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.

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.

Plant powder teabags: a novel and practical approach to resolve culturability and diversity of rhizobacteria.

We have developed teabags packed with dehydrated plant powders, without any supplements, for preparation of plant infusions necessary to develop media for culturing rhizobacteria. These bacteria are efficiently cultivated on such plant teabag culture media, with better progressive in situ recoverability compared to standard chemically synthetic culture media. Combining various plant-based culture media and incubation conditions enabled us to resolve unique denaturing gradient gel electrophoresis (DGGE) bands that were not resolved by tested standard culture media. Based on polymerase chain reaction PCR-DGGE of 16S rDNA fingerprints and sequencing, the plant teabag culture media supported higher diversity and significant increases in the richness of endo-rhizobacteria, namely Gammaproteobacteria (Enterobacteriaceae) and predominantly Alphaproteobacteria (Rhizobiaceae). This culminated in greater retrieval of the rhizobacteria taxa associated with the plant roots. We conclude that the plant teabag culture medium by itself, without any nutritional supplements, is sufficient and efficient for recovering and mirroring the complex and diverse communities of rhizobacteria. Our message to fellow microbial ecologists is: simply dehydrate your plant canopy, teabag it and soak it to prepare your culture media, with no need for any additional supplementary nutrients.

Starch filler and osmoprotectants improve the survival of rhizobacteria in dried alginate beads.

This work deals with optimising the cell survival of rhizobacteria encapsulated in alginate beads filled with starch. Immobilisation of rhizobacteria was done by dripping alginate-starch solution mixed with rhizobacteria into a calcium solution. Beads were analysed based on matrix formulation, bacteria growth phase, osmoprotectants and nature of calcium solution. Maximum cell recovery was obtained on Raoultella terrigena grown in medium supplemented with trehalose and calcium gluconate as gelling agent. Furthermore, dried beads containing Azospirillum brasilense presented 76% of viable cells after one year of storage. The survival of rhizobacteria during the bioencapsulation process can be improved by incorporating starch on beads composition, varying the growth phase of cells and using trehalose in growth culture medium. This work provides a selection of appropriate methods to improve the surviving rate of encapsulated cells during their production and long-term storage (∼1 year at 4°C).

Use of potato extract broth for culturing root-nodule bacteria.

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.

Assess suitability of hydroaeroponic culture to establish tripartite symbiosis between different AMF species, beans, and rhizobia.

BACKGROUND: Like other species of the Phaseoleae tribe, common bean (Phaseolus vulgaris L.) has the potential to establish symbiosis with rhizobia and to fix the atmospheric dinitrogen (N2) for its N nutrition. Common bean has also the potential to establish symbiosis with arbuscular mycorrhizal fungi (AMF) that improves the uptake of low mobile nutrients such as phosphorus, from the soil. Both rhizobial and mycorrhizal symbioses can act synergistically in benefits on plant. RESULTS: The tripartite symbiosis of common bean with rhizobia and arbuscular mycorrhizal fungi (AMF) was assessed in hydroaeroponic culture with common bean (Phaseolus vulgaris L.), by comparing the effects of three fungi spp. on growth, nodulation and mycorrhization of the roots under sufficient versus deficient P supplies, after transfer from initial sand culture. Although Glomus intraradices Schenck & Smith colonized intensely the roots of common bean in both sand and hydroaeroponic cultures, Gigaspora rosea Nicolson & Schenck only established well under sand culture conditions, and no root-colonization was found with Acaulospora mellea Spain & Schenck under either culture conditions. Interestingly, mycorrhization by Glomus was also obtained by contact with mycorrhized Stylosanthes guianensis (Aubl.) sw in sand culture under deficient P before transfer into hydroaeroponic culture. The effect of bean genotype on both rhizobial and mycorrhizal symbioses with Glomus was subsequently assessed with the common bean recombinant inbreed line 7, 28, 83, 115 and 147, and the cultivar Flamingo. Significant differences among colonization and nodulation of the roots and growth among genotypes were found. CONCLUSION: The hydroaeroponic culture is a valuable tool for further scrutinizing the physiological interactions and nutrient partitioning within the tripartite symbiosis.

Plant performance, dioxygenase-expressing rhizosphere bacteria, and biodegradation of weathered hydrocarbons in contaminated soil.

Two greenhouse pot experiments were conducted to investigate the potential of 13 plant species (grasses, cruciferes, legumes, herbs) to thrive in a long-term contaminated soil from a former manufactured gas plant (MGP) site, to promote the proliferation of total and aromatic ring dioxygenase-expressing bacteria (ARDB) in the root zone, and to foster the biodegradation of petrol hydrocarbons (PHCs) and polycyclic aromatic hydrocarbons (PAHs). PHCs at 23200 mg kg(-1) and PAHs at 2194 mg kg(-1) reduced seed germination, plant survival, and shoot yields for most plants. Total bacteria and ARDB were generally more abundant in contaminated soil and were most numerous in the rhizosphere of mustard. During 68 d, the loss of total petrol hydrocarbons (TPHs) and total US EPA priority PAHs (TPAHs) was greatest in soil planted with hemp and mustard. Pea, cress, and pansy increased the amounts of PAHs extracted from soil, including an almost 60% increase for dibenzo(ah)anthracene. Plants may enhance the chemical extractability and perhaps biological availability of initially unextractable molecules.

Poly-beta-hydroxybutyrate turnover in Azorhizobium caulinodans is required for growth and affects nifA expression.

Azorhizobium caulinodans is able to fix nitrogen in the free-living state and in symbiosis with the tropical legume Sesbania rostrata. The bacteria accumulate poly-beta-hydroxybutyrate (PHB) under both conditions. The structural gene for PHB synthase, phbC, was inactivated by insertion of an interposon. The mutant strains obtained were devoid of PHB, impaired in their growth properties, totally devoid of nitrogenase activity ex planta (Nif-), and affected in nucleotide pools and induced Fix- nodules devoid of bacteria. The Nif- phenotype was the consequence of the lack of nifA transcription. Nitrogenase activity was partially restored to a phbC mutant by constitutive expression of the nifA gene. However, this constitutive nifA expression had no effect on the nucleotide content or on growth of the phbC mutant. It is suggested that PHB is required for maintaining the reducing power of the cell and therefore the bacterial growth. These observations also suggest a new control of nifA expression to adapt nitrogen fixation to the availability of carbon and reducing equivalents.

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.

Levels and identities of nonrhizobial microorganisms found in commercial legume inoculant made with nonsterile peat carrier.

Sixty samples of commercial North American legume inoculants manufactured for sale in 1994 using nonsterile peat as carrier were tested for Rhizobium (or Bradyrhizobium) content and non-Rhizobium biological contaminant load. Products of three major producers of such inoculants for sale in Canada were examined. Viable Rhizobium content varied from 5.6 x 10(5) to 8.1 x 10(9) cells/g, while the contaminant load varied from 1.8 x 10(8) to 5.5 x 10(10) cfu/g. Most of the inoculants contained more nonrhizobial organisms than they did rhizobia. Identifications were made of the most numerous nonrhizobial bacteria occurring in 100 samples of inoculants collected in 1993 and 1994. The most commonly identified contaminant was Xanthomonas maltophilia. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloacae were also found at high levels in some products. Contaminant organisms capable of inhibiting rhizobial growth in plate culture were found in the products of all three manufacturers.

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source.

When Azorhizobium caulinodans was grown in chemostat cultures with N2 as the N source at a constant dilution rate of 0.1 h-1 in media with a constant concentration (50 mM) of succinate and variable concentrations (1.5 to 585 microM) of nicotinate, neither the growth yield on succinate, the specific rate of O2 consumption, nor the specific rate of CO2 production showed linear regression with the concentration of nicotinate. Moreover, for transient continuous cultures in which the nicotinate concentration was gradually lowered, growth parameters remained unchanged until an apparently critical level of 0.7 microM nicotinate was reached. Below this nicotinate level, an immediate washout of the chemostat population began. A. caulinodans nicotinate hydroxylase-negative mutant 61007, unable to catabolize nicotinate, and the wild type behaved similarly. Thus, for continuous cultures supplied with N2 as the N source, submicromolar concentrations of nicotinate both sustained pyridine nucleotide biosynthesis at sufficient levels and precluded the use of nicotinate as a catabolic substrate. Furthermore, when more nicotinate was provided, dual succinate-nicotinate limitation in continuous cultures did not occur. Finally, when nicotinate is present in suboptimal concentrations, the specific growth rate is directly proportional to the amount of nicotinate present per unit of biomass. By contrast, in batch cultures with different nicotinate concentrations and with either succinate or lactate as the carbon and energy source, anomalous growth curves were obtained. With a low concentration (1.5 microM) of nicotinate, growth on N2 occurred, albeit at low rates. With a high concentration (195 microM) of nicotinate, growth on N2 was temporarily stimulated, but nicotinate was quickly exhausted and growth was thereafter nicotinate limited. Continuous supplementation of batch cultures with nicotinate allowed only transient exponential growth followed by linear growth. Thus, also for batch cultures, nicotinate catabolism is dispensable, although a high concentration of nicotinate temporarily stimulates growth on N2. Ut us concluded that A. caulinodans is a true diazotroph.

Fast-growing root nodule bacteria produce a novel polyamine, aminobutylhomospermidine.

Polyamines in various root nodule bacteria including Bradyrhizobium japonicum, Rhizobium fredii, R. leguminosarum, R. meliloti and R. loti were identified by capillary gas chromatography. Homospermidine was the polyamine present in highest concentration in all the rhizobia tested. In addition to putrescine and homospermidine, fast-growing type of rhizobial cells contained a novel polyamine, aminobutylhomospermidine, NH2(CH2)4NH(CH2)4NH(CH2)4NH2. The unusual tetraamine was not found in the cells of slow-growing type of rhizobia throughout their growth period, indicating a difference in polyamine metabolism between fast-growing type and slow-growing type of root nodule bacteria.

Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme.

We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.