小麦全蚀病菌致病性遗传规律研究*

利用弱致病性白化突变菌株与强致病菌株进行有性重组,研究了小麦全蚀病菌(禾顶囊壳小麦变种,Gaeumannomyces gramimis var. tritici)致病性的遗传规律。结果表明,以接种株地上部干重为致病性指标时,该菌对小麦的致病性为数量遗传性状,估计控制致病性的基因数为5 ~ 7个。弱致病菌株与强致病菌株杂交F1代致病性由弱至强呈连续分布,子代致病性的平均水平接近种亲代致病性的平均值。致病性的遗传力为54.36% ~ 71.00%,平均为62.32%。致病性表型易受环境因素的影响。菌落颜色与致病性无明显相关。     

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.     

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.     

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.     

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 (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.     

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.     

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。     

Variation in Sensitivity of Gaeumannomyces graminis to Antibiotics Produced by Fluorescent Pseudomonas spp. and Effect on Biological Control of Take-All of Wheat

Isolates of Gaeumannomyces graminis var. tritici, the causal agent of take-all of wheat, varied in sensitivity in vitro to the antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) produced by fluorescent Pseudomonas spp. shown previously to have potential for biological control of this pathogen. None of the four isolates of G. graminis var. avenae examined were sensitive to either of the antibiotics in vitro at the concentrations tested. The single isolate of G. graminis var. graminis tested was insensitive to PCA at 1.0 (mu)g/ml. Pseudomonas fluorescens 2-79 and Pseudomonas chlororaphis 30-84, both of which produce PCA, effectively suppressed take-all caused by each of two PCA-sensitive isolates of G. graminis var. tritici. PCA-producing strains exhibited a reduced ability or complete inability to suppress take-all caused by two of three isolates of G. graminis var. tritici that were insensitive to PCA at 1.0 (mu)g/ml. P. fluorescens Q2-87, which produces Phl, suppressed take-all caused by three Phl-sensitive isolates but failed to provide significant suppression of take-all caused by two isolates of G. graminis var. tritici that were insensitive to Phl at 3.0 (mu)g/ml. These findings affirm the role of the antibiotics PCA and Phl in the biocontrol activity of these fluorescent Pseudomonas spp. and support earlier evidence that mechanisms in addition to PCA are responsible for suppression of take-all by strain 2-79. The results show further that isolates of G. graminis var. tritici insensitive to PCA and Phl are present in the pathogen population and provide additional justification for the use of mixtures of Pseudomonas spp. that employ different mechanisms of pathogen suppression to manage this disease.     

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.     

Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici.

Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G. graminis var. tritici and other fungal root pathogens. Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots. Six independent, prototrophic Phz- mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings. Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library. These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear. These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.     

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.     

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.