and Arizona and was introduced into South Africa for ornamental purposes. It subsequently became highly invasive, necessitating control. The cochineal insect, Dactylopius tomentosus (Lamarck) (Hemiptera: Dactylopiidae), was selected as a potential biological control agent based on its restricted host range among Cylindropuntia species and previous success in controlling C. imbricata (DC.) F. Knuth (Ci). Eight D. tomentosus provenances (Cholla, Cholla E, Fulgida, Mamillata, Imbricata, Tunicata U, Tunicata V and Rosea) from Cylindropuntia species in their native ranges were reared on Cff, whilst Cholla and Imbricata were also reared on Ci. Large differences were found in the development and survival of crawlers, and in the reproductive capacity of females. Three subjective categories of provenance interaction with host plants were identified based on a fitness index (FI) calculated from data relating to crawler survival, female development time and fecundity: (i) thriving (FI > or = 1) - insects had shorter developmental times, high crawler survival and highly fecund females (Cholla); (ii) surviving (FI<1 but >0) - insects had extended development times, low crawler survival and low fecundity (Imbricata, Fulgida and Mamillata); and (iii) dying (FI = 0) - insects died before or at the second instar (Rosea, Tunicata U and Tunicata V). Cholla, therefore, is highly suitable for biological control of Cff in South Africa. In addition, Cholla thrived on Cff but only survived on Ci whilst, in contrast, Imbricata thrived on Ci but only survived on Cff. This differential ability of provenances to thrive or survive on different host plants demonstrated that host adapted biotypes of D. tomentosus exist; therefore, biotypes should be taken into account when considering this species as a biological control agent of cactus weeds.

Dactylopius tomentosus (Lamarck) (Hemiptera: Dactylopiidae) is a cochineal insect whose host range is restricted to Cylindropuntia species (Caryophyllales: Cactaceae). This insect has been utilized successfully for biological control of Cylindropuntia imbricata (Haw.) F.M. Knuth in Australia and South Africa. Despite this, its biology has not been studied previously, probably due to the widely held belief that the biology of all Dactylopius species is similar. This study investigated the life cycle and the morphological and reproductive characteristics of D. tomentosus. Results revealed some unique characteristics of D. tomentosus: (i) eggs undergo a much longer incubation period, an average of 17 days compared to <1 day in its congeners; (ii) eggs are laid singly but are retained as an egg mass secured in a mesh of waxy threads attached to the female; (iii) the developmental times of males and females are longer compared to other Dactylopius spp. due to a longer egg incubation period; (iv) D. tomentosus does not undergo parthenogenesis; (v) D. tomentosus is smaller in size than its congeners; and (vi) male mating capacity and reproductive potential were both high and variable between males. There was a significant, strong, positive relationship (r = 0.93) between female mass and fecundity, whereas the relationship between the number of females mated per male that became gravid and their fecundity was negative (r = -0.68). Besides contributing to our knowledge of this economically important species, the finding of unique characteristics of D. tomentosus biology underlines the need to study each species in this genus.

Occasional pesticide application in integrated pest management to at least part of a crop requires that any biological control agents must re-invade previously sprayed areas in order that resurgent pests can be constrained. The ability of the phytoseiid predatory mite Phytoseiulus persimilis to feed on adult two-spotted spider mite (TSSM) Tetranychus urticae on excised leaf discs in both control conditions and in a treatment with a sub lethal residue of agricultural mineral oil (AMO) was assessed. The predator exhibited a Type II functional response with the asymptote significantly higher in the AMO conditions due to the fact that the prey grew slower and reached a smaller size in this treatment. In terms of prey volume eaten, the satiation level of the predator was unchanged by the AMO deposits. The numbers of eggs produced by adult P. persimilis females at densities of 4, 8 and 16 TSSM adult females/disc in the control were significantly higher than those in the AMO treatment, but were similar for the higher density levels, 32 and 64 prey per disc. Thus the functional response in terms of volume of prey eaten explained the numerical response in terms of predator eggs produced. The presence of AMO deposits when the prey were at high density had no effect on predator efficiency (volume eaten) but resulted in a lower intake than that in control conditions when there was a greater distance between prey.

The physiological mechanisms by which plants limit the growth of bacterial pathogens during gene-for-gene resistance are poorly understood. We characterized early events in the Arabidopsis thaliana-Pseudomonas syringae pathosystem to identify physiological changes for which the kinetics are consistent with bacterial growth restriction. Using a safranine-O dye solution to detect vascular activity, we demonstrated that A. thaliana Col-0 resistance to P. syringae pv. tomato DC3000 cells expressing avrRpm1 involved virtually complete cessation of vascular water movement into the infection site within only 3 h postinoculation (hpi), under the conditions tested. This vascular restriction preceded or was simultaneous with precipitous decreases in photosynthesis, stomatal conductance, and leaf transpiration, with the latter two remaining at detectable levels. Microscopic plant cell death was detected as early as 2 hpi. Interestingly, suppression of bacterial growth during AvrRpm1-mediated resistance was eliminated by physically blocking leaf water loss through the stomata without altering plant cell death and was nearly eliminated by incubating plants at high relative humidity. The majority of the population growth benefit from blocking leaf water loss occurred early after inoculation, i.e., between 4 and 8 hpi. Collectively, these results support a model in which A. thaliana suppresses P. syringae growth during gene-for-gene resistance, at least in part, by coupling restricted vascular flow to the infection site with water loss through partially open stomata; that is, the plants effectively starve the invading bacteria for water.

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O-, but not the C23O-, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.