From microbes to microbiomes: Applications for plant health and sustainable agriculture.

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 over-promising, 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.

First report of cucurbit yellow vine disease caused by Serratia marcescens on cucurbit crops in Iowa.

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 (~108 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.

An atypical 3-ketoacyl ACP synthase III required for acyl homoserine lactone synthesis in Pseudomonas syringae pv. syringae B728a.

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 (C4-C10) or acyl-ACPs (C6-C10). 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 ß-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.

Light cues induce protective anticipation of environmental water loss in terrestrial bacteria.

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.

Ornamental tobacco floral nectar is a rich source of antimicrobial peptides.

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.

Expression and Functional Analysis of the Type III Secretion System Effector Repertoire of the Xylem Pathogen Erwinia tracheiphila on Cucurbits.

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.

Characterization of Xanthomonas citri pv. glycines Population Genetics and Virulence in a National Survey of Bacterial Pustule Disease in Korea.

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.

Pan-Genome Analysis of Effectors in Korean Strains of the Soybean Pathogen Xanthomonas citri pv. glycines.

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.

The Contributions to Virulence of the Effectors Eop1 and DspE Differ Between Two Clades of Erwinia tracheiphila Strains.

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.

Characterization of Erwinia tracheiphila Bacteriophage FBB1 Isolated from Spotted Cucumber Beetles that Vector E. tracheiphila.

Erwinia tracheiphila, the causal pathogen of bacterial wilt of cucurbit crops, is disseminated by cucumber beetles. A bacteriophage, designated FBB1 (Fu-Beattie-Beetle-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.

Biological Control and Microbial Ecology Draft Genome Sequence Data of Glutamicibacter sp. FBE-19, a Bacterium Antagonistic to the Plant Pathogen Erwinia tracheiphila.

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 ß-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.

Seeing the Light: The Roles of Red- and Blue-Light Sensing in Plant Microbes.

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.

Mining Halophytes for Plant Growth-Promoting Halotolerant Bacteria to Enhance the Salinity Tolerance of Non-halophytic Crops.

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.

Pseudomonas syringae pv. syringae B728a Regulates Multiple Stages of Plant Colonization via the Bacteriophytochrome BphP1.

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 Psyringae 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 Psyringae 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.

Horizontal Gene Acquisitions, Mobile Element Proliferation, and Genome Decay in the Host-Restricted Plant Pathogen Erwinia Tracheiphila.

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.