Interpretive value of symptoms of infection by Gaeumannomyces graminis in wheat seedlings grown in sand culture

Symptoms of infection by the fungal pathogen Gaeumannomyces graminis were studied in roots of wheat seedlings grown in sand culture in order to assess resistance. Three sets of wheat were studied, 10 cultivars previously thought to differ in infection, and 44 and 54 segregating families from two crosses between four of these wheats, Aus1080 × Condor and Termu89–72 × Bayonet respectively. The isolates of G. graminis employed had previously been selected for differences in virulence. The interpretive value of symptoms of infection were considered, by comparison with direct measurement of the spread of hyphae from a fixed point of inoculation, and by repeatability of measurements over experiments. Two symptoms of infection, cortical browning and stelar blackening, are thought to be host mechanisms to resist hyphal invasion and were suppressed by rapid colonisation of root tissues. A third symptom, general discolouring of tissue, appeared to be a necrotic symptom of aggressive colonisation. Since only root segments distal to the point of infection were studied, stelar blackening appeared the least flexible measure of severity of infection. Cortical browning appeared to be influenced by nutritive status of seedlings, and was negatively associated with the incidence of necrotic discolorations. Wheats differed consistently and mostly in cortical browning, and while also differing in extent of necrotic discoloration, difficulty in quantifying the latter character limited its value in assessing resistance to G. graminis in this study.     

Signal Molecules Inducing Systemic Resistance and Susceptibility in Barley Seedlings

Exudate collected from the cut end of barley seedlings exhibited both activities that induced systemic resistance and susceptibility against Erysiphe graminis f. sp. hordei race Hh4 depending on the time after pruning. Exudates collected between 3–6 h after pruning showed maximum activity that induced systemic resistance, whereas those during 9–12 h conversely induced susceptibility in barley seedlings. The accumulation of antifungal substances in barley leaves correlates to the timing, of induced resistance. The antifuntingal substances were watersoluble and severely affected the infection of E. graminis f. sp. hordei.     

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.     

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.     

Further properties of peanut clump virus and studies on its natural transmission

Purified preparations of particles of peanut clump virus (PCV) had A₂₆₀/A₂₈₀ values (corrected for light scattering) of 1.00. They contained rod-shaped particles with sedimentation coefficients of 183 S and 224 S, and a density in CsCl of 1.32 g/ml. PCV infected 36 species in 8 plant families. No serological relationship was detected between PCV and barley stripe mosaic, beet necrotic yellow vein, Nicotiana velutina mosaic and tobacco mosaic viruses. PCV was seed-borne for two generations in groundnut (Arachis hypogaea) but was not seed-borne in great millet (Sorghum arundinaceum), Phaseolus mungo or Nicotiana benthamiana. Seedlings of groundnut, great millet and wheat (Triticum aestivum) became infected when grown in soil from groundnut fields with outbreaks of clump disease, and the infectivity of soil survived air-drying at 25°C for 3 months. Groundnut seedlings became infected when grown in sterilised soil contaminated with washed roots of naturally-infected S. arundinaceum but not in soil to which roots of naturally infected groundnut or shoots of infected groundnut were added, or in which mechanically inoculated groundnut seedlings were grown at the same time. The patchy distribution of PCV in a crop was related to the infectivity of the soil for groundnut and to the presence of Polymyxa graminis resting spores which could be detected in the roots of S. arundinaceum bait seedlings, but not in those of groundnut. The results indicate that PCV is transmitted by a vector that is resistant to air-drying and closely associated with S. arundinaceum roots. For these reasons P. graminis is thought to be the vector of PCV.     

Fusarium oxysporum f. sp. radicis-lycopersici can Induce Systemic Resistance in Barley Against Powdery Mildew

Drench inoculation of the undisturbed roots of barley seedlings with Fusarium oxysporum f. sp. radicis‐lycopersici (FORL) significantly reduced the primary infection frequency of Blumeria graminis f. sp. hordei (BGH) on the first leaves. The length of secondary hyphae and subsequent conidial production of BGH were also found to be significantly reduced by preinoculation with FORL. The reduction in infection frequency was observed as early as 48 h after inducer treatment, namely when plants were challenge‐inoculated immediately following inoculation with FORL. The induced resistance continued up to 16 days after treatment as indicated by the reduction in infection frequency, up to 22 days after treatment when evaluated as a reduction in the length of secondary hyphae, and up to 35 days after treatment when evaluated as a reduction in conidial production. Characteristics of FORL that may explain its success as an inducer of resistance against barley powdery mildew are discussed.     

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.     

Confirmation of the transmission of barley yellow mosaic virus (BaYMV) by the fungus Polymyxa graminis

Resting spores (cystosori) of Polymyxa graminis, selected from roots of barley plants infected with barley yellow mosaic virus (BaYMV), were used to start mono-fungal sand cultures. Out of 20 attempts using over 800 cystosori, P. graminis became established in 12, and in two of these BaYMV symptoms also occurred. BaYMV was detected by ELISA in extracts of dried roots heavily infected with cystosori and in zoospores of P. graminis. Calculations suggested that, on average, each zoospore carried less than 100 virus particles. In two virus acquisition experiments, non-viruliferous isolates of P. graminis failed to acquire BaYMV from roots of mechanically-inoculated plants. In two further experiments, non-viruliferous isolates were grown on rooted tillers produced from healthy plants and those infected with BaYMV by either vector or mechanical inoculation. Zoospores and cystosori of P. graminis subsequently transmitted the virus, but only from plants where it had been introduced by the vector. Repeated mechanical transmission appeared to have selected a strain of virus that could not be acquired and/or transmitted by the vector. The results provide convincing evidence that P. graminis is a vector of BaYMV but suggest that, in natural populations, only a small proportion of spores may be viruliferous.     

Regulation of resistance and susceptibility in wheat–powdery mildew pathosystem with exogenous cytokinins

Dose–response relationship between resistance of wheat seedlings (Triticum aestivum, cultivar Zarya) to Erysiphe graminis f. sp. tritici Marchal. (Syn. Blumeria graminis), a causal organism of wheat powdery mildew and exogenous zeatin has been investigated. Two-week-old seedlings were inoculated with the pathogen. Zeatin or zeatinriboside were added to the nutrient solution immediately after inoculation. The dose–response curve of cytokinin in the most cases was multiphasic, with peaks of increased susceptibility occurring at 0.25–1.5 and 1.5–9 μM cytokinin, separated by a region of increased resistance at 0.5–3 μM cytokinin. The change in mineral nutrition or simultaneous treatment with thidiazuron revealed alterations of the dose–response curve ranging from a curve with maximum of resistance to a curve with maximum of susceptibility. Both multiphase nature of dose–response and its variability were proposed as possible explanations for earlier observed discrepancies in experimental data on modification of disease resistance by cytokinins. A mathematical model for two metabolic processes with substrate inhibition connected in-series was suggested to explain the multiphase dose–response. In this model, the product of the first reaction was used as substrate for the second reaction. Numerical experiments showed the changes in the shape of dose–response curve with changes in parameters dependent of cytokinin metabolism.     

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.     

Direct and indirect fluorescent antibody staining ofOphiobolus graminis Sacc. in culture and in the rhizosphere of cereal plants

Fluorescein isothiocyanate (FITC) conjugates were prepared to whole-cell and cell-wall fractions of four cultures ofOphiobolus graminis W1, W2, O1 and O₂. Homologous and heterologous direct and indirect FA staining of the four cultures ofO. graminis gave positive staining in all reactions. This indicated that the four cultures could not be differentiated by fluorescent antibody (FA) staining. Species specificity of the conjugates was shown by the staining ofO. graminis hyphae in the rhizosphere of wheat and oat roots. Plant tissues were not stained. Furthermore out of 52 rhizosphere isolates stained with whole-cell and cell-wall conjugates of the four cultures ofO. graminis, only 7 cross-reacted with the whole-cell conjugates whereas none cross-reacted with the cell-wall conjugates.These results indicate the potentiality of the FA staining technique as a serological tool in localizing, and identifyingO. graminis amongst mixed fungal populations in the rhizosphere of roots.     

Subtractive Cloning of DNA from Polymyxa graminis– an Obligate Parasitic Plasmodiophorid

A simple subtractive hybridization was applied for cloning of Polymyxa graminis deoxyribonucleic acid (DNA). Total DNA preparations from concentrated P. graminis resting spores and from roots of non‐infested (healthy) barley were digested with different restriction enzymes and batch hybridized, followed by cloning in a plasmid vector. Sequencing and blast analysis of coincidentally selected clones enabled construction of polymerase chain reaction primers specific to P. graminis DNA. Four Polymyxa‐specific primers showed different affinities to DNA of type I and type II P. graminis and to DNA of P. betae.     

Investigations on fungicides: The systemic fungicidal activity of certain N-carboxymethyl dithiocarbamic acid derivatives

The systemic activity of thirty-three N-substituted S-esters derived from dithiocarbamic acid was investigated by assessing their ability to reduce the infection of broad-bean seedlings by Botrytis fabae and of wheat seedlings by Erysiphe graminis, following application to the roots or cut shoots of the host. Marked systemic activity against mildews was shown by the N-carboxymethyl dithiocarbamates, by S-carboxymethyl-N, N-dimethyl-dithiocarbamate (G₃₃) and by procaine and 6-azauracil. The effect was not very host-specific since most compounds showing high activity in wheat also showed activity in pea, cucumber and, to a smaller extent, apple. All the materials tested showed a much lower level of systemic activity in broad-bean seedlings against B. fabae. Measurements were also made of the uptake, translocation, phytotoxicity and the stability within plant tissues of some of these compounds. The degree of systemic activity which they show is discussed in relation to these and other properties of the compounds.     

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.     

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.     

Effect of Ophiobolus graminis infection on the assimilation and distribution of 14C in wheat

The uptake of ¹⁴C and movement of ¹⁴C-labelled assimilates in wheat plants inoculated with Ophiobolus graminis was examined following exposure of the second youngest leaf to ¹⁴CO₂. Autoradiographs of plants with infected seminal roots showed that assimilates were not translocated past the sites of root infection but accumulated in the undamaged portions of infected root systems, in particular the developing crown roots. There was no evidence that assimilates accumulated in the vicinity of O. graminis lesions. The net assimilation of ¹⁴CO₂ by wheat plants over a 5 h feeding period was not significantly affected by O. graminis infection. However, infection reduced the amount of ¹⁴C lost through respiration. Infection delayed the transfer of labelled assimilates from the fed leaf to the remainder of the plant but increased the proportion translocated to the roots. The latter effect was not apparent when infected plants were continuously irrigated during, and for 20 h following, the feeding period.     

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.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

A venacin, the resistance factor in oat roots against Ophio-bolus graminis var. graminis, and a related triterpeneglycoside, aescin, induced a rapid release of K⁺ from mycelia of Opbio-bolus graminis and Neurospora crassa, suspended in phosphate buffer. N. crassa also released Mg²⁺ whereas no outflux of Mg²⁺ was found from O. graminis. The inhibitors induced a release of inorganic phosphate into acetate buffer from Neurospora crassa. The amount of inorganic phosphate in the mycelia decreased when O. graminis and N. crassa were treated with the inhibitors in phosphate buffer. In other media the inhibitors had weak or no effects on the ion contents of the mycelia. The effect of aescin was low in Aspergillus niger and nil in Pythium irregulare. However, high amounts of K⁺, Mg²⁺, and phosphate ions were lost to the medium when the mycelium of P. irregulare, washed with distilled water, was suspended in different buffers. The ions lost were reabsorbed during the experimental period. The leakage of ions indicates that the plasma membrane of the growth sensitive fungi is severely affected by the inhibitors, while a corresponding effect on the growth insensitive fungi does not take place.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism 3. Effect on Uptake of Potassium, Magnesium and Inorganic Phosphate

A venacin, the resistance factor in oat roots against Ophiobolus graminis var. graminis, and a related triterpeneglycoside, aescin, inhibited the uptake of K⁺ and Mg²⁺ in the fungal mycelium both in phosphate and succinate buffers. The uptake of the cations in Neurospora crassa was similarly inhibited when the inhibitors were dissolved in phosphate or acetatebuffer, while no decrease in the uptake of K⁺ and Mg²⁺ was observed when the inhibitors were dissolved in succinate buffer. The uptake of cations in Aspergillus niger and Pythium irregulare was more or less unaffected by aescin. The uptake of inorganic phosphate was in no case inhibited, but some decrease of the accumulation of inorganic phosphate in Ophiobolus graminis and Ncurospora crassa due to inhibitor treatment in phosphate buffer was observed. No accumulation of Ca²⁺ was observed in any of the tested fungi.