牛蒡寡糖-钕配合物对西瓜枯萎病菌(Fusarium oxysporum f. sp. niveum)的影响

[目的]确定牛蒡寡糖－钕配合物对病原菌的最佳作用浓度。[方法] 采用平板培养和液体培养法，研究了不同浓度牛蒡寡糖－钕配合物对尖镰孢菌菌落直径、果胶酶活性、菌丝生长量和菌丝细胞膜透性的影响。[结果] 在平板培养试验中，2～180 mg/L的牛蒡寡糖－钕配合物促进菌落扩展，但菌丝稀疏，200～400 mg/L 的牛蒡寡糖－钕配合物抑制菌落扩展，菌丝稀而薄。在液体培养试验中，2～400 mg/L的牛蒡寡糖－钕配合物抑制菌丝生长，抑制率为4.3%～64.4%，随着牛蒡寡糖－钕配合物浓度的升高，其抑制作用加强；2～400 mg/L 的牛蒡寡糖－钕配合物降低了果胶酶活性；当测定时间为15、30、60、90、120和180 min 时，2～400 mg/L的牛蒡寡糖－钕配合物增加了菌丝细胞膜透性。[结论] 该研究为牛蒡寡糖-钕配合物在植物上的合理应用提供了理论依据。

Biological Characteristics of Fusarium oxysporum f. sp.niveum Isolated from Watermelon Cultivated in Plastic Greenhouse and Indoor Toxicity Determination of Different Fungicides in Hainan Province of China

[ Objective] The paper was to study biological characteristics of Fusarium oxysporum f. sp. niveum in watermelon cultivated in plastic greenhouse in Hainan Province of China and to determine indoor toxicity of fungicides. [ Method] Effects of various factors on mycelial growth and spore germination were stud- ied, and the lethal temperature of spores and indoor toxicity of fungicides were also determined. [Result] The fungal mycelium had a better growth rate on water- melon juice medium than the others. The optimum temperature for mycelial growth and spore germination were 28 ℃ and 30 ℃, and the optimum pH values were 7 - 8 and 7 - 9, respectively. Light-dark alternation was conducive for mycelial growth. The lethal temperature of spore was 60 ℃ for 5 min. The fungal mycelium grew better while taking fructose and glucose as carbon sources and yeast extract and peptone as nitrogen sources. Toxicity determination showed that 50% pro- chloraz-manganese chloride complex WP had the greatest inhibition effect on mycelial growth, with the ECso value of 0.730 9 μg/mL; followed by 32.5% benzoic azoxystrobin SC, 30% benzoylate· propieanazol EC, 10% difenoconazole WG, 25% bromothalonil · carbendazim WP and 25% bromothalonil WP, with the ECso values from 1.884 7μg/mL to 8. 161 0μg/mL. [Conclusion] The study provided basic data for field control against F. oxysporum f. sp. niveum on watermelon cultivated in plastic greenhouse in Hainan Province.

Biochemical and Physiological Changes of Three Watermelon Cultivars Infested with Fusarium oxysporum f.sp.niveum

The dynamic changes in membrane permeability, activities of disease-related enzymes, and contents of pathogenesis-relevant chemical compounds and photosynthetic pigments in root cells of three watermelon cultivars were studied after inoculation with Fusarium oxysporum f. sp. niveum at seedling stage. The results showed that the capacity of self-regulating and returning to normal status of cultivar Kelunsheng (resistant) was greater than that of a susceptible cultivar, Zaohua, in terms of malonaldehyde (MDA) content, relative conductivity, and activity of superoxide dismutase (SOD) and catalase (CAT). The resistant cultivar maintained a higher activity of dehydrogenase, higher content of vitamin C(Vc) , and relatively lower content of soluble sugar than the susceptible cultivar. The content of soluble protein was higher in the resistant cultivar than that in the susceptible one at day 1 after inoculation. The capacities of the resistant cultivar to inhibit chlorophyll deterioration and maintain a higher carotenoid content were significantly stronger than those of the susceptible cultivar.

河北省西瓜枯萎病菌生理小种鉴定与AFLP分析

【目的】明确河北省西瓜枯萎病菌生理小种类型和遗传多样性,为西瓜抗病育种和西瓜枯萎病的综合防治提供依据。【方法】本文对采自河北省12个西瓜种植地区的46个西瓜枯萎病菌菌株和4个已知生理小种的菌株进行了致病性测定和AFLP分析。【结果】根据菌株对3个鉴别寄主表现出的致病性强弱,将河北省46个西瓜枯萎病菌菌株划分为3个不同的生理小种,即0号、1号和2号,分别占供试菌株的17.4%、65.2%和17.4%,其中1号生理小种为优势小种,分布在河北省所有西瓜种植区。21对AFLP引物对供试菌株扩增出1157条带,其中多态性带389条,占总带数的33.6%。河北省西瓜枯萎病菌的遗传距离变化在0.33～0.95之间,平均为0.66,群体遗传多样性比较丰富。基于AFLP标记聚类分析表明,50个菌株被划分为3个类群(AFLP Groups,AGs)。AGI包含4个菌株,均为2号生理小种;AGII包括4个菌株,均为0号生理小种;AGIII包括42个菌株,以1号生理小种为主(32个),占该类群的76.2%。【结论】河北省西瓜枯萎病菌存在明显的致病性分化,其AFLP类群划分与致病性鉴定划分的生理小种之间存在一定相关性,但与菌株的地理来源无关。

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。

西瓜尖孢镰刀菌FOV-135的绿色荧光蛋白基因转化

建立并优化了PEG～CaCl2介导的原生质体转化体系，成功地将绿色荧光蛋白基因转入到西瓜尖孢镰刀菌FOV-135菌株中。转化子连续转接5代能够稳定遗传，荧光强度良好，每微克质粒DNA可获得4～6个性状稳定的转化子。PCR验证也表明gfp基因已转入到菌株FOV-135中。

GFP标记的尖孢镰刀菌西瓜专化型侵染西瓜过程观察

将绿色荧光蛋白基因转入西瓜尖孢镰刀菌FON中,并利用荧光共聚焦显微镜观察GFP标记菌株侵染西瓜的过程。结果显示,转化子连续转接4代能够稳定遗传,荧光强度良好,PCR验证gfp基因已转入菌株FON中;利用GFP标记菌株在荧光共聚焦显微镜下观察其侵染西瓜苗的过程,发现在1/2 MS培养的西瓜苗中,FON经过48 h侵染即可进入西瓜的根维管束,第3天便进入茎维管束,第4天进入叶维管束(包括叶柄和叶脉);在土壤盆栽条件下,侵染后2 d FON进入西瓜的根维管束,第9天进入茎维管束,第11天进入叶维管束。培养基培养与土壤盆栽相比,培养基栽培FON侵染的速度更快。

Changes of microbiome in response to sugars in a wilt pathogen-infested soil

Sugars are frequently and abundantly found in root exudates,but influence of specific sugars on the fate of soil-borne pathogens,microbiome structure,and particularly microbial interactions are not well understood.A 42-day of microcosm incubation was conducted with two soils:a natural watermelon Fusarium wilt pathogen(i.e.,Fusarium oxysporum f.sp.niveum(FON))-infested soil(Low-FON soil)and the soil further receiving the wilt pathogen inocula(High-FON soil).Both soils were supplemented with four simple sugars before incubation.The results show that,in both soils,FON was enriched by all sugars although co-living with tremendously diverse microbes;and bacterial richness,evenness,and diversity were decreased and bacterial community structure was changed by all sugars.Bacterial richness and evenness were negatively correlated with FON quantity in both Low-FON and High-FON soils,indicating that FON may tend to live in soil with low alpha-diversity.In both Low-FON and High-FON soils,the sugar-spiked networks had more links,higher density,larger modules,and shorter harmonic geodesic distance,suggesting greater potentials for microbial interaction and niche-sharing.The positive links between some of the keystone taxa and FON indicates that these keystone taxa may have promoted FON.This may be one of reasons why FON could proliferate vigorously after sugar supplementation.

Antifungal Activity of a Synthetic Oil Compound against Fusarium oxysporum,the Causal Agent of Tagetes

This paper evaluated the antifungal activity and mechanism of the main active component of the root extract of Tagetes(Tagetes fungicide),and an emulsion was developed to improve water solubility of Tagetes fungicide,thus improving its application.The results of in vitro antifungal effect assay and scanning electron microscopy(SEM)showed that the Tagetes fungicide had a wide spectrum of fungicidal ability and the antifungal mechanism of fungicide might be associated with a change in hyphae morphology.The total protein expression of Fusarium oxysporum f.sp.Niveum changed after being cultured with fungicide,showing the decreased expression of high molecular protein while increasing expression of low molecular protein.When the hydrophilic-lipophilic balance value was 10,the emulsion was stable.The intracellular organelles of plant leaves infected by F.oxysporum f.sp.Niveum showed some changes after cultured with the fungicide emulsion.

西瓜枯萎病拮抗菌株筛选及田间防效试验

本试验通过大量分离筛选 ,共获得对西瓜枯萎病菌具有较强拮抗作用的木霉 9株及细菌 1 0株 ,其中以绿色木霉TR 8菌株和芽孢杆菌B6 7菌株的拮抗作用最强。将两菌株分别发酵后 ,制备成TR 8制剂和B6 7制剂 ,并将两菌株发酵物混合后制备成健根宝粉剂。室内人工接种试验表明 ,3种拮抗菌制剂对西瓜枯萎病均有较好的防治效果 ,尤以健根宝粉剂的防治效果最理想。田间试验表明 ,健根宝粉剂以 1∶50拌土在播种时穴施 ,对西瓜枯萎病的防效达80 %以上 ,显著高于常用药剂多菌灵和施特灵。此外 。

西瓜不同品种苗期感染枯萎病菌后的生理生化变化

研究了西瓜不同品种苗期感染枯萎病菌 (FusariumoxysporumSchlecht.f.sp .niveum)后根部细胞的质膜透性、某些酶类和化合物 ,以及光合色素含量的动态变化。结果表明 ,受枯萎病菌侵染后 ,抗病品种克伦生在染病后的短时间内 (48h左右 )能使细胞内MDA、相对电导率、SOD酶和CAT酶的代谢基本恢复到正常状态 ,其自我调节恢复正常状态的能力显著大于感病品种早花 ;抗病品种比感病品种具有保持较高脱氢酶活性的能力 ;抗病品种Vc含量明显高于感病品种 ;抗病品种可溶性糖的相对含量要比感病品种低 ;接菌 1d后抗病品种可溶性蛋白含量高于感病品种 ;抗病品种抑制叶绿素含量下降的能力和保持较高类胡萝卜素含量的能力明显强于感病品种。

茼蒿中绿原酸抑制西瓜枯萎病菌活性研究

为了确定茼蒿中抑制西瓜枯萎病菌的物质,以西瓜枯萎病菌为指示菌株,对从茼蒿茎叶组织中分离纯化并鉴定出的3-O-咖啡酰奎宁酸(绿原酸)的离体抑菌活性进行了研究。结果表明,绿原酸对西瓜枯萎病菌的最不抑菌浓度(MIC)和最不杀菌浓度(MFC)分别为31.25和125 mg·m L^(-1),经室内毒力测定,其对西瓜枯萎病菌的凌半最大效应浓度(EC50)为53.05 mg·m L^(-1)。在绿原酸浓度分别为31.25、125、53.05 mg·m L^(-1)时,即MIC、EC_(50)、MFC的绿原酸浓度处理下,西瓜枯萎病菌的孢子萌发率分别为64%、33%、12%,而绿原酸浓度为0时的孢子萌发率则达到92%,经邓肯氏差异分析,各处理间孢子萌发率差异均显著。随着绿原酸浓度的增大,西瓜枯萎病菌的菌丝干、鲜重均呈现出下降的趋势。此外,绿原酸处理能使细胞膜通透性增加,从而导致菌体细胞内电解质外渗速度加快。本研究结果为茼蒿的深度开发和利用提供了科学依据,也为其他天然抑菌活性成分的筛选及抑菌机理的研究提供可行的试验方法和理论依据,对研究和开发新的天然植物源杀菌剂具有重要的学术价值和应用前景。

西瓜枯萎病生防菌XG-1的筛选、鉴定及其抑菌作用

从湖北省荆州西瓜植株内分离出内生菌97株,通过平板对峙和抑菌圈法筛选出对西瓜枯萎病菌(Fusari-um oxysporum f.sp.niveum)具有较强拮抗作用的菌株XG-1。该菌株对棉花枯萎病菌(Fusarium oxysporum f.sp.vasinfectum)、棉花黄萎病菌(Verticillium dahliae Kleb)、棉花立枯病菌(Rhizoctonia solani Kühn)等5种病原真菌均具有抑制作用。通过形态特征观察及16S rDNA序列分析将菌株XG-1鉴定为枯草芽孢杆菌(Bacillussubtilis)。菌株XG-1能使西瓜枯萎病菌菌丝产生明显的畸形,对其孢子的萌发具有较强的抑制作用。

西瓜枯萎病菌专化型鉴定及防治研究

1982～1987年间自黑龙江、北京等10个省市采集到60份西瓜病株,分别进行分离和培养鉴定,结果有尖孢镰刀菌、茄病镰刀菌、木贼镰刀菌。致病性试验是将3个镰刀菌分别接种于西瓜品种郑州3号,结果尖孢镰刀菌致病,茄病镰刀菌和木贼镰刀菌不侵染。分别从黄瓜和甜瓜枯萎病株分离出来的尖孢镰刀菌,与西瓜病株分离得的尖孢镰刀菌进行交互接种于西瓜、甜瓜和黄瓜的幼苗上。鉴定的结果是不同的专化型,即尖孢镰刀菌西瓜专化型〔Fusarium oxysporum. f. sp. niveum(E. F. Smith)Snyder et. Hans.〕。在防治上,采用综合防治措施,经过1989～1990年试验,防效达90.91％～92.70％,增产58.95％～64.93％。

西瓜枯萎病菌遗传分化研究

本试验利用21个RAPD随机引物对50个西瓜枯萎病菌系进行了RAPD扩增，研究了病菌遗传多样性。结果表明，对供试菌系扩增出134条带，其中多态性带113条，占总带数的84．3％。菌系间的遗传相似系数变化在0．45-0．93之间，多数菌系间的同源程度较低。基于RAPD标记聚类分析表明，50个菌系划分为6个RAPD群（RAPD groups，简称RGs），即RGⅠ，RGⅡ，RGⅢ，RGⅣ，RGV和RGⅥ。其中RGⅠ和RGⅡ各含有一个菌系，分别来自秦皇岛和新疆；RCⅢ和RGⅤ包括2个菌系，分别来自秦皇岛、承德和廊坊、沧州；RGⅣ包括唐山、石家庄、邯郸和新疆菌系；其余的菌系属于RGⅥ。分类结果进一步说明西瓜枯萎病菌间存在着较大的遗传分化。

镰刀菌酸毒素对西瓜幼苗根细胞跨膜电位及叶细胞有关抗逆酶的抑制

【目的】了解引起连作西瓜严重枯萎病的尖孢镰刀菌毒素对西瓜幼苗根和叶细胞有关生理功能的影响。【方法】用从尖孢镰刀菌西瓜专化型菌株提取的真菌毒素镰刀菌酸处理西瓜幼苗,考察其对细胞根系跨膜电位和叶片中相关抗逆酶的影响。【结果】高浓度镰刀菌酸处理西瓜幼苗12h后,根部细胞跨膜电位比对照下降61.9%～81.8%。不同浓度的毒素处理24h后,根系脱氢酶活性减少35.9%～90.9%。镰刀菌酸明显加速了叶片细胞膜脂质过氧化,24h后叶片丙二醛含量是对照的5.2～11.0倍。叶片中的苯丙氨酸解氨酶活性先增加,然后减少,6～12h活性最高,为对照的7.2～10.5倍。过氧化氢酶、超氧化物岐化酶、过氧化物酶也是先上升然后下降。过氧化氢酶活性在毒素处理3h后达到高峰,但再过3h后下降18.6%～52.5%。超氧化物岐化酶和过氧化物酶在毒素处理12h后达到最大,然后下降。叶片中的β-1,3-葡聚糖酶和几丁质酶在毒素处理后先快速上升,然后下降,最大活性分别出现在处理后12h和3h,分别是对照的10和15～30倍。【结论】镰刀菌酸强烈抑制了西瓜幼苗的根和叶细胞正常生理功能。破坏了叶片细胞的正常防卫系统和根细胞吸水和运输功能,引起细胞膜脂质过氧化生成大量的丙二醛,细胞结构遭受严重破坏,导致发病甚至细胞死亡。

西瓜枯萎病拮抗细菌的筛选

采用梯度稀释法对23份土样进行筛选,共获得114株不同类型且长势良好的细菌,其中8株细菌对西瓜枯萎病病菌的拮抗性能较好。对此8株细菌进行革兰氏染色,有7株为革兰氏阳性菌;进行芽孢染色,其中6株细菌产生芽孢。

西瓜枯萎病高效拮抗菌XJUL-12的筛选与鉴定

目的:分离获得西瓜枯萎病高效拮抗菌,为研究西瓜枯萎病高效拮抗菌的拮抗机制奠定基础。方法:用碾碎法从新疆有毒植物麻(Urtica cannabina L.)、亚洲薄荷(Mentha asiatica Boris.)、阿尔泰藜芦(Veratrum lobelianum Bernh.)中分离内生菌,并用琼脂扩散法筛选出对西瓜枯萎病具有较强抗性的内生菌XJUL-12,通过PCR方法扩增XJUL-12的16S rDNA,并与GeneBank中已鉴定菌的16S rDNA序列对比,用N-J方法构建XJUL-12进化树,用Bootstraping法对其评估,同时结合其形态特征、生理生化检测、G+C mol%含量对XJUL-12进行鉴定。结果:XJUL-12的16S rDNA序列与Bacillus subtilisstrain CGMCC1869同源性为99%,G+C mol%含量为46.72mol%。结论:筛选获得的内生菌XJUL-12对西瓜枯萎病具有较强抗性,并将XJUL-12鉴定为枯草芽孢杆菌。

几种生防放线菌离体颉颃作用测定

采用菌饼对峙法测定4种放线菌对7种病原真菌的离体颉颃作用。结果发现,SC1,SC11和SE2菌株对所有靶标菌均有不同程度的抑制作用。菌株SC11对各供试靶标菌均有良好稳定的抑制作用,接种后5 d测量靶标菌菌落半径均小于4 mm,菌丝日生长速率低于0.75 mm/d。菌株SE2对粉红聚端孢、立枯丝核菌、番茄叶霉菌的抑制作用比其它3种放线菌要好,抑制率高达100%。在所有测定的靶标菌中,番茄灰霉菌、粉红聚端孢菌、西瓜枯萎菌、立枯丝核菌是较易于被放线菌抑制的靶标菌。显微观察发现受,SC1,SC11,SE2抑制后,靶标菌菌丝出现不同程度的畸形。

西瓜枯萎病菌T-DNA插入转化子库的构建及其质量评价

为研究西瓜枯萎病菌的致病分子机理,开展了病原菌的转化子库构建工作。采用携带潮霉素抗性p TFCM双元载体的农杆菌AGL-1介导的ATMT转化方法 ,获得菌株FON-01的转化子,通过表型观察筛选突变体。结果表明:当乙酰丁香酮(AS)浓度为200μmol/L、农杆菌OD600为0.4、分生孢子浓度为1×106个/m L时,于28℃共培养48 h转化效率高达(663.33±24.95)个/106个孢子。构建了总数为3 832个的转化子库并对其进行了质量评价,从2 201个转化子中筛选出菌落形态、生长速度及产孢量等方面有所变异的突变体73个。病原菌转化子库的构建为进一步开展致病分子机理及相关基因克隆等方面研究奠定基础。