‘Candidatus Liberibacter asiaticus’-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress in Planta

The lipopolysaccharides (LPS) of Gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid (VLCFA) modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on non-host tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host citrus (Citrus sinensis, confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione (GSH) pool, callose deposition and activation of the salicylic acid (SA) and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ BCP peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (a) attenuates NO-mediated SAR signaling and (b) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.

Metabolomics Insights into Chemical Convergence in Xanthomonas perforans and Metabolic Changes Following Treatment with the Small Molecule Carvacrol

Microbes are natural chemical factories and their metabolome comprise diverse arrays of chemicals. The genus Xanthomonas comprises some of the most important plant pathogens causing devastating yield losses globally and previous studies suggested that species in the genus are untapped chemical minefields. In this study, we applied an untargeted metabolomics approach to study the metabolome of a globally spread important xanthomonad, X. perforans. The pathogen is difficult to manage, but recent studies suggest that the small molecule carvacrol was efficient in disease control. Bacterial strains were treated with carvacrol, and samples were taken at time intervals (1 and 6 h). An untreated control was also included. There were five replicates for each sample and samples were prepared for metabolomics profiling using the standard procedure. Metabolomics profiling was carried out using a thermo Q-Exactive orbitrap mass spectrometer with Dionex ultra high-performance liquid chromatography (UHPLC) and an autosampler. Annotation of significant metabolites using the Metabolomics Standards Initiative level 2 identified an array of novel metabolites that were previously not reported in Xanthomonas perforans. These metabolites include methoxybrassinin and cyclobrassinone, which are known metabolites of brassicas; sarmentosin, a metabolite of the Passiflora-heliconiine butterfly system; and monatin, a naturally occurring sweetener found in Sclerochiton ilicifolius. To our knowledge, this is the first report of these metabolites in a microbial system. Other significant metabolites previously identified in non-Xanthomonas systems but reported in this study include maculosin; piperidine; β-carboline alkaloids, such as harman and derivatives; and several important medically relevant metabolites, such as valsartan, metharbital, pirbuterol, and ozagrel. This finding is consistent with convergent evolution found in reported biological systems. Analyses of the effect of carvacrol in time-series and associated pathways suggest that carvacrol has a global effect on the metabolome of X. perforans, showing marked changes in metabolites that are critical in energy biosynthesis and degradation pathways, amino acid pathways, nucleic acid pathways, as well as the newly identified metabolites whose pathways are unknown. This study provides the first insight into the X. perforans metabolome and additionally lays a metabolomics-guided foundation for characterization of novel metabolites and pathways in xanthomonad systems.

Microbial Diversity Analysis and Genome Sequencing Identify Xanthomonas perforans as the Pathogen of Bacterial Leaf Canker of Water Spinach (Ipomoea aquatic)

Ipomoea aquatica is a leafy vegetable widely cultivated in tropical Asia, Africa, and Oceania. Bacterial leaf canker disease has been attacking the planting fields and seriously affecting the quality of I. aquatica in epidemic areas in China. This study examined the microbial composition of I. aquatica leaves with classical symptoms of spot disease. The results showed that Xanthomonas was overwhelmingly dominant in all four diseased leaf samples but rarely present in rhizospheric soil or irrigation water samples. In addition, Pantoea was also detected in two of the diseased leaf samples. Pathogen isolation, identification, and inoculation revealed that both Xanthomonas sp. TC2-1 and P. ananatis were pathogenic to the leaves of I. aquatic, causing crater-shaped ulcerative spots and yellowing with big brown rot lesions on leaves, respectively. We further sequenced the whole genome of strain TC2-1 and showed that it is a member of X. perforans. Overall, this study identified X. perforans as the causal pathogen of I. aquatica bacterial leaf canker, and P. ananatis as a companion pathogen causing yellowing and brown rot on leaves. The correct identification of the pathogens will provide important basis for future efforts to formulate targeted application strategy for bacterial disease control.

A mutation of a single core gene, tssM, of Type VI secretion system of Xanthomonas perforans influences virulence, epiphytic survival and transmission during pathogenesis on tomato

Xanthomonas perforans is a seed-borne hemi-biotrophic pathogen that successfully establishes infection in the phyllosphere of tomato. While the majority of the studies investigating mechanistic basis of pathogenesis have focused on successful apoplastic growth, factors important during asymptomatic colonization in the early stages of disease development are not well understood. In this study, we show that tssM gene of the type VI secretion system cluster i3* (T6SS-i3*) plays a significant role during initial asymptomatic epiphytic colonization at different stages during the life cycle of the pathogen. Mutation in a core gene, tssM of T6SS-i3*, imparted higher aggressiveness to the pathogen, as indicated by higher overall disease severity, higher in planta growth as well as shorter latent infection period compared to the wild-type upon dip-inoculation of 4-5-week-old tomato plants. Contribution of tssM towards aggressiveness was evident during vertical transmission from seed-to-seedling with wild-type showing reduced disease severity as well as lower in planta populations on seedlings compared to the mutant. Presence of functional TssM offered higher epiphytic fitness as well as higher dissemination potential to the pathogen when tested in an experimental setup mimicking transplant house high-humidity conditions. We showed higher osmotolerance being one mechanism by which TssM offers higher epiphytic fitness. Taken together, these data reveal that functional TssM plays a larger role in offering ecological advantage to the pathogen. TssM prolongs the association of hemi-biotrophic pathogen with the host, minimizing overall disease severity, yet facilitating successful dissemination.

Whole genome sequences reveal the Xanthomonas perforans population is shaped by the tomato production system

Modern agricultural practices increase the potential for plant pathogen spread, while the advent of affordable whole genome sequencing enables in-depth studies of pathogen movement. Population genomic studies may decipher pathogen movement and population structure as a result of complex agricultural production systems. We used whole genome sequences of 281 Xanthomonas perforans strains collected within one tomato production season across Florida and southern Georgia fields to test for population genetic structure associated with tomato production system variables. We identified six clusters of X. perforans from core gene SNPs that corresponded with phylogenetic lineages. Using whole genome SNPs, we found genetic structure among farms, transplant facilities, cultivars, seed producers, grower operations, regions, and counties. Overall, grower operations that produced their own transplants were associated with genetically distinct and less diverse populations of strains compared to grower operations that received transplants from multiple sources. The degree of genetic differentiation among components of Florida’s tomato production system varied between clusters, suggesting differential dispersal of the strains, such as through seed or contaminated transplants versus local movement within farms. Overall, we showed that the genetic variation of a bacterial plant pathogen is shaped by the structure of the plant production system.

Harnessing Eco-Evolutionary Dynamics of Xanthomonads on Tomato and Pepper to Tackle New Problems of an Old Disease

Bacterial spot is an endemic seedborne disease responsible for recurring outbreaks on tomato and pepper around the world. The disease is caused by four diverse species, Xanthomonas gardneri, Xanthomonas euvesicatoria, Xanthomonas perforans, and Xanthomonas vesicatoria. There are no commercially available disease-resistant tomato varieties, and the disease is managed by chemical/biological control options, although these have not reduced the incidence of outbreaks. The disease on peppers is managed by disease-resistant cultivars that are effective against X. euvesicatoria but not X. gardneri. A significant shift in composition and prevalence of different species and races of the pathogen has occurred over the past century. Here, I attempt to review ecological and evolutionary processes associated with the population dynamics leading to disease emergence and spread. The goal of this review is to integrate the knowledge on population genomics and molecular plant–microbe interactions for this pathosystem to tailor disease management strategies. Expected final online publication date for the Annual Review of Phytopathology, Volume 59 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.