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.     

Modelling and Estimation of the Relative Potential for Infection of Winter Wheat by Inoculum of Gaeumannomyces graminis Derived from Propagules and Infected Roots

Abstract A model was developed to estimate the mean number of infections of seminal roots of wheat exposed to two sources of inoculum of the take-all fungus Gaeumannomyces graminis var. tritici , in an experimental system. The sources comprise discrete propagules of initial, soil inoculum and infected roots of volunteer plants that had been infected by the initial inoculum, prior to the growth of crop plants. The model was based on the probability of escape from infection by individual roots ofthe crop plants. Parameter estimation was by maximum likelihood. A model was first fitted to data for infection of roots from the soil inoculum. This yielded estimates for the efficiency of soil inoculum to cause infection in the absence of volunteer plants. The parameter for efficiency of infection by soil inoculum was resolved into components for inoculum density, survival of inoculum and the probability of success of individual propagules. The model was extended to include simultaneous exposure of crop roots to soil inoculum and to root inoculum on the volunteer plants. The presence of volunteer seedlings prior to sowing of crop plants resulted in an increase in the effectiveness of inoculum to cause disease. Sowing date and soil condition, as affected by addition of sand, were shown to have significant effects on the efficiency of both sources of inoculum.     

Control of Gaeumannomyces graminis Var. tritici in Wheat by Isolates of the G. graminis var. graminis/Phialophora sp. (Lobed Hyphopodia) Complex under Field Conditions

In an experiment carried out under field conditions, wheat inoculated with Gaeumanmyces graminis var. tritics had less take-all and yielded significantly more grain when simultaneously inoculated with G. graminis var. graminis and a lobed hyphopodiate phialophora sp. then when unprotected with these fungi.     

Studies on the spread of Gaeumannomyces graminis var. tritici in wheat.

The effect of cultivations on the spread of Gaeumannomyces graminis var. tritici from a line source of inoculum, consisting of naturally infected stubble and roots, was recorded in the field over 2 yr. With the aid of cultivations, spread in the first wheat crop occurred frequently to a distance of 0·9 m and occasionally to a distance of 2·5 m. However, the following wheat crop was uniformly infected, probably as a result of a rapid build-up of background inoculum in the first wheat, so that any spread by cultivations was masked.     

Survival, growth and pathogenicity of Gaeumannomyces graminis var. graminis with different methods of long-term storage

The fungal plant pathogen Gaeumannomyces graminis var. graminis was preserved with 12 different storage methods. Five strains, each with unique morphological and pathological characteristics, were used for comparison of the methods. The storage treatments included potato-dextrose agar slants, with or without mineral oil, stored at either 4 C, 28 C or ambient temperature; colonized agar plugs placed in glycerol solution at either -75 C or -20 C; colonized agar plugs placed in sterile deionized water at either 4 C or ambient temperature; and mycelial growth on intact or precut pieces of filter paper, desiccated and stored at ambient temperature. Survival was evaluated at 6, 12, 24, 36, 48 and 120 mo. The three best treatments for survival were PDA slants, with or without mineral oil, and colonized agar plugs stored in water, all at ambient temperature. All five fungal strains were recovered from all four replicates at each sampling date for agar plugs stored in water at ambient temperature. The worst treatments were agar slants and agar plugs in water stored at 4 C and agar plugs stored in glycerol at -20 C. Morphological characteristics were not affected by storage treatments. In general, there were minimal or no effects on growth and pathogenicity for all strains for all storage treatments with survival. Colonized agar plugs stored in water at ambient temperature provides an economical storage method (materials and labor) that does not need an electrical power for long-term maintenance.     

Purification and Characterization of a Secreted Laccase of Gaeumannomyces graminis var. tritici

We purified a secreted fungal laccase from filtrates of Gaeumannomyces graminis var. tritici cultures induced with copper and xylidine. The active protein had an apparent molecular mass of 190 kDa and yielded subunits with molecular masses of 60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2,6-dimethoxyphenol (K_m = 2.6 × 10⁻⁵ ± 7 × 10⁻⁶ M), catechol (K_m = 2.5 × 10⁻⁴ ± 1 × 10⁻⁵ M), pyrogallol (K_m = 3.1 × 10⁻⁴ ± 4 × 10⁻⁵ M), and guaiacol (K_m = 5.1 × 10⁻⁴ ± 2 × 10⁻⁵ M). In addition, the laccase catalyzed the polymerization of 1,8-dihydroxynaphthalene, a natural fungal melanin precursor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These findings indicate that this laccase may be involved in melanin polymerization in this phytopathogen’s hyphae and/or in lignin depolymerization in its infected plant host.     

Disease in wheat genotypes naturally infected with Gaeumannomyces graminis var. tritici

Wheat genotypes consisting of seven homozygous lines and 40 segregate families were studied at two sites naturally infested with the take-all pathogen, Gaeumannomyces graminis var. tritici. The numbers of seminal and coronal roots infected with G. graminis and other root pathogens were recorded. The genotypes differed in infection with G. graminis, with little evidence of genotype × environment interactions. The incidence of G. graminis and Rhizoctonia solani was negatively associated, but the association did not greatly influence differences between wheats in infection with G. graminis. The distribution of R. solani was negatively associated with the severity of take-all at only one site. Of symptoms of infection with G. graminis, wheat genotypes differed most in incidence of deadheads, but differences were not consistent over environments, and were associated with earliness of maturity. Wheats differed more in expression of disease than in infection with G. graminis. The course of disease was deduced from associations between the incidence of pathogens and components of plant growth and yield. G. graminis was the dominant pathogen at both sites, and caused a yield loss of 0–15% at one site, and an average 62% loss at the other. More components of yield were affected where disease was most severe.     

Purification and characterization of a secreted laccase of Gaeumannomyces graminis var. tritici.

We purified a secreted fungal laccase from filtrates of Gaeumannomyces graminis var. tritici cultures induced with copper and xylidine. The active protein had an apparent molecular mass of 190 kDa and yielded subunits with molecular masses of 60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2, 6-dimethoxyphenol (Km = 2.6 x 10(-5) +/- 7 x 10(-6) M), catechol (Km = 2.5 x 10(-4) +/- 1 x 10(-5) M), pyrogallol (Km = 3.1 x 10(-4) +/- 4 x 10(-5) M), and guaiacol (Km = 5.1 x 10(-4) +/- 2 x 10(-5) M). In addition, the laccase catalyzed the polymerization of 1, 8-dihydroxynaphthalene, a natural fungal melanin precursor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These findings indicate that this laccase may be involved in melanin polymerization in this phytopathogen's hyphae and/or in lignin depolymerization in its infected plant host.     

Incidence of Gaeumannomyces graminis var. tritici and K deficiency increase rhizospheric denitrification

Wheat inoculated with the root pathogen Gaeumannomyces graminis var. tritici (Ggt) was grown in quartz silt at two levels of potassium nutrition. While in plants well supplied with K the incidence of Ggt did not affect plant growth, it reduced shoot and root weight of K deficient plants. Denitrification, measured by the acetylene inhibition technique and expressed as N₂O/mg root weight, was increased either by low K nutrition or by Ggt infection. Highest denitrification in the rhizosphere of plants was found with a combination of both, K deficiency and Ggt attack.     

小麦全蚀病菌原生质体制备的条件及再生菌株的致病性

分别利用崩溃酶、溶壁酶、纤维素酶和蜗牛酶酶解小麦全蚀病菌,进行原生质体的制备试验。结果显示,4种酶均能消化该菌细胞壁,获得一定数量的原生质体;产生原生质体效率最高的是溶壁酶,该酶在浓度为16 mg/mL时产生的原生质体数量最多,最佳的酶解时间为2~3 h,最适作用温度为28℃。制备的原生质体可以再生并与原始出发菌株具有相同的致病能力。     

Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici

Bacillus subtilis strain Z-14 has biological control activity against the take-all fungus Gaeumannomyces graminis var. tritici (Ggt). In Petri dishes, the crude extract from B. subtilis Z-14 culture filtrate reduced take-all severity in roots of wheat seedlings by 91.3% and potted plants by 69.8% compared to the Ggt-inoculated control. Treatment with the crude extract also significantly (P?<?.05) increased growth of roots’ average length, and fresh weight in comparison with those of the Ggt-inoculated control. B. subtilis Z-14 culture filtrate was relatively thermally stable with 88.2% of the antifungal activity being retained after being heated at 100°C for 30?min. Meanwhile, the antifungal activity remained almost unchanged (>95%) when the culture filtrate was exposed to a pH ranging from 3 to 8, but significantly reduced in basic conditions. This activity was not transferred to the organic solvent phase after treatment with organic extraction agents. B. subtilis Z-14 culture filtrate exhibited a broad spectrum of antifungal activities against various phytopathogenic fungi. Three homologs of iturin A (C14–16) were characterised by liquid chromatography-mass spectroscopy (LC-MS) and electrospray ionisation mass spectrometry/mass spectrometry collision-induced dissociation (ESI-MS/MS CID).     

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.     

Colonization of barley roots by endophytic fungi and their reduction of take-all caused by Gaeumannomyces graminis var. tritici

Fungal root endophytes obtained from natural vegetation were tested for antifungal activity in dual culture tests against the root pathogen Gaeumannomyces graminis var. tritici. Fifteen isolates, including Acremonium blochii, Acremonium furcatum, Aspergillus fumigatus, Cylindrocarpon sp., Cylindrocarpon destructans, Dactylaria sp., Fusarium equiseti, Phoma herbarum, Phoma leveillei, and a sterile mycelium, selected based on the dual culture test, were inoculated on barley roots in growth tubes under axenic conditions, both in the absence and presence of G. graminis var. tritici. All isolates colonized the rhizosphere and very often the root cortex without causing disease symptoms and without affecting plant growth. Eight isolates significantly reduced the symptoms caused by G. graminis var. tritici, and 6 of them reduced its presence in the roots.     

Changes in population structure of the soilborne fungus Gaeumannomyces graminis var. tritici during continuous wheat cropping

A method was developed to assess the genetic structure of Gaeumannomyces graminis var. tritici (Ggt) populations and test the hypothesis of an association between disease level in the field with changes in pathogen populations. A long-term wheat monoculture experiment, established since 1994, generated different take-all epidemics with varying the number of wheat crop successions in the 1999-2000 cropping season. Genetic polymorphism in Ggt populations was investigated over natural, local epidemics. Four populations of 30 isolates were isolated from necrotic wheat roots in a first, third, fourth, and sixth wheat crop in the same year. Each Ggt isolate was characterized with RAPD (Random Amplification Polymorphism DNA) markers and AFLP (Amplified Fragment Length Polymorphism) fingerprinting. Seventeen multilocus genotypes based on the combination of RAPD and AFLP markers were identified among all these populations. The 120 isolates were divided into two main groups, G1 and G2, according to bootstrap values higher than 86%, except for an unique isolate from the third wheat crop. Within each group, populations ranged between 93 and 100% similarity. Both groups included isolates collected from the first, third, fourth or sixth wheat crop. However, G1 group profiles dominated amongst isolates sampled in the first and the sixth wheat crops, whereas G2 group profiles largely dominated amongst isolates collected from the third and fourth wheat crops. Aggressiveness of group G2 (38%) was significantly greater than that of G1 (29.5%). These results suggest that changes in Ggt population structure occur during continuous wheat cropping. The distinction of two Ggt groups provides a simple basis for further spatio-temporal analysis of Ggt population during polyetic take-all decline.