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.     

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.     

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.     

Water use and phosphorus and potassium status of wheat seedlings colonized byGaeumannomyces graminis orPhialophora radicicola

Summary The water consumption and levels of phosphorus, potassium, and total minerals were measured for wheat seedlings colonized byGaeumannomyces graminis var.tritici, Phialophora radicicola var.radicicola, orPhialophora radicicola var.graminicola. Infection byG. graminis resulted in a considerable reduction in water consumption, and reduced level of phosphorus when the supply of phosphorus to the seedlings was plentiful. Colonization byP. radicicola var.radicicola increased levels of phosphorus and potassium, but these increases varied according to the isolate of the fungus and the supply of phosphorus and potassium available to the seedlings. Colonization byP. radicicola var.graminicola resulted in reduced water consumption by the seedlings.The results are discussed in relation to stelar cell wall thickening in wheat roots colonized byP. radicicola, and the effects on nutrient uptake of mycorrhizal root systems.     

Failure of in vitro growth inhibition by cysteine to differentiate between Australian Gaeumannomyces graminis isolates pathogenic and non-pathogenic to oats

Radial growth of oat and non oat-attacking Australian isolates of Gaeumannomyces graminis was greatly inhibited by increasing concentration of DL-cysteine in basal medium agar, and growth was completely inhibited by cysteine concentrations of 3 μM. As a group, isolates of G. graminis var. tritici (both oat and non oat-attacking forms) were more inhibited than isolates of G.graminis var.avenae at 1 μM cysteine, but differences did not occur at other concentrations. Isolates of a lobed-hyphodiate fungus similar to G. graminis var. graminis were more tolerant of cysteine than other isolates. The findings indicate that in vitro inhibition of Australian G. graminis isolates by cysteine is not useful for differentiation between oat and non oat-attacking types, and is unlikely to be fundamentally related to the ability of isolates to attack oats.     

Detoxification of Benzoxazolinone Allelochemicals from Wheat by Gaeumannomyces graminis var. tritici, G. graminis var. graminis, G. graminis var. avenae, and Fusarium culmorum

The ability of phytopathogenic fungi to overcome the chemical defense barriers of their host plants is of great importance for fungal pathogenicity. We studied the role of cyclic hydroxamic acids and their related benzoxazolinones in plant interactions with pathogenic fungi. We identified species-dependent differences in the abilities of Gaeumannomyces graminis var. tritici, Gaeumannomyces graminis var. graminis, Gaeumannomyces graminis var. avenae, and Fusarium culmorum to detoxify these allelochemicals of gramineous plants. The G. graminis var. graminis isolate degraded benzoxazolin-2(3H)-one (BOA) and 6-methoxy-benzoxazolin-2(3H)-one (MBOA) more efficiently than did G. graminis var. tritici and G. graminis var. avenae. F. culmorum degraded BOA but not MBOA. N-(2-Hydroxyphenyl)-malonamic acid and N-(2-hydroxy-4-methoxyphenyl)-malonamic acid were the primary G. graminis var. graminis and G. graminis var. tritici metabolites of BOA and MBOA, respectively, as well as of the related cyclic hydroxamic acids. 2-Amino-3H-phenoxazin-3-one was identified as an additional G. graminis var. tritici metabolite of BOA. No metabolite accumulation was detected for G. graminis var. avenae and F. culmorum by high-pressure liquid chromatography. The mycelial growth of the pathogenic fungi was inhibited more by BOA and MBOA than by their related fungal metabolites. The tolerance of Gaeumannomyces spp. for benzoxazolinone compounds is correlated with their detoxification ability. The ability of Gaeumannomyces isolates to cause root rot symptoms in wheat (cultivars Rektor and Astron) parallels their potential to degrade wheat allelochemicals to nontoxic compounds.     

The potential of non-pathogenic Gaeumannomyces spp., occurring naturally or introduced into wheat crops or preceding crops, for controlling take-all in wheat

Take‐all disease (Gaeumannomyces graminis var. tritici) in wheat crops is known to be suppressed by naturally occurring antagonistic fungi, closely related to the pathogen, that infect grasses and cereals. This form of suppression was re‐investigated because of the changing importance and role of grass weeds and grass covers in arable farming. Natural populations of the competitive fungus Gaeumannomyces cylindrosporus, allowed to develop under rye‐grass, were more effective than artificially introduced populations in suppressing the development of take‐all in following wheat crops. To be effective, the antagonist needs to be present before the start of wheat cropping. Introducing G. cylindrosporus, but not G. graminis var. graminis (a potential antagonist that is faster growing), into a previous crop, or just after the previous crop, sometimes suppressed take‐all, but the effect was small. It is concluded that, for any future attempts at biocontrol by these fungi, they should be introduced into a preceding crop not susceptible to take‐all. Take‐all inoculum in the soil should be at a minimum and effective hosts of the take‐all pathogen must not be present as weeds or volunteers.     

DNA Probe for Identification of the Take-All Fungus, Gaeumannomyces graminis

A 4.3-kilobase mitochondrial DNA fragment was cloned from Gaeumannomyces graminis var. tritici, the causative agent of take-all disease of wheat. Although this DNA fragment hybridized with all three varieties of G. graminis, it showed little homology with DNA from other fungi and thus should be useful for identification of Gaeumannomyces sp. recovered from infected plants.     

Virus-like particles in the take-all fungus, Gaeumannomyces graminis

Isometric virus-like particles (VLP) measuring 35 nm and 27 nm occurred in cultured mycelium of Gaeumannomyces graminis var. tritici and G. graminis var. avenae. These VLP had, respectively, sedimentation coefficients (s°_{20, W}) 148S and 110S and ultraviolet absorption (maximum 260 nm, minimum 240 nm) typical of nucleoprotein (A260:280 = 1.6, A260:240 = 1.2). Preparations of the 35 nm particles had two major and one minor component in caesium chloride, and 27 nm particles had two components (buoyant densities 1.37, 1.36, 1.30, 1.35, and 1.29 g/cm³ respectively). Preparations of the 35 nm particles or 35 nm plus 27 nm particles had one major protein species with estimated molecular weight 70000 daltons. The 35 nm VLP were absent from 11 isolates of G. graminis var. tritici from first cereal crops after fallow or non-susceptible break crops; two of these contained the 27 nm particles. More than half of 145 isolates, from cereals after 2–12 consecutive susceptible crops, contained either 35 nm or 27 nm VLP. VLP were not confined to G. graminis isolates from soils exhibiting ‘take-all decline’ nor consistently associated with weak pathogenicity or with isolates of unusual growth, morphology, pigmentation, lysis or readiness to form perithecia. Isolates with one kind of particle were mostly more pathogenic and those with both kinds less pathogenic than isolates without VLP. The proportion of isolates with 27 nm and 35 nm particles increased progressively in samples from different consecutive crops during the first 9 years of cropping, then decreased. Isolates did not gain or lose VLP during infection and re-isolation from wheat seedlings grown in sand. Four ‘infected’ isolates were freed from VLP either by culturing ascospores or by growing hyphal tips excised from colonies kept near their thermal death point. Both VLP appeared in cultures which had undergone anastomosis with infected isolates.     

Thickening and browning of cortical cell walls in seminal roots of wheat seedlings infected with Gaeumannomyces graminis var. tritici

This paper examines a little studied reaction of wheat roots to invasion by Gaeumannomyces graminis var. tritici, namely the thickening and browning of cortical cell walls. Examination was confined to distal segments of young seminal roots grown in sand. Browning and thickening of walls of cells were associated with the lignification of tissue and appeared to be a response of living or recently live cells to invasion by weakly virulent isolates. Wheat genotypes differed in their ability to thicken and lignify cell walls, and the difference between two wheat cultivars appeared to be under simple genetic control. There was some evidence that cortical browning temporarily retarded radial invasion by hyphae of G. graminis into young seminal roots of wheats.     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

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.     

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.     

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.     

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.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.     

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.     

Intermittent wetting of soils at high temperature reduces survival of the take-all fungus

Two pot experiments using naturally infested soil and a range of watering regimes were conducted to study the possible effect of level and frequency of wetting of hot soil (to simulate the period between growing seasons in Western Australia) on inoculum of the take-all fungus (Gaeumannomyces graminis var.tritici). In combination with the high soil temperatures, all watering regimes reduced infectivity and propagule number of the take-all fungus, this reduction being absent in dry soils.     

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.