Whole genome analysis of Gluconacetobacter azotocaptans DS1 and its beneficial effects on plant growth.

Plant-associated bacteria play an important role in the enhancement of plant growth and productivity. Gluconacetobacter azotocaptans is an exceptional bacterium considering that till today it has been isolated and reported only from Mexico and Canada. It is a plant growth-promoting bacterium and can be used as biofertilizer for different crops and vegetables. The objective of the current study was to evaluate the inoculation effect of Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, Azosprillium zeae N7, Azosprillium brasilense N8, and Azosprillium canadense DS2, on the growth of vegetables including cucumber, sweet pepper, radish, and tomato. All strains increased the vegetables' growth; however, G. azotocaptans DS1 showed better results as compared to other inoculated and control plants and significantly increased the plant biomass of all vegetables. Therefore, the whole genome sequence of G. azotocaptans DS1 was analyzed to predict genes involved in plant growth promotion, secondary metabolism, antibiotics resistance, and bioremediation of heavy metals. Results of genome analysis revealed that G. azotocaptans DS1 has a circular chromosome with a size of 4.3 Mbp and total 3898 protein-coding sequences. Based on functional analysis, genes for nitrogen fixation, phosphate solubilization, indole acetic acid, phenazine, siderophore production, antibiotic resistance, and bioremediation of heavy metals including copper, zinc, cobalt, and cadmium were identified. Collectively, our findings indicated that G. azotocaptans DS1 can be used as a biofertilizer and biocontrol agent for growth enhancement of different crops and vegetables. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02996-1.

Effect of Chemical Fertilization on the Impacts of Plant Growth-Promoting Rhizobacteria in Maize Crops.

The use of chemical fertilizers strongly promotes productivity in agricultural crops; therefore, large amounts of chemical fertilizers have been used. The use of plant growth-promoting bacteria may be a strategy to reduce the use of chemical fertilizers; however, little is known about the effect of chemical fertilization on the performance of these bacteria through plant-microbe interactions. The present study aimed to verify the performance of Bacillus subtilis, Azospirillum brasilense, B. pumilus, B. amyloliquefaciens, Herbaspirillum seropedicae, Gluconacetobacter diazotrophicus, and the mixtures A. brasilense + B. subtilis, B. pumilus + B. amyloliquefaciens, and H. seropedicae + G. diazotrophicus on parameters such as nitrogen and phosphorus extraction from soil, the concentrations of these nutrients in maize plants, and plant growth in both fertilized and unfertilized soil. The results showed that H. seropedica increased the nitrogen content by 6.6 g kg-1 in leaves and 2.2 g kg-1 in the root when comparing the unfertilized with the fertilized condition. G. diazotrophicus increased the nitrogen content by 3.7 g kg-1 in leaves and 2.4 g kg-1 in the root. B. pumilus increased the phosphorous content by 1.7 g kg-1 in leaves, and B. amyloliquefaciens increased the phosphorous content by 0.61 g kg-1. The present study showed that even though the bacteria presented good performance related to plant growth under fertilized conditions, H. seropedicae, G. diazotrophicus, B. pumilus, and B. amyloliquefaciens could be used in the maize crop with a reduced chemical fertilization dose.

Corn microbial diversity and its relationship to yield.

This study aimed to identify possible relationships between corn (Zea mays L.) productivity and its endosphere microbial community. Any insights would be used to develop testable hypotheses at the farm level. Sap was collected from 14 fields in 2014 and 10 fields in 2017, with a yield range of 10.1 to 21.7 tonnes per hectare (t/ha). The microbial sap communities were analyzed using terminal restriction fragment length polymorphism (TRFLP) and identified using an internal pure culture reference database and BLAST. This technique is rapid and inexpensive and is suitable for use at the grower level. Diversity, richness, and normalized abundances of each bacterial population in corn sap samples were evaluated to link the microbiome of a specific field to its yield. A negative trend was observed (r = -0.60), with higher-yielding fields having lower terminal restriction fragment (TRF) richness. A partial least square regression analysis of TRF intensity and binary data from 2014 identified 10 TRFs (bacterial genera) that positively, or negatively, correlated with corn yields, when either absent or present at certain levels or ratios. Using these observations, a model was developed that accommodated criteria for each of the 10 microbes and assigned a score for each field out of 10. Data collected in 2014 showed that sites with higher model scores were highly correlated with larger yields (r = 0.83). This correlation was also seen when the 2017 data set was used (r = 0.87). We were able to conclude that a positive significant effect was seen with the model score and yield (adjusted R2 = 0.67, F[1,22] = 46.7, p < 0.001) when combining 2014 and 2017 data. The results of this study are being expanded to identify the key microbes in the corn sap community that potentially impact corn yield, regardless of corn variety, geographic factors, or edaphic factors.

Functional characterization of potential PGPR exhibiting broad-spectrum antifungal activity.

This study describes the biocontrol potential of rhizobacteria against a range of fungal phytopathogens. Out of 227 bacteria isolated from the rhizosphere of maize, rice, wheat, potato, sunflower and soybean crops cultivated in different agro-ecological regions of Pakistan, 48 exhibited >60 % antifungal activity against Fusarium oxysporum, Fusarium moniliforme, Rhizoctonia solani, Colletotrichum gloeosporioides, Colletotrichum falcatum, Aspergillus niger, and Aspergillus flavus. The rhizobacteria inhibiting >65 % pathogen growth were selected for detailed molecular and in planta studies most of which were identified as Pseudomonas and Bacillus species based on 16S rRNA gene sequence analysis. Antifungal metabolites produced by these rhizobacteria analyzed through LCMS were identified as antibiotics (iturin, surfactins, fengycin, DAPG, Phenazine, etc.), cell wall degrading enzymes (protease, chitinase, and cellulase), plant growth promotion enzymes and hormones (indole-3-acetic acid, ACC-deaminase, phosphates, nitrogen fixation), N-acyl-homoserine lactones and siderophores. The growth room experiment validated the potential of these bacteria as biofertilizer and biopesticide agents. Of all, P. aeruginosa strain FB2 and B. subtilis strain RMB5 showed significantly higher potential as antagonistic plant-beneficial bacteria effective against a range of fungal phytopathogens. Both these bacteria can be used to develop a dual-purpose bacterial inoculum as biopesticide and biofertilizer. Rest of the antagonistic PGPR may be exploited for disease control in less-infested soils.

Introducing key microbes from high productive soil transforms native soil microbial community of low productive soil.

This study aimed to understand the changes in rhizosphere microbial structure and diversity of an average corn yielding field site soil with the introduced microbial candidates from a high-yielding site. Soils used in this study were from two growers' fields located in Dunnville, Ontario, Canada, where one of the farms has an exceptional high corn yield (G-site soil; ca 20 tons/acre) and the other yields an average crop (H-site soil; 12 tons/acre) (8 years of unpublished A & L data). In growth room experiments using wheat as the indicator crop, calcium alginate beads with microbes composed of Azospirillum lipoferum, Rhizobium leguminosarum, Burkholderia ambifaria, Burkholderia graminis, Burkholderia vietnamiensis, Pseudomonas lurida, Exiguobacterium acetylicum, Kosakonia cowanii, and Paenibacillus polymyxa was introduced into the soil at planting to the average-yielding soil. These bacteria had been isolated from the high-yielding farm soil. Among the nine microbial candidates tested, three (P. polymyxa, E. acetylicum and K. cowanii) significantly impacted the plant health and biometrics in addition to microbial richness and diversity, where the microbial profile became very similar to the high productive G-site soil. One hundred and forty-two bacterial terminal restriction fragments (TRFs) were involved in the community shift and 48 of them showed significant correlation to several interacting soil factors. This study indicates the potential of shifting microbial profiles of average-yielding soils by introducing key candidates from highly productive soils to increase biological soil health.

Genetic, physiological and biochemical characterization of Bacillus sp. strain RMB7 exhibiting plant growth promoting and broad spectrum antifungal activities.

BACKGROUND: Plant growth promoting rhizobacteria (PGPR) are functionally diverse group of bacteria having immense potential as biofertilizers and biopesticides. Depending upon their function, they may serve as partial replacements for chemical fertilizer or pesticides as an eco-friendly and cost-effective alternatives as compared to their synthetic counterparts. Therefore, isolation, characterization and practical evaluation of PGPRs having the aforementioned multifaceted beneficial characteristics, are essentially required. This study describes the detailed polyphasic characterization of Bacillus sp. strain RMB7 having profound broad spectrum antifungal activity and plant growth promoting potential. RESULTS: Based on 16S rRNA gene sequencing, strain RMB7 was identified as Bacillus specie. This strain exhibited the production of 8 mg. L(-1)of indole-3-acetic acid (IAA) in tryptophan-supplemented medium. It was able to solubilize 50.6 mg. L(-1) tri-calcium phosphate, reduced 601ηmol acetylene h(-1)/vial and inhibited >70% growth of nine fungal phytopathogens tested in vitro. Under natural pathogen pressure, inoculation with strain RMB7 and RMB7-supernatant conferred resistance by arugula plant against Pythium irregulare with a concurrent growth improvement over non-inoculated plants. The T-RFLP analysis based on 16S rRNA gene showed that inoculation with RMB7 or its supernatant have a major impact on the indigenous rhizosphere bacterial population. Mass spectrometric analysis revealed the production of lipopeptide surfactins as well as iturin A presence in crude extract of RMB7. PCR-amplification further confirmed the presence of genes involved in the biosynthesis of these two bioactive lipopeptide compounds. CONCLUSIONS: The data show that Bacillus sp. strain RMB7 has multifaceted beneficial characteristics. It may be an ideal plant growth promoting as well as biocontrol agent, for its integrated use in disease and nutrient management strategies.

Impact of swapping soils on the endophytic bacterial communities of pre-domesticated, ancient and modern maize.

BACKGROUND: Endophytes are microbes that live within plants such as maize (corn, Zea mays L.) without causing disease. It is generally assumed that most endophytes originate from soil. If this is true, then as humans collected, domesticated, bred and migrated maize globally from its native Mexico, they moved the species away from its native population of endophyte donors. The migration of maize persists today, as breeders collect wild and exotic seed (as sources of diverse alleles) from sites of high genetic diversity in Mexico for breeding programs on distant soils. When transported to new lands, it is unclear whether maize permits only selective colonization of microbes from the Mexican soils on which it co-evolved, tolerates shifts in soil-derived endophytes, or prevents colonization of soil-based microbes in favour of seed-transmitted microbes. To test these hypotheses, non-sterilized seeds of three types of maize (pre-domesticated-Mexican, ancient-Mexican, modern-temperate) were planted side-by-side on indigenous Mexican soil, Canadian temperate soil or sterilized sand. The impact of these soil swaps on founder bacterial endophyte communities was tested using 16S-rDNA profiling, culturing and microbial trait phenotyping. RESULTS: Multivariate analysis showed that bacterial 16S-rDNA TRFLP profiles from young, surface-sterilized maize plants were more similar when the same host genotype was grown on the different soils than when different maize genotypes were grown on the same soil. There appeared to be two reasons for this result. First, the largest fraction of bacterial 16S-signals from soil-grown plants was shared with parental seeds and/or plants grown on sterilized sand, suggesting significant inheritance of candidate endophytes. The in vitro activities of soil-derived candidate endophytes could be provided by bacteria that were isolated from sterile sand grown plants. Second, many non-inherited 16S-signals from sibling plants grown on geographically-distant soils were shared with one another, suggesting maize can select microbes with similar TRFLP peak sizes from diverse soils. Wild, pre-domesticated maize did not possess more unique 16S-signals when grown on its native Mexican soil than on Canadian soil, pointing against long-term co-evolutionary selection. The modern hybrid did not reject more soil-derived 16S-signals than did ancestral maize, pointing against such rejection as a mechanism that contributes to yield stability across environments. A minor fraction of 16S-signals was uniquely associated with any one soil. CONCLUSION: Within the limits of TRFLP profiling, the candidate bacterial endophyte populations of pre-domesticated, ancient and modern maize are partially buffered against the effects of geographic migration --- from a Mexican soil associated with ancestral maize, to a Canadian soil associated with modern hybrid agriculture. These results have implications for understanding the effects of domestication, migration, ex situ seed conservation and modern breeding, on the microbiome of one of the world's most important food crops.

Involvement of hexokinase1 in plant growth promotion as mediated by Burkholderia phytofirmans.

Potato plantlets inoculated with strain PsJN of the bacterium Burkholderia phytofirmans exhibit consistent and significant increases in plant growth under in vitro conditions, when compared with uninoculated plants. The greatest influence on the degree and type of growth enhancement that develops has been shown to be mediated by the sugar concentration in the agar media. Bacterial growth promotion has been suggested in other studies to be regulated by the sugar sensor enzyme hexokinase1, the role of which is activation of glucose phosphorylation. In this present study, we examined the co-relationship between root and stem development in potato plants treated with PsJN and the activity of hexokinase1. Plants grown in the presence of 1.5% and 3% sucrose showed increased levels of hexokinase1 activity only in the roots of inoculated plants, suggesting that the increased enzyme levels may be associated with root growth. Analysis for mRNA using reverse transcriptase did not reveal any significant differences in transcription levels of the gene between inoculated and uninoculated plants. When PsJN-inoculated plants were grown in 1.5% and 3% concentrations of glucose and fructose, stem height and mass, leaf number, root mass, and overall biomass increased. No growth promotion occurred when PsJN-inoculated plants were grown in 3% maltose. Subsequently, a hexokinase1 activity assay showed that PsJN-induced growth of potato plants was found to only occur when plants were grown in the presence of sugars that are recognized by the plant hexokinase1. The results suggest that PsJN may enhance sugar uptake in plants by direct or indirect stimulation of hexokinase1 activity in roots and this results in enhanced overall plant growth.

Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in pakistan.

Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to 41degrees C and at pH 11.

A Nylon Membrane Bag Assay for Determination of the Effect of Chemicals on Soilborne Plant Pathogens in Soil.

A new nylon membrane bag (NMB) assay was developed for studies to determine the effect of chemicals added to soil on survival of soilborne plant pathogens. The rapid and effective assay can be used to study organisms for which there are no selective media or for which a selective medium is expensive or difficult to prepare. This assay consists of placing pathogens inside a bag made of small-pore (0.22-µm) nylon filtration membrane, which is placed in soil and later retrieved to determine survival of the pathogens on nonselective media. Chemicals but not other microorganisms can enter the bag from the soil. Using this assay, Streptomyces scabies, Fusarium oxysporum f. sp. lycopersici race 3, and Ralstonia solanacearum were successfully recovered from soil after 72 h as demonstrated by growth on a semiselective Streptomyces medium (S. scabies) or nonselective potato dextrose agar medium (F. oxysporum f. sp. lycopersici race 3 and R. solanacearum) with minimal microbial contamination. Addition of acetic acid (200 mM) to soil killed 100% of S. scabies. SPK (a mixture of organic chemicals) at a concentration of 1,500 mg kg-1 of soil killed 83.3% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 100% of F. oxysporum f. sp. lycopersici race 3 spores, and 97.2% of R. solanacearum cells. SPK at 1,000 mg kg-1 of soil killed 50% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 68.2% of F. oxysporum f. sp. lycopersici race 3 spores, and 12% of R. solanacearum. Benlate (500 to 1,500 mg kg-1 of soil) did not kill the culture plugs of F. oxysporum f. sp. lycopersici race 3 but reduced the growth rate of F. oxysporum f. sp. lycopersici race 3. Benlate (500, 1,000, and 1,500 mg kg-1 of soil) reduced F. oxysporum f. sp. lycopersici race 3 spore germination by 39.4, 49.3, and 50.4%, respectively. Streptomycin sulfate (1,500, 800, 400, and 200 mg kg-1 of soil) caused 75.3, 21, 11.9, and 0.9% mortality, respectively, of R. solanacearum.

Isolation, characterization, and effect of fluorescent pseudomonads on micropropagated sugarcane.

In this study, we report on the isolation, identification, and characterization of seven fluorescent pseudomonads isolated from the roots, shoots, and rhizosphere soil of sugarcane and their impacts on the growth of sugarcane plantlets. 16S rRNA gene sequence of five isolates showed close homology with Pseudomonas putida, one with Pseudomonas graminis, and one with Pseudomonas fluorescens. Physiological and biochemical characterizations were determined using API50CH and QTS24 identification kits. The isolates were also subjected to tests for various known growth promoting properties including production of indole acetic acid, the ability to fix nitrogen via the presence of the nifH gene, and ability to solubilize phosphate. Biological control potential was determined from agar diffusion assays of HCN production and production of antifungal compounds against local isolates of Colletotrichum falcatum (that induces red-rot disease of sugarcane). Direct plant growth promoting effects were tested on sugarcane plantlets in tissue culture under gnotobiotic conditions. All seven isolates provided significant increases in fresh and dry masses but only five strains increased shoot height.

Detection of high concentrations of organic acids in fish emulsion and their role in pathogen or disease suppression.

Fish emulsion (FE) added to a sandy-loam soil at 1 and 2% rates reduced the viability of Verticillium dahliae microsclerotia by 39 and 74% in 1 day, 87 and 98% in 3 days, and 95 and 99% in 6 days, respectively. The immediate kill of microsclerotia indicated that FE contains toxic substances. We found in FE high concentrations (400 mmol/liter) of organic acids, including some known toxicants. Glycolic, acetic, formic, n-butyric, and propionic acids were the major organic acids detected in FE at the proportions of 52.5, 26.9, 7.9, 7.2, and 4.7%, respectively. In solution assays, the viability of V. dahliae microsclerotia treated for 24 h in 1, 2, 5, and 10% FE (pH 3.6 to 3.0) or a mixture of organic acids (pH 4.1 to 3.9) equivalent to the proportions in FE was reduced by 74, 94, 97, and 99% or 81, 91, 98, and 99%, respectively. The viability of microsclerotia was increased when the treatment solutions were buffered to pH 6.0. The organic acids mixtures and formic (0.025%) and acetic (0.1%) acids were toxic to Pythium ultimum. A mixture of organic acids (1, 2, and 4%) provided immediate protection of cucumber seedlings from damping-off in P. ultimum-infested muck and sandy-loam soils but not in peat-based mix. FE (1 and 2%) provided immediate protection of cucumber seedlings from damping-off in an infested muck soil, and disease protection was consistent when planting was delayed for 7, 14, and 28 days after adding FE. FE (1, 2, and 4%) did not provide immediate protection of cucumber seedlings from damping-off in a P. ultimum-infested peat-based mix; however, disease suppression was evident when planting was delayed for 7, 14, and 21 days after adding FE. Real-time polymerase chain reaction analyses of the peat-based mix indicated that the P. ultimum populations in the FE-amended mix declined over time. This study suggests that these organic acids in FE played a major role in pathogen or disease suppression, depending on the soil and substrate.

Characterization of a phenazine and hexanoyl homoserine lactone producing Pseudomonas aurantiaca strain PB-St2, isolated from sugarcane stem.

A novel strain of fluorescent pseudomonad (PB-St2) was isolated from surface-sterilized stems of sugarcane grown in Pakistan. The bacterium was identified as Pseudomonas aurantiaca on the basis of 16S rRNA gene sequence analysis and results from physiological and biochemical characteristics carried out with API50 CH and QTS 24 bacterial identification kits. Assays using substrate specific media for enzymes revealed lipase and protease activities but cellulase, chitinase, or pectinase were not detected. The bacterium was unable to solubilize phosphate or produce indole acetic acid. However, it did produce HCN, siderophores, and homoserine lactones. In dual culture assays on agar, the bacterium showed antifungal activity against an important pathogen of sugarcane in Pakistan, namely Colletotrichum falcatum, as well as for pathogenic isolates of Fusarium oxysporium, F. lateritium but not against F. solani. The antifungal metabolites were identified using thin-layer chromatography, UV-spectra, and MALDI-TOFF spectra and shown to be phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine (2-OH-PHZ), and N-hexanoyl homoserine lactone (HHL) (assessed using only TLC data). The capacity of this bacterium to produce HCN and 2-OH-PHZ, as well as to inhibit the growth of C. falcatum, has not been previously reported.

Azospirillum zeae sp. nov., a diazotrophic bacterium isolated from rhizosphere soil of Zea mays.

Two free-living nitrogen-fixing bacterial strains, N6 and N7(T), were isolated from corn rhizosphere. A polyphasic taxonomic approach, including morphological characterization, Biolog analysis, DNA-DNA hybridization, and 16S rRNA, cpn60 and nifH gene sequence analysis, was taken to analyse the two strains. 16S rRNA gene sequence analysis indicated that strains N6 and N7(T) both belonged to the genus Azospirillum and were closely related to Azospirillum oryzae (98.7 and 98.8 % similarity, respectively) and Azospirillum lipoferum (97.5 and 97.6 % similarity, respectively). DNA-DNA hybridization of strains N6 and N7(T) showed reassociation values of 48 and 37 %, respectively, with A. oryzae and 43 % with A. lipoferum. Sequences of the nifH and cpn60 genes of both strains showed 99 and approximately 95 % similarity, respectively, with those of A. oryzae. Chemotaxonomic characteristics (Q-10 as quinone system, 18 : 1omega7c as major fatty acid) and G+C content of the DNA (67.6 mol%) were also similar to those of members of the genus Azospirillum. Gene sequences and Biolog and fatty acid analysis showed that strains N6 and N7(T) differed from the closely related species A. lipoferum and A. oryzae. On the basis of these results, it is proposed that these nitrogen-fixing strains represent a novel species. The name Azospirillum zeae sp. nov. is suggested, with N7(T) (=NCCB 100147(T)=LMG 23989(T)) as the type strain.

Sphingobacterium canadense sp. nov., an isolate from corn roots.

A free-living Gram-negative bacterial strain CR11(T) was isolated from corn roots. Polyphasic taxonomy was performed, including API20 NE and API50 CH bacterial identification kits, Biolog analysis, lipids and fatty acid analysis, DNA-DNA hybridization, 16S rRNA and cpn60 gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain CR11(T) belonged to the genus Sphingobacterium and was closely related to Sphingobacterium multivorum IFO 14947(T) (98% similarity) and Sphingobacterium. thalpophilum ATCC 43320(T) (97% similarity). DNA-DNA hybridization showed 11% and 13% DNA re-association with S. multivorum LMG 8342(T) and S. thalpophilum LMG 11520(T), respectively. Major fatty acids (16:0, 15:0 iso and 17:0 iso 3-OH) and the G+C content of the DNA (40.5 mol%), were also similar to those of the genus Sphingobacterium. The predominant respiratory quinone was MK-7. In all analyses, including phenotypic characterization, this isolate was found to be different from the closely related species, S. multivorum and S. thalpophilum. On the basis of these results, this strain represents a new species within the genus Sphingobacterium. The name Sphingobacterium canadense sp. nov. is suggested and the type strain is CR11(T) (=NCCB 100125(T)=LMG 23727(T)).

Monitoring in planta bacterial infection at both cellular and whole-plant levels using the green fluorescent protein variant GFPuv.

* Green fluorescent protein (GFP) labeling of bacteria has been used to study their infection of and localization in plants, but strong autofluorescence from leaves and the relatively weak green fluorescence of GFP-labeled bacteria restrict its broader application to investigations of plant-bacterial interactions. * A stable and broad-host-range plasmid vector (pDSK-GFPuv) that strongly expresses GFPuv protein was constructed not only for in vivo monitoring of bacterial infection, localization, activity, and movement at the cellular level under fluorescence microscopy, but also for monitoring bacterial disease development at the whole-plant level under long-wavelength ultraviolet (UV) light. * The presence of pDSK-GFPuv did not have significant impact on the in vitro or in planta growth and virulence of phytobacteria. A good correlation between bacterial cell number and fluorescence intensity was observed, which allowed us to rapidly estimate the bacterial population in plant leaf tissue. We demonstrated that GFPuv-expressing bacteria can be used to screen plants that are compromised for nonhost disease resistance and Agrobacterium attachment. * The use of GFPuv-labeled bacteria has a wide range of applications in host-bacterial interaction studies and bacterial ecology-related research.

Azospirillum canadense sp. nov., a nitrogen-fixing bacterium isolated from corn rhizosphere.

A free-living diazotrophic strain, DS2(T), was isolated from corn rhizosphere. Polyphasic taxonomy was performed including morphological characterization, Biolog analysis, and 16S rRNA, cpn60 and nifH gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain DS2(T) was closely related to the genus Azospirillum (96 % similarity). Chemotaxonomic characteristics (DNA G+C content 67.9 mol%; Q-10 quinone system; major fatty acid 18 : 1omega7c) were also similar to those of the genus Azospirillum. In all the analyses, including phenotypic characterization using Biolog analysis and comparison of cellular fatty acids, this isolate was found to be different from the closely related species Azospirillum lipoferum, Azospirillum oryzae and Azospirillum brasilense. On the basis of these results, a novel species is proposed for this nitrogen-fixing strain. The name Azospirillum canadense sp. nov. is suggested with the type strain DS2(T) (=NCCB 100108(T)=LMG 23617(T)).

Factors influencing the concentration of volatile fatty acids, ammonia, and other nutrients in stored liquid pig manure.

In order to minimize odor and manage nutrients in liquid pig manure we need to be able to predict what operational practices most influence the concentrations of volatile fatty acids (VFAs), ammonium nitrogen (NH(4)(+)-N), and other nutrients present in the manure. To determine this, we collected manure from 15 pig operations in southwestern Ontario in the fall of 2001 and 2002 and spring of 2002 and 2003. The manure was stored in concrete tanks at all operations. Manure from finishing pigs had the highest concentration of VFAs, NH(4)(+)-N, and other nutrients, followed by manure from mixed operations, and then manure from sow operations. The average concentration of total VFAs and NH(4)(+)-N in finishing pig manure was 166 mM compared with 36 and 99 mM, respectively, in sow manure. Total N, P, and K were 2.3, 2.5, and 1.7 times greater, respectively, in finishing pig compared with sow manure. There was no seasonal or year to year variation in amount. The diet of the pigs, use of feed additives or antibiotics, location of tanks, and whether the tanks were covered or mixed were not significant factors contributing to the difference in manure chemistry. The main reason for the differences between the three types of manure was manure dilution. The average dry matter content of finishing pig manure was 4.5 times that of sow manure. This was due to larger density of pigs in finishing compared with sow operations, less manure storage capacity per pig for finishing compared with sow operations, and more wash water being used for sow operations.

Edaphic Soil Levels of Mineral Nutrients, pH, Organic Matter, and Cationic Exchange Capacity in the Geocaulosphere Associated with Potato Common Scab.

ABSTRACT In order to determine possible relationships between geocaulosphere soil properties and severity of common scab of potato caused by Streptomyces scabies, soils were collected from representative commercial potato fields in Canada: in Simcoe and Dufferin Counties, Ontario and across Prince Edward Island (PEI) in August 2004. Soils immediately adjacent to tubers were sampled and analyzed for select edaphic factors and for pathogen presence using polymerase chain reaction (PCR) tests with primers that amplify a region of the TxtA gene involved in regulating the biosynthesis of the thaxtomin toxin family. Individual tubers were assessed visually for scab severity. The relationships between soil chemical factors and disease severity were investigated for each region to detect the strongest relationships. Principal component analysis revealed a distinctive clustering of samples with respect to disease severity in PEI but not in Ontario soils. Total and percent saturation of K (%K) were the only factors found associated with high disease severity in soils from both provinces. In PEI soils, pH, Mg, Ca, Cu, and %K, %Mg, %Ca, and %Na were associated with high disease severity, whereas cation exchange capacity (CEC) and Al were correlated with low disease severity soils. In Ontario, high Mn content was strongly correlated with low disease severity soils, whereas %K and organic matter content were correlated with disease severity. Partitioning samples into presence or absence of the TxtA PCR product with corresponding high or low severity showed further significant relationships in the data. There was an excellent correlation between Streptomyces spp. presence as detected by PCR and disease severity in PEI soils; however, the relationship was not as clear in Ontario soils, where many PCR-positive soils had low disease incidence. Principal component and partial least square analysis indicated that disease severity was predicted by soil factors such as organic matter, CEC, pH, Al, %Ca, %Mg, and %K for PEI but not for Ontario soils. The data reveal that the relationship between scab severity and soil chemical components is complex and potentially soil specific.

Inoculation effects of Pseudomonas putida, Gluconacetobacter azotocaptans, and Azospirillum lipoferum on corn plant growth under greenhouse conditions.

Alcohol production from corn is gaining importance in Ontario, Canada, and elsewhere. A major cost of corn production is the cost of chemical fertilizers and these continue to increase in price. The competitiveness of alcohol with fossil fuels depends on access to low-cost corn that allows growers to earn a sustainable income. In this study we set out to determine if we can identify root-associated microorganisms from Ontario-grown corn that can enhance the nutrient flow to corn roots, directly or indirectly, and help minimize the use of extraneous fertilizer. Bacteria were isolated from corn rhizosphere and screened for their capacity to enhance corn growth. The bacteria were examined for their ability to fix nitrogen, solubilize phosphate, and produce indole acetic acid (IAA) and antifungal substances on potato dextrose agar. Bacterial suspensions were applied to pregerminated seed of four corn varieties (39D82, 39H84, 39M27, and 39T68) planted in sterilized sand and unsterilized cornfield soil. The plants were grown under greenhouse conditions for 30 days. Three isolates were identified as having growth-promoting effect. These bacteria were identified as to species by biochemical tests, fatty acid profiles, and 16S rDNA sequence analysis. Corn rhizosphere isolates, Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, and Azospirillum lipoferum N7, provided significant plant growth promotion expressed as increased root/shoot weight when compared to uninoculated plants, in sand and/or soil. All strains except P. putida CQ179 were capable of nitrogen fixation and IAA production. Azospirillum brasilense, however, produced significantly more IAA than the other isolates. Although several of the strains were also able to solubilize phosphate and produce metabolites inhibitory to various fungal pathogens, these properties are not considered as contributing to growth promotion under the conditions used in this study. These bacteria will undergo field tests for their effect on corn growth.