<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry11Ba works synergistically with Cry4Aa but not with Cry11Aa for toxicity against mosquito <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera: Culicidae) larvae

A 2,175-bp modified gene (cry11Ba-S1) encoding Cry11Ba from Bacillus thuringiensis subsp. jegathesan was designed and the recombinant protein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant Cry11Ba was highly toxic against Culex pipiens mosquito larvae, being nine and 17 times more toxic than mosquitocidal Cry4Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis, respectively. Interestingly, a further increase in the toxicity of the recombinant Cry11Ba was achieved by mixing with Cry4Aa, but not with Cry11Aa. These findings suggested that Cry11Ba worked synergistically with Cry4Aa, but not with Cry11Aa, in exhibiting toxicity against C. pipiens larvae. On the other hand, the amount of Cry toxin bound to brush border membrane vesicles (BBMVs) did not significantly change between individual toxins and the toxin mixtures, suggesting that the increase in toxins binding to BBMVs was not a reason for the observed synergistic effect. It is generally accepted that synergism of toxins is a potentially powerful tool for enhancing insecticidal activity and managing Cry toxin resistance in mosquitoes. The mixture of Cry4Aa and Cry11Ba in order to increase toxicity would be very valuable in terms of mosquito control.     

Interaction between toxin crystals and vegetative insecticidal proteins of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in lepidopteran larvae

Insecticides based on crystalline toxins of Bacillus thuringiensis are very good biological plant protection products. However, the spectrum of activity of some toxins is narrow or resistance among insects has been developed. We tested the insecticidal activity of crystals of the B. thuringiensis MPU B9 strain alone and supplemented with Vip3Aa proteins against important pests: Spodoptera exigua Hübner (Lepidoptera: Noctuidae), Cydia pomonella L. (Lepidoptera: Tortricidae) and Dendrolimus pini L. (Lepidoptera: Lasiocampidae). The Cry toxins were more active for D. pini but less active against S. exigua and C. pomonella than Vip3Aa. Supplementation of Cry toxins by small amounts of vegetative insecticidal proteins demonstrated synergistic effect and significantly enhanced the toxicity of the insecticide. The results indicate the utility of Cry and Vip3Aa toxins mixtures to control populations of crops and forests insect pests.     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.     

Trypsinized Cry1Fa and Vip3Aa have no detrimental effects on the adult green lacewing <Emphasis Type="Italic">Chrysopa pallens</Emphasis> (Neuroptera: Chrysopidae)

An exposure bioassay was established for green lacewing Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae) adults using a suitable artificial diet and honey–water solution (20%) to assess the toxicity of trypsinized Cry1Fa and Vip3Aa. Lethal and sub-lethal life table parameters were unaffected after C. pallens adults were given honey–water solutions containing Cry1Fa and Vip3Aa (50 µg/ml) and an artificial diet. In contrast, life table parameters of C. pallens adults were significantly affected when boric acid was mixed with the honey–water solution as a positive control. The uptake, temporal stability and bioactivity of Cry1Fa and Vip3Aa before and after C. pallens access to honey–water were confirmed using double antibody sandwich enzyme-linked immunosorbent assays and a bioactivity verification bioassay. The results prove that trypsinized Cry1Fa and Vip3Aa are safe for C. pallens adults, thus it is speculated that transgenic crops expressing Cry1Fa and Vip3Aa have no detrimental effects on lacewings and are compatible with biological control programs. This study describes a robust experimental design for evaluating the potential toxicity of alkaline gut-activated Bacillus thuringiensis (Bt) proteins on C. pallens adults which can be used to determine the potential toxicity of other Bt proteins on this species.     

Enhanced biosynthesis of phenazine-1-carboxamide by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> strains using statistical experimental designs

Phenazine-1-carboxamide (PCN) is one of the major biocontrol agents produced by plant growth-promoting rhizosphere (PGPR) pseudomonads including Pseudomonas chlororaphis. In this study, a combined strategy of genetic modification and statistical experimental designs was applied to obtain mutants of P. chlororaphis strains with high-yield PCN production. To achieve this, the lon gene was knocked out in wild-type P. chlororaphis HT66 and the breeding mutant P3 strain with a non-scar deletion strategy. The resulting HT66Δlon and P3Δlon mutants produced a significantly higher PCN production in shake-flask cultures which was 5- and  9-folds greater than their native counterparts. The potential ability of strain P3Δlon for PCN production was further optimized by statistical designs. A two-level Plackett–Burman (PB) experimental design with six variables was employed to scrutinize medium components that significantly influence PCN production. Notably, glycerol, tryptone, and soy peptone were identified to be the most significant factors (p?<?0.05). Response surface methodology (RSM) based on the central composite design (CCD) was adopted to determine these factors optimal levels and their interactive effects between culture components for PCN production. The predicted maximum PCN production was 9002 mg/L, whereas an actual PCN production of 9174 mg/L was recorded in the validation experiments using the optimal medium containing glycerol 37.08 mL/L, tryptone 20.00 g/L, and soy peptone 25.03 g/L, which was nearly threefolds higher than without optimization and 20-folds higher than the wild-type strain. In conclusion, the results revealed that P. chlororaphis display a high potential for industrial-scale production for phenazine biopesticides.     

Suggested interrelationships of RNA-polymerase sigma S subunit and nitrogen control system in <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis>

The effect of mutation in rpoS gene encoding sigma S subunit of RNA-polymerase on the capacity of Pseudomonas chlororaphis 449 to assimilate nitrogen was investigated. It has been shown that mutant cells with knocked-out rpoS gene had significantly lower capacity to utilize the nitrogen sources such as alanine, proline, histidine, arginine, urea, and ammonium and glutamine synthetase was downregulated in their cell free extracts. Both defects were abolished by glutamine supplementation to the medium. It is suggested that in Pseudomonas chlororaphis the association of the nitrogen control system and the system of gene expression is regulated by RNA-polymerase sigma S subunit, which can be responsible for cell adaptation at nitrogen supply limitation.     

Quorum sensing systems of regulation,synthesis of phenazine antibiotics,and antifungal activity in rhizospheric bacterium <Emphasis Type="Italic">pseudomonas chlororaphis</Emphasis> 449

Strain Pseudomonas chlororaphis 449, an antagonist of a broad spectrum of phytopathogenic microorganisms isolated from the maize rhizosphere, was shown to produce three phenazine antibiotics: phenazine-1-carboxylic acid (PCA), 2-hydroxylphenazine-1-carboxylic acid (2-OH-PCA), and 2-hydroxylphenazine (2-OH-PHZ). Two Quorum Sensing (QS) systems of regulation were identified: Phz/R and CsaI/R. Genes phzI and csaI were cloned and sequenced. Cells of strain 449 synthesize at least three types of AHL: N-butanoyl-L-homoserine lactone (C₄-AHL), N-hexanoyl-L-homoserine lactone (C₆-AHL), and N-(3-oxo-hexanoyl)-L-homoserine lactone (30C₆-AHL). Transposon mutagenesis was used to generate mutants of strain 449 deficient in synthesis of phenazines, which carried inactivated phzA and phzB genes of the phenazine operon and gene phzO. Mutations phzA ^? and phzB ^? caused a drastic reduction in the antagonistic activity of bacteria toward phytopathogenic fungi. Both mutants lost the ability to protect cucumber and leguminous plants against phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. These results suggest a significant role of phenazines in the antagonistic activity of P. chlororaphis 449.     

Bt Proteins Have No Detrimental Effects on Larvae of the Green Lacewing, <Emphasis Type="Italic">Chrysopa pallens</Emphasis> (Rambur) (Neuroptera: Chrysopidae)

Biosafety of a genetically modified crop is required to be assessed prior to its commercialization. For this, a suitable artificial diet was developed and used to establish a dietary exposure test for assessing the toxicity of midgut-active Bt insecticidal proteins on Chrysopa pallens (Rambur). Subsequently, this dietary exposure test was used to evaluate the toxicity of the proteins Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa on C. pallens larvae. Temporal stability, bioactivity, and the intake of the insecticidal proteins were confirmed by enzyme-linked immunosorbent assay and a sensitive-insect bioassay. The life history characteristics, such as survival, pupation, adult emergence, 7-day larval weight, larval developmental time, and emerged male and female fresh weights remained unaffected, when C. pallens were fed the pure artificial diet (negative control) and the artificial diets containing 200 μg/g of each purified protein: Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, or Vip3Aa. On the contrary, all of the life history characteristics of C. pallens larvae were adversely affected when fed artificial diet containing boric acid (positive control). The results demonstrate that diets containing the tested concentrations of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, and Vip3Aa have null effects on C. pallens larvae. The outcome indicates that genetically modified crops expressing the tested Bt proteins are safe for the lacewing, C. pallens.     

Evaluation of <Emphasis Type="Italic">Nastus fausti</Emphasis> Reitter (Coleoptera: Curculionidae: Entiminae: Nastini) for biological control of invasive giant hogweeds (<Emphasis Type="Italic">Heracleum</Emphasis> spp.)

The weevil Nastus fausti Reitter (Coleoptera, Curculionidae) was evaluated for its potential in the biological control of invasive giant hogweeds (Heracleum spp.). Quantitative sampling suggested that at a high population density (more that 3–4 mature larvae per plant) damage by N. fausti larvae could have some negative impact on the above-ground part of the plant. However, no-choice laboratory tests showed that N. fausti females were able to feed on a number of Apiaceae genera, including such important cultivated crops as carrot, parsnip, and celeriac. Feeding on these plants did not cause any significant decrease in female survival or fecundity. Moreover, at least part of N. fausti larvae may feed and develop on roots of these plants, and the rate of their growth and development does not differ significantly from that in larvae fed on roots of H. mantegazzianum. N. fausti adult and larval feeding on Angelica purpurascens, representative of related genus of the same tribe, was recorded under natural conditions, too. In combination, these data suggest that N. fausti is an oligophagous species connected with plants from at least several genera of Apiaceae and thus it cannot be considered a potential agent for biological control of invasive Heracleum species.     

Salt tolerance conferred by expression of a global regulator IrrE from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in oilseed rape

As salinity is a major threat to sustainable agriculture worldwide, cultivation of salt-tolerant crops becomes increasingly important. IrrE acts as a global regulator and a general switch for stress resistance in Deinococcus radiodurans. In this study, to determine whether the irrE gene can improve the salt tolerance of Brassica napus, we introduced the irrE gene into B. napus by the Agrobacterium tumefaciens-mediated transformation method. Forty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) analyses confirmed that the irrE gene had integrated into the plant genome. Northern as well as Western blot analyses revealed that the transgene was expressed at various levels in transgenic plants. Analysis for the T₁ progenies derived from four independent transformants showed that irrE had enhanced the salt tolerance of T₁ in the presence of 350 mM NaCl. Furthermore, under salt stress, transgenic plants accumulated more compatible solutes (proline) and a lower level of malondialdehyde (MDA), and they had higher activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). However, agronomic traits were not affected by irrE gene overexpression in the transgenic B. napus plants. This study indicates that the irrE gene can improve the salt tolerance of B. napus and represents a promising candidate for the development of crops with enhanced salt tolerance by genetic engineering.     

Dynamic variation of toxic and non-toxic <Emphasis Type="Italic">Microcystis</Emphasis> proportion in the eutrophic Daechung Reservoir in Korea

This study was conducted to determine the environmental factors affecting the level of potentially toxic Microcystis. The long-term tendencies of temperature, precipitation, and water quality factors were analyzed to determine the environmental characteristics of the Daechung Reservoir in Korea, and water samples were directly collected to analyze the dynamics of toxic and non-toxic Microcystis at weekly intervals from May to October 2012. Microcystis was the dominant genus during the study period, and it was composed of potentially toxic and non-toxic Microcystis. The fraction of potentially toxic Microcystis ranged from 6.0% to 61.1%. The amount of toxic Microcystis was highly related to the intracellular microcystin concentration (r = 0.760, P < 0.01). Therefore, the fraction of potentially toxic Microcystis is an important concern in Microcystis blooming because the intracellular microcystin concentration may reflect microcystin levels in the water. The prevalence of potentially toxic Microcystis was highly related to water temperature in Daechung Reservoir (r = 0.585, P < 0.01). Thus, temperature increase during Microcystis blooming may lead to more frequent toxic Microcystis blooms in eutrophic water bodies.     

Effects of Naphthalene Degradative Plasmids on the Physiological Characteristics of Rhizosphere Bacteria of the Genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

We studied the specific growth rate, duration of the lag phase, stability of plasmids, and activities of the key enzymes involved in naphthalene biodegradation in rhizosphere pseudomonades carrying the structurally similar plasmids pOV17 and pBS216. It was demonstrated that these plasmids determined various levels of catechol-2,3-dioxygenase activities. The structural rearrangements in the plasmid pBS216 could “switch off” the genes of the catechol oxidation meta-pathway. It was shown that certain combinations of degradative plasmids and bacterial hosts, such as Pseudomonas chlororaphis PCL1391(pBS216), P. chlororaphis PCL1391(pOV17), and P. putida 53a(pOV17), were considerably more efficient than natural variants in their growth characteristics and the stability of the biodegradation activity, having a potential for bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs).     

The fitness of the cotton mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis> (Tinsley) is reduced after feeding on virus-infected <Emphasis Type="Italic">Hibiscus rosa</Emphasis>-<Emphasis Type="Italic">sinensis</Emphasis>

The cotton mealybug Phenacoccus solenopsis (Tinsley) and Cotton leaf curl Multan virus (CLCuMV), serious threats to economic crops and garden plants, have invaded southern China and widely infected Hibiscus rosa-sinensis. Whether an inter-species connection has facilitated the invasion process is unclear. In this study the interaction between P. solenopsis and H. rosa-sinensis infected with CLCuMV was investigated in the laboratory. We observed that 1st and 2nd instar nymphs of P. solenopsis preferred to feed on healthy H. rosa-sinensis leaves, whereas 3rd instar nymphs and female adults had no preference between healthy and virus-infected H. rosa-sinensis leaves. The developmental time of each P. solenopsis developmental stage increased significantly after feeding on infected H. rosa-sinensis leaves (p < 0.05). In particular, the development time for 2nd instar female and male nymphs and 3rd instar female nymphs increased by approximately twofold. The generation time of female mealybugs increased from 25.84 d on healthy H. rosa-sinensis to 32.12 d when feeding on CLCuMV-infected H. rosa-sinensis, and the survival rate decreased from 71.04 % on healthy H. rosa-sinensis to 5.80 % on infected plants. Nymph survival was most affected by feeding on infected plants. Additionally, the fecundity of female mealybugs feeding on infected H. rosa-sinensis decreased by 47.8 %. Thus, feeding on CLCuMV-infected H. rosa-sinensis significantly decreased the biological fitness and invading and colonizing abilities of P. solenopsis.     

Strain-specific response to ampicillin in <Emphasis Type="Italic">Wolbachia-</Emphasis>infected mosquito cell lines

Wolbachia pipientis (Rickettsiales; Anaplasmataceae) is an obligate intracellular alpha proteobacterium that occurs in arthropods and filarial worms. Some strains of Wolbachia can be maintained as persistent infections in insect cell lines. C/wStr1 cells from the mosquito Aedes albopictus maintain a robust infection with Wolbachia strain wStr, originally isolated from the planthopper, Laodelphax striatellus. To explore possible functions of penicillin-binding proteins expressed from the wStr genome, C/wStr1 cells were exposed to ampicillin. Absolute levels of Wolbachia increased 3.5-fold in ampicillin-treated cells and fivefold in naive cells newly infected with wStr. Because cell numbers were depressed by ampicillin treatment, Wolbachia yield on a per-cell basis increased by 15-fold. The absence of a similar effect on wAlbB in Aa23 host cells suggests that the Wolbachia strain, the presence/absence of genes encoding penicillin-binding proteins, or the interaction between wAlbB and its host cells may modulate the effects of ampicillin.     

Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.     

Findings of the leaf beetles <Emphasis Type="Italic">Chrysolina tundralis</Emphasis> and <Emphasis Type="Italic">Chrysolina roddi</Emphasis> (Coleoptera,Chrysomelidae) in the middle part of European Russia

Two species of leaf beetles, Chrysolina tundralis and Ch. roddi, were found in the “Galich’ya Gora” Nature Reserve (the middle part of European Russia), 1200 and 700 km, respectively, from the main part of the ranges of these species. Adults and larvae of Ch. tundralis feed on Lamium purpureum, those of Ch. roddi feed on Seseli intermedia there. Diagnostic characters of the adults of both species are reported.     

Allelic differentiation at the <Emphasis Type="Italic">Sg-1</Emphasis> locus for the terminal sugar of the C-22 position of group A saponin in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &amp; Zucc.)

Group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean (Glycine max), and the elimination of group A saponins is an important breeding objective. The group A saponins include two main Aa and Ab types, controlled by codominant alleles at the Sg-1 locus that is one of several key loci responsible for saponin biosynthesis in the subgenus Glycine soja. However, A0 mutant lacking group A saponin is a useful gene resource for soybean quality breeding. Here, eight Chinese wild soybean A0 accessions were sequenced to reveal the mutational mechanisms, and the results showed that these mutants were caused by at least three kinds of mechanisms involving four allelic variants (sg-1^0-b2, sg-1^0-b3, Sg-1^b-0, and Sg-1^b-01). The sg-1^0-b2 had two nucleotide deletions at positions +?72 and +?73 involving in the 24th and 25th amino acids. The sg-1^0-b3 contained a stop codon (TGA) at the 254th residue. The Sg-1^b-0 and Sg-1^b-01 were two novel A0-type mutants, which likely carried normal structural alleles, and nevertheless did not encode group A saponin due to unknown mutations beyond the normal coding regions. In addition, to reveal the structural features, allelic polymorphism, and mechanisms of the abiogenetic absence of group A (i.e., A0 phenotype), nucleotide sequence analysis was performed for the Sg-1 locus in wild soybean (Glycine soja). The results showed that Sg-1 alleles had a lower conservatism in the coding region; as high as 18 sequences were found in Chinese wild soybeans in addition to the Sg-1^a (Aa) and Sg-1^b (Ab) alleles. Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons and nine amino acid substitutions. Two evolutionarily transitional allelic sequences (Sg-1^a7 and Sg-1^b2) from Sg-1^a toward Sg-1^b were detected.     

Diversity and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> isolates obtained from rice rhizosphere soils in Taiwan

Azotobacter species, free-living nitrogen-fixing bacteria, have been used as biofertilizers to improve the productivity of non-leguminous crops, including rice, due to their various plant growth-promoting traits. The purposes of this study were to characterize Azotobacter species isolated from rice rhizospheres in Taiwan and to determine the relationship between the species diversity of Azotobacter and soil properties. A total of 98 Azotobacter isolates were isolated from 27 paddy fields, and 16S rRNA gene sequences were used to identify Azotobacter species. The characteristics of these Azotobacter strains were analyzed including carbon source utilization and plant growth-promoting traits such as nitrogen fixation activity, indole acetic acid production, phosphate-solubilizing ability, and siderophore secretion. Of the 98 strains isolated in this study, 12 were selected to evaluate their effects on rice growth. Four species of Azotobacter were identified within these 98 strains, including A. beijerinckii, A. chroococcum, A. tropicalis, and A. vinelandii. Of these four species, A. chroococcum was predominant (51.0%) but A. beijerinckii had the highest level of nucleotide diversity. Strains within individual Azotobacter species showed diverse profiles in carbon source utilization. In addition, the species diversity of Azotobacter was significantly related to soil pH, Mn, and Zn. Members of the same Azotobacter species showed diverse plant growth-promoting traits, suggesting that the 98 strains isolated in this study may not equally effective in promoting rice growth. Of the 12 strains evaluated, A. beijerinckii CHB 461, A. chroococcum CHB 846, and A. chroococcum CHB 869 may be used to develop biofertilizers for rice cultivation because they significantly promoted rice growth. This study contributes to the selection of suitable Azotobacter strains for developing biofertilizer formulations and soil management strategies of Azotobacter for paddy fields.