Evaluation of populations of fluorescent pseudomonads related to decline of take-all patch on turfgrass

Take-all on turfgrass caused by Gaeumannomyces graminis var. avenae (Gga) occurs as patches of yellowish plants. On some patches the central zone was recolonized by the same grass species, Festuca sp., previously damaged by the fungus despite the centrifugal extension of the disease. This disease remission was assimilated to decline. Rhizosphere bacterial counts showed that total population of bacteria was nearly the same in all zones across the patches. However, the ratio of fluorescent Pseudomonas spp./ total bacteria was 1/22, 1/15.4, 1/3.5 and 1/2.9 in the disease free area, the front margin of the patch, in the damaged part of the patch, and in the recolonized central part respectively. Furthermore, in this last mentioned zone, 44 to 82% of the fluorescent Pseudomonas spp. were antagonistic in vitro to Gga, whereas only 12 to 34% from the disease free area were antagonistic. So the development of take-all on turf induced quantitative and qualitative changes in populations of fluorescent pseudomonads. The remission of the disease in the center was correlated to higher amount of antagonistic fluorescent pseudomonads in this part of the patches. This typical patch with the well defined zones can provide a good model for the study of changes in bacterial populations related to the build up of take-all decline.     

Transformation of Gaeumannomyces graminis with β‐Glucuronidase Reporter and Hygromycin Resistance Genes

Take‐all disease is caused by Gaeumannomyces graminis, (Sacc.) Arx & D. Olivier, a soil‐borne fungus, which colonizes the root and crown tissue of many members of the Poaceae plant family. This fungus is able to grow along the surface of roots as darkly pigmented runner hyphae, which has the ability to penetrate the root. Here, we describe a genetic transformation of G. graminis var. graminis by using polyethylene glycol (PEG)‐based protoplast transformation. Fungus cells were transformed with a plasmid, pHPG, containing the gusA reporter gene that codes for β‐glucuronidase (GUS) and the hph gene for hygromycin resistance as the selectable marker. A de novo transformant selection assay was developed to identify the putative transformants that were expressing the hph gene. In addition, the transformed cells maintained the ability to infect the plant tissues. The GUS‐expressing fungus can be used to study fungal infection processes including fungal penetration, colonization and the role(s) of melanin during pathogenesis. Thus, this study is the first report of G. graminis var. graminis transformed with a visibly detectable reporter gene that provides a useful tool to a better understanding of host–Gaeumannomyces interactions.     

Comparison of natural and artificial epidemics of take-all in sequences of winter wheat crops

Winter wheat was grown for six successive years (Expt 1) and for three successive years (Expt 2) in field experiments on different soil types. Artificial inoculum of the take-all fungus (Gaeumannomyces graminis var. tritici cultured on autoclaved oat grains) was incorporated in the soil of some of the plots just before, or at, sowing of the first winter wheat crop. Expt 1 tested the incorporation of similar amounts of inoculum (212 kg ha^-1) at different depths. Expt 2 tested different amounts of inoculum at the same, shallow depth. Early sowing (September), late sowing (October) and spring inoculation were additional treatments, applied to the first crop only, in Expt 2. Seasonal factors apart, the disease outcome in the first year after inoculation depended on amounts and placement of applied inoculum, as well as date of sowing. Deeper inoculum resulted in less disease (Expt 1). Severe take-all was produced in Expt 2 by incorporating inoculum shallowly in sufficient quantities (400 kg ha^-1 or more). Less inoculum (200 kg ha^-1) generated less disease, especially in earlier-sown plots. Differences in disease amongst inoculum treatments were greatest in the first year and diminished subsequently, particularly where sowing had been early in the first year. In Expt 1, where first crops exposed to artificial inoculum developed moderate-to-severe disease, disease in subsequent second and/or third crops was less. In the fourth crop a second peak of disease occurred, coinciding with a first peak in sequences without added inoculum. Take-all decline (TAD) appeared to be expressed in all sequences thereafter. In Expt 2 in sequences without added inoculum, TAD occurred after a peak of disease in the second crop. Where 400 kg ha^-1 or more of inoculum were added, disease was severe in the first year and decreased progressively in successive years. Disease was less patchy in plots that received artificial inoculum. However, it remains uncertain mat severe disease caused by artificial inoculation achieved an early onset of true TAD. The infectivity of the top 12 cm of soil in the first 3 yr of Expt 1, determined by bioassay, depended on the depth of added inoculum and amount of disease in subsequent crops. However, at the time of the naturally occurring peak of disease severity (in either inoculated or non-inoculated plots) it did not predict either disease or TAD. Differences and similarities amongst epidemics developing naturally and those developing from different amounts and placement of applied inoculum have been revealed. The epidemiological implications of adding inoculum and the potential value of artificially-created epidemics of take-all in field trials are discussed.     

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.     

Analysis of Mycelial Growth Rates and RAPD-PCR Profiles in a Population of Gaeumannomyces graminis var. tritici Originating from Wheat Plants Grown from Fungicide-treated Seed

Linear mycelial growth rates of 70 isolates of Gaeumannomyces graminis var. tritici on agar medium amended or unamended with the fungicide silthiofam were not correlated. Mycelial growth rate was not influenced by the fungicide applied to the seed of the plants from, which the isolates originated. DNA polymorphism determined by randomly amplified polymorphic DNA (RAPD) polymerase chain reaction was used to assess genetic variation among isolates. Thirty RAPD markers generated with five arbitrary 10‐mer primers revealed DNA polymorphism suitable for assessing variability in this fungal population. Cluster analysis of RAPD data identified two groups at the 54% similarity level. There was a significant relationship between the presence of 11 markers and sensitivity to silthiofam.     

Use of a root assessment tray for the detection of take-all on lateral roots of wheat seedlings

A root assessment tray was designed for the meticulous assessment of take-all on wheat seedling roots from soil bioassays. Subsequently, the detection of lateral root infections (in addition to the more obvious infections on main axes of seminal roots) resulted in increased estimates of propagule numbers of the take-all fungus (Gaeumannomyces graminis var.tritici) for 196 of the 368 soil samples bioassayed in a field study conducted in Western Australia between 1984 and 1986.     

Control of Mn status and infection rate by genotype of both host and pathogen in the wheat take-all interaction

Take-all is a world-wide root-rotting disease of cereals. The causal organism of take-all of wheat is the soil-borne fungus Gaeumannomyces graminis var tritici (Ggt). No resistance to take-all, worthy of inclusion in a plant breeding programme, has been discovered in wheat but the severity of take-all is increased in host plants whose tissues are deficient for manganese (Mn). Take-all of wheat will be decreased by all techniques which lift Mn concentrations in shoots and roots of Mn-deficient hosts to adequate levels. Wheat seedlings were grown in a Mn-deficient calcareous sand in small pots and inoculated with four field isolates of Ggt. Infection by three virulent isolates was increased under conditions which were Mn deficient for the wheat host but infection by a weakly virulent isolate, already low, was further decreased. Only the three virulent isolates caused visible oxidation of Mn in vitro. The sensitivity of Ggt isolates to manganous ions in vitro did not explain the extent of infection they caused on wheat hosts. In a similar experiment four Australian wheat genotypes were grown in the same Mn-deficient calcareous sand and inoculated with one virulent isolate of Ggt. Two genotypes were inefficient at taking up manganese and were very susceptible to take-all, one was very efficient at taking up manganese and was resistant to take-all, and the fourth genotype was intermediate for both characters. All genotypes were equally resistant under Mn-adequate conditions.     

Comparison of rates of natural senescence of the root cortex of wheat (with and without mildew infection), barley,oats and rye

Summary In comparative tests in a glasshouse, the cortex of oat and rye roots senesced more slowly than the cortex of wheat and barley roots. Of the cereals tested, wheat showed the most rapid rate of root cortical senescence, and the rate was unaffected by inoculation of leaves withErysiphe graminis. The results are discussed in relation to infection by root pathogens.     

An antifungal and plant growth promoting metabolite from a sterile dark ectotrophic fungus

A sterile dark ectotrophic fungus isolated from roots of an Australian native grass, Neurachne alopecuroidea produces compound 1 in liquid cultures. The structure of the metabolite was determined by spectroscopic and X-ray diffraction studies. The metabolite shows activity against phytopathogens and plant growth promoting activity, properties that are also expressed in vivo by the ectotrophic fungus.     

Relationships between take-all,soil conduciveness to the disease,populations of fluorescent pseudomonads and nitrogen fertilizers

Take-all of wheat, caused by Gaeumannomyces graminis var tritici (Ggt), is reduced by ammoniacal fertilizers as compared to nitrate sources. This influence of nitrogen on the disease is only observed on nodal roots at flowering. But soil conduciveness to take-all, as measured in a soil bioassay, is modified earlier. Forty days after nitrogen application at early tillering, the NH₄-treated soil became less conducive than the NO₃-treated one. When nitrogen applications are done at sowing and at tillering, differences in disease propagation between the two soils are enhanced. Results from four years of experimentation show that when the level of natural soil inoculum is high, disease severity is reduced by ammonium, showing an effect on the parasitic phase of Ggt. At a low level of natural inoculum the effect of the source of nitrogen is mainly observed on the percent of infected plants, indicating that the saprophytic and preparasitic phases are affected. Rhizospheric bacterial populations increase from sowing to tillering, but differences on take-all conduciveness after tillering are not correlated with differences in the amounts of aerobic bacteria or fluorescent pseudomonads isolated from soils treated with different sources of nitrogen. Qualitative changes in fluorescent Pseudomonas spp. populations, like in vitro antagonism, are more likely to explain differences in soil conduciveness to take-all than are quantitative changes in this group. Nevertheless, the introduction of Ggt in a cropped soil leads to a greater increase in fluorescent pseudomonads populations than in total aerobic bacteria.The delay between reducing soil conduciveness and reducing disease in the field with ammonium nitrogen fertilization, the qualitative change of fluorescent pseudomonads populations and the role of necroses in rhizobacteria multiplication, provide information leading to our representation of a dynamic model based on the differentiation of the wheat root system into seminal and nodal roots.