Plant-Growth-Promoting Rhizobacteria Emerging as an Effective Bioinoculant to Improve the Growth, Production, and Stress Tolerance of Vegetable Crops.

Vegetable cultivation is a promising economic activity, and vegetable consumption is important for human health due to the high nutritional content of vegetables. Vegetables are rich in vitamins, minerals, dietary fiber, and several phytochemical compounds. However, the production of vegetables is insufficient to meet the demand of the ever-increasing population. Plant-growth-promoting rhizobacteria (PGPR) facilitate the growth and production of vegetable crops by acquiring nutrients, producing phytohormones, and protecting them from various detrimental effects. In this review, we highlight well-developed and cutting-edge findings focusing on the role of a PGPR-based bioinoculant formulation in enhancing vegetable crop production. We also discuss the role of PGPR in promoting vegetable crop growth and resisting the adverse effects arising from various abiotic (drought, salinity, heat, heavy metals) and biotic (fungi, bacteria, nematodes, and insect pests) stresses.

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.

Anatomical study of the female reproductive system and bacteriome of Diaphorina citri Kuwayama, (Insecta: Hemiptera, Liviidae) using micro-computed tomography.

Huanglongbing (HLB) (citrus greening disease) is one of the most serious bacterial diseases of citrus. It is caused by (1) Candidatus Liberibacter africanus, transmitted by Trioza erytreae and (2) C.L. asiaticus and C.L. americanus, transmitted by Diaphorina citri. As part of a multidisciplinary project on D. citri (www.citrusgreening.org), we made a detailed study, using micro-computed tomography, of the female abdominal terminalia, reproductive system (ovaries, accessory glands, spermatheca, colleterial (= cement) gland, connecting ducts, and ovipositor) and bacteriome, which we present here. New terms and structures are introduced and described, particularly concerning the spermatheca, ovipositor and bacteriome. The quality of images and bacteriome reconstructions are comparable, or clearer, than those previously published using a synchrotron or fluorescence in situ hybridisation (FISH). This study: reviews knowledge of the female reproductive system and bacteriome organ in D. citri; represents the first detailed morphological study of D. citri to use micro-CT; and extensively revises existing morphological information relevant to psylloids, hemipterans and insects in general. High quality images and supplementary videos represent a significant advance in knowledge of psylloid anatomy and are useful tools for future research and as educational aids.

Functional and Comparative Genomic Analysis of Integrated Prophage-Like Sequences in "Candidatus Liberibacter asiaticus".

Huanglongbing disease (HLB; yellow shoot disease) is a severe worldwide infectious disease for citrus family plants. The pathogen "Candidatus Liberibacter asiaticus" is an alphaproteobacterium of the Rhizobiaceae family that has been identified as the causative agent of HLB. The virulence of "Ca. Liberibacter asiaticus" has been attributed, in part, to prophage-carried genes. Prophage and prophage-like elements have been identified in 12 of the 15 available "Ca. Liberibacter asiaticus" genomes and are classified into three prophage types. Here, we reexamined all 15 "Ca. Liberibacter asiaticus" genomes using a de novo prediction approach and expanded the number of prophage-like elements from 16 to 33. Further, we found that all of the "Ca. Liberibacter asiaticus" genomes contained at least one prophage-like sequence. Comparative analysis revealed a prevalent, albeit previously unknown, prophage-like sequence type that is a remnant of an integrated prophage. Notably, this remnant prophage is found in the Ishi-1 "Ca. Liberibacter asiaticus" strain that had previously been reported as lacking prophages. Our findings provide both a resource for data and new insights into the evolutionary relationship between phage and "Ca. Liberibacter asiaticus" pathogenicity.IMPORTANCE Huanglongbing (HLB) disease is threatening citrus production worldwide. The causative agent is "Candidatus Liberibacter asiaticus." Prior work using mapping-based approaches identified prophage-like sequences in some "Ca. Liberibacter asiaticus" genomes but not all. Here, we utilized a de novo approach that expands the number of prophage-like elements found in "Ca. Liberibacter asiaticus" from 16 to 33 and identified at least one prophage-like sequence in all "Ca. Liberibacter asiaticus" strains. Furthermore, we identified a prophage-like sequence type that is a remnant of an integrated prophage-expanding the number of prophage types in "Ca. Liberibacter asiaticus" from 3 to 4. Overall, the findings will help researchers investigate the role of prophage in the ecology, evolution, and pathogenicity of "Ca. Liberibacter asiaticus."

No Evidence of Apoptotic Response of the Potato Psyllid Bactericera cockerelli to "Candidatus Liberibacter solanacearum" at the Gut Interface.

"Candidatus Liberibacter solanacearum" is a pathogen transmitted by the potato psyllid Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) in a persistent manner. In this study, we investigated the molecular interaction between "Ca. Liberibacter solanacearum" and the potato psyllid at the gut interface. Specifically, we focused on the apoptotic response of potato psyllids to the infection by two "Ca. Liberibacter solanacearum" haplotypes, LsoA and LsoB. To this end, we first quantified and localized "Ca. Liberibacter solanacearum" in the gut of adult psyllids. We then evaluated the existence of an apoptotic response in the insect gut using microscopy analyses to visualize the nuclei and the actin cytoskeleton of the gut cells and DNA fragmentation analyses by agarose gel electrophoresis. We also performed annexin V cell death assays to detect apoptosis. Finally, we annotated apoptosis-related genes from the potato psyllid transcriptome and evaluated their expression in response to "Ca. Liberibacter solanacearum" infection. The results showed no cellular markers of apoptosis despite the large amount of "Ca. Liberibacter solanacearum" present in the psyllid gut. In addition, only three genes potentially involved in apoptosis were regulated in the psyllid gut in response to "Ca. Liberibacter solanacearum": the apoptosis-inducing factor AIF3 was downregulated in LsoA-infected psyllids, while the inhibitor of apoptosis IAPP5 was downregulated and IAP6 was upregulated in LsoB-infected psyllids. Overall, no evidence of apoptosis was observed in the gut of potato psyllid adults in response to either "Ca. Liberibacter solanacearum" haplotype. This study represents a first step toward understanding the interactions between "Ca. Liberibacter solanacearum" and the potato psyllid, which is crucial to developing approaches to disrupt their transmission.

Growth parameters of Liberibacter crescens suggest ammonium and phosphate as essential molecules in the Liberibacter-plant host interface.

BACKGROUND: Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus. L. asiaticus, L. americanus, L. africanus cause citrus greening disease, while Ca. L. solanacearum causes potato Zebra chip disease. None of the pathogens grows in axenic culture. L. crescens grows in three media: a BM-7, a serum-free Hi® Grace's Insect Medium (Hi-GI), and a chemically-defined medium called M15. To date, no optimal growth parameters of the model species L. crescens have been reported. Studying the main growth parameters of L. crescens in axenic culture will give us insights into the lifestyle of the Ca. Liberibacter pathogens. RESULTS: The evaluation of the growth parameters-pH, aeration, temperature, and buffering capacity-reflects the optimal living conditions of L. crescens. These variables revealed that L. crescens is an aerobic, neutrophilic bacterium, that grows optimally in broth in a pH range of 5.8 to 6.8, in a fully oxygenated environment (250 rpm), at 28 °C, and with monosodium phosphate (10 mM or 11.69 mM) as the preferred buffer for growth. The increase of pH in the external media likely results from the deamination activity within the cell, with the concomitant over-production of ammonium in the external medium. CONCLUSION: L. crescens and the Ca. Liberibacter pathogens are metabolically similar and grow in similar environments-the phloem and the gut of their insect vectors. The evaluation of the growth parameters of L. crescens reveals the lifestyle of Liberibacter, elucidating ammonium and phosphate as essential molecules for colonization within the hosts. Ammonium is the main driver of pH modulation by active deamination of amino acids in the L. crescens amino acid rich media. In plants, excess ammonium induces ionic imbalances, oxidative stress, and pH disturbances across cell membranes, causing stunted root and shoot growth and chlorosis-the common symptoms of HLB-disease. Phosphate, which is also present in Ca. L. asiaticus hosts, is the preferred buffer for the growth of L. crescens. The interplay between ammonium, sucrose, potassium (K+), phosphate, nitrate (NO3-), light and other photosynthates might lead to develop better strategies for disease management.

Growth Dynamics and Survival of Liberibacter crescens BT-1, an Important Model Organism for the Citrus Huanglongbing Pathogen "Candidatus Liberibacter asiaticus".

Liberibacter crescens is the only cultured member of its genus, which includes the devastating plant pathogen "Candidatus Liberibacter asiaticus," associated with citrus greening/Huanglongbing (HLB). L. crescens has a larger genome and greater metabolic flexibility than "Ca Liberibacter asiaticus" and the other uncultured plant-pathogenic Liberibacter species, and it is currently the best model organism available for these pathogens. L. crescens grows slowly and dies rapidly under current culture protocols and this extreme fastidiousness makes it challenging to study. We have determined that a major cause of rapid death of L. crescens in batch culture is its alkalinization of the medium (to pH 8.5 by the end of logarithmic phase). The majority of this alkalinization is due to consumption of alpha-ketoglutaric acid as its primary carbon source, with a smaller proportion of the pH rise due to NH3 production. Controlling the pH rise with higher buffering capacity and lower starting pH improved recoverability of cells from 10-day cultures by >1,000-fold. We have also performed a detailed analysis of L. crescens growth with total cell numbers calibrated to the optical density and the percentage of live and recoverable bacteria determined over 10-day time courses. We modified L. crescens culture conditions to greatly enhance survival and increase maximum culture density. The similarities between L. crescens and the pathogenic liberibacters make this work relevant to efforts to culture the latter organisms. Our results also suggest that growth-dependent pH alteration that overcomes medium buffering should always be considered when growing fastidious bacteria.IMPORTANCELiberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by "Candidatus Liberibacter asiaticus" and carried by the Asian citrus psyllid. L. crescens is the only close relative of "Ca Liberibacter asiaticus" that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens.

Possible cross-feeding pathway of facultative methylotroph Methyloceanibacter caenitepidi Gela4 on methanotroph Methylocaldum marinum S8.

Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M. caenitepidi Gela4 co-cultured with M. marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M. caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M. caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M. marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M. caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M. marinum S8 produced acetate (< 16 µM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M. caenitepidi Gela4 cross-fed by M. marinum S8. Co-culture of M. caenitepidi Gela4 and M. marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria.

Biodegradation of phenol by cold-tolerant bacteria isolated from alpine soils of Binaloud Mountains in Iran.

Degradation of phenol is considered to be a challenge because of harsh environments in cold regions and ground waters. Molecular characterization of phenol degrading bacteria was investigated to gain an insight into the biodegradation in cold areas. The psychrotolerant and psychrophiles bacteria were isolated from alpine soils in the northeast of Iran. These strains belonged to Pseudomonas sp., Stenotrophomonas spp. and Shinella spp. based on analysis of the 16S rRNA gene. These strains were capable of the complete phenol degradation at a concentration of 200 mg L-1 at 20 °C. Moreover, the strains could degrade phenol at a concentration of 400 and 600 mg L-1 at a higher time. Effects of environmental factors were studied using one factor at a time (OFAT) approach for Pseudomonas sp.ATR208. When the bacterium was grown in a liquid medium with 600 mg L-1 of concentration supplemented with optimum carbon and nitrogen sources, more than 99% of phenol removal was obtained at 20 °C and 24 h. Therefore, the present study indicated the potential of the local cold tolerant bacteria in the phenol bioremediation.

Developing Citrus Huanglongbing (HLB) Management Strategies Based on the Severity of Symptoms in HLB-Endemic Citrus-Producing Regions.

Citrus Huanglongbing (HLB), also known as greening, is a destructive disease caused by the fastidious, phloem-colonizing bacteria Candidatus Liberibacter spp.; 'Ca. Liberibacter asiaticus' (Las) is the most prevalent of the species causing HLB. The Asian citrus psyllid (ACP, Diaphorina citri) transmits Las. HLB is threatening citrus production worldwide, and there is no cure for infected trees. Management strategies targeting diseased trees at different stages of colonization by Las are needed for sustainable citrus production in HLB-endemic regions. We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost on mildly diseased trees. We tested thermotherapy on severely diseased trees and assessed tree protectors to prevent feeding by ACP, thus preventing Las from being transmitted to new plantings that replaced HLB-moribund trees. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors ß-aminobutyric acid, plus ascorbic acid and potassium phosphite with or without salicylic acid, slowed down the progression of HLB and reduced disease severity by approximately 18%, compared with the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared with the untreated control. Thermotherapy treatment (55°C for 2 min) exhibited a suppressive effect on growth of Las and progress of HLB in severely diseased trees for 2 to 3 months after treatment. The tree protectors prevented feeding by ACP, and therefore young replant trees remained healthy and free from infection by Las over the 2-year duration of the experiment. Taken together, these results may contribute to a basis for developing a targeted approach to control HLB based on stage of host colonization, application of plant defense elicitors, N fertilizer, compost, thermotherapy, and tree protectors. There is potential to implement these strategies in conjunction with other disease control measures to contribute to sustainable citrus production in HLB-endemic regions.

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.

Bacterial mediated alleviation of heavy metal stress and decreased accumulation of metals in plant tissues: Mechanisms and future prospects.

Heavy metal pollution of agricultural soils is one of main concerns causing some of the different ecological and environmental problems. Excess accumulation of these metals in soil has changed microbial community (e.g., structure, function, and diversity), deteriorated soil, decreased the growth and yield of plant, and entered into the food chain. Plants' tolerance to heavy metal stress needs to be improved in order to allow growth of crops with minimum or no accumulation of heavy metals in edible parts of plant that satisfy safe food demands for the world's rapidly increasing population. It is well known that PGPRs (plant growth-promoting rhizobacteria) enhance crop productivity and plant resistance to heavy metal stress. Many recent reports describe the application of heavy metal resistant-PGPRs to enhance agricultural yields without accumulation of metal in plant tissues. This review provides information about the mechanisms possessed by heavy metal resistant-PGPRs that ameliorate heavy metal stress to plants and decrease the accumulation of these metals in plant, and finally gives some perspectives for research on these bacteria in agriculture in the future.

Imperative roles of halotolerant plant growth-promoting rhizobacteria and kinetin in improving salt tolerance and growth of black gram (Phaseolus mungo).

The salinity stress causes a major threat for plant growth, yield, and biomass production. The present study was designed to assess the effect of exogenously applied kinetin and halotolerant plant growth-promoting rhizobacteria (H-PGPR) on alleviation of salt stress in black gram (Phaseolus mungo). A total of 15 rhizobacterial isolates obtained from a salt-affected area were analyzed for their capability to improve growth of P. mungo plants growing in greenhouse conditions. Out of the tested rhizobacteria, the two bacterial isolates which exhibited maximum growth potential were screened and their growth-promoting attributes were evaluated. The role of screened H-PGPR and/or kinetin (8 and 10 µM) was evaluated in P. mungo plants irrigated with three levels of brackish water (S1 = 3, S2 = 5, and S3 = 7 dSm-1) under field condition. Salt stress reduced transpiration rate, stomatal conductance, salt tolerance index, growth, leaf area, photosynthetic pigments, leaf relative water content (LRWC), biomass production, and seed yield in subjected plants. Conversely, the salinized plants treated with kinetin and/or H-PGPR exhibited improved levels of chlorophyll contents, LRWC, root growth, shoot growth, biomass production, and seed yield. The H-PGPR and/or kinetin supplementation also reduced electrolyte leakage in salt-stressed plants. Overall, the present findings will be of great value to recognize the mechanism of salt stress alleviation in P. mungo plants under the influence of H-PGPR and/or kinetin.

'Candidatus Liberibacter asiaticus' Accumulates inside Endoplasmic Reticulum Associated Vacuoles in the Gut Cells of Diaphorina citri.

Citrus greening disease known also as Huanglongbing (HLB) caused by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus' (CLas) has resulted in tremendous losses and the death of millions of trees worldwide. CLas is transmitted by the Asian citrus psyllid Diaphorina citri. The closely-related bacteria 'Candidatus Liberibacter solanacearum' (CLso), associated with vegetative disorders in carrots, is transmitted by the carrot psyllid Bactericera trigonica. A promising approach to prevent the transmission of these pathogens is to interfere with the vector-pathogen interactions, but our understanding of these processes is limited. It was recently reported that CLas induced changes in the nuclear architecture, and activated programmed cell death, in D. citri midgut cells. Here, we used electron and fluorescent microscopy and show that CLas induces the formation of endoplasmic reticulum (ER)-associated bodies. The bacterium recruits those ER structures into Liberibacter containing vacuoles (LCVs), in which bacterial cells seem to propagate. ER- associated LCV formation was unique to CLas, as we could not detect these bodies in B. trigonica infected with CLso. ER recruitment is hypothesized to generate a safe replicative body to escape cellular immune responses in the insect gut. Understanding the molecular interactions that undelay these responses will open new opportunities for controlling CLas.

Dynamics of Huanglongbing-associated Bacterium Candidatus Liberibacter asiaticus in Citrus aurantifolia Swingle (Mexican Lime).

BACKGROUND: The bacterial disease citrus huanglongbing (HLB), associated with "Candidatus Liberibacter asiaticus" (C.Las) has severely impacted the citrus industry, causing a significant reduction in production and fruit quality. In the present study, it was monitored the C.Las population dynamics in symptomatic, HLB-positive Mexican lime trees (Citrus aurantifolia Swingle) in a tropical, citrus-producing area of Mexico. The objective of this study was to identify the dynamics of the population of huanglongbing-associated bacterium Candidatus Liberibacter asiaticus and its insect vector in Citrus aurantifolia Swingle (Mexican lime). MATERIALS AND METHODS: Leaf samples were collected every 2 months over a period of 26 months for quantification of bacterial titers and young and mature leaves were collected in each season to determine preferential sites of bacterial accumulation. The proportion of living and dead bacterial cells could be determined through the use of quantitative real-time PCR in the presence of ethidium monoazide (EMA-qPCR). RESULTS: It was observed a lower bacterial titer at high temperatures in the infected trees relative to titers in mild weather, despite a higher accumulation of the insect vector Diaphorina citri in these conditions. This study also revealed seasonal fluctuations in the titers of bacteria in mature leaves when compared to young leaves. No statistically significant correlation between any meteorological variable, C.Las concentration and D. citri population could be drawn. CONCLUSION: Although, HLB management strategies have focused on vector control, host tree phenology may be important. The evaluation of citrus phenology, C.Las concentration, ACP population and environmental conditions provides insights into the cyclical, seasonal variations of both the HLB pathogen and its vector. These findings should help in the design of integrative HLB control strategies that take into account the accumulation of the pathogen and the presence of its vector.

A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development.

The coordinated control of Ca2+ signaling is essential for development in eukaryotes. Cyclic nucleotide-gated channel (CNGC) family members mediate Ca2+ influx from cellular stores in plants (Charpentier et al., 2016; Gao et al., 2016; Frietsch et al., 2007; Urquhart et al., 2007). Here, we report the unusual genetic behavior of a quantitative gain-of-function CNGC mutation (brush) in Lotus japonicus resulting in a leaky tetrameric channel. brush resides in a cluster of redundant CNGCs encoding subunits which resemble metazoan voltage-gated potassium (Kv1-Kv4) channels in assembly and gating properties. The recessive mongenic brush mutation impaired root development and infection by nitrogen-fixing rhizobia. The brush allele exhibited quantitative behavior since overexpression of the cluster subunits was required to suppress the brush phenotype. The results reveal a mechanism by which quantitative competition between channel subunits for tetramer assembly can impact the phenotype of the mutation carrier.

Monitoring the colonization and infection of legume nodules by Micromonospora in co-inoculation experiments with rhizobia.

The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation. Over 50% of nodules from each legume housed Micromonospora, and using 16S rRNA gene sequence identification, we verified that the reisolated strains corresponded to the microorganisms inoculated. Entry of the bacteria and colonization of the plant hosts were monitored using a GFP-tagged Lupac 08 mutant together with rhizobia, and by using immunogold labeling. Strain Lupac 08 was localized in plant tissues, confirming its capacity to enter and colonize all hosts. Based on studying three different plants, our results support a non-specific relationship between Micromonospora and legumes. Micromonospora Lupac 08, originally isolated from Lupinus re-enters root tissue, but only when coinoculated with the corresponding rhizobia. The ability of Micromonospora to infect and colonize different legume species and function as a potential plant-growth promoting bacterium is relevant because this microbe enhances the symbiosis without interfering with the host and its nodulating and nitrogen-fixing microbes.

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.

A Plant Bacterial Pathogen Manipulates Its Insect Vector's Energy Metabolism.

Insect-transmitted plant-pathogenic bacteria may alter their vectors' fitness, survival, behavior, and metabolism. Because these pathogens interact with their vectors on the cellular and organismal levels, potential changes at the biochemical level might occur. "Candidatus Liberibacter asiaticus" (CLas) is transmitted in a persistent, circulative, and propagative manner. The genome of CLas revealed the presence of an ATP translocase that mediates the uptake of ATP and other nucleotides from medium to achieve its biological processes, such as growth and multiplication. Here, we showed that the levels of ATP and many other nucleotides were significantly higher in CLas-infected than healthy psyllids. Gene expression analysis showed upregulation for ATP synthase subunits, while ATPase enzyme activity showed a decrease in ATPase activity. These results indicated that CLas stimulated Diaphorina citri to produce more ATP and many other energetic nucleotides, while it may inhibit their consumption by the insect. As a result of ATP accumulation, the adenylated energy charge (AEC) increased and the AMP/ATP and ADP/ATP ratios decreased in CLas-infected D. citri psyllids. Survival analysis confirmed a shorter life span for CLas-infected D. citri psyllids. In addition, electropenetrography showed a significant reduction in total nonprobing time, salivation time, and time from the last E2 (phloem ingestion) to the end of recording, indicating that CLas-infected psyllids were at a higher hunger level and they tended to forage more often. This increased feeding activity reflects the CLas-induced energetic stress. In conclusion, CLas alters the energy metabolism of its psyllid vector, D. citri, in order to secure its need for energetic nucleotides.IMPORTANCE Insect transmission of plant-pathogenic bacteria involves propagation and circulation of the bacteria within their vectors. The transmission process is complex and requires specific interactions at the molecular and biochemical levels. The growth of the plant-pathogenic bacteria in the hemolymph of their vectors indicated that the hemolymph contains all the necessary nutrients for their growth. In addition to nutrients, "Candidatus Liberibacter asiaticus" (CLas) can take up energetic nucleotides, such as ATP, from its vector, Diaphorina citri, using ATP translocase. In this study, we found that the CLas pathogen manipulates the energy metabolism of its insect vector. The accumulation of ATP in CLas-infected D. citri psyllids indicated that CLas induces ATP production to fulfill its need for this energetic compound. As a result of ATP accumulation, a shorter life span and altered feeding behavior were observed. These findings increase our knowledge of insect transmission of the persistent-circulative-propagative type of plant pathogens vectored by insects.

Building the bacterial cell wall at the pole.

Polar growth is the predominant mode of cell wall extension in the Actinobacteria and the alphaproteobacterial clade Rhizobiales. The observation of polar elongation in taxonomically diverse bacteria suggests that polar growth may have evolved independently. Indeed, the regulatory mechanisms governing the assembly of cell wall biosynthesis machinery at the pole are distinct in the Actinobacteria and Rhizobiales. Here we highlight recent advances in our understanding of polar growth mechanisms in bacteria, with an emphasis on Streptomyces and Agrobacterium. This review illustrates that common themes are emerging in the regulation of polar growth in diverse bacteria. Emerging themes include the use of landmark proteins to direct growth to the pole and coordination of polar growth with cell-cycle progression.