Search

A multifunctional surface, subsurface and systemic therapeutic (MS3T) formulation comprised of two bactericides, both didecyldimethylammonium chloride (DDAC) and a zinc (Zn)-chelate, was developed as an alternative to copper pesticides for crop protection. Agricultural grade chemicals were used to prepare MS3T formulations. Minimal inhibitory concentration (MIC) was determined to be tested in vitro against Xanthomonas alfalfae subsp. citrumelonis (herein called Xa), Escherichia coli (E. coli), and Pseudomonas syringae (Ps). Assessment of the phytotoxic potential was carried out on tomato under greenhouse conditions. Moreover, field trials were conducted during three consecutive years on grapefruit (Chrysopelea paradise) groves to evaluate efficacy against citrus canker (Xanthomonas citri subsp. citri), scab (Elsinoe fawcetti), and melanose (Diaporthe citri). In addition to disease control, improvements to both fruit yield and quality were observed likely due to the nutritional activity of MS3T via the sustained release of plant nutrients (Zn and nitrogen). Zn residues of leaf tissues were analyzed via atomic absorption spectroscopy (AAS) at various time points before and after MS3T foliar applications throughout the duration of the 2018 field trial. Field trial results demonstrated MS3T to be an effective alternative to copper (Cu)-based formulations for the control of citrus canker.

Currently, little is known about the host pathogen interaction between Phytophthora spp. and citrus roots versus leaves. Therefore, we compared the molecular events occurring in citrus roots and leaves after zoospore inoculation with Phytophthora nicotianae. We analyzed the physical characteristics and genetic responses to P. nicotianae infection of leaves and roots for susceptible and tolerant citrus rootstocks to examine the potential for leaves to model root responses to P. nicotianae infection. Leaves responded faster and stronger to P. nicotianae infection than roots, and leaves showed greater differential response than roots. In addition to differences in hormonal responses, sugar, phospholipase D (PLD), and phospholipase A (PLA) involvement in the interaction between citrus and P. nicotianae was identified. This work, for the first time, creates a solid P. nicotianae zoospore infection protocol, reports P. nicotianae infection on citrus leaves through stomata, and provides evidence that different host organs respond to the pathogen differentially in timing and magnitude. This work identifies the hormones, sugars, pathogenesis-related genes, PLDs, and PLAs that are involved in the molecular events occurring in citrus under infection of P. nicotianae zoospore, and advances our understanding of the mechanisms underlying the interaction.

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (CLas), is a devastating disease of citrus. After initial infection, CLas quickly colonizes the root system before canopy symptoms develop. There is limited understanding of CLas movement from roots to canopy and local and systemic effects on root dynamics. Using split-root rhizoboxes and late summer below-the-split bud inoculation, effects of local infection on systemic disease development were studied. Upward bacterial movement from roots is linked to seasonal flushes and CLas population in roots. CLas stayed isolated to one side of the roots for at least 8 months, until the spring flush. HLB caused differential root responses depending on tree age at infection. Systemic effects, independent of CLas movement, occur very early after infection. Stimulation of root growth occurred on noninfected roots prior to CLas detection in 1.5-year-old trees but decreased in 2.5-year-old trees. Independent of tree age, root growth was stimulated during spring root flushes after CLas population stabilized. Root dieback began simultaneously with detection of CLas in roots (6 weeks postinoculation). Infection and tree age altered root lifespan. In total, 1.5-year-old CLas-infected roots from summer and fall flushes had 3 and 6 weeks reduced lifespan. In contrast, 2.5-year-old CLas-infected plants lifespan was unaffected. Season affected root lifespan with late summer root flush lifespan was three times shorter than fall or spring root flushes. Split-root inoculation allowed study of local and systemic effects of CLas infection in roots, information crucial to prolonging the productivity of HLB-affected trees. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Accumulation of toxic copper in soil and development of copper-resistant pests are emerging challenges currently faced by the agricultural community worldwide. As an alternative, we have developed a ternary zinc chelate solution (TSOL) pesticide where zinc ions are the primary active ingredient. The material is composed of zinc, urea, and hydrogen peroxide. Urea was chosen as it is widely used as a plant fertilizer and can also bind to both zinc and hydrogen peroxide. No phytotoxicity was observed with TSOL on Meyer lemon (Citrus × meyeri) seedlings at a field spray rate of 800 μg/mL Zn metal concentration. Antimicrobial studies showed that TSOL exhibited improved killing efficacy against Escherichia coli and Xanthomonas alfalfae compared to Zn ions alone. Citrus canker field trials in a grapefruit (Chrysopelea paradisi) grove over three years showed that TSOL provided comparable disease protection to copper products at an equivalent or lower metal content.

Reviewed March 2021. There are no changes in recommendation from 2020.

Background The symbiotic interactions that occur between humans and organisms in our environment have a tremendous impact on our health. Recently, there has been a surge in interest in understanding the complex relationships between the microbiome and human health and host immunity against microbial pathogens, among other things. To collect and manage data about these interactions and their complexity, scientists will need ontologies that represent symbiotic interactions as they occur in reality. Methods We began with two papers that reviewed the usage of ‘symbiosis’ and related terms in the biology and ecology literature and prominent textbooks. We then analyzed several prominent standard terminologies and ontologies that contain representations of symbiotic interactions, to determine if they appropriately defined ‘symbiosis’ and related terms according to current scientific usage as identified by the review papers. In the process, we identified several subtypes of symbiotic interactions, as well as the characteristics that differentiate them, which we used to propose textual and axiomatic definitions for each subtype of interaction. To both illustrate how to use the ontological representations and definitions we created and provide additional quality assurance on key definitions, we carried out a referent tracking analysis and representation of three scenarios involving symbiotic interactions among organisms. Results We found one definition of ‘symbiosis’ in an existing ontology that was consistent with the vast preponderance of scientific usage in biology and ecology. However, that ontology changed its definition during the course of our work, and discussions are ongoing. We present a new definition that we have proposed. We also define 34 subtypes of symbiosis. Our referent tracking analysis showed that it is necessary to define symbiotic interactions at the level of the individual, rather than at the species level, due to the complex nature in which organisms can go from participating in one type of symbiosis with one organism to participating in another type of symbiosis with a different organism. Conclusion As a result of our efforts here, we have developed a robust representation of symbiotic interactions using a realism-based approach, which fills a gap in existing biomedical ontologies.

In 2019, citrus production in Florida declined by more than 70%, mostly because of Huanglongbing (HLB), which is caused by the bacterium '

WOS:000561413700007 PubMed ID:32561578 Phloem-limited bacterial "Candidatus Liberibacter" species are associated with incurable plant diseases worldwide. Antimicrobial treatments for these pathogens are challenging due to the difficulty of reaching the vascular tissue they occupy at bactericidal concentrations. Here, in vitro antimicrobial mechanisms of Zinkicide TMN110 (ZnK), a nonphytotoxic zinc oxide (Zn0)-based nanoformulation, were compared to those of bulk ZnO (b-ZnO) using as a model the only culturable species of the genus, Liberibacter crescens. Minimum bactericidal concentration (MBC) determination and time-kill assays showed that ZnK has a bactericidal effect against L. crescens, whereas b-ZnO is bacteriostatic. When ZnK was used at the MBC (150 ppm), its antimicrobial mechanisms included an increase in Zn solubility, generation of intracellular reactive oxygen species, lipid peroxidation, and cell membrane disruption; all of these were of greater intensity than those of b-ZnO. Inhibition of biofilms, which are important during insect vector colonization, was stronger by ZnK than by b-ZnO at concentrations between 2.5 and 10 ppm in batch cultures; however, neither ZnK nor b-ZnO removed L. crescens preformed biofilms when applied between 100 and 400 ppm. In microfluidic chambers simulating source-to-sink phloem movement, ZnK significantly outperformed b-ZnO in Zn mobilization and bactericidal activity against L. crescens planktonic cells in sink reservoirs. In microfluidic chamber assays assessing antibiofilm activity, ZnK displayed a significantly enhanced bactericidal activity against L. crescens individual attached cells as well as preformed biofilms compared to that of b-ZnO. The superior mobility and antimicrobial activity of ZnK in microenvironments make this formulation a promising product to control plant diseases caused by "Candidatus Liberibacter" species and other plant vascular pathogens. IMPORTANCE "Candidatus Liberibacter" species are associated with incurable plant diseases that have caused billions of dollars of losses for United States and world agriculture. Chemical control of these pathogens is complicated, because their life cycle combines intracellular vascular stages in plant hosts with transmission by highly mobile insect vectors. To date, "Candidatus Liberibacter" species are mostly unculturable, except for Liberibacter crescens, a member of the genus that has been used as a model for in vitro assays. Here, we evaluated the potential of Zinkicide (ZnK) as an antimicrobial against "Candidatus Liberibacter" species in batch cultures and under flow conditions, using L. crescens as a biological model. ZnK displayed bactericidal activity against L. crescens in batch cultures and showed increased mobility and bactericidal activity in microfluidic devices resembling "Candidatus Liberibacter" species natural habitats. ZnK performance observed here against L crescens makes this compound a promising candidate to control plant diseases caused by vascular pathogens.

Huanglongbing (HLB), associated with the phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’ (Las), is devastating trees in Florida citrus orchards. Phytophthora nicotianae (P.n.), omnipresent in citrus soils, causes root rot that reduces water and nutrient uptake by fibrous roots. To investigate fibrous root damage and replacement and canopy size in relation to infection of fibrous roots by P.n. and Las, rootstock seedlings of Swingle citrumelo (C. paradisi Macfad. × Poncirus trifoliata [L.] Raf.) were inoculated with Las or P.n. in two greenhouse pot trials. P.n. caused root damage within 5 weeks post inoculation, which led to greater reduction of canopy size than for Las-infected seedlings by the end of the experiment. Las increased fibrous root biomass accumulation at 5 weeks post root trimming (wpt) in the 2014 trial and at 11 wpt in the 2015 trial. New root length was not consistently increased by Las. Reduced total leaf area of asymptomatic Las-infected seedlings compared to non-inoculated controls might be due to the combined effect of altered carbohydrate allocation between shoots and roots and altered leaf morphology. This article is protected by copyright. All rights reserved.

Antimicrobial activity of experimental formulations of two structurally different nano-zinc oxide materials, plate-like Zinkicide SG4 and particulate Zinkicide SG6, was evaluated against Xanthomonas citri subsp. citri, the cause of citrus canker. In vitro assay demonstrated Zinkicide SG4 had a twofold lower minimum inhibitory concentration (MIC) against Escherichia coli and X. alfalfae subsp. citrumelonis (62.5 to 250 µg/ml) compared with copper sulfate (250 µg/ml), copper hydroxide (250 to 500 µg/ml), or cuprous oxide/zinc oxide (125 to 250 µg/ml). Zinkicide SG6 had a sevenfold to eightfold lower MIC against Escherichia coli and X. alfalfae subsp. citrumelonis (31 to 250 μg/ml). Leaves of sweet orange (Citrus sinensis) and fruit of ‘Ruby Red’ grapefruit (C. paradisi) were evaluated for citrus canker disease control. A greenhouse assay with foliage demonstrated that spray treatment with Zinkicide reduced citrus canker lesion development after injection-infiltration of X. citri subsp. citri into the leaf intercellular space. In field trials conducted in Southeast Florida in 2014 and 2015, Zinkicide SG4 and SG6 reduction of grapefruit canker incidence exceeded that of cuprous oxide and cuprous oxide/zinc oxide bactericides. Zinkicide formulations were also effective against the fungal diseases, citrus scab (Elsinoe fawcetti) and melanose (Diaporthe citri), on grapefruit. No sign of phytotoxicity to the fruit rind was observed during either season. Antimicrobial activity of Zinkicide for protection of leaves and fruit against X. citri subsp. citri was comparable or exceeded that for commercial copper and zinc oxide formulations which may be attributed to translaminar movement of Zinkicide.

Caribbean fruit fly, also known as Caribfly or Anastrepha suspensa , is a major tephritid pest of guavas. A virulent entomopathogenic nematode (EPN) species was investigated to suppress the fruit-to-soil stages of Caribflies, which are also attacked by the koinobiont parasitoid Diachasmimorpha longicaudata in south Florida. The main objective was to develop a feasible and cost-effective EPN-application method for integrated pest management (IPM) of Caribfly to improve guava production. Naturally infested guavas were treated with increasing Heterorhabditis bacteriophora infective juvenile (IJ) concentration or rate (0, 25, 50, …, 1,600 IJs cm -2 ) in field trials to measure the optimum IJ rate and then examine sensitivity of producing guavas to inclusion of Heterorhabditis bacteriophora in Caribfly IPM plans. Relative survival of Caribfly in treatments significantly decreased with increasing IJ rate from 0 to 100 IJs cm -2 . Similarly, probability of observing large numbers of parasitoid wasps ( Diachasmimorpha longicaudata ) in EPN treatments significantly declined with increasing IJ rate (0-100 IJs cm -2 ), even though the non-target effects of Heterorhabditis bacteriophora on relative survival of Diachasmimorpha longicaudata could not be determined because of few emerging parasitoid wasps. Optimum suppression (⩾ 60%) of Caribfly was consistently achieved at 100 IJs cm -2 or 17,500 IJs fruit -1 . Profitability analysis showed that Heterorhabditis bacteriophora can be included in Caribfly IPM tactics to produce guavas. Costs of EPNs in Caribfly IPM are minimized if Heterorhabditis bacteriophora is strategically applied by spot treatment of fruit. Repayment of costs of EPN-augmentation by spot treatments appears achievable by recovering 5.71% of the annual yield losses (⩾1,963 kg ha -1 ≈ US$ 8,650 ha -1 ), which are largely due to Caribfly infestation. Hectare-wide EPN-augmentation (or broadcasting) method requires more fruit recovery than the total annual yield losses to repay its high costs. Profitability of guava production in south Florida will not be very sensitive to marginal costs of the spot treatment method, when compared to the field-wide broadcasting of Heterorhabditis bacteriophora .

Copper (Cu) bactericides/fungicides are used extensively for crop protection in agriculture. Concerns for Cu accumulation in soil, Cu leaching into the surrounding ecosystem, and development of Cu resistance in phytopathogenic bacteria are evident. While there is no suitable alternative to Cu available to date for agricultural uses, it is possible to reduce Cu per application by supplementing with Zn and improving Cu bioavailability using nanotechnology. We have prepared a non-phytotoxic composite material consisting of generally recognized as safe ZnO 800 particles and nanocopper-loaded silica gel (ZnO-nCuSi). The morphology of the ZnO-nCuSi material was characterized using scanning electron microscopy, showing ZnO particles dispersed in the silica gel matrix. ZnO-nCuSi demonstrated strong in vitro antimicrobial properties against several model plant bacterial species. Two consecutive year field efficacy results showed that agri-grade ZnO-nCuSi was effective in controlling citrus canker disease at less than half the metallic rate of the commercial cuprous oxide/zinc oxide pesticide.

Huanglongbing (HLB) is associated with the phloem-limited bacterium, Candidatus Liberibacter asiaticus (Las). Phytophthora nicotianae (P.n.) causes root rot of citrus, which reduces water and nutrient uptake by citrus fibrous roots. The discovery that Las damage to fibrous roots occurs before tree canopy symptoms develop led to the prediction that Las root infection directly damages roots and may interact with soil-borne pathogens to cause further damage. Hence, comparison of root damage by Las and P.n. alone or in combination was carried out on seedlings of Cleopatra mandarin (Citrus reticulata Blanco) rootstock to evaluate the possible interaction of Las and P.n. and their relative contribution to fibrous root loss. The results demonstrated that i) roots of seedlings have a similar level of damage when inoculated with Las or P.n., and coinoculation causes comparable damage as each pathogen alone; and ii) Las infection increases and decreases P.n. infection incidence overtime without following a clear progression.

Parker, J. K., Wisotsky, S. R., Johnson, E. G., Hijaz, F. M., Killiny, N., Hilf, M. E., and De La Fuente, L. 2014. Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium. Phytopathology 104:15-26. Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’. Infection with ‘Ca. L. asiaticus’ is incurable; therefore, knowledge regarding ‘Ca. L. asiaticus’ biology and pathogenesis is essential to develop a treatment. However, ‘Ca. L. asiaticus’ cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of ‘Ca. L. asiaticus’ in vitro, ‘Ca. L. asiaticus’ inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King’s B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air–liquid interface of juice cultures contained ‘Ca. L. asiaticus’ cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining ‘Ca. L. asiaticus’ viability in vitro, which will contribute to future development of a culture medium for ‘Ca. L. asiaticus’.

Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.

Huanglongbing (HLB) is a systemic disease of citrus caused by phloem-limited bacteria ‘Candidatus Liberibacter’ spp. with ‘Ca. Liberibacter asiaticus’ (Las) the most widespread. Phloem-limited bacteria such as liberibacters and phytoplasmas are emerging as major pathogens of woody and herbaceous plants. Little is known about their systemic movement within a plant and the disease process in these tissues. Las movement after initial infection was monitored in leaves and roots of greenhouse trees. Root density, storage starch content, and vascular system anatomy in relation to Las presence in field and greenhouse trees, both with and without symptoms, showed the importance of root infection in disease development. Las preferentially colonized roots before leaves, where it multiplied and quickly invaded leaves when new foliar flush became a sink tissue for phloem flow. This led to the discovery that roots were damaged by root infection prior to development of visible foliar symptoms and was not associated with carbohydrate starvation caused by phloem-plugging as previously hypothesized. The role of root infection in systemic insect-vectored bacterial pathogens has been underestimated. These findings demonstrate the significance of early root infection to tree health and suggest a model for phloem-limited bacterial movement from the initial insect feeding site to the roots where it replicates, damages the host root system, and then spreads to the rest of the canopy during subsequent leaf flushes. This model provides a framework for testing movement of phloem-limited bacteria to gain greater understanding of how these pathogens cause disease and spread.

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains. Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of lesions and protection from bactericide treatments in the field or in the fruit disinfection process.

14 páginas, ilustraciones y tablas estadísticas, Quantitative real-time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species-complex in the Steinernema glaseri- group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species-specific primers and TaqMan® probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8-ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000–01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baitingmethod for detecting the EPN species composition in population mixtures.Moreover, the spatial patterns of EPN in this orchardwere remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.

Streptomyces species are best known for their ability to produce a wide array of medically and agriculturally important secondary metabolites. However, there is a growing number of species which, like Streptomyces scabies, can function as plant pathogens and cause scab disease on economically important crops such as potato. All of these species produce the phytotoxin thaxtomin, a nitrated dipeptide which inhibits cellulose synthesis in expanding plant tissue. The biosynthesis of thaxtomin involves conserved non-ribosomal peptide synthetases, P450 monooxygenases, and a nitric oxide synthase, the latter being required for nitration of the toxin. This nitric oxide synthase is also responsible for the production of diffusible nitric oxide by scab-causing streptomycetes at the host-pathogen interface, suggesting that nitric oxide production might play an additional role during the infection process. The thaxtomin biosynthetic genes are transcriptionally regulated by an AraC/XylS family regulator, TxtR, which is conserved in pathogenic streptomycetes and is encoded within the thaxtomin biosynthetic gene cluster. The TxtR protein specifically binds cellobiose, a known inducer of thaxtomin biosynthesis, and cellobiose is required for expression of the biosynthetic genes. A second virulence gene in pathogenic Streptomyces species, nec1, encodes a novel secreted protein that may suppress plant defence responses. The thaxtomin biosynthetic genes and nec1 are contained on a large mobilizable pathogenicity island; the transfer of this island to recipient streptomycetes likely explains the rapid emergence of new pathogenic species. The newly available genome sequence of S. scabies will provide further insight into the mechanisms utilized by pathogenic streptomycetes during plant-microbe interactions.

SummaryNitric oxide (NO) is a potent intercellular signal for defense, development, and metabolism in animals and plants. In mammals, highly regulated nitric oxide synthases (NOSs) generate NO. NOS homologs exist in some prokaryotes, but direct evidence for NO production by these proteins has been lacking. Here, we demonstrate that a NOS in plant-pathogenic Streptomyces species produces diffusible NO. NOS-dependent NO production increased in response to cellobiose, a plant cell wall component, and occurred at the host-pathogen interface, demonstrating induction by host signals. These data document in vivo production of NO by prokaryotic NOSs and implicate pathogen-derived NO in host-pathogen interactions. NO may serve as a signaling molecule in other NOS-containing bacteria, including the medically and environmentally important organisms Bacillus anthracis, Staphylococcus aureus, and Deinococcus radiodurans.

Thaxtomin, a phytotoxic dipeptide that inhibits cellulose synthesis in expanding plant cells, is a pathogenicity determinant in scab-causing Streptomyces species. Cellobiose and cellotriose, the smallest subunits of cellulose, stimulated thaxtomin production in a defined medium, while other oligosaccharides did not. Cellobiose upregulated transcription of thaxtomin biosynthetic genes. Streptomyces scabies, Streptomyces acidiscabies, and Streptomyces turgidiscabies did not hydrolyze cellulose, suggesting that these cello-oligosaccharides are plant-derived. Cellotriose was released from rapidly growing plant seedlings growing in vitro. These data support a model in which scab-causing pathogens upregulate thaxtomin production in response to cellotriose released from thaxtomin-sensitive plant tissue.

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N'-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O(2).

Xanthomonas citri subsp. citri (Xcc) strain A is the causal agent of citrus bacterial canker (CBC) on most Citrus spp.;close relatives. Two restricted host range strains of CBC, Aw and A*, from Florida and southwest Asia, respectively, infect Mexican lime. Several studies have linked biofilm formation by Xcc to bacterial colonization prior to and after plant ingress, but none have evaluated connections between biofilm formation and the behaviour of different strains of Xcc on citrus hosts and non-hosts. In this study biofilm formation and swimming motility were evaluated for citrus pathogenic xanthomonads including wide and restricted host range strains of Xcc, X. alfalfae subsp. citrumelonis (Xac) (the causal agent of citrus bacterial spot) and X. campestris pv. campestris (Xc). Differential biofilm formation was observed in vitro and in planta among the Xanthomonas strains assayed. Minimal medium XVM2 increased biofilm formation, especially for those strains with a host range restricted to Mexican lime. In planta, strains produced more biofilm on leaves or fruits of their host than on non-hosts. Scanning electron microscopy of biofilms on leaf and fruit surfaces revealed differences in structure of bacterial aggregates with respect to the strain's host range. In addition, swimming motility varied widely depending on the host range of the strain. It was concluded that biofilm formation in vitro and in planta for strains of Xcc and Xac was related to their host range, as these processes affect colonization at the early stages of the infection process. © 2014 British Society for Plant Pathology.