Identification and Manipulation of Soil Properties To Improve the Biological Control Performance of Phenazine-Producing Pseudomonas fluorescens

Pseudomonas fluorescens 2-79RN₁₀ protects wheat against take-all disease caused by Gaeumannomyces graminis var. tritici; however, the level of protection in the field varies from site to site. Identification of soil factors that exert the greatest influence on disease suppression is essential to improving biocontrol. In order to assess the relative importance of 28 soil properties on take-all suppression, seeds were treated with strain 2-79RN₁₀ (which produces phenazine-1-carboxylate [PCA⁺]) or a series of mutants with PCA⁺ and PCA⁻ phenotypes. Bacterized seeds were planted in 10 soils, representative of the wheat-growing region in the Pacific Northwest. Sixteen soil properties were correlated with disease suppression. Biocontrol activity of PCA⁺ strains was positively correlated with ammonium-nitrogen, percent sand, soil pH, sodium (extractable and soluble), sulfate-sulfur, and zinc. In contrast, biocontrol was negatively correlated with cation-exchange capacity (CEC), exchangeable acidity, iron, manganese, percent clay, percent organic matter (OM), percent silt, total carbon, and total nitrogen. Principal component factor analysis of the 16 soil properties identified a three-component solution that accounted for 87 percent of the variance in disease rating (biocontrol). A model was identified with step-wise regression analysis (R² = 0.96; Cp statistic = 6.17) that included six key soil properties: ammonium-nitrogen, CEC, iron, percent silt, soil pH, and zinc. As predicted by our regression model, the biocontrol activity of 2-79RN₁₀ was improved by amending a soil low in Zn with 50 μg of zinc-EDTA/g of soil. We then investigated the negative correlation of OM with disease suppression and found that addition of OM (as wheat straw) at rates typical of high-OM soils significantly reduced biocontrol activity of 2-79RN₁₀.     

Variation in Sensitivity of Gaeumannomyces graminis to Antibiotics Produced by Fluorescent Pseudomonas spp. and Effect on Biological Control of Take-All of Wheat

Isolates of Gaeumannomyces graminis var. tritici, the causal agent of take-all of wheat, varied in sensitivity in vitro to the antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) produced by fluorescent Pseudomonas spp. shown previously to have potential for biological control of this pathogen. None of the four isolates of G. graminis var. avenae examined were sensitive to either of the antibiotics in vitro at the concentrations tested. The single isolate of G. graminis var. graminis tested was insensitive to PCA at 1.0 (mu)g/ml. Pseudomonas fluorescens 2-79 and Pseudomonas chlororaphis 30-84, both of which produce PCA, effectively suppressed take-all caused by each of two PCA-sensitive isolates of G. graminis var. tritici. PCA-producing strains exhibited a reduced ability or complete inability to suppress take-all caused by two of three isolates of G. graminis var. tritici that were insensitive to PCA at 1.0 (mu)g/ml. P. fluorescens Q2-87, which produces Phl, suppressed take-all caused by three Phl-sensitive isolates but failed to provide significant suppression of take-all caused by two isolates of G. graminis var. tritici that were insensitive to Phl at 3.0 (mu)g/ml. These findings affirm the role of the antibiotics PCA and Phl in the biocontrol activity of these fluorescent Pseudomonas spp. and support earlier evidence that mechanisms in addition to PCA are responsible for suppression of take-all by strain 2-79. The results show further that isolates of G. graminis var. tritici insensitive to PCA and Phl are present in the pathogen population and provide additional justification for the use of mixtures of Pseudomonas spp. that employ different mechanisms of pathogen suppression to manage this disease.     

Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici.

Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G. graminis var. tritici and other fungal root pathogens. Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots. Six independent, prototrophic Phz- mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings. Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library. These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear. These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.     

Relative importance of fluorescent siderophores and other factors in biological control of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens 2-79 and M4-80R.

Pseudomonas fluorescens 2-79 suppresses take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. The bacteria produce an antibiotic, phenazine-1-carboxylic acid (PCA), and a fluorescent pyoverdin siderophore. Previous studies have established that PCA has an important role in the biological control of take-all but that antibiotic production does not account fully for the suppressiveness of the strain. To define the role of the pyoverdin siderophore more precisely, mutants deficient in production of the antibiotic, the siderophore, or both factors were constructed and compared with the parental strain for control of take-all on wheat roots. In all cases, strains that produced PCA were more suppressive than those that did not, and pyoverdin-deficient mutant derivatives controlled take-all as effectively as their respective fluorescent parental strains. Thus, the phenazine antibiotic was the dominant factor in disease suppression and the fluorescent siderophore had little or no role. The siderophore also was of minor importance in a second strain, P. fluorescens M4-80R, that does not produce PCA. Strains 2-79 and M4-80R both produced substances distinct from the pyoverdin siderophore that were responsible for fungal inhibition in vitro under iron limitation, but these substances also had, at most, a minor role in disease suppression in situ.     

Relative importance of fluorescent siderophores and other factors in biological control of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens 2-79 and M4-80R.

Pseudomonas fluorescens 2-79 suppresses take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. The bacteria produce an antibiotic, phenazine-1-carboxylic acid (PCA), and a fluorescent pyoverdin siderophore. Previous studies have established that PCA has an important role in the biological control of take-all but that antibiotic production does not account fully for the suppressiveness of the strain. To define the role of the pyoverdin siderophore more precisely, mutants deficient in production of the antibiotic, the siderophore, or both factors were constructed and compared with the parental strain for control of take-all on wheat roots. In all cases, strains that produced PCA were more suppressive than those that did not, and pyoverdin-deficient mutant derivatives controlled take-all as effectively as their respective fluorescent parental strains. Thus, the phenazine antibiotic was the dominant factor in disease suppression and the fluorescent siderophore had little or no role. The siderophore also was of minor importance in a second strain, P. fluorescens M4-80R, that does not produce PCA. Strains 2-79 and M4-80R both produced substances distinct from the pyoverdin siderophore that were responsible for fungal inhibition in vitro under iron limitation, but these substances also had, at most, a minor role in disease suppression in situ.     

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.     

Isolation, identification, and accumulation of 2-acetamidophenol in liquid cultures of the wheat take-all biocontrol agent Pseudomonas fluorescens 2-79

Pseudomonas fluorescens strain 2-79 (NRRL B-15132) is a classic biological control agent known to produce phenazine-1-carboxylic acid (PCA) as its primary means of suppressing take-all disease of wheat. In addition to PCA, an unknown metabolite was discovered in a liquid culture used to produce the biocontrol agent. The objective of the current study was to isolate, identify, and evaluate the accumulation of this compound in production cultures. Upon centrifugal fractionation of a production culture, thin-layer chromatography (TLC) analyses of extracts of the cells and cell-free supernatant indicated the compound to be primarily in the supernatant. Purified compound was obtained by extraction of culture supernatant, followed by flash chromatography of the extract and preparative TLC. The ¹H and ¹³C nuclear magnetic resonance and electron impact mass spectra indicated the compound to be 2-acetamidophenol (AAP). Measured by reversed-phase HPLC, the accumulations of AAP and PCA in cultures of strain 2-79 reached 0.05 g/l and 1 g/l, respectively. The accumulations of AAP and PCA in liquid cultures were linearly correlated (P < 0.001), as shown by studies of cultures stimulated to yield varying levels of PCA by controlling levels of oxygen transfer, pH, and growth medium composition. In this study, oxygen limitation, a defined amino-acid-free medium, and neutral pH stimulated maximal production of both AAP and PCA. Furthermore, a transposon mutant of 2-79 [2A40 2-79 (phz–)] unable to produce PCA did not accumulate AAP. These findings indicate that AAP and PCA are likely to share a common segment of biosynthetic pathway. This is the first report of AAP production by a strain of P. fluorescens. Possible routes of AAP production are discussed relative to current knowledge of the phenazine biosynthetic pathway of strain 2-79. The pertinence of AAP to the design of commercial seed inoculants of phenazine-producing bacteria for controlling wheat take-all is also considered. Received: 2 November 1999 / Received revision: 3 April 2000 / Accepted: 14 April 2000     

Production of the Antibiotic Phenazine-1-Carboxylic Acid by Fluorescent Pseudomonas Species in the Rhizosphere of Wheat

Pseudomonas fluorescens 2-79 and P. aureofaciens 30-84 produce the antibiotic phenazine-1-carboxylic acid and suppress take-all, an important root disease of wheat caused by Gaeumannomyces graminis var. tritici. To determine whether the antibiotic is produced in situ, wheat seeds were treated with strain 2-79 or 30-84 or with phenazine-nonproducing mutants or were left untreated and then were sown in natural or steamed soil in the field or growth chamber. The antibiotic was isolated only from roots of wheat colonized by strain 2-79 or 30-84 in both growth chamber and field studies. No antibiotic was recovered from the roots of seedlings grown from seeds treated with phenazine-nonproducing mutants or left untreated. In natural soils, comparable amounts of antibiotic (27 to 43 ng/g of root with adhering soil) were recovered from roots colonized by strain 2-79 whether or not the pathogen was present. Roots of plants grown in steamed soil yielded larger bacterial populations and more antibiotic than roots from natural soils. In steamed and natural soils, roots from which the antibiotic was recovered had significantly less disease than roots with no antibiotic, indicating that suppression of take-all is related directly to the presence of the antibiotic in the rhizosphere.     

祁连山西水林区土壤阳离子交换量及盐基离子的剖面分布

以祁连山西水林区分布的棕钙土、灰褐土、栗钙土和高山草甸土为对象,研究了阳离子交换量和盐基离子(K+、Na+、Ca2+、Mg2+)的剖面分布规律及其与土壤理化因子的关系。结果表明:土壤阳离子交换量(CEC,介于4.80—48.10 cmol/kg)和盐基总量(TEB,介于4.67—21.34 cmol/kg)随剖面深度的增加逐渐减小,不同土壤类型的大小顺序为:灰褐土>高山草甸土>栗钙土>棕钙土;土壤盐基组成以Ca2+、Mg2+为主(占TEB的比例平均为71.6%、22.9%),K+、Na+所占比例较低(占TEB的比例平均为3.3%、2.2%);棕钙土、灰褐土和栗钙土盐基离子的剖面分布由浅至深呈现:K+≈Ca2+>Na+≈Mg2+,高山草甸土盐基离子则呈现:K+>Na+>Mg2+>Ca2+。不同土壤类型间盐基离子的含量及饱和度随发生层次不同存在较大差异。土壤有机质是CEC的主要贡献因素,粉粒对CEC也有显著的促进作用,而砂粒、CaCO3对CEC有显著抑制作用。土壤生物复盐基作用弱于淋溶作用,造成盐基饱和度较大(BSP,介于44.4%—97.2%),并随剖面深度的增加逐渐增大。相关性分析表明,土壤交换性Na+、Mg2+的含量及饱和度均呈极显著正相关,交换性Na+、Mg2+饱和度与CaCO3含量呈极显著正相关;pH值与BSP呈极显著正相关;土壤速效P含量与CEC呈极显著正相关,速效K含量与交换性K+含量呈极显著正相关。     

Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.     

Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.     

Effect of Soil Chemical and Physical Properties on Sorption and Desorption Behavior of Lead in Different Soils of India

Lead (Pb) is a non-biodegradable contaminant, present in the environment, especially near lead-based industrial sites, agricultural lands, and roadside soils. Bioavailability of Pb in the soil is controlled by the sorption and desorption behavior of Pb, which are further controlled by the soil chemical and physical properties. In this study, sorption and desorption amounts of Pb in soil were compared with soil physical (sand, silt, clay content) and chemical (pH; electrical conductivity, EC; percent organic carbon, (%OC); cation exchange capacity, CEC) properties. Twenty-six surface soils (0–5cm), expected to vary in physical and chemical properties, were collected from different parts of India and were treated with known concentration of Pb solution (40 μg/L). The amount of Pb sorbed and desorbed were measured and correlated with soil properties using simple linear regressions. Sorption was significantly (p ≤ 0.05) and positively correlated with pH, and %OC; desorption was significantly (p ≤ 0.05) negatively correlated with the same two factors. Stepwise multiple regressions were performed for better correlations. Predicted sorption and desorption amounts, based on multiple regression equations, showed reasonably good fit (R² = 0.79 and 0.83, respectively) with observed values. This regression model can be used for estimation of sorption and desorption amounts at contaminated sites.     

菜园土壤微生物生态特征与土壤理化性质的关系

对广州白云区64个菜园土壤样本的研究表明,微生物碳与土壤全氮、碱解氮、有效钾、阳离子交换量和有机质,微生物氮与土壤全氮、全磷、阳离子交换量及有机质,土壤基础呼吸与土壤全氮、碱解氮、全钾、阳离子交换量及有机质,AWCD值与全氮及有机质含量,以及Shannon多样性指数与全氮和阳离子交换量均呈显著正相关关系.较低的碱解氮含量使土壤微生物碳、土壤基础呼吸和呼吸商的值升高,过高的碱解氮则使呼吸商下降;过高的土壤有效磷降低微生物碳、微生物氮和微生物呼吸商.有效磷/碱解氮比值过高降低了土壤微生物碳、微生物氮、微生物碳氮比及土壤基础呼吸.土壤微生物生态特征与土壤理化性质关系密切,有效养分过高及养分比例不适当对土壤微生物均存在不利影响.     

Natural suppression of take-all disease of wheat in Montana soils

This research was initiated to determine whether soils suppressive to take-all of wheat caused by Gaeumannomyces graminis var. tritici (Ggt) occur in Montana, and to identify the organisms most likely involved in this suppression. From an initial screening of eight soils collected from different wheat growing areas of Montana, two were highly suppressive to take-all. Microbial characterization of these soils indicated that different mechanisms were involved in the suppression. In Larslan soil, mycoparasitism appeared to be the main mechanism. Two different fungi with exceptional ability to reduce the severity of take-all were isolated from this soil. One of these fungi could parasitize the hyphae of Ggt. Field tests with these fungi in Ggt infested soil showed increases of over 100% in both harvestble tillers and grain yield as compared to treatments without these two fungi. In tests with 48 different bacteria and 10 actinomycetes from Larslan soil, none were able to consistently reduce severity of take-all alone, or in mixtures. In Toston soil, antibiosis by actinomycetes and perhaps the involvement of Pseudomonas spp. in production of antibiotics and/or siderophores appeared to be the most likely mechanisms involved in take-all suppression. Increases in shoot dry weight over that in the Ggt infested control using mixtures of pseudomonads and actinomycetes ranged from 25% to 87%. Actinomycetes added individually or in mixtures to soil infested with Ggt consistently reduced the severity of the disease to a greater extent than did mixtures of Pseudomonas spp.     

Liquid-culture pH,temperature, and carbon (not nitrogen) source regulate phenazine productivity of the take-all biocontrol agentPseudomonas fluorescens 2-79

 Strain 2-79 is a biocontrol agent againsttake-all, an important disease of wheat caused by Gaeumannomyces graminis var. tritici. In the rhizosphere, it produces the antibiotic phenazine 1-carboxylic acid (PCA) as the primary means of disease suppression. One barrier to commercial use of phenazine-producing pseudomonads, like strain 2-79, is the lack of liquid-culture technology for mass production. For instance, there is little published research concerning the impact of liquid-culture secondary metabolism on the biocontrol qualities of the cell harvest, i.e., efficacy, phytotoxicity, and storage survival. Yet it is important to know whether the fermentation process should be designed to enhance or eliminate secondary metabolite accumulation. To enable future exploration of this issue, we identified liquid-culture parameters that could be manipulated to controlthe phenazine productivity of strain 2-79. Our results indicated that PCA accumulation was very sensitive to the culture pH and temperature. It was possible to produce large cell populations with either high or low phenazine productivity by choosing to control culture pH at 7 and 8 respectively. Although high cell accumulations were achieved over the broad 25–34°C range studied, high, moderate, or low PCA productivities were observed at 25–27°C, 29–32.5°C, or 34°C respectively. When pH was controlled at 7, specific PCAproductions at 25°C could be modulated by the choice of carbon source supplied. PCA accumulation per unit biomass reached 0.31 g/g on glucose, 0.16 g/g on glycerol and xylose, and only 0.09 g/g on fructose. Although the nitrogen source was also tested as a variable, it had little influence on culture PCA productivity under controlled pH. Received: 12 July 1994 / Received revision: 8 October 1994/Accepted: 22 November 1994     

Nutritional factors regulating growth and accumulation of phenazine 1-carboxylic acid by Pseudomonas fluorescens 2-79

Summary Pseudomonas fluorescens strain 2-79 (NRRL B-15 132) is a biocontrol agent of take-all of wheat caused by the fungus Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine 1-carboxylic acid, which acts as the primary mechanism of disease suppression. As a first step toward designing efficient methods of mass producing and formulating this biocontrol agent, the regulation of growth and antibiotic production by growth factors (including purines, pyrimidines, vitamins) and minerals (supplying B³⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Mo⁶⁺, Zn²⁺) was examined in defined liquid culture. Additions of boric acid and sulfates of iron and magnesium enhanced both cell and associated antibiotic accumulation. However, accumulation of the antibiotic alone improved with additions of zinc sulfate, ammonium molybdate, and cytosine, but worsened with addition of adenine. Interactive effects involving the sulfates of iron, magnesium, and zinc were observed, and optimal iron-magnesium and iron-zinc combinations were demonstrated with respect to biomass and antibiotic accumulation, respectively.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned Correspondence to: P. J. Slininger     

Gluconic acid: an antifungal agent produced by Pseudomonas species in biological control of take-all

Pseudomonas strain AN5 (Ps. str. AN5), a non-fluorescent Australian bacterial isolate, is an effective biological control (biocontrol) agent of the take-all disease of wheat caused by the fungus Gaeumannomyces graminis var. tritici (Ggt). Ps. str. AN5 controls Ggt by producing an antifungal compound which was purified by thin layer and column chromatography, and identified by NMR and mass spectroscopic analysis to be d-gluconic acid. Commercially bought pure gluconic acid strongly inhibited Ggt. Two different transposon mutants of Ps. str. AN5 which had lost take-all biocontrol did not produce d-gluconic acid. Gluconic acid production was restored, along with take-all biocontrol, when one of these transposon mutants was complemented with the corresponding open reading frame from wild-type genomic DNA. Gluconic acid was detected in the rhizosphere of wheat roots treated with the wild-type Ps. str. AN5, but not in untreated wheat or wheat treated with a transposon mutant strain which had lost biocontrol. The antifungal compounds phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol, produced by other Pseudomonads and previously shown to be effective in suppressing the take-all disease, were not detected in Ps. str. AN5 extracts. These results suggest that d-gluconic acid is the most significant antifungal agent produced by Ps. str. AN5 in biocontrol of take-all on wheat roots.     

Effect of growth culture physiological state,metabolites, and formulation on the viability,phytotoxicity, and efficacy of the take-all biocontrol agent Pseudomonas fluorescens 2–79 stored encapsulated on wheat seeds

Strain 2–79 is a biocontrol agent of take-all, an important root disease of wheat caused by Gaeumannomyces graminis var. tritici. In the rhizosphere, strain 2–79 produces the antibiotic phenazine-1-carboxylic acid as the primary means of disease suppression. Barriers to the commercial use of phenazine-producing pseudomonads, such as strain 2–79, include the lack of liquid-culture and formulation technologies needed to optimize cost-effective mass production and application. For instance, there is little published research concerning the impact of growth culture physiological state and associated metabolites on the biocontrol qualities of the cells harvested and formulated in seed coatings, i.e., efficacy, phytotoxicity, and storage survival. To enable exploration of these issues, cells of strain 2–79 in various physiological states were obtained by harvesting fermentors at 24-h intervals after inoculation. Cells formulated in 0.5% methylcellulose suspended in either water (MW) or metabolite-bearing, spent culture broth (MSB) were applied as wheat-seed coatings, air dried, and stored at 4°C. Younger cells (24–48?h) had twice the drying survival rate but only half of the storage life demonstrated by older cells (72–96?h) (P?0.05). Cell populations surviving drying were 3.5 times higher in MW than in MSB formulations and they remained viable up to 3 times longer (P?0.05). This effect of formulation on viability was attributable to the culture nutrients but not the metabolites present in the spent broth. Disease suppression in bacterized seed treatments was significant (P?0.05) relative to unbacterized controls and averaged 9.1%, but did not vary significantly (P?0.24) with culture age, encapsulation medium, or storage time. Relative seedling height improvement increased with relative disease suppression (P=0.003) and significantly decreased with lengthening storage time (P=0.004). This latter decline in plant growth promotion coincided with the deterioration of biocontrol agent viability during storage. Seed batches inoculated with cells in both MW and MSB encapsulations suffered significant germination losses due to phytotoxic metabolites. The extent of loss was an interactive result of encapsulation medium and storage time (P?0.01), and the rate of loss was much higher for seeds with MSB than with MW coatings, i.e. 54% compared to 11% loss after 6 months storage.     

Reduction of Take-all Inoculum by Rotation with Lupins, Oats or Field Peas

The feasibility of use of lupins, oats and field peas as alternative rotation crops to reduce inoculum of the take-all fungus (Gaeumannomyces graminis var. tritici) (under Western Australian field conditions) and disease in following wheat was investigated with a one year field trial, the soil from which was used in two succeeding pot experiments. The possible mechanisms of reduction of inoculum and disease by these crops were examined testing the soil for pathogen and disease suppression. Rotation with lupins or oats for two seasons reduced (P <0.05) inoculum of the take-all fungus and lupins, oats or field peas reduced (P <0.05) disease in following wheat. Lupins alone reduced inoculum and disease, (P <0.1) after one season. No apparent suppression of the pathogen in the absence of host plants was recorded after one season of rotation, but after two seasons, lupins, oats or field peas all suppressed (P <0.02) growth of the pathogen within soil. However only field pea soil suppressed take-all in comparison with the wheat control. Although after two seasons all rotation crops were effective in reducing inoculum and disease the mechanisms of reduction appear to differ between the rotation crops used in this study.     

Evaluation of fungicides applied to soil to control naturally-occurring take-all using a balanced-incomplete-block design and very small plots

Six sterol biosynthesis-inhibiting fungicides representing several combinations of properties were applied to soil to control naturally-occurring take-all (caused by Gaeumannomyces graminis var. tritici) in winter wheat in field experiments in two successive years. The average take-all severity category was never more than moderate in the different clay-loam and sandy loam sites used in each year. At each site in each year there were six treatments and an untreated control in an arrangement based on a balanced-incomplete-block design for six treatments in 10 blocks each with three treatments. Each block had three treated plots and a control plot and was paired with the complementary block of three treatments (plus control) to form a complete replicate, of which there were 30 per site. Take-all assessments in June or July showed that after incorporation into the seed bed (at 2 kg ha“¹and sometimes at 1 kg ha”¹) in autumn, two non-volatile, strongly lipophilic compounds, nuarimol and triadimenol, with good intrinsic toxicity to the take-all fungus and slow rates of degradation, partially controlled take-all. However, another compound, flutriafol, with similar properties to nuarimol and triadimenol, controlled take-all less. Two slightly volatile, strongly lipophilic compounds, flusilazole and penconazole, with good intrinsic activity, were less effective (at 2 kg ha^-1). A volatile, less lipophilic compound, PP 969, with less intrinsic activity, also partially controlled take-all, but only after application as a drench in the spring (2 kg ha^-1). The most effective treatments were generally more effective the greater the level of disease (as indicated by assessments of disease in control plots), especially in spring assessments of disease. Although flutriafol did not perform as expected, it still seems reasonable to conclude that the requirements for a soil-applied fungicide to control take-all are likely to be: (i) good intrinsic fungitoxicity, (ii) some mobility in soil water (i.e. not strongly lipophilic), and (iii) season-long persistence.