Biological Control of Take-all Disease of Wheat Caused by Gaeumannomyces graminis var. tritici Under Field Conditions Using a Phialophora sp.

A Phialophora sp. (isolate I-52), originally isolated from soil in a wheat field exhibiting suppression of take-all disease caused by Gaeumannomyces graminis var. tritici , was tested under field conditions for its ability to suppress this disease in winter and spring wheat. I-52 was grown on a variety of autoclaved organic substrates, including oat, millet and canola seed. All of these gave significant disease control when added to the seed furrow with inoculum of the take-all fungus. W hole seed of I-52 substrate was as effective as particles < 0.5 mm in diameter. Placing I-52 in powdered form directly on to wheat seed was ineffective in controlling take-all. Rates as low as 2 g of I-52/3.3 m of row added with the seed provided some control of take-all, and nearly complete control in winter wheat was obtained using 15 g/3.3 m. The winter wheat host cultivar did not influence the degree of control of take-all by I-52.     

Effects of wheat volunteers and blackgrass in set-aside following a winter wheat crop on soil infectivity and soil conduciveness to take-all

Two experiments were carried out in France in which disease indices were used to evaluate the effects of wheat volunteers and blackgrass (Alopecurus myosuroides) on soil infectivity and soil conduciveness to take-all caused by Gaeumannomyces graminis var. tritici. Soil infectivity was evaluated by measuring the disease index on susceptible wheat plants grown on soil samples collected from the field. Soil conduciveness to the disease was obtained by measuring disease indices on plants grown on soil samples to which different amounts of take-all fungus inoculum were added. One experiment (Expt. 1) was carried out using soils from farmers' fields (two fields in 1994 and two in 1995); soil infectivity and soil conduciveness were evaluated for three experimental situations: bare soil, soil with wheat volunteers and soil with blackgrass plants. In 1994 the soil infectivity was zero in bare soil, high with the wheat cover, and intermediate with the blackgrass cover. In 1995 the soil infectivity was uniformly low for all three conditions. Soils bearing wheat were less conducive than bare soil, soils bearing blackgrass and bare soils were similarly conducive. A second experiment (Expt. 2) carried out in 1995 compared the soil infectivity and soil conduciveness to take-all of soils planted with wheat or blackgrass in set-aside land after periods of wheat monoculture of 0–6 yr. The soil infectivity was low for all treatments. The soil was more conducive after blackgrass than after wheat. In both cases, the soil conduciveness was less when the monoculture had continued for more than 4 yr. The decline was less after blackgrass than after wheat. Thus, whenever set-aside is set up during the increase phase of the disease in fields with cereal successions, abundant wheat volunteers might hinder the expected positive effect of a break in cereal successions on take-all development. The presence of blackgrass in a set-aside field, with significant soil infectivity and high soil conduciveness, might increase the risks of take-all development in a wheat crop following set-aside.     

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.     

Experiments on soil drenching with fungicides against take-all in wheat

In short term pot experiments benomyl, iprodione and KWG 0599 applied as soil drenches in several types of soil significantly suppressed take-all symptoms from inoculum placed just below wheat seeds planted 1×5 cm deep, and in sand but not other soils when seeds were 5 cm deep. Benomyl was, however, effective against inoculum below seed planted 5 cm deep in a loam-sand mixture when the drench contained an alcohol ethoxylate surfactant. Computer simulations of fungicide distributions in the soils correlated well with disease control observations. In long term outdoor pot experiments two drenches with benomyl (without surfactant) controlled disease significantly for at least 3 months against inoculum placed 15 cm deep. The significance of these results for the practical control of take-all by fungicides is discussed.     

The use of ammonium fertilisers to reduce the severity of take-all (Gaeumannomyces graminis var. tritici) on wheat in Western Australia

In field experiments in Western Australia ammonium fertilisers significantly reduced the severity of root damage to wheat ( Triticum aestivum ) caused by Gaeumannomyces graminis var. tritici. A series of response lines depicting the effect of sources and rates of nitrogen on take-all are presented. In general, the higher the rate of ammonium nitrogen used, the greater the reduction in the severity of take-all. Ammonium sulphate and ammonium sulphate/mono-ammonium phosphate mixture were more effective in reducing disease than di-ammonium phosphate, urea or ammonium nitrate. Sodium nitrate did not reduce take-all.     

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.     

Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization of superior root-colonizing P. fluorescens strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed(-1) or soil(-1)) to establish high rhizosphere population densities (10(7) CFU g of root(-1)) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10(5) CFU g of root(-1) after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

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.     

Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat

Endophytic actinobacteria isolated from healthy cereal plants were assessed for their ability to control fungal root pathogens of cereal crops both in vitro and in planta. Thirty eight strains belonging to the genera Streptomyces, Microbispora, Micromonospora, and Nocardioidies were assayed for their ability to produce antifungal compounds in vitro against Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease in wheat, Rhizoctonia solani and Pythium spp. Spores of these strains were applied as coatings to wheat seed, with five replicates (25 plants), and assayed for the control of take-all disease in planta in steamed soil. The biocontrol activity of the 17 most active actinobacterial strains was tested further in a field soil naturally infested with take-all and Rhizoctonia. Sixty-four percent of this group of microorganisms exhibited antifungal activity in vitro, which is not unexpected as actinobacteria are recognized as prolific producers of bioactive secondary metabolites. Seventeen of the actinobacteria displayed statistically significant activity in planta against Ggt in the steamed soil bioassay. The active endophytes included a number of Streptomyces, as well as Microbispora and Nocardioides spp. and were also able to control the development of disease symptoms in treated plants exposed to Ggt and Rhizoctonia in the field soil. The results of this study indicate that endophytic actinobacteria may provide an advantage as biological control agents for use in the field, where others have failed, due to their ability to colonize the internal tissues of the host plant.     

Plant genotype, micronutrient fertilization and take-all infection influence bacterial populations in the rhizosphere of wheat

The relationship between micronutrient efficiency of four wheat (Triticum aestivum L.) genotypes, tolerance to take-all disease (caused by Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker), and bacterial populations in the rhizosphere was tested in soil fertilized differentially with Zn and Mn. Plant growth was reduced by Mn or Zn deficiency and also by take-all. There was an inverse relationship between micronutrient efficiency of wheat genotypes when grown in deficient soils and the length of take-all lesions on roots (efficient genotypes had shorter lesions than inefficient ones). In comparison to the rhizosphere of control plants of genotypes Aroona and C8MM receiving sufficient Mn and Zn, the total numbers of bacterial cfu (colony forming units) were greater in the rhizosphere of Zn-efficient genotype Aroona under Zn deficiency and in Mn-efficient genotype C8MM under Mn deficiency. These effects were not observed in other genotypes. Take-all decreased the number of bacterial cfu in the rhizosphere of fully-fertilized plants but not of those subjected to either Mn or Zn deficiency. In contrast, the Zn deficiency treatment acted synergistically with take-all to increase the number of fluorescent pseudomonads in the rhizosphere. Although numbers of Mn-oxidising and Mn-reducing bacteria were generally low, take-all disease increased the number of Mn reducers in the rhizosphere of Mn-efficient genotypes Aroona and C8MM. Under Mn-deficiency conditions, the number of Mn reducers in the rhizosphere increased in Aroona but not in C8MM wheat. The results suggest that bacterial microflora may play a role in the expression of Mn and Zn efficiency and tolerance to take-all in some wheat genotypes.     

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.     

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.     

Soil-applied fungicides for controlling take-all in field experiments with winter wheat

Soil treatment fungicides were tested against take-all (Gaeumannomyces graminis var. tritici) in three field experiments with winter wheat. Fungicides were applied as drenches either before sowing in autumn, and incorporated by rotary harrowing, or to the crop in spring. The most effective treatments were autumn applied benomyl (20 kg/ha) and nuarimol (0·55-4·4 kg/ha). However, the highest nuarimol concentration depressed yield. Benomyl sometimes induced a resurgence of take-all in the second wheat crop after treatment. Nuarimol had no adverse effects in subsequent crops, and neither fungicide hindered the onset of take-all decline in a third crop after treatment. The possible value of soil treatment in future control strategies is discussed.     

Take-all in spring-sown cereals under continuous cultivation: disease progress and decline in relation to crop succession and nitrogen

Incidence and severity of the take-all disease in spring wheat and spring barley caused by Gaeumannomyces graminis (syn. Ophiobolus graminis) were studied during seven years of monoculture. The fungus apparently survived for much longer periods in the soil under non-susceptible break-crops than previously recorded. The incidence and severity of infection increased progressively with each successive cereal crop from initially low levels to a maximum within 3–7 years, which was followed by a progressive but limited decline in the disease. Spring wheat was more susceptible to take-all than spring barley and the development of take-all decline (TAD) was recorded earlier in the sequences of wheat than of barley crops. Nitrogen did not influence the disease until the point of maximum incidence and severity, when it caused a reduction in disease levels in addition to that associated with TAD. Factors influencing the time of onset and the rate of development of take-all and of TAD are discussed and possible explanations for TAD are suggested.     

Frequency of Antibiotic-Producing Pseudomonas spp. in Natural Environments

The antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) are major determinants of biological control of soilborne plant pathogens by various strains of fluorescent Pseudomonas spp. In this study, we described primers and probes that enable specific and efficient detection of a wide variety of fluorescent Pseudomonas strains that produce various phenazine antibiotics or Phl. PCR analysis and Southern hybridization demonstrated that specific genes within the biosynthetic loci for Phl and PCA are conserved among various Pseudomonas strains of worldwide origin. The frequency of Phl- and PCA-producing fluorescent pseudomonads was determined on roots of wheat grown in three soils suppressive to take-all disease of wheat and four soils conducive to take-all by colony hybridization followed by PCR. Phenazine-producing strains were not detected on roots from any of the soils. However, Phl-producing fluorescent pseudomonads were isolated from all three take-all-suppressive soils at densities ranging from approximately 5 x 10(sup5) to 2 x 10(sup6) CFU per g of root. In the complementary conducive soils, Phl-producing pseudomonads were not detected or were detected at densities at least 40-fold lower than those in the suppressive soils. We speculate that fluorescent Pseudomonas spp. that produce Phl play an important role in the natural suppressiveness of these soils to take-all disease of wheat.     

Predisposition to Septoria nodorum as a result of take-all (Gaeumannomyces graminis) infection of wheat

In a series of experiments excised leaves from take-all infected wheat plants and from control plants were inoculated with Septoria nodorum. Larger lesions, more lesions/leaf and more pycnidia/unit area of lesion were produced from take-all plants. Significant effects of predisposition were demonstrated when only 3% of the area of the total root system was infected by take-all. Microscopical investigations revealed that germ-tubes of S. nodorum grew more rapidly on leaves from take-all plants, but the time of penetration was not affected. It is proposed that the observed effects of predisposition arose because more germ-tubes produced successful infections and host tissue was more rapidly colonised. The importance of these results for the epidemiology of glume blotch is discussed.     

Control of the take-all fungus byMicrodochium bolleyi,and interactions involvingM. bolleyi,Phialophora graminicola andPericonia macrospinosa on cereal roots

Summary In glasshouse experiments,Microdochium bolleyi (Mb) significantly reduced infection of wheat roots by the take-all fungus,Gaeumannomyces graminis vartritici (Ggt), when inocula were dispersed in soil at ratios of 10∶1 (Mb:Ggt) or more. Spread of take-all lesions up roots from a layer of inoculum also was reduced when Mb was inoculated immediately below the crown. In contrast,Periconia macrospinosa did not control take-all even at an inoculum ratio of 100∶1. M. bolleyi interfered with growth on roots byPhialophora graminicola, a known biocontrol agent of take-all. It is suggested that this phenomenon and control of take-all by these fungi occur by competition for cortical cells that senesce in the normal course of root development.     

Exploiting Genotypic Diversity of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp.: Characterization of Superior Root-Colonizing P. fluorescens Strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed⁻¹ or soil⁻¹) to establish high rhizosphere population densities (10⁷ CFU g of root⁻¹) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10⁵ CFU g of root⁻¹ after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

Occurrence of Phialophora radicicola var. graminicola and Gaeumannomyces graminis var. tritici on roots of wheat in field crops

Assessments of Phialophora radicicola var. graminicola (PRG) and Gaeumannomyces graminis var. tritici (GGT) were made by culturing and by direct microscopic examination of pieces of seminal roots from 16 winter wheat crops grown in different cropping sequences and with different phosphate manuring. PRG occurred on all wheat crops, but was abundant only on wheat after grass, where it seemed to delay the onset of damaging take-all by 1 yr. Delayed occurrence of take-all by phosphate fertiliser was not related to differences in populations of PRG. Wheat grown in ‘take-all decline’ soils had only small amounts of PRG, indicating that the development and the decline of take-all epidemics may be influenced by different biological control mechanisms; breaking sequences of wheat crops by 1 yr grass leys might harness the advantages of both mechanisms.