轮枝菌诱导棉花细胞的氧化迸发

大丽轮枝菌（Verticilium dahliae kleb.)是引起棉花黄萎病的病原真菌。使用从大丽轮枝菌V44（高毒）和V64（低毒）的菌丝体制备的各市县导物（I44和I64）作用悬浮培养的豫棉6号（感性）和豫棉8号（耐性）细胞系,用过氧化物酶法测定诱导后30min内的反应性氧变化,发现仅有不亲和性较高的体系,即弱毒力的大丽轮枝菌（V64）和耐性的豫棉8号所组成的体系（I64-Y8）表现最高的反应性氧迸发,3min-6min时增加61.8%。使用显著低于杀菌浓度的剂量的水杨酸（SA）和H2O2作用上述大丽轮枝菌,发现水杨酸和H2O2都能影响微生物,经1mmol/L水杨酸和0.2mmol/LH2O2作用后的微生物,其所产生的诱导物对植物细胞反应性氧的诱导作用要高于未被作用的微生物的诱导物,两种化学性质的影响的共同点是使反应性氧迸发的峰值时间提前,峰值增加,水杨酸提前12min,H2O2的影响还突出表现在使反应性氧迸发的曲线锐化,即峰使时间范围的平均每分钟增加率表现显著增加,9min-12min时达到109%。     

大丽轮枝菌毒素的多糖组份对棉花致萎作用的研究

用β-葡萄糖苷酶水解大丽轮枝菌(Verticillium dahliae Kleb.)毒素中的多糖组份。发现经酶处理后的5个不同致病力类型的棉花黄萎病菌(大丽轮枝菌)菌株毒素都不能影响整个毒素复合物对棉花的致萎作用,表明毒素中的多糖组份在棉花的致萎作用中不占有重量地位。     

大丽轮枝菌毒素的脂肪组分对棉花致萎活性研究

用脂肪酶(37℃,6小时)水解不同致病力类型的5个大丽轮枝菌(Verticillium dahliaeKleb.)毒素中的脂肪组分。试验结果表明经过水解后的5个菌株的毒素都不影响对棉花的致萎活性,说明毒素中的脂肪组分在对棉花的致萎作用中不占重要地位。     

大丽轮枝菌分泌糖蛋白的分离及其致萎性研究

利用伴刀豆球蛋白（ＣｏｎＡ） 亲和层析、Ｓｅｐｈａｄｅｘ Ｇ＿１５０ 凝胶层析、双向电泳、ＳＤＳ梯度电泳等手段对大丽轮枝菌（Ｖｅｒｔｉｃｉｌｌｉｕｍ ｄａｈｌｉａｅ Ｋｌｅｂ．）分泌的糖蛋白复合物进行分离,对其中一约２６ ｋＤ 的组分进行了Ｎ 端氨基酸序列分析。以棉花（ Ｇｏｓｓｙｐｉｕｍ ｂａｒｂａｄｅｎｓｅ Ｌ．）叶片为材料,进行了糖蛋白致萎性实验。结果表明,沸水浴或ＣｏｎＡ 处理的蛋白致萎性消失,Ｚｅａｔｉｎ 使糖蛋白致萎性减弱。该糖蛋白能够诱导海岛棉培养细胞中棉酚等倍半萜的合成,棉酚的积累随着糖蛋白浓度的增加而增加,但到一定程度后下降,此时较高浓度的大丽轮枝菌分泌糖蛋白引起植物细胞死亡     

大丽轮枝菌毒素致萎活性成分的研究

测定大丽轮枝菌(Verticillium dahliae)Kleb．)不同致病力类型的5个菌株毒素的成分,试验结果表明各菌株的毒素都含有蛋白质、多糖和脂类物质,并发现致病力强的菌株毒素所含的蛋白质和多糖的量高于致病力弱的菌株毒素,而脂类物质的量却低于弱菌株毒素。经过高温(100℃,10分钟)和蛋白酶(37℃,6小时)处理后的各菌株毒素则不能使棉花致萎,说明毒素中的蛋白质组分在毒素对棉花的致萎作用中占有重要的地位。     

大丽轮枝菌微菌核形成能力的遗传

带有硝酸盐利用缺陷型遗传标记的大丽轮技菌Verticilliumdahliae黑色菌核型和白色菌丝型菌株在25℃下配对培养,形成野生型融合菌落带,对融合带的分生孢子后代进行遗传分析的结果表明,融合带中的异核体表现不稳定,分布不均匀。微菌核遗传因子可随亲本细胞质在异核体中的运动和交换而发生迁移。     

大丽轮枝菌硝酸盐利用缺陷型和抗杀菌剂突变体的遗传研究

初步研究了大丽轮枝菌硝酸盐利用缺陷型和抗杀菌剂突变体的遗传特征。结果表明,大丽轮枝菌对三环唑的抗性不能稳定遗传,抗性菌株经低温或室温保顾一个月及转代培养后无丧失抗性,５株抗多菌灵的突变体,有一株在第二代单胞后代培养时丧失抗性,１株在单孢后代中发生分离;     

大丽轮枝菌微菌核的萌发条件及致死温度

微菌核是大丽轮枝菌在土壤中的主要存活结构和初侵染源,在土壤中可存活14年之久,其数量及存活状况直接影响着大丽轮枝菌型黄萎病的发生为害程度。以棉花黄萎病菌Verticillium dahliae XJ2008菌株为试材,研究了微菌核萌发的最佳条件、致死温度及土壤温度对微菌核存活的影响。结果表明,20℃、pH8.0是微菌核萌发的最佳条件。大丽轮枝菌微菌核具有很强的耐高温特性,随着处理时间的延长,微菌核的萌发率呈下降趋势,在55℃及以上处理时其萌发率下降迅速,而在50℃及以下处理时其萌发率下降相对较慢,在55℃处理360min可使微菌核完全致死,而在40℃、45℃和50℃处理1,440min,仍然有少量的微菌核存活,但其萌发率随着时间的推移呈下降趋势。土壤微菌核模拟试验结果表明,土壤温度对微菌核有很强的致死作用,40℃条件下处理4d后土壤中的微菌核已全部死亡。该研究结果为通过覆膜增温防治作物黄萎病提供了理论依据。     

4种木霉菌对棉花黄萎病菌抑制作用的测定

以绿色木霉(Trichoderma viride)、康氏木霉(Trichoderma koningii)和二株未知木霉菌株(Trichoderma spp.)为供试木霉菌株,采用对峙培养法测定了不同温度处理下对棉花黄萎病菌(Verticilliam dahliae)的拮抗作用。结果表明,木霉在不同温度下对棉花黄萎病菌的抑制作用不一,其中以30℃和25℃黑暗条件下木霉对棉花黄萎病菌菌丝生长的抑制作用最强,对峙培养5d后,绿色木霉、康氏木霉和二株未知木霉菌在30℃条件下对棉花黄萎病菌的抑制率分别达到67.3%、65.9%、56.8%、65.9%,25℃条件下的抑制率分别为65.9%、72.9%、65.9%、78.8%;20℃下木霉菌株对棉花黄萎病菌的抑制作用次之,10℃、15℃和35℃条件下木霉菌丝扩散速度较慢,且孢子产生量少,不能有效地抑制棉花黄萎病菌菌丝的扩展,表明环境温度过高或过低对木霉菌丝的生长及分生孢子的产生均有较大影响。该研究为筛选棉花黄萎病菌更为有效的生防木霉菌株提供理论依据。     

甘肃省甜瓜黄萎病的病原鉴定

【背景】2013年11月在甘肃省兰州市皋兰县的日光温室秋冬茬甜瓜种植棚发现黄萎症状的甜瓜植株,病株率约为1%。【目的】明确甜瓜黄萎病的病原。【方法】采用组织分离法进行病原菌分离;通过科赫氏法则(Koch’s法则)明确分出病菌的致病性;采用形态学和分子生物学方法对病原菌进行种类鉴定。【结果】分离得到轮枝菌属真菌8株,轮枝菌属真菌的病株分出率达100%;2个代表性菌株GLTG-2和GLTG-5(显微特征相似但菌落形态和生长速率不同),在温度18-24℃及昼/夜光周期为11.5 h/12.5 h的试验条件下,人工接种可引起甜瓜苗矮化、枯萎;接种后40 d,枯死株率分别为70%和40%;BLASTn分析结果显示,菌株GLTG-2的rDNA-ITS序列与Verticillium dahliae菌株MRHf7的序列相似性达99.78%,菌株GLTG-5的rDNA-ITS序列与V.dahliae菌株MRHf7和Vd414的序列相似性达100.00%。【结论】引起甜瓜黄萎病的病原菌被鉴定为大丽轮枝菌(V. dahliae),这是大丽轮枝菌引起甜瓜黄萎病在我国和亚洲地区的首次报道。     

Screening of Clone Lines with High Yield of Oligosaccharins from Culture Cells of Panax ginseng

Cell plating clone technique was employed to screen clone lines with high yield of oligosaccharins from culture cells of Panax ginseng C. A. Mey. Near 300 clone lines were obtained. The results from some clone lines analysed implied that these clone lines were significantly different in cell growth rate, oligosaccharins content and yield. Furthermore, there was a distinct correlation between oligosaccharins productivity and cell growth. A more stable high-yield oligosaccharin clone line PG-180 had been selected according to the characteristics of growth rate, oligosaccharin yield and peroxidases isozyme patterns during successive subculturing of 11 generations of clone lines. The mean growth rate of clone line PG-180 was 0. 495 g dry wt/L · d, and was 1.39 folds higher than to the original strain. Its mean content and yield of oligosaccharins were 14. 69 % dry wt and 2.183 g/L, which were 65 % and 132% respectively higher than those of the original strain. In comparing the time course of cell suspension culture between clone line PG-180 and the original strain, the optimal period for high oligosaccharin production from P. ginseng culture cells was approximately three weeks.     

Isolation of Glycoproteins from Verticillium dahliae and Their Phytotoxicity

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes cotton wilt-disease. Glycoproteins secreted by V. dahliae have been found to play an important role in wilting syndrome. In this study the glycoproteins were purified consecutively by ConA-Sepharose 4B affinity chromatography, Sephadex G-150 gel filtration, two-dimensional gel electrophoresis and SDS gradient gel electrophoresis. The N-terminal residual sequence of a 26 kD glycoprotein was analyzed. Plant-wilting tests were carried out by injection of glycoproteins, and those treated by heat, ConA and zeatin, into cotton leaves, respectively. Results showed that heat and ConA treatment abolished the wilt-causing activity of the glycoproteins, and zeatin alleviated the wilt syndrome of cotton. Furthermore, the glycoproteins were found to be effective elicitors in inducing the biosynthesis of sesquiterpene aldehyde phytoalexins in suspension cell cultures of Gossypium barbadense L., and heat-treatment lowered, but not abolished the elicitor activity. However, application of native glycoproteins at the concentration higher than 5 mg/L resulted in cell death.     

大丽轮枝菌分泌蛋白激发子的分离纯化及生物功能研究

利用硫酸铵沉淀、(A)KTA explorer 10蛋白纯化仪、非变性电泳、割胶电洗脱等方法,从大丽轮枝菌(Verticillium dahliae)发酵液中分离纯化出一种蛋白激发子,经SDS-PAGE电泳检测单一条带,相对分子量为20 kD.该蛋白激发子能够诱导烟草的过敏反应,处理6h后,处理部位出现水溃状,24h后出现坏死斑.该激发子可以诱导烟草细胞在较短时间内产生防卫反应信号分子H2O2和NO,并引起活性氧爆发.     

Reaction of Cotton Cultivars and an F2 Population to Stem Inoculation with Isolates Verticillium dahliae

Four Verticillium dahliae isolates (V76, TS‐2, PH, and V44) were used in screening four cotton cultivars (Pima S‐7, Acala Prema, M‐315 and Acala 44). Pima S‐7 and Acala Prema gave the highest resistance reactions and Acala 44 was the most susceptible. Isolate V76 of V. dahliae was the most virulent. An interspecific cross between the resistant cv Pima S‐7 (Gossypium barbadense) and the susceptible cv. Acala 44 (G. hirsutum) was made and the F₂ population phenotyped for Verticillium wilt effect. Phenotyping of plant reaction to the disease was quantified by using a set of six growth parameters (number of healthy leaves, number of nodes, leaf weight, stem weight, leaf to stem ratio, and total shoot weight) measured 3 weeks after inoculation. The F₂ phenotypic distribution of these parameters suggests that distribution is towards resistance and polygenic. Transgressive segregation also was observed. The number of healthy leaves and total shoot weight were found to be the best indicators of resistance. Results obtained in this study will be useful to quantify resistance to V. dahliae and identify the best parameters to phenotype in genetic studies.     

Occurrence of Verticillium dahliae on sunflower (Helianthus annus) in the UK

In August 1994 verticilliosis-like symptoms were noticed in a maturing crop of sunflower at IACR-Rothamsted. Wilting plants with chlorotic areas on some leaves, and dark areas of microsclerotia at the base of stems, were first observed on 11 August. As the plants matured, dark stripes were also observed on the stems. Many stems became brittle and later collapsed. Infected xylem showed a brown discolouration, later turning to black, and microsclerotia developed. Verticillium dahliae was isolated from all parts of the plant.
In general, once symptoms were present, the incidence of verticilliosis-affected plants increased, and the disease developed, more rapidly in early maturing varieties than in the later maturing types. At harvest, incidence ranged between 24.5% and 89.8%, depending on variety. Early maturing varieties appeared to be more susceptible than later maturing types.
Isolates of V. dahliae were cultured in the laboratory. Sunflower plants were artificially inoculated with these isolates and developed verticilliosis symptoms when grown under controlled environmental conditions.     

Isolation of microsatellite markers from an interspecific hybrid isolate of the fungal plant pathogen Verticillium dahliae

Ten microsatellite markers have been isolated from an amphihaploid isolate of Verticillium dahliae using genomic libraries enriched for (AGT), (GAC), (GCC), (TAC) and (TTA) repetitive elements. These were characterized on four amphihaploid isolates and two haploid isolates each of V. dahliae and Verticillium albo‐atrum. Nine were polymorphic, with between two and five alleles, and the tenth was polymorphic when tested on a further 20 haploid V. dahliae isolates (three alleles). Only one primer pair gave the expected double bands from the amphihaploid isolates, supporting the view that V. albo‐atrum is not one of the parents of the interspecific hybrid amphihaploid isolates.     

抑制茄子黄萎菌的植物提取物的筛选

从蔬菜、花卉、树木、作物和杂草等植物中选取29科73种植物材料,通过室内生物测定和田间试验,筛选出的葡萄叶、大葱叶提取物对黄萎菌具有较强的抑制作用,菌丝生长抑制率达68.44%和61.31%,孢子萌发抑制率达到95%以上,田间防病效果与茄子"病菌净"药剂达到同一显著水平,防效分别达到了75.76%和71.72%。丁香枝叶、薄荷叶、辣椒叶、番茄叶、醉蝶根、南瓜叶、韭菜叶、核桃叶、银杏叶、大蓟叶、藿香叶、苣卖菜叶、万寿菊叶、柳枝叶、花椒果和橘皮等也具有较好的抑菌活性,可作为其它病原菌的天然抗菌剂筛选研究试材。