Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem.

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2'-deoxy-2'-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp.

A major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

Inoculation of Ensifer fredii strain LP2/20 immobilized in agar results in growth promotion and alteration of bacterial community structure of Chinese kale planted soil.

In our former research, we succeeded in using agar, alginate, and perlite as immobilization materials to maintain long-term survival of the inoculant, Ensifer fredii LP2/20, in a controlled glasshouse. Therefore the information on the establishment and activity of the inoculant to promote plant growth under field conditions, the effects of the inoculant on the soil microbial communities and specific microbial taxa, and the association between the inoculant and soil elements merit further studies. Here, we found that agar was the most suitable material that supported the establishment of the inoculant under field conditions. RNA-based analysis showed that E. fredii LP2/20 immobilized in agar was still metabolically active at day 50 after being introduced into soil. Inoculation of E. fredii LP2/20 immobilized in agar conferred the highest plant dry weight (up to 89.94%) and all plant elements including total N (9.55%), P (17.94%), K (68.42%), Ca (39.77%), Mg (30.76%), Fe (29.85%), and Zn (22.44%). Inoculation of E. fredii LP2/20 immobilized in agar increased soil chemicals including soil organic matter (99.02%), total N (272.48%), P (31.75%), K (52.74%), Fe (51.06%), and Zn (63.10%). High-throughput next-generation sequencing of bacterial 16S rRNA amplicons showed that the Proteobacteria, Acidobacteria, Bacteroidetes, and Firmicutes were dominant phyla in Chinese kale field soil. Inoculation of E. fredii LP2/20 significantly affected the soil bacterial community structure by decreasing total bacterial richness and diversity. The numbers of alpha- and gamma-Proteobacteria were significantly increased while the number of delta-Proteobacteria was significantly decreased due to E. fredii LP2/20 establishment. Soil total P, K, and Ca and soil pH were the important factors that shaped the soil bacterial community composition.

Zinkicide Is a ZnO-Based Nanoformulation with Bactericidal Activity against Liberibacter crescens in Batch Cultures and in Microfluidic Chambers Simulating Plant Vascular Systems.

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 (ZnO)-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.

Assessment of unconventional antimicrobial compounds for the control of 'Candidatus Liberibacter asiaticus', the causative agent of citrus greening disease.

In this study, newly identified small molecules were examined for efficacy against 'Candidatus Liberibacter asiaticus' in commercial groves of sweet orange (Citrus sinensis) and white grapefruit (Citrus paradisi) trees. We used benzbromarone and/or tolfenamic acid delivered by trunk injection. We evaluated safety and efficacy parameters by performing RNAseq of the citrus host responses, 16S rRNA gene sequencing to characterize citrus-associated microbial communities during treatment, and qRT-PCR as an indirect determination of 'Ca. L. asiaticus' viability. Analyses of the C. sinensis transcriptome indicated that each treatment consistently induced genes associated with normal metabolism and growth, without compromising tree viability or negatively affecting the indigenous citrus-associated microbiota. It was found that treatment-associated reduction in 'Ca. L. asiaticus' was positively correlated with the proliferation of several core taxa related with citrus health. No symptoms of phytotoxicity were observed in any of the treated trees. Trials were also performed in commercial groves to examine the effect of each treatment on fruit productivity, juice quality and efficacy against 'Ca. L. asiaticus'. Increased fruit production (15%) was observed in C. paradisi following twelve months of treatment with benzbromarone and tolfenamic acid. These results were positively correlated with decreased 'Ca. L. asiaticus' transcriptional activity in root samples.

Quercus leaf extracts display curative effects against Candidatus Liberibacter asiaticus that restore leaf physiological parameters in HLB-affected citrus trees.

Citrus greening, also called Huanglongbing (HLB), is one of the most destructive citrus diseases worldwide. It is caused by the fastidious gram-negative α-proteobacteria bacterium Candidatus Liberibacter asiaticus (CLas) and vectored by the Asian citrus psyllid (ACP), Diaphorina citri. Currently, there is no cure for HLB, no compounds have been successful in controlling HLB, and no sustainable management practices have been established for the disease. Thus, searching for alternative citrus greening disease mitigation strategies is considered an urgent priority for a sustainable citrus industry. The aim of this study was to use compounds extracted from oak, Quercus hemisphaerica, and to assess the antibacterial effects of these against CLas-infected citrus plants. The application of aqueous oak leaf extracts showed substantial inhibitory effects against CLas in citrus plants and the activity of genes related to starch. Significant differences were also observed in plant phenotypic and physiological traits after treatments. Citrus plants treated with oak extracts displayed an increase in stomatal conductance, chlorophyll content and nutrient uptake concurrently with a reduction of CLas titer, when compared to citrus plants treated with just water. The information provided from this study suggests a new management treatment program to effectively deal with the HLB disease.

Identification and Antibiotic Resistance Assessment of Ensifer adhaerens YX1, a Vitamin B12 -Producing Strain Used as a Food and Feed Additive.

This study provides phenotypic and molecular analyses of the antibiotic resistance of Ensifer adhaerens strain YX1 (CICC 11008s), a strain that was identified using a polyphasic taxonomy approach. The antibiotic resistance profile of E. adhaerens YX1 was assessed using the Clinical & Laboratory Standards Inst. (CLSI) method. The strain was susceptible to ciprofloxacin, levofloxacin, norfloxacin, ofloxacin, gentamicin, tobramycin, chloramphenicol, tetracycline, imipenem, and ceftazidime, and resistant to kanamycin, streptomycin, fosfomycin, and nitrofurantoin. The antibiotic resistance genes nsfA, nsfB, fosA, aph, and aadA1 were not detected in E. adhaerens YX1 via PCR using gene-specific primers. Subsequently, the genome sequence of E. adhaerens was screened for antibiotic genes. Although no antibiotic resistance genes were identified using the ResFinder database, five genes copies of one resistance gene, adeF, were detected using the Comprehensive Antibiotic Resistance Database (CARD). The results of this study will be useful for understanding the phenotypic and genotypic aspects of E. adhaerens antibiotic resistance. No safety issues were identified for E. adhaerens YX1 in terms of antibiotic resistance. Performing similar studies will be conducive to the safety assessment and control of the use of E. adhaerens in the food and feed industry. PRACTICAL APPLICATION: Few relevant reports are currently available regarding antibiotic resistance assessments or other safety evaluations for Ensifer adhaerens. Because of a lack of relevant information on the safety of this bacterium, including the genetic basis of antibiotic resistance in the production strain, it has not been recommended for use in the "qualified presumption of safety" (QPS) list and subsequent updated lists. The current study shows no safety issue of E. adhaerens YX1 in terms of its antibiotic resistance. These results are important as they provide an initial basis for an understanding of the antibiotic resistance/susceptibility of E. adhaerens YX1 (CICC 11008s), which produces vitamin B12 and is widely used in the food and feed industry.

The in Planta Effective Concentration of Oxytetracycline Against 'Candidatus Liberibacter asiaticus' for Suppression of Citrus Huanglongbing.

Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium 'Candidatus Liberibacter asiaticus' (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.

A novel Zn chelate (TSOL) that moves systemically in citrus plants inhibits growth and biofilm formation of bacterial pathogens.

Ternary solution (TSOL) is a novel Zn chelate-based systemic antimicrobial formulation designed for treating citrus bacterial pathogens 'Candidatus Liberibacter asiaticus' and Xanthomonas citri subsp. citri. TSOL is a component of MS3T, a novel multifunctional surface/sub-surface/systemic therapeutic formulation. Antimicrobial activity of TSOL was compared with the antimicrobial compound ZnO against X. citri subsp. citri and 'Ca. L. asiaticus' surrogate Liberibacter crescens in batch cultures. X. citri subsp. citri and L. crescens were also introduced into microfluidic chambers, and the inhibitory action of TSOL against biofilm formation was evaluated. The minimum inhibitory concentration of TSOL for both X. citri subsp. citri and L. crescens was 40ppm. TSOL was bactericidal to X. citri subsp. citri and L. crescens above 150 ppm and 200 ppm, respectively. On the contrary, ZnO was more effective as a bactericidal agent against L. crescens than X. citri subsp. citri. TSOL was more effective in controlling growth and biofilm formation of X. citri subsp. citri in batch cultures compared to ZnO. Time-lapse video imaging microscopy showed that biofilm formation of X. citri subsp. citri was inhibited in microfluidic chambers treated with 60 ppm TSOL. TSOL also inhibited further growth of already formed X. citri subsp. citri and L. crescens biofilms in microfluidic chambers. Leaf spraying of TSOL showed higher plant uptake and systemic movement in citrus (Citrus reshni) plants compared to that of ZnO, suggesting that TSOL is a promising antimicrobial compound to control vascular plant pathogens such as 'Ca. L. asiaticus'.

Antimicrobial Zn-Based "TSOL" for Citrus Greening Management: Insights from Spectroscopy and Molecular Simulation.

Huanglongbing (HLB), also known as citrus greening, is a bacterial disease that poses a devastating threat to the citrus industry worldwide. To manage this disease efficiently, we developed and characterized a ternary aqueous solution (TSOL) that contains zinc nitrate, urea, and hydrogen peroxide. We report that TSOL exhibits better antimicrobial activity than commercial bactericides for growers. X-ray fluorescence analysis demonstrates that zinc is delivered to citrus leaves, where the bacteria reside. FTIR and Raman spectroscopy, molecular dynamics simulations, and density functional theory calculations elucidate the solution structure of TSOL and reveal a water-mediated interaction between Zn2+ and H2O2, which may facilitate the generation of highly reactive hydroxyl radicals contributing to superior antimicrobial activity of TSOL. Our results not only suggest TSOL as a potent antimicrobial agent to suppress bacterial growth in HLB-infected trees, but also provide a structure-property relationship that explains the superior performance of TSOL.

Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of "Candidatus Liberibacter asiaticus" in planta.

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide.

Zinc is an inhibitor of the LdtR transcriptional activator.

LdtR is a master regulator of gene expression in Liberibacter asiaticus, one of the causative agents of citrus greening disease. LdtR belongs to the MarR-family of transcriptional regulators and it has been linked to the regulation of more than 180 genes in Liberibacter species, most of them gathered in the following Clusters of Orthologous Groups: cell motility, cell wall envelope, energy production, and transcription. Our previous transcriptomic evidence suggested that LdtR is directly involved in the modulation of the zinc uptake system genes (znu) in the closely related L. crescens. In this report, we show that LdtR is involved in the regulation of one of the two encoded zinc uptake mechanisms in L. asiaticus, named znu2. We also show that LdtR binds zinc with higher affinity than benzbromarone, a synthetic effector inhibitory molecule, resulting in the disruption of the LdtR:promoter interactions. Using site-directed mutagenesis, electrophoretic mobility shift assays (EMSAs), and isothermal titration calorimetry, we identified that residues C28 and T43 in LdtR, located in close proximity to the Benz1 pocket, are involved in the interaction with zinc. These results provided new evidence of a high-affinity effector molecule targeting a key player in L. asiaticus' physiology and complemented our previous findings about the mechanisms of signal transduction in members of the MarR-family.

Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics.

Citrus huanglongbing (HLB) or greening is a devastating disease of citrus worldwide and no effective control measure is currently available. Plant defense activators environmentally friendly compounds capable of inducing resistance against many plant pathogens. Earlier studies showed that foliar spray of plant defense inducers could slow down HLB disease progress. In this study, eight plant defense activators and three antibiotics were evaluated in three field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that four trunk injections of several activators, including salicylic acid, oxalic acid, acibenzolar-S-methyl, and potassium phosphate, provided significant control of HLB by suppressing 'Candidatus Liberibacter asiaticus' titer and disease progress. Trunk injection of penicillin, streptomycin, and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of 'Ca. L. asiaticus' titer and HLB symptom expressions than plant defense activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of pathogenesis-related (PR) genes PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision making regarding developing an effective HLB management program.

Challenges for Managing Candidatus Liberibacter spp. (Huanglongbing Disease Pathogen): Current Control Measures and Future Directions.

Huanglongbing (HLB; "citrus greening" disease) has caused significant damages to the global citrus industry as it has become well established in leading citrus-producing regions and continues to spread worldwide. Insecticidal control has been a critical component of HLB disease management, as there is a direct relationship between vector control and Candidatus Liberibacter spp. (i.e., the HLB pathogen) titer in HLB-infected citrus trees. In recent years, there have been substantial efforts to develop practical strategies for specifically managing Ca. Liberibacter spp.; however, a literature review on the outcomes of such attempts is still lacking. This work summarizes the greenhouse and field studies that have documented the effects and implications of chemical-based treatments (i.e., applications of broad-spectrum antibiotics, small molecule compounds) and nonchemical measures (i.e., applications of plant-beneficial compounds, applications of inorganic fertilizers, biological control, thermotherapy) for phytopathogen control. The ongoing challenges associated with mitigating Ca. Liberibacter spp. populations at the field-scale, such as the seasonality of the phytopathogen and associated HLB disease symptoms, limitations for therapeutics to contact the phytopathogen in planta, adverse impacts of broad-spectrum treatments on plant-beneficial microbiota, and potential implications on public and ecosystem health, are also discussed.

Huanglongbing Control: Perhaps the End of the Beginning.

Huanglongbing (HLB) is one of the most destructive citrus plant diseases worldwide. It is associated with the fastidious phloem-limited α-proteobacteria 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' and 'Ca. Liberibacter americanus'. In recent years, HLB-associated Liberibacters have extended to North and South America. The causal agents of HLB have been putatively identified, and their transmission pathways and worldwide population structure have been extensively studied. However, very little is known about the epidemiologic relationships of Ca. L. asiaticus, which has limited the scope of HLB research and especially the development of control strategies. HLB-affected plants produce damaged fruits and die within several years. To control the disease, scientists have developed new compounds and screened existing compounds for their antibiotic and antimicrobial activities against the disease. These compounds, however, have very little or even no effect on the disease. The aim of the present review was to compile and compare different methods of HLB disease control with newly developed integrative strategies. In light of recent studies, we also describe how to control the vectors of this disease and the biological control of other citrus plant pathogens. This work could steer the attention of scientists towards integrative control strategies.

Essential oils of Origanum compactum and Thymus vulgaris exert a protective effect against the phytopathogen Allorhizobium vitis.

Allorhizobium (Agrobacterium) vitis is a host-specific pathogenic bacterium that causes grapevine crown gall disease, affecting vine growth and production worldwide. The antibacterial activities of different aromatic plant essential oils were tested in vitro and in planta against A. vitis. Among the essential oils tested, those of Origanum compactum and Thymus vulgaris showed the most significant in vitro antibacterial activities, with a MIC of 0.156 and 0.312 mg/mL, respectively. A synergistic effect of these two essential oils (1:1) was observed and confirmed by the checkerboard test. Carvacrol (61.8%) and thymol (47.8%) are, respectively, the major compounds in the essential oils of O. compactum and T. vulgaris and they have been shown to be largely responsible for the antibacterial activities of their corresponding essential oils. Results obtained in vitro were reinforced by an in planta pathogenicity test. A mixture of O. compactum and T. vulgaris essential oils (1:1), inoculated into the injured stem of a tomato plant and a grapevine at 0.312 mg/mL as a preventive treatment, reduced both the number of plants developing gall symptoms and the size of the tumors.

Plant-fed versus chemicals-fed rhizobacteria of Lucerne: Plant-only teabags culture media not only increase culturability of rhizobacteria but also recover a previously uncultured Lysobacter sp., Novosphingobium sp. and Pedobacter sp.

In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts.

Characterisation of a gene encoding a membrane protein that affects exopolysaccharide production and intracellular Mg2+ concentrations in Ensifer meliloti.

Exopolysaccharides play an important role in the physiology of a bacterial cell. Ensifer meliloti is capable of producing at least two types of exopolysaccharides (EPS): succinoglycan and galactoglucan. In E. meliloti, EPS are best known for their role in mediating interaction with its symbiotic hosts. It was previously shown that high concentrations of Mg2+ or K+ were capable of suppressing the mucoid phenotype associated with galactoglucan production in an expR+ derivative of Rm1021. In an attempt to determine how Mg2+ regulates galactoglucan production, SRmD363 was mutagenised and screened for mutants which were visibly mucoid at high concentrations of magnesium. Tn5 mutations in genes exoX, emmB, phoC and SMc00722 were isolated. SMc00722 is annotated as a hypothetical transmembrane protein that is conserved in the α-proteobacteria. Characterisation of SMc00722 in Rm1021 showed that the increased mucoidy was due to succinoglycan. Strains carrying mutations in SMc00722 showed increased biofilm production, and were more sensitive to high Mg2+ concentrations and deoxycholate. In addition, we show that strains carrying a mutation in SMc00722 have elevated intracellular Mg2+ concentrations. Taken together, the data are consistent with the hypothesis that SMc0722 may play a role in maintaining intracellular magnesium concentration, and we suggest that this gene be tentatively annotated as mhrA (magnesium homeostasis related).

Evaluation of the Spatiotemporal Dynamics of Oxytetracycline and Its Control Effect Against Citrus Huanglongbing via Trunk Injection.

Citrus huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing 'Candidatus Liberibacter asiaticus' (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected 'Hamlin' sweet orange trees on 'Swingle' citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by high-performance liquid chromatography method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days postinjection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e., 1.76 and 2.19 log reduction) between 2 and 28 days postinjection. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.

Disruption of Vector Host Preference with Plant Volatiles May Reduce Spread of Insect-Transmitted Plant Pathogens.

Plant pathogens can manipulate the odor of their host; the odor of an infected plant is often attractive to the plant pathogen vector. It has been suggested that this odor-mediated manipulation attracts vectors and may contribute to spread of disease; however, this requires further broad demonstration among vector-pathogen systems. In addition, disruption of this indirect chemical communication between the pathogen and the vector has not been attempted. We present a model that demonstrates how a phytophathogen (Candidatus Liberibacter asiaticus) can increase its spread by indirectly manipulating the behavior of its vector (Asian citrus psyllid, Diaphorina citri Kuwayama). The model indicates that when vectors are attracted to pathogen-infected hosts, the proportion of infected vectors increases, as well as, the proportion of infected hosts. Additionally, the peak of infected host populations occurs earlier as compared with controls. These changes in disease dynamics were more important during scenarios with higher vector mortality. Subsequently, we conducted a series of experiments to disrupt the behavior of the Asian citrus psyllid. To do so, we exposed the vector to methyl salicylate, the major compound released following host infection with the pathogen. We observed that during exposure or after pre-exposure to methyl salicylate, the host preference can be altered; indeed, the Asian citrus psyllids were unable to select infected hosts over uninfected counterparts. We suggest mechanisms to explain these interactions and potential applications of disrupting herbivore host preference with plant volatiles for sustainable management of insect vectors.