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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.

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.

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.

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., (Copyright © 2020 American Society for Microbiology.)

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.

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., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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.

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 B 12 and is widely used in the food and feed industry., (© 2019 Institute of Food Technologists®.)

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 Zn 2+ and H 2 O 2 , 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.

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'., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

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.

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.

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.

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)., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Aims: Huanglongbing (HLB)-affected citrus often display zinc deficiency symptoms. In this study, supplemental zinc was applied to citrus to determine its effect on Candidatus Liberibacter asiaticus (Las) titre, HLB symptoms, and leaf microbiome., Methods and Results: HLB-affected citrus were treated with various amounts of zinc. The treatments promoted Las growth and affected microbiomes in citrus leaves. Phylochip(™) -based results indicated that 5475 of over 50 000 known Operational Taxonomic Units (OTUs) in 52 phyla were detected in the midribs of HLB-affected citrus, of which Proteobacteria was the most abundant, followed by Firmicutes and Actinobacteria. In comparison, the microbiomes of zinc-treated diseased plants had overall more OTUs with higher amounts of Proteobacteria, but decreased percentages of Firmicutes and Actinobacteria. In addition, more OTUs of siderophore-producing bacteria were present. Only zinc-sensitive Staphylococcaceae had higher OTU's in the diseased plants without zinc treatments., Conclusions: Although HLB-affected citrus appear zinc deficient, zinc amendments increased the pathogen levels and shifted the microbiome., Significance and Impact of the Study: HLB is currently the most devastating disease of citrus worldwide. Zinc is often applied to HLB-affected citrus due to zinc deficiency symptoms. This study provided new insights into the potential effects of zinc on HLB and the microbial ecology of citrus., (© 2016 The Society for Applied Microbiology.)

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.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

In this study, we developed and validated a method for the extraction, identification, and quantitation of penicillin G and its metabolites (penilloic acid and penillic acid) in a variety of citrus fruits by employing sequential liquid/liquid and solid-phase extraction techniques in conjunction with UHPLC-MS/MS. Two product ion transitions per analyte were required for identification, which contributes to a high degree of selectivity. Corrected recoveries of penicillin G using an isotopically labeled internal standard were 90-100% at fortification levels of 0.1, 0.25, 1, and 10 ng/g. Absolute recoveries for penillic acid and penilloic acid were 50-75% depending on the matrix used. The limit of detection (LOD) of penicillin G and its metabolites was found to be 0.1 ng/g when 2 g of citrus was extracted. This method is useful in determining residue levels of penicillin G and its metabolites in citrus trees infected with huanglongbing bacteria after antibiotic treatment.

By 2014, huanglongbing (HLB), the most destructive disease of citrus, and its insect vector, the Asian citrus psyllid (ACP), Diaphorina citri (Kuwayama), became established in all major citrus-growing regions of the world, including the United States, with the exception of California. At present, application of insecticides is the most widely followed option for reducing ACP populations, while application of antibiotics for suppressing HLB disease/symptoms is being practiced in some citrus-growing regions. Application of insecticides during the dormant winter season, along with cultivation of HLB-free seedlings and early detection and removal of symptomatic and asymptomatic trees, has been very effective in managing ACP. Area-wide management of ACP by application of insecticides at low volume in large areas of citrus cultivation has been shown to be effective in managing HLB and reducing management costs. As insecticide resistance is a major problem in sustainable management of ACP, rotation/alternation of insecticides with different chemistries and modes of action needs to be followed. Besides control of the insect vector, use of antibiotics has temporarily suppressed the symptoms of HLB in diseased trees. Recent efforts to discover and screen existing as well as new compounds for their antibiotic and antimicrobial activities have identified some promising molecules for HLB control. There is an urgent need to find a sustainable solution to the HLB menace through chemical control of ACP populations and within HLB-infected trees through the judicious use of labeled insecticides (existing and novel chemistries) and antibiotics in area-wide management programs with due consideration to the insecticide resistance problem., (© 2014 Society of Chemical Industry.)

There is evidence that many plant secondary metabolites may act as signal molecules to trigger the bacterial ability to metabolize polychlorinated biphenyls (PCBs) during the rhizoremediation process. However, the bases for the PCB rhizoremediation process are still largely unknown. The rhizobacterium Rhodococcus erythropolis U23A is unable to use flavanone as a growth substrate. However, on the basis of an assay that monitors the amount of 4-chlorobenzoate produced from 4-chlorobiphenyl by cells grown co-metabolically on flavanone plus sodium acetate, this flavonoid was previously found to be a potential inducer of the U23A biphenyl catabolic pathway. In this work, and using the same assay, we identified ten other flavonoids that did not support growth, but that acted as inducers of the U23A biphenyl pathway, and we confirmed flavonoid induction of the biphenyl catabolic pathway using quantitative real-time polymerase chain reaction (RT-qPCR) on the bphA gene. We also examined the effect of the growth co-substrate on flavonoid induction. Sodium acetate was replaced by glucose, mannose, sucrose, or mannitol, which are sugars found in plant root exudates. The data showed that the level of induction of strain U23A biphenyl-degrading enzymes was significantly influenced by the nature and concentration of the flavonoid in the growth medium, as well as by the substrate used for growth. Sucrose allowed for an optimal induction response for most flavonoids. Some flavonoids, such as flavone and isoflavone, were better inducers of the biphenyl catabolic enzymes than biphenyl itself. We also found that all flavonoids tested in this work were metabolized by strain U23A during co-metabolic growth, but that the metabolite profiles, as well as the level of efficiency of degradation, differed for each flavonoid. To obtain insight into how flavonoids interact with strain U23A to promote polychlorinated biphenyl (PCB) degradation, we determined the concentration of flavanone at which optimal PCB-degrading performance of strain U23A was achieved. We showed that it corresponded to the concentration required to fully induce the biphenyl catabolic pathway of the strain. Together, our data demonstrate that optimal PCB degradation during the rhizoremediation process will require the adjustment of several parameters, including the presence of the appropriate flavonoids at the proper concentrations and the presence of proper growth substrates that positively influence the ability of flavonoids to induce the pathway.

Here we report on the growth, accumulation performances of, and leaf proteomic changes in Eucalyptus camaldulensis plants harvested for different periods of time in an industrial, heavy metals (HMs)-contaminated site in the presence or absence of soil microorganism (AMs/PGPRs) additives. Data were compared to those of control counterparts grown in a neighboring nonpolluted district. Plants harvested in the contaminated areas grew well and accumulated HMs in their leaves. The addition of AMs/PGPRs to the polluted soil determined plant growth and metal accumulation performances that surpassed those observed in the control. Comparative proteomics suggested molecular mechanisms underlying plant adaptation to the HMs challenge. Similarly to what was observed in laboratory-scale investigations on other metal hyperaccumulators but not on HMs-sensitive plants, eucalyptus grown in the contaminated areas showed an over-representation of enzymes involved in photosynthesis and the Calvin cycle. AMs/PGPRs addition to the soil increased the activation of these energetic pathways, suggesting the existence of signaling mechanisms that address the energy/reductive power requirement associated with augmented growth performances. HMs-exposed plants presented an over-representation of antioxidant enzymes, chaperones, and proteins involved in glutathione metabolism. While some antioxidant enzymes/chaperones returned to almost normal expression values in the presence of AMs/PGPRs or in plants exposed to HMs for prolonged periods, proteins guaranteeing elevated glutathione levels were constantly over-represented. These data suggest that glutathione (and related phytochelatins) could act as key molecules for ensuring the effective formation of HMs-chelating complexes that are possibly responsible for the observed plant tolerance to metal stresses. Overall, these results suggest potential genetic traits for further selection of phytoremediating plants based on dedicated cloning or breeding programs.

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'.

Huanglongbing is the most devastating disease of citrus caused by Candidatus Liberibacter asiaticus (Las). In the present study, we report the discovery of novel small molecule inhibitors against SecA ATPase of Las by using structure based design methods. We built the homology model of SecA protein structure of Las based on the SecA of Escherichia coli. The model was used for in-silico screening of commercially available compounds from ZINC database. Using the glide flexible molecular docking method, twenty structures were chosen for in vitro studies. Five compounds were found to inhibit the ATPase activity of SecA of Las at nano molar concentrations and showed antimicrobial activities against Agrobacterium tumefaciens with MBC ranging from 128 to 256 μg/mL. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Las., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Huanglongbing (HLB) is the most devastating disease of citrus. The global citrus industry is in urgent need of effective chemical treatments for HLB control because of its rapid spreading worldwide. Due to the fastidious nature of the pathogens, and the poor permissibility of citrus leaf surfaces, effective screening of chemicals for the HLB control can be challenging. In this study, we developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the best HLB-affected scion-rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared with grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. These findings are useful not only for chemical treatments but also for graft-based transmission studies in HLB and other Liberibacter diseases.

Thirty two rhizobia were isolated from the fresh healthy root nodules of horse gram. They were found to be highly salt tolerant. They were identified as rhizobia by cultural, biochemical and 16S rRNA sequence. The sequences of the four selected isolates were deposited in the NCBI GenBank. The obtained accession numbers were GQ483457, GQ483458, GQ483459 and GQ483460. All the rhizobia were able to grow at 10 ppm mercuric chloride concentration. Four isolates HGR-11, 16, 30 and 31 were used to study the effect of different concentrations of mercuric chloride on the growth of rhizobia. These isolates were able to grow at 30 ppm concentration also. In these isolates, HGR-11 and HGR-30 showed maximum growth at 20 ppm than at control. These isolates contained one mega plasmid (-22 kb) at 20 ppm mercuric chloride concentration.

Some plant pathogens form obligate relationships with their insect vector and are vertically transmitted via eggs analogous to insect endosymbionts. Whether insect endosymbionts manipulate plant defenses to benefit their insect host remains unclear. The tomato psyllid, Bactericerca cockerelli (Sulc), vectors the endosymbiont "Candidatus Liberibacter psyllaurous" (Lps) during feeding on tomato (Solanum lycopersicum L.). Lps titer in psyllids varied relative to the psyllid developmental stage with younger psyllids harboring smaller Lps populations compared to older psyllids. In the present study, feeding by different life stages of B. cockerelli infected with Lps, resulted in distinct tomato transcript profiles. Feeding by young psyllid nymphs, with lower Lps levels, induced tomato genes regulated by jasmonic acid (JA) and salicylic acid (SA) (Allene oxide synthase, Proteinase inhibitor 2, Phenylalanine ammonia-lyase 5, Pathogenesis-related protein 1) compared to feeding by older nymphs and adults, where higher Lps titers were found. In addition, inoculation of Lps without insect hosts suppressed accumulation of these defense transcripts. Collectively, these data suggest that the endosymbiont-like pathogen Lps manipulates plant signaling and defensive responses to benefit themselves and the success of their obligate insect vector on their host plant.

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide and is threatening the survival of the Floridian citrus industry. Currently, there is no established cure for this century-old and emerging disease. As a possible control strategy for citrus HLB, therapeutic compounds were screened using a propagation test system with 'Candidatus Liberibacter asiaticus'-infected periwinkle and citrus plants. The results demonstrated that the combination of penicillin and streptomycin (PS) was effective in eliminating or suppressing the 'Ca. L. asiaticus' bacterium and provided a therapeutically effective level of control for a much longer period of time than when administering either antibiotic separately. When treated with the PS, 'Ca. L. asiaticus'-infected periwinkle cuttings achieved 70% of regeneration rates versus <50% by other treatments. The 'Ca. L. asiaticus' bacterial titers in the infected periwinkle plants, as measured by quantitative real-time polymerase chain reaction, decreased significantly following root soaking or foliar spraying with PS. Application of the PS via trunk injection or root soaking also eliminated or suppressed the 'Ca. L. asiaticus' bacterium in the HLB-affected citrus plants. This may provide a useful tool for the management of citrus HLB and other Liberibacter-associated diseases.

The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 μg MC ml(-1). Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Liquid media containing potato extract and 1% of glucose or sucrose were used to culture root-nodule bacteria (rhizobia) in shaken Erlenmeyer flasks. For comparison, these bacteria were also cultured in yeast extract-mannitol broth (YEMB) as a standard medium. Proliferation of rhizobia was monitored by measuring optical densities (OD550) of the cultures and by plate counting of the viable cells (c.f.u) of the bacteria. In general, multiplication of the rhizobia in potato extract-glucose broth (PEGB) and potato extract-sucrose broth (PESB) was markedly faster, as indicated by higher values of OD550, than in YEMB. The numbers of R. leguminosarum by. vicae GGL and S. meliloti 330 in PEGB and PEGB were high and ranged from 1.2 x 10(10) to 4.9 x 10(10) mL(-1) after 48 h of incubation at 28 degrees C. B. japonicum B3S culture in PEGB contained 6.4 x 10(9) c.f.u. ml(-1) after 72 h of incubation. PEGB and YEMB cultures of the rhizobia were similar with respect to their beneficial effects on nodulation of the host-plants of these bacteria.

In order to determine their tolerance to pesticides, 122 strains of rhizobia isolated from different geographical regions, and belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium and Bradyrhizobium were tested against eight herbicides, four fungicides and five insecticides. Sensitivity to the pesticides was measured by using the filter paper disk method at four concentrations, 0.45, 4.5, 45 and 450 μg per disk. When the pesticides were used at 0.45 μg per disk, a concentration similar to that found when pesticides are applied under field conditions, no inhibition was observed. Strains growth was affected at concentrations of 45 and 450 μg pesticide per disk. These higher concentrations can be encountered when seeds are treated with pesticides. Pesticides tolerance level was correlated to pesticide function, i.e rhizobial strains were more tolerant to insecticides, followed by herbicides and then fungicides. Two fungicides, captan and mancozeb, inhibited the highest number of strains. Only one insecticide, carbaryl, affected the growth of some rhizobial strains. Strains isolated from the arctic (Mesorhizobium spp. and R. leguminosarum bv. viciae), a putative pesticides-free environment, were either less or equally affected by pesticides compared to strains isolated from agricultural regions.

Background, Aim, and Scope: Selenium is a trace metalloid of global environmental concern. The boundary among its essentiality, deficiency, and toxicity is narrow and mainly depends on the chemical forms and concentrations in which this element occurs. Different plant species-including Brassica juncea-have been shown to play a significant role in Se removal from soil as well as water bodies. Furthermore, the interactions between such plants, showing natural capabilities of metal uptake and their rhizospheric microbial communities, might be exploited to increase both Se scavenging and vegetable biomass production in order to improve the whole phytoextraction efficiency. The aim of the present study was to evaluate the capability of selenite removal of B. juncea grown in hydroponic conditions on artificially spiked effluents. To optimize phytoextraction efficiency, interactions between B. juncea and rhizobacteria were designedly elicited., Materials and Methods: Firstly, B. juncea was grown on water-filtering agriperlite beds in the presence of three different selenite concentrations, namely, 0.2, 1.0, and 2.0 mM. Plant growth was measured after 3 and 6 weeks of incubation in order to establish the selenite concentration at which the best plant biomass production could be obtained. Afterwards, water-filtering agriperlite beds were inoculated either with a selenium-acclimated microbial community deriving from the rhizosphere of B. juncea grown, erstwhile, in a selenite-amended soil or with axenic cultures of two bacterial strains, vicelike Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02, previously isolated and described for their high resistance to selenite. These latter were seeded separately or as a dual consortium. Selenite was amended at a final concentration of 1.0 mM. Total Se content in plant tissues (both shoots and roots), plant biomass production, and persistence of bioaugmented microbial inocula during the experimental time were monitored. Moreover, parameters such as bioconcentration factor (BF) and phytoextraction efficiency (PE) were determined at the end of the testing run to evaluate the effects of the different bioaugmentation strategies adopted on selenite phytoextraction efficiency of B. juncea., Results: A general but significant increase in capacity to extract and transport selenium to the epigeous plant compartments was recorded in B. juncea grown in beds augmented with microbial inocula, except for the treatment with B. mycoides SeITE01 alone. Nevertheless, a severe decrease in vegetable biomass production was observed after all microbial treatments with the exception of the plants that had received only S. maltophilia SeITE02. Actually, an increase in selenium phytoextraction efficiency up to 65% was observed in B. juncea, when this bacterial strain was inoculated., Discussion: Emendation of B. juncea grown in water-filtering beds with a Se(IV)-acclimated microbial community caused a higher Se uptake along with a reduction of plant biomass yield with respect to plants grown without addition of the same bacterial inoculum. The increase of selenium BF in shoots suggests that the Se(IV)-acclimated microbial community not only elicited the plant capacity to absorb selenite, but also did improve the capacity to transport the metalloid to the epigeous compartments. On the other hand, the reduction in plant biomass yield might be related exactly to this improved capability of B. juncea to accumulate selenium at concentrations that are actually toxic for plants. Differently, addition of two selenite-resistant bacterial strains, namely, S. maltophilia SeITE02 and B. mycoides SEITE01, had weaker effects on plant biomass production when compared to those recorded in the presence of the Se(IV)-adapted microbial community. In particular, inoculation of water-filtering beds with the SeITE02 strain alone was the sole strategy resulting in a positive effect on both plant biomass production in stressful conditions and the capacity of shoots to accumulate selenium. In fact, its putative ability of reducing Se(IV) to organo-Se compounds significantly enhanced either selenium absorption by the plants or active metalloid translocation to epigeous parts., Conclusions: Bioaugmentation with the bacterial strain S. malthophila SeITE02 is suggested to elicit selenite phytoextraction efficiency in B. juncea., Recommendations: Manipulation of synergistic interactions between plants having phytoextraction capabilities and their associated rhizobacteria may enhance already consolidated treatment processes aimed to detoxify selenite laden wastewater.

A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined. Colonies, positive for 'Ca. L. asiaticus' by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect 'Ca. L. asiaticus' and 'Ca. L. americanus' cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of 'Ca. L. asiaticus' and one of 'Ca. L. americanus' grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of 'Ca. L. asiaticus' and 'Ca. L. americanus' associated with symptoms of HLB.

The use of pesticides in agricultural soils may affect the soil microbiota. The effect of repeated application of copper sulfate in soil on indigenous populations of rhizobia was assessed in a medium-term field experiment. Copper sulfate was applied over 8 years at two different rates, 12.5 and 50 kg of CuSO4 ha(-1) year(-1), in the field. The concentrations of total copper in soil varied between 14.0 (control plots that did not receive copper sulfate) and 91.0 mg kg(-1) (the most contaminated plots) at the time of sampling, 3 years after the end of the copper treatments. All the other physicochemical parameters were similar among the plots that also shared the same cropping history. The target rhizobia were monospecific populations of Rhizobium leguminosarum bv. viciae nodulating Vicia sativa and communities of rhizobial species nodulating Phaseolus vulgaris. The size of the vetch rhizobial populations was significantly reduced in the soils with the higher Cu content, whereas the size of the Phaseolus rhizobial populations was not significantly affected. However, the number of nodules formed on both vetches and common beans were reduced for the plants grown in the most contaminated soils, suggesting an additional toxic effect of copper on plant physiology. The diversity (Simpson's indices) of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer (IGS), was not influenced by copper application. Also, the genetic structure of the R. leguminosarum bv. viciae populations was not modified by copper treatments. By contrast, a shift was observed in the composition of the Phaseolus-nodulating communities in relation to soil copper content. The communities were composed of three 16S rDNA haplotypes: one corresponding to the R. leguminosarum (biovar phaseoli) species, the two others forming a new lineage of Phaseolus rhizobia based on 16S rDNA sequence analysis. The reduced frequency of the R. leguminosarum species in the Phaseolus-nodulating communities from the copper-treated soils was linked to its higher sensitivity to copper as compared to the higher tolerance of isolates belonging to the other rhizobial lineage. The new lineage was functionally efficient for symbiotic nitrogen fixation with P. vulgaris. Our results suggest that functional redundancy among species exhibiting variability for copper tolerance preserved the size of Phaseolus-nodulating communities. In contrast, the abundance of the vetch-nodulating rhizobia, which was a monospecific functional group mainly constituted by copper-sensitive genotypes, was adversely affected by repeated application of copper sulfate.

The diffusion of metallo-beta-lactamases (MBLs) among clinically important human pathogens represents a therapeutic issue of increasing importance. However, the origin of these resistance determinants is largely unknown, although an important number of proteins belonging to the MBL superfamily have been identified in microbial genomes. In this work, we analyzed the distribution and function of genes encoding MBL-like proteins in the class Rhizobiales. Among 12 released complete genomes of members of the class Rhizobiales, a total of 57 open reading frames (ORFs) were found to have the MBL conserved motif and identity scores with MBLs ranging from 8 to 40%. On the basis of the best identity scores with known MBLs, four ORFs were cloned into Escherichia coli for heterologous expression. Among their products, one (blr6230) encoded by the Bradyrhizobium japonicum USDA110 genome, named BJP-1, hydrolyzed beta-lactams when expressed in E. coli. BJP-1 enzyme is most closely related to the CAU-1 enzyme from Caulobacter vibrioides (40% amino acid sequence identity), a member of subclass B3 MBLs. A kinetic analysis revealed that BJP-1 efficiently hydrolyzed most beta-lactam substrates, except aztreonam, ticarcillin, and temocillin, with the highest catalytic efficiency measured with meropenem. Compared to other MBLs, BJP-1 was less sensitive to inactivation by chelating agents.

Chelate-assisted phytoremediation has been proposed as an effective tool for the extraction of heavy metals from soil by plants. However, side effects of the addition of chelate to soil microbial community are usually neglected. We studied the potential effects of chelate (glucose and citric acid) amendment on phytoextraction of copper and microbial community composition in soil under laboratory conditions. A copper (Cu) accumulator, Elsholtzia splendens, and a non-accumulator, Trifolium repens, were grown on a sandy loam soil containing 317 mg kg(-1) Cu. Microbial community compositions were analyzed by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that the biomass of E. splendens grown with the chelate did not differ from that of the control. Addition of citric acid decreased the biomass of T. repens in comparison to that of glucose treatment. Application of glucose or citric acid significantly increased the extractable Cu concentration in planted and unplanted soils. Concentrations of Cu in the shoots of E. splendens were 2.6, 1.9 and 2.9 times of those of T. repens under no chelate, citric acid and glucose treatments, respectively. PCR-DGGE fingerprint analysis revealed that there were negative correlations between bacteria diversity and NH4NO3 extractable Cu under glucose or citric acid treatment. It was indicated the amendment of glucose to the plant T. repens increased the bacteria diversity in soil as compared to that in soils of non-chelate treatment. The above results indicated chelate addition facilitated phytoremediation of soil Cu and did not have a negative effect on microbial community.

This study investigates the impact of long-term heavy metal contamination on the culturable, heterotrophic, functional and genetic diversity of rhizobacterial communities of perennial grasses in water meadow soil. The culturable heterotrophic diversity was investigated by colony appearance on solid LB medium. Genetic diversity was measured as bands in denaturing gradient gel electrophoresis (DGGE) obtained directly from rhizosphere soil and rhizoplane DNA extracts, and from the corresponding culturable communities. In the two rhizospheric fractions the DGGE profiles of the direct DNA extracts were similar and stable among replicates, whereas in the enriched cultures the profiles of the fractions differed, but among the replicates they were similar. One hundred isolates were collected into 33 different operational taxonomic units by use of amplified internal transcribed spacers and into 19 heavy metal-resistant phenotypes. The phylogenetic position of strains belonging to 18 operational taxonomic units, representing more than 80% of the isolates, was determined by 16S rRNA gene sequencing. Several heavy metal-resistant strains were isolated from rhizoplane. Finally, metal-resistant rhizobacteria were tested for plant growth-promoting characteristics; some were found to contain 1-aminocyclopropane-1-carboxylic acid deaminase and/or to produce indole acetic acid and siderophores. Two strains resistant to cadmium and zinc, Pseudomonas tolaasii RP23 and Pseudomonas fluorescens RS9, had all three plant growth-promoting characteristics. Our findings suggest that bacteria can respond to soil metal contamination, and the described methodological approach appears promising for targeting potential plant growth-promoting rhizobacteria.

The laboratory experiments tested the influence of selected pesticides on the symbiotic efficiency and nitrogenase activity of Rhizobium leguminosarumin bv. trifolii KGL, Sinorhizobiuni melilotii Bp and Badyrhizobium sp. Ornithopus B bacteria entering into symbiosis with clover, lucerne and serradella, respectively. The results obtained indicate that the pesticides used in the experiments (Funaben T seed dressing and Pivot 100SL herbicide) caused reduced nitrogenase activity in active strains tested. In addition, a toxic effect of the applied pesticides on the nodulation and root growth of the tested plants was observed.

In this study, silicate-dissolving bacteria NBT strain was grown with 0.25% maltose as carbon source, and the rifampicin-resistance was generated by ultraviolet mutagenesis and streak naturalized to 200 microg x ml(-1). Through triparental cross, the luxAB genes were introduced into NBT-R200 at the help of pRK2013. The luminescence activity of the hybrid strain was tested, which indicated that all colonies had a high luminescence activity and kanamycin-, tetracyctine-and rifampicin-resistance. The NBT-R200 cells prepared from initial logarithmic growth phage were more likely to be sensitive to foreign DNA, and the maximum translocation frequency was up to 6.70 x 10(-5). In addition, the optimal mating ratio was 1:1:1. The potassium release ability from feldspar and the luminescence of luxAB genes marked silicate-dissolving bacteria RL85 strain were stable, and hence, the RL85 was available to rhizosphere microecology researches.

The strain NBT of silicate bacterium was labelled with streptomycin, and a stable streptomycin resistance strain NBT was obtained. Its colonization dynamics and affecting factors in wheat rhizosphere were studied in agar plates and greenhouse pots were studied by counting the method with selective medium. The results of pot culture experiment showed that strain NBT could successfully colonize in the rhizosphere of wheat. In pot cultures of sterile soil, the highest colonization level (3.4 x 10(7) cfu.g-1 root soil) was reached on 9th day after seeds sown; at 54th day, the population of strain NBT tended to stable, and decreased to 1.4 x 10(4) cfu.g-1 root soil. In pot cultures of unsterile soil, the highest colonization level (3.8 x 10(7) cfu.g-1 root soil) was reached at 9th day, and the population of strain NBT tended to a stationary state at 60th day, with the numbers being 1.4 x 10(4) cfu.g-1 root soil. Some biological and abiotic factors could greatly influence the colonization of the beneficial microorganism.

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA'-'lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA'-'lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA(+)) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA(-)) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.

The species and strain sensitivity of free and symbiotic nitrogen-fixing agents to heavy metals: cadmium, copper, lead and zinc in the gradient of concentration from 0.5 to 20.0 MPC/l has been studied under the laboratory conditions. Azotobacter (35 strains) and rhizobia (63 strains) have been tested. It has been established that zinc and copper are more toxical for free nitrogen-fixing agents than lead and cadmium. Among symbiotrophic nitrogen-fixing agents the nodule bacteria for alfalfa were most sensitive to cadmium; nodule bacteria of pea and lupine--to copper; those of galega and soya--to zinc. Lead proved to be the least toxic for rhizobia. Negative effect of the heavy-metal mix on diasotrophs was higher than that of each separate metal. The cultures resistant to the effect of heavy metals have been found which may be used when producing preparation for biological reclamation of polluted soils.

Low-molecular-weight RNA analysis was performed for the identification and classification of 20 Argentinian strains isolated from the root nodules of Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA base composition, DNA-DNA reassociation experiments and physiological and biochemical tests were also carried out for these strains and the whole 16S rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results of the genotypic and phenotypic characterization showed that the strains isolated in this study belong to a group that clustered in the genus Mesorhizobium. The results of DNA-DNA hybridizations showed that this group is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is proposed for this species. The type strain is LMG 19008T (= CECT 5336T).

The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested.