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。     

全蚀病菌在玉米上的新变种

本文报道了玉米全蚀病菌禾顶囊壳菌(Gaeumannomyces graminis)的新变种——玉米变种[Gaeumannomyces graminis(Sacc.)Arx et Olivler var.maydis Yao Wang et Zhu var.nov.]。该变种在形态学、致病性、生物学及可溶性蛋白电泳谱带等方面,均不同于禾顶囊壳菌小麦变种[G.graminis var,tritici J.Walker)、水稻变种(G.graminis var.graminis Trans.)和燕麦变种[G.graminis var.avenae(Turner)Dennis]。模式标本保存在沈阳农业大学真菌标本室。     

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。     

小麦全蚀病菌胞外β—1，3—葡聚糖酶的产生和部分特性的研究

本文就小麦全蚀病菌胞外β—1,3-葡聚糖酶的产生和部分酶学特性进行了研究。结果表明,小麦全蚀病菌能够产生胞外β-1,3-葡聚糖酶。在供试的三种培养基中,最佳产酶培养基为改进的MS培养基。当以改进的MS为基础培养基时,最佳碳源为麦麸皮;最佳氮源为牛肉浸膏;产酶的最适条件为培养基初始pH为6,培养温度为26℃,250ml三角瓶中装培养基量为50ml时接菌量为4块菌饼(直径5mm)。另外,对酶的部分性质的研究结果表明,酶最适作用温度和pH分别为60℃和7．0,在50℃以下以及pH=5．5～7．5范围内稳定。     

小麦全蚀病菌胞外β-1,3-葡聚糖酶的产生和部分特性的研究

本文就小麦全蚀病菌胞外-1,3-葡聚糖酶的产生和部分酶学特性进行了研究。结果表明,小麦全蚀病菌能够产生胞外-1,3-葡聚糖酶。在供试的三种培养基中,最佳产酶培养基为改进的MS培养基。当以改进的MS为基础培养基时,最佳碳源为麦麸皮;最佳氮源为牛肉浸膏;产酶的最适条件为培养基初始pH为6,培养温度为26℃,250ml三角瓶中装培养基量为50ml时接菌量为4块菌饼(直径5mm)。另外,对酶的部分性质的研究结果表明,酶最适作用温度和pH分别为60℃和7.0,在50℃以下以及pH 5.5～7.5范围内稳定。     

小麦全蚀病菌胞外β-1,3-葡聚糖酶的产生和部分特性的研究*

本文就小麦全蚀病菌胞外-1,3-葡聚糖酶的产生和部分酶学特性进行了研究。结果表明,小麦全蚀病菌能够产生胞外-1,3-葡聚糖酶。在供试的三种培养基中,最佳产酶培养基为改进的MS培养基。当以改进的MS为基础培养基时,最佳碳源为麦麸皮;最佳氮源为牛肉浸膏;产酶的最适条件为培养基初始pH为6,培养温度为26℃,250ml三角瓶中装培养基量为50ml时接菌量为4块菌饼(直径5mm)。另外,对酶的部分性质的研究结果表明,酶最适作用温度和pH分别为60℃和7.0,在50℃以下以及pH 5.5～7.5范围内稳定。     

提高小麦原生质体再生植株频率的研究

从小麦徐州211的成熟种子诱导愈伤组织,建立了胚性悬浮细胞系。酶解悬浮细胞获得原生质体,用含o．8％琼脂糖的改良Ms培葬基进行琼脂糖珠培养,再生细胞分裂,并形成愈伤组织。诱导再生愈伤组织分化．得到了完整的再生植株。原生质体培养两周后,加入降渗培养液可促进克隆的形成。在分化培养基中,低浓度蔗糖可提高植株分化率。高浓度的激动索和玉米素对芽的分化有效并能抑制愈伤化。再生愈伤组织诱导分化时期的早晚影响植林分化频率。     

桃褐腐病菌(Monilia fructigena)原生质体制备及再生条件

以桃褐腐病菌(Monilia fructigena)为供试菌株,研究了酶系组成、液体培养基、菌龄、酶解温度、酶解时间对原生质体制备的影响,以及等渗液、固体再生培养基、酶解时间对原生质体再生的影响。结果表明:Fries(1/2)液体培养基培养24h,在10mg/mL崩溃酶+5mg/mL纤维素酶+20mg/mL蜗牛酶+10mg/mL溶菌酶的混合酶液中28°C酶解4h为桃褐腐病菌原生质体制备的最佳条件。采用液体再生涂布平板法,以含Ca2+的STC为等渗液的液体培养基和含蔗糖及Ca2+的Fries(1/2)固体培养基为桃褐腐病菌原生质体再生的最佳条件。经过观察与测定,再生菌株保持了原有的培养性状和致病性,接种桃果实后发病率为100%。     

花脸香蘑原生质体的制备及再生条件

以珍稀食用菌花脸香蘑菌丝为原材料,对原生质体制备与再生条件进行系统研究,并通过响应面法优化酶解液种类、酶解温度、酶解时间和稳渗剂等影响因素。结果表明以0.6mol/mL甘露醇作稳渗剂,在以1%溶菌酶+1%蜗牛酶+1%纤维素酶为复合酶解液,酶解温度为30℃,60-70r/min摇床振荡培养条件下,酶解4h,原生质体产量达到2.31×10⁷CFU/mL,在以蔗糖为稳渗剂的液体培养基上再生率达到25%。研究结果可为花脸香蘑后期研究提供依据。     

转化脂介导小麦原生质体转化及转基因白化苗的再生

应用转化脂(Lipofectin)介导转化技术成功地将含有潮霉素磷酸转移酶(Hygromycinphosphotransferase,Hpt)基因的E．Coli质粒pCGNl055导入到了小麦(徐州211)原生质体内,并由此获得了转化愈伤组织。根据Hpt抗性及DNA分子杂交证实转化频率高达6．O－8．8％,在分化培养基上转化愈伤组织再生出了白化植株,通过Southern杂交和酶活性检测证明外源Hpt基因存在于转化植株(白化苗)内,并进行了有效的表达。     

Genetic variation, pathogenicity and mycelial growth rate differentiation between Gaeumannomyces graminis var. tritici isolates derived from winter and spring wheat

The variability of Gaeumannomyces graminis var. tritici ( Ggt ) isolates was evaluated at an intravarietal level using non-molecular and molecular methods. Pathogenicity and linear growth rate of the pathogen were estimated. Very high pathogenicity was found in 44% of the isolates, medium in 20% and low only in 8%. Significant differences in mycelial growth rate were observed. The quickest linear growth rate of Ggt mycelium was observed at 25°C. Isolates derived from winter wheat grew faster than those obtained from spring wheat. The correlation between growth rates and pathogenicity was not significant. DNA polymorphism determined by random amplified polymorphic DNA (RAPD)–PCR was used to assess genetic variation among isolates. Thirty-two RAPD markers revealed DNA polymorphism suitable for assessing variability among isolates examined. Cluster analysis of RAPD data identified a few groups of isolates. RAPD markers associated with pathogenicity as well as mycelium growth rate were found.     

禾顶囊壳4个变种对禾本科牧草的致病性研究

采用人工接种法测定了禾顶囊壳小麦变种、燕麦变种、玉米变种和禾谷变种对4种禾本科牧草鸭茅、披碱草、苇状羊茅和无芒雀麦的致病性。结果表明,除禾顶囊壳玉米变种Ggm01菌株对披碱草没有致病性外,4个变种对供试的其它牧草都有致病性,且能产生全蚀病的典型症状。其中小麦变种和燕麦变种对供试牧草的致病性强于禾谷变种,玉米变种致病性最弱。各变种的菌株存在着致病性分化。小麦变种Ggt9813菌株对苇状羊茅和披碱草的致病性强于燕麦变种,发病严重度均达到50％以上。目前在我国尚未发现燕麦变种,小麦变种主要出现在北方地区,在牧草上的发生亦不广泛,因此二者具有检疫重要性。     

Effects of sowing date and volunteers on the infectivity of soil infested with Gaeumannomyces graminis var. tritici and on take-all disease in successive crops of winter wheat

In a field experiment on winter wheat, take‐all on plants and the infectivity of the soil were studied in crop sequences with different combinations of sowing dates. Take‐all was negligible in the first wheat crop, but thereafter the mean disease intensity (measured using a take‐all rating, TAR, with a maximum of 300) was 108, 190, 118 and 251 in the second to fifth successive crops. In each growing season, the disease differed amongst sequences and built up more rapidly and was more intense on plants sown in mid‐September than on plants sown in mid‐October. In late‐sown plots, where volunteers had been present during the mid‐September to mid‐October period, take‐all reached an intensity intermediate between that in early‐sown plots and that in late‐sown plots that had been kept free of volunteers. Volunteers, therefore, partially offset the expected beneficial effect of decreased disease with later sowing. Differences in take‐all amongst sequences were most pronounced in the second wheat crop and early sowing of the previous wheat increased intensity of disease. In the following (third) crop, differences in disease intensity amongst sequences were smaller. Soil infectivity (measured by seedling bioassay after harvest) built up progressively from a low level after the first crop to peak after the third crop. In this build‐up phase, soil infectivity estimates were always numerically greater after harvest of early‐sown treatments than after later‐sown treatments, although never significant at P= 0.05. The greatest difference (P= 0.06) was recorded in October before sowing of the third crop, where the comparison was between soil after two previous early sowings and soil after two previous later sowings and control of volunteers. In the same autumn, presence of green cover (i.e. volunteers) was associated with a smaller loss of soil infectivity between harvest and later sowing than occurred in an absence of green cover. In 2^nd–4^th crops, where comparisons were available and mean TARs indicated moderate levels of take‐all, sowing later had no yield benefit, despite more take‐all and greater soil infectivity associated with early sowing. Important considerations for the management of crops at risk of take‐all are 1) choosing appropriate sowing dates to minimize take‐all or to encourage take‐all decline and 2) controlling volunteers and weed hosts where crops are sown late to minimise take‐all.     

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).     

Analysis of Mycelial Growth Rates and RAPD-PCR Profiles in a Population of Gaeumannomyces graminis var. tritici Originating from Wheat Plants Grown from Fungicide-treated Seed

Linear mycelial growth rates of 70 isolates of Gaeumannomyces graminis var. tritici on agar medium amended or unamended with the fungicide silthiofam were not correlated. Mycelial growth rate was not influenced by the fungicide applied to the seed of the plants from, which the isolates originated. DNA polymorphism determined by randomly amplified polymorphic DNA (RAPD) polymerase chain reaction was used to assess genetic variation among isolates. Thirty RAPD markers generated with five arbitrary 10‐mer primers revealed DNA polymorphism suitable for assessing variability in this fungal population. Cluster analysis of RAPD data identified two groups at the 54% similarity level. There was a significant relationship between the presence of 11 markers and sensitivity to silthiofam.     

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.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.