Control of Ophiobolus patch in Agrostis turf using avirulent fungi and take-all suppressive soils in pot experiments

The incorporation of avirulent fungi such as Gaeumannomyces graminis var. graminis, an avirulent isolate of G. graminis var. tritici, a Phialophora sp. with lobed hyphopodia synonymous with Phialophora radiciola var. radicicola sensu Deacon and P. radicicola var. graminicola at the time of seeding Agrostis turf in pots of sterilised soil completely controlled Ophiobolus patch disease. The addition of a 5 mm layer of take-all suppressive (TAS) soils, artifically developed by the repeated addition of live mycelium of the varieties avenae, tritici and graminis of G. graminis to soil, controlled the disease to a lesser extent. However, a 20 mm layer of a TAS soil developed from live mycelium of G. g. avenae almost completely suppressed the disease. A survey of 66 golf and bowling greens in four states of Australia showed that P. r. graminicola was the most prevalent avirulent fungus.     

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.     

Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.     

A comparative study of Phialophora radicicola, an avirulent fungal root parasite of grasses and cereals

The biology and infection-behaviour of a typical isolate of Phialophora radicicola Cain have been compared with those of a representative isolate of Ophiobolus graminis (Sacc.) Sacc. Both species can utilize a nitrate source of nitrogen and both require thiamine and biotin for growth on inorganic nitro-gen; P. radicicola, but not O. graminis, was able to synthesize biotin when grown on asparagine as a nitrogen source. The pH range for good growth of P. radicicola in nutrient solution was narrower than that for O. graminis, and its growth rate on agar was only one-third. P. radicicola was the more active decomposer of cellulose, and its cellulolysis adequacy index was I.66 as com-pared with a value of 0.33 for 0. graminis. In agreement with prediction from Garrett's (I966) hypothesis on the cellulolysis adequacy index, saprophytic survival of P. radicicola in wheat straw was shortened by additional soil nitrogen, which prolongs survival of O. graminis.P. radicicola was found to spread ectotrophically over the roots of wheat, oats and barley by runner hyphae indistinguishable from those of O. graminis, but cortical infection caused no necrosis and no discernible check to growth of the infected cereals, nor any significant decrease in grain yield of inoculated wheat grown to maturity. Pre-existing infection of wheat roots by P. radicicola retarded spread of infection by O. graminis; inoculation of several grass species with P. radicicola reduced the extent of infection by O. graminis of wheat following the grasses.     

Effect of temperature on growth of some avirulent fungi and cross-protection against the wheat take-all fungus

The linear growth rates of Gaeumannomyces graminis var. graminis, G. graminis var. tritici, Phialophora radicicola var. graminicola and a lobed hyphopodiate Phialophora sp. were studied on agar at various temperatures between 5 and 30 °C and on wheat roots at two temperature regimes (12 h at 7°/12 h at 13 °C and 12 h at 17°/12 h at 23 °C). On agar at 30 °C, the isolates of G. graminis graminis grew faster than those of G. graminis tritici and Phialophora sp. but three isolates of G. g. graminis grew more slowly than the other two fungi at 5 and 10 °C. Two other isolates of G. g. graminis were cold-tolerant and had growth rates comparable to those of G. g. tritici and Phialophora sp. at 10 °C. The growth rates of Australian isolates of P. radicicola graminicolu were similar to that of a British isolate and were about a third to a half those of the other three fungi at most temperatures. The growth rates of the fungi on wheat roots at the low and high temperature regimes were correlated with the growth rates on agar at 10 and 20 °C respectively. The correlation was better at low temperatures r= 0.81) than at high temperatures (r = 0.62). Cross-protection experiments using two G. g. graminis isolates which grow poorly at temperatures below 15 °C and a cold-tolerant isolate each of G. g. graminis and Phialophora sp. showed that, while all four fungi protected wheat against take-all at high temperatures (17/23 °C) as evidenced by less severe disease and significantly greater dry weights, only the cold-tolerant fungi were effective at low temperatures (7/13 °C). The use of cold-tolerant isolates of avirulent fungi in field experiments may result in better protection in the early stages of wheat growth when Australian soil temperatures are mostly below 15 °C.     

Effect of osmotic potential on the growth of Gaeumannomyces graminis and Phialophora spp.

The linear growth of 10 isolates each of Gaeumannomyces graminis var. graminis, G. graminis var. tritici and Phialophora graminicola and five isolates each of G. graminis var. avenae and a lobed-hyphopodiate Phialophora sp. was studied on osmotically adjusted agar at 20 °C. While most isolates of G. graminis var. avenae ceased growing at osmotic potentials of -60 bars (1 bar = 10⁵ Pa), six out of 10 isolates of G. graminis var. tritici grew at that potential. The growth of all isolates of G. graminis var. tritici and var. avenae ceased at -70 bars. In contrast, four out of 10 isolates of P. graminicola grew at -70 bars, but all stopped growing at -80 bars. Most of the isolates of G. graminis var. graminis and the lobed-hyphopodiate Phialophora sp. grew at -70 bars while three out of 10 isolates of G. graminis var. graminis and one out of five isolates of the lobed-hyphopodiate Phialophora sp. were capable of growth at -80 bars. None of the fungi grew at -90 bars. Detailed studies of the growth of two or three isolates each of the five fungi at 10, 20, 30 and 35 °C were carried out on osmotic agar controlled by the addition of either sodium chloride or potassium chloride. In general, similar reductions in growth occurred with decreasing osmotic potential regardless of the solute used. At 10 and 20 °C., all three isolates of P. graminicola showed optimal growth at about -5 bars while the other fungi grew fastest at -1²middot; bars. At 30 °C., one isolate of the lobed hyphopodiate Phialophora sp. and two isolates each of P. graminicola, G. graminis var. tritici and G. graminis var. avenae grew optimally at osmotic potentials of -10 to -15 bars. The other isolate of the Phialophora sp. and two isolates of G. graminis var. graminis studied grew optimally at the highest potential (-1·2 bars). However, at 35 °C the last three fungi exhibited optimal growth at osmotic potentials of-10 to -20 bars. The ecological significance of these results is discussed in relation to cross-protection against the take-all fungi by the avirulent fungi.     

Saprophytic survival of Gaeumannomyces graminis and Phialophora spp. at various temperature-moisture regimes

The saprophytic survival of the pathogen, Gaeumannomyces graminis var. tritici and two isolates each of three avirulent fungi, G. graminis var. graminis, Phialophora graminicola and a lobed-hyphopodiate Phialophora sp. was studied in two soil types under controlled temperature and moisture conditions in the laboratory. In general, the fungi survived longest in the cool, dry soil (15°C, < -10 MPa) followed by the warm dry soil (30°C, < -10 MPa). All the fungi were virtually eliminated from the warm, moist soil (30°C, -0.3 MPa) after 3 months. Survival was intermediate under cool, moist conditions (15°C, -0.3 MPa). Under cool, moist conditions, G. graminis var. graminis survived better than the other three fungi in the first 3 months in both soil types and continued to do so for a further 3 months in one soil. Both isolates of the lobed-hyphopodiate Phialophora sp. survived poorly in the two soil types being almost eliminated after 3 months. There were considerable differences between the survival of the two isolates each of G. graminis var. graminis and P. graminicola, especially under cool, moist conditions. Of the six avirulent isolates studied, one isolate of G. graminis var. graminis (DAR24167) survived best under the three temperature-moisture regimes which showed differences. It also survived better than the take-all fungus under moist, cool conditions and at a comparable rate under dry conditions. Therefore, this variation in survival should be considered when selecting antagonists for the biological control of take-all.     

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.     

Evidence for resistance in wheat cultivars grown in sand culture to the take-all pathogen, Gaeumannomyces graminis var. tritici

A laboratory method to inoculate seedlings uniformly with Gaeumannomyces graminis var. tritici is described. Resistance is defined via the rate of hyphal entry into the vascular tissue of host seedlings, and is measured by direct observation and by early stelar lesion development in seminal roots. The two scores for resistance are compared and evaluated, for infection with an isolate of low virulence. Evidence was obtained for resistance in the roots of wheat seedlings to G. graminis.     

Natural (non-pathogenic) death of the cortex of wheat and barley seminal roots,as evidenced by nuclear staining with acridine orange

Summary Nuclear staining with acridine orange was used to assess cell viability in the cortex of wheat and barley seminal roots from glasshouse and field experiments. Results from this method correlated well with nuclear assessments made in unstained or Feulgen-stained roots, and other evidence is presented to support the validity of the method. The pattern of root cortex death (RCD) was similar in wheat and barley and consistent over a wide range of conditions. Behind the extending root tip and zone of nucleate root hairs, nuclei disappeared progressively from the outer five (of six) cortical cell layers of the root axes, starting in the epidermis. Stainable nuclei remained in the sixth cell layer, next to the endodermis, and in most cell layers around the bases of root laterals and in a small region immediately below the grain. The onset of cell death was apparently related more to the age of a root region than to its distance behind the root tip, and it was not closely correlated with endodermal or stelar development assessed by staining with phloroglucinol/HCl. The rate of RCD was much faster in wheat than barley in both glasshouse and field conditions, and faster in some spring wheat cultivars than in others in the glasshouse. RCD occurred in sterile vermiculite and perlite and was not enhanced by the presence of soil microorganisms; nor was it enhanced in soil by the addition of the non-pathogenic fungal parasitesPhialophora radicicola var..graminicola orMicrodochium bolleyi. RCD is suggested to be endogenously controlled by the amount of photosynthate reaching the cortex. Its implications for growth of soil microorganisms and especially for growth and biological control of root-infecting fungi are discussed.     

Infection process of Gaeumannomyces graminis var. graminis on the roots and culms of rice

Crown sheath rot, caused by the ascomycete Gaeumannomyces graminis var. graminis that infects the root and the base of the culm of rice, causes early grains maturation, tiller death and reduced yield. As a paucity of information exists in the literature on the rice‐G. graminis var. graminis interaction at the microscopic level, this study aimed to gain novel insights into the infection process of this pathogen in the root and culm of rice using both light and scanning electron microscopy. In the roots, the fungus initially colonized the epidermal, exodermal and sclerenchyma cells. At 15 days after inoculation (dai), fungal hyphae colonized the cortex and clusters of perithecia were observed in the roots. At 20 dai, the fungus reached the central cylinder, and an intense fungal colonization at the base of the culm was observed that resulted in the formation of a mycelial mat on both adaxial and abaxial surfaces of the leaf sheaths. At 25 dai, fungal growth was noticed in the parenchyma cells, vascular bundles and airspaces. Perithecia emerged through the base of prophyllum and from the first leaf sheath at 30 dai. The results of this study provide new insights into the infection process of G. graminis var. graminis in rice and may help to find better control measures in reducing crown sheath rot development.     

Effects of shading and powdery mildew infection on senescence of the root cortex and coleoptile of wheat and barley seedlings,and implications for root- and foot-rot fungi

Summary Nuclear and cytoplasmic staining methods were used to study natural senescence of the root cortex and coleoptile of wheat and barley seedlings grown in glasshouse conditions. Coleoptiles of barley senesced more slowly than those of wheat, paralleling the known difference in rates of root cortex senescence in these cereals. The coleoptiles and root cortices of both cereals senesced more slowly in shaded than in unshaded conditions, but infection of the shoots of barley byErysiphe graminis had little effect on root cortex senescence. The results are discussed in relation to infection by root- and foot-rot fungi. Previous reports on the effects of illumination on take-all infection (Gaeumannomyces graminis) are explained. It is suggested that natural senescence of the coleoptile might affect establishment of infection by the eyespot fungus,Pseudocercosporella herpotrichoides, either directly or through the activities of competing microorganisms.     

Pathogenicity of ‘take-all’ fungus to oats: Its relationship to the concentration and detoxification of the four avenacins

Data for inhibition of the growth of Gaeumannomyces graminis var. tritici (Ggt) and var. avenae (Gga), Phialophora radicicola and Fusarium avenaceum, caused by avenacins, are presented. The avenacins found in all oat species examined are sufficient in quantity to totally suppress growth of wheat ‘take-all’ (Ggt), even old roots containing 25 μg/g (fr. wt). Fungal variants that can also attack oats [var. avenae (Gga)] show considerable variations in their tolerance to avenacin A-1, ec₅₀ values being 5–80 μg/ml. Nevertheless, all Gga isolates maintained some growth at avenacin A-1 concentrations as high as 200 μg/ml and it is this ability to grow, albeit slowly, at high concentrations that is the critical difference between Gga and Ggt strains. The pathogenicity towards oats of a range of isolates of Gga is related to the fungicidal activity of avenacins. Gga pathogenicity is shown to increase with poor nutrition of the oat hosts (poor illumination, lack of minerals). Fungal detoxification of avenacins produces mono-deglucosylavenacin A-1, bis-deglucosylavenacin A-1 and, in one case, tris-deglycosylavenacin A-1. Ggt strains left avenacin A-1 almost unaffected giving only traces of mono-deglucosyl product. Gga strains bring about mono- and bis-deglucosylation whilst Fusarium avenaceum causes mainly bis-deglucosylation. Mono-deglucosylavenacin is shown to be less inhibitory to Gga than is avenacin A-1, whilst the bis-deglucosyl compound is still less inhibitory.     

Rhizosphere microflora and colonization of wheat roots byGaeumannomyces graminis var.tritici after foliar application of urea and benomyl

The effect of foliar application of 2% urea and 0.6% benomyl on changes in colonization of the rhizosphere by microorganisms and of roots by the fungusGaeumannomyces graminis (Sacc.)Arx etOlivier var.tritici Walker was followed in vegetation glass-house experiments. Treatment with a urea solution resulted in increased counts of bacteria (82 %),Pseudomonas fluorescens (46 %),Agrobacterium sp. (31 %) and antagonistic bacteria with respect to the used fungus isolate and in a decreased occurrence of micromycetes (63 %). Treatment of wheat with a benomyl solution resulted in an increased count of bacteria (43 %) and a decreased occurrence ofP. fluorescens (16 %),Agrobacterium sp. (50 %) and fungi (67 %). After treatment with both compounds the infection of roots byG. graminis considerably decreased as compared with untreated plants. The results are discussed from the point of view of the effect of application of the studied compounds to upper parts of wheat on the microflora colonizing its roots.     

Methoxyhydroquinone, a Growth Inhibitor of Ophiobolus graminis, in Leaves of Oat Seedlings

A methanol extract of leaves of oat seedlings grown in sand cultures in the dark contained a compound which inhibited the growth of Ophiobolus graminis. The inhibitory factor was isolated and proved to be present in the plant as methoxyhydroquinone glucoside. The glucoside was readily hydrolysed to the corresponding aglucone. The methoxyhydroquinone, or possibly its oxydation product, methoxy-P-benzoquinone, was inhibitory to both Ophiobolus graminis var. graminis and Ophiobolus graminis var. avenae, whereas Fusarmm oxysporum var. lycopcrsici was not affected. Synthetic methoxyhydroquinone at 80 mg/l gave a 100% inhibition of Ophiobolus graminis var. graminis. After being exposed to 80 mg/l of the inhibitor for 24 h the mycelium was unable to initiate growth when transferred to a fresh nutrient solution. Only extracts from young leaves showed inhibitory activity, extracts from mature leaves giving no inhibition. The hydroquinone, or its glucoside, was not detected in roots of young seedlings, where avenacin was the only antifungal compound present.     

Wheat responses to aggressive and non-aggressive arbuscular mycorrhizal fungi

In southwestern Australia fields, colonization of wheat roots by arbuscular mycorrhizal fungi (AMF) is reduced due to repeated use of phosphate (P) fertilizers. We predicted AMF that aggressively colonize wheat roots at low P supply would also aggressively colonize at high P supply, but provide no additional P uptake benefit and reduce growth. Wheat (cv. Kulin) seedlings were non-mycorrhizal (NM) or inoculated separately with 10 isolates of AMF from wheat-belt soils in a glasshouse experiment. Kojonup loamy sand was supplied with P to provide suboptimal and supraoptimal P for growth of NM wheat in this soil. At low P supply, wheat growth was limited by P availability. All AMF isolates colonized wheat roots at 14 days after emergence of seedlings. At 42 days, percentage root length colonized (%RLC) was highest for two isolates of Scutellospora calospora, WUM 12(2) and WUM 12(3), followed by Glomus sp. WUM 51, G. invermaium WUM 10(1), Acaulospora laevis WUM 11(4) and Gigaspora decipiens WUM 6(1). These isolates, designated as `aggressive colonizers', ranged from 50 to 89%RLC. A second group of AMF ranged from 1 to 19%RLC at 42 days. This group, termed `non-aggressive colonizers', included Acaulospora spp. WUM 11(1), WUM 46, and WUM 49 and Glomus sp. WUM 44. High soil P supply increased seedling growth 2–3 fold, but reduced%RLC. Grouping of aggressive and non-aggressive AMF based on colonization rate at high P supply was similar to that at low P. At low P supply, only the two isolates of S. calospora increased wheat growth compared to the NM plant. The remaining aggressive and non-aggressive AMF reduced growth of wheat at low P, while aggressive colonizers reduced growth at high P. At low P supply, the aggressive colonizers increased shoot P concentration, while at high P, shoot P was not affected by AMF. Growth depression by aggressive colonizers was associated with reduced sucrose concentration in roots. Based on the negative growth response under low and high P fertility in the glasshouse, AMF could be expected to produce non-beneficial effects on wheat in the field depending on the P status of the soil and the aggressiveness of AMF in the community. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microbiota in Wheat Roots Evaluated by Cloning of ITS1/2 rDNA and Sequencing

Fungi (17 species), oomycetous organisms (four species of Pythium) and a plasmodiophorid (Polymyxa graminis) were recorded in wheat roots analysed by cloning of the total ITS1/2 rDNA and sequencing of representative clones. Roots of a fourth successive wheat crop were inhabited mostly by fungal pathogens including Gaeumannomyces graminis var. tritici, Monographella nivalis var. nivalis, Ophiosphaerella sp. and Helgardia anguioides. Roots of a first wheat crop were inhabited mostly by P. graminis and saprotrophic Pythium species. Results on fungal diversity and density were compared with those obtained by pure culture isolation and morphotyping. Only M. nivalis var. nivalis and H. anguioides were identifed in wheat roots by both the molecular and the pure culture isolation methods. New and additional evidence for the ecological roles of the species recorded is discussed.     

Leaf water and carbohydrate status of VA mycorrhizal rose exposed to drought stress

Summary Shoot water relations and carbohydrate levels were compared for droughted nonmycorrhizal and vesicular-arbuscular (VA) mycorrhizalRosa hybrida L. cv ‘Samantha’ plants grown with high and low phosphorus fertilization. Leaf diffusive conductance (g _i ) of plants colonized byGlomus intraradices Schenk and Smith andGlomus deserticola Trappe, Bloss and Menge were 2 × and 1.5× greater, respectively, than in nonmycorrhizal plants. Regardless of P fertilization, leaf osmotic and bulk water potentials were 0.5 to 1.1 MPa higher in mycorrhizal than in nonmycorrhizal plants. Leaf starch, chlorophyll and water contents while fructose, glucose and total soluble carbohydrates were lower. Level of P fertilization had no effect on water relations or soluble carbohydrate content of nonmycorrhizal roses. The water status of droughted rose was impoved more byG. intraradices than byG. deserticola. Washington State University College of Agriculture and Home Economics Research Center Scientific Paper No. 7375.     

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.     

Microassay for biological and chemical control of infection of tobacco byPhytophthora parasitica var.nicotianae

We developed a simple, rapid, small-scale assay for infection of tobacco seedlings byPhytophthora parasitica var.nicotianae. One 7-day-old tobacco seedling was placed in each well of a 96-well microtiter plate and inoculated with 500 zoospores ofP. parasitica var.nicotianae. After 72 h all of the inoculated seedlings of the susceptible cultivar, KY14, were infected, and the pathogen had produced sporangia that were visible on the surfaces of the seedlings. Sporangia did not develop on seedlings that were inoculated simultaneously with zoospores and either 1 µg/mL of the chemical fungicide metalaxyl or 5 µL of filtrate of a sporulated culture of the biocontrol agent,Bacillus cereus UW85. Seedlings of tobacco cultivar KY17 were infected byP. parasitica var.nicotianae, although mature plants of this variety are resistant to the pathogen. This microassay may facilitate the rapid screening of potential biological and chemical control agents and may be useful for studying mechanisms of infection and control ofPhytophthora spp. under hydroponic conditions.