重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子

重寄生真菌盾壳霉Coniothyrium minitans是核盘菌Sclerotinia sclerotiorum的重要生防菌。为了探讨盾壳霉胞外蛋白酶在寄生核盘菌过程中的作用,采用明胶平板法对盾壳霉寄生核盘菌菌核产生的蛋白酶活性进行了检测,并进一步采用福林酚法定量测定蛋白酶活性,研究盾壳霉产生胞外蛋白酶的培养条件及影响蛋白酶活性的因子。试验结果表明,在被盾壳霉寄生的核盘菌菌核中检测到蛋白酶活性,表明蛋白酶可能参与盾壳霉重寄生作用。发现核盘菌菌核浸出液培养基适合盾壳霉产生胞外蛋白酶,摇培(20℃、200r/min)5d时蛋白酶活性最高,达到0.22U/mL。盾壳霉胞外蛋白酶酶促反应的最适温度为60℃,最适pH7.0。当温度不高于40℃时,蛋白酶酶活较稳定。5mmol/L的金属离子Mg²⁺、Zn²⁺、Ca²⁺、Cu²⁺、Mn²⁺、Li⁺和K⁺等对蛋白酶酶活没有显著影响(P>0.05),而Fe²⁺(5mmol/L)显著(P<0.05)提高了蛋白酶活性。盾壳霉蛋白酶对苯甲基磺酰氟(PMSF)敏感,说明盾壳霉产生的胞外蛋白酶可能主要是丝氨酸蛋白酶。这些结果为盾壳霉胞外蛋白酶的分离纯化和功能研究奠定了基础。     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum is a major disease of oilseed rape (Brassica napus). During infection, large, white/grey lesions form on the stems of the host plant, perturbing seed development and decreasing yield. Due to its ability to produce long‐term storage structures called sclerotia, S. sclerotiorum inoculum can persist for long periods in the soil. Current SSR control relies heavily on cultural practices and fungicide treatments. Cultural control practices aim to reduce the number of sclerotia in the soil or create conditions that are unfavourable for disease development. These methods of control are under increased pressure in some regions, as rotations tighten and inoculum levels increase. Despite their ability to efficiently kill S. sclerotiorum, preventative fungicides remain an expensive gamble for SSR control, as their effectiveness is highly dependent on the ability to predict the establishment of microscopic infections in the crop. Failure to correctly time fungicide applications can result in a substantial cost to the grower. This review describes the scientific literature pertaining to current SSR control practices. Furthermore, it details recent advances in alternative SSR control methods including the generation of resistant varieties through genetic modification and traditional breeding, and biocontrol. The review concludes with a future directive for SSR control on oilseed rape.     

抑制向日葵核盘菌菌核形成的生防菌S-16的鉴定及其生防作用研究

本研究从内蒙古包头市萨拉齐向日葵根围土壤中分离得到1株生防细菌 S-16,拮抗试验表明,菌株S-16能够显著抑制向日葵核盘菌的菌丝生长。显微镜观察发现,核盘菌菌丝生长点附近出现明显的异常分枝和囊泡状畸形,并且在距菌株S-16一定范围内核盘菌不能形成菌核。培养基内添加1%的菌株S-16发酵滤液能够有效抑制向日葵核盘菌菌核的形成。室内生测表明,2＆#215;106 cfu/mL浓度的菌株S-16菌液在离体叶片及幼苗上对向日葵菌核病的防效分别为94.62%和94.21%。通过细菌形态特征和生理生化反应,并结合API鉴定和16S rDNA序列分析,将菌株S-16鉴定为枯草芽孢杆菌Bacillus subtilis。     

乳茄3种新病害病原菌鉴定

 乳茄(Solanum mammosum),又叫多头乳茄、五指茄、五代同堂、黄金果,为茄科茄属常绿小灌木。乳茄是一种珍贵的观果植物,原产美洲,近年引入我国广东、广西及云南种植,因其果色鲜艳,且观果期长,果形奇特,有“五子拜寿”和“五子登科”的寓意,在切花和盆栽花卉上广泛应用。     

‘浙油50’油菜菌核病发生规律与流行模型研究

为了探索‘浙油50’油菜菌核病入侵扩散与灾变流行规律,更好地促进油菜产业的发展,通过采用人工接种与田间发病调查等方法,研究油菜菌核病发生规律与流行模型。结果表明：机直播油菜播期迟、密度大有利油菜菌核病发生,植株从出现水渍状病斑到萎蔫枯死为15天左右,自然菌丝入侵总体潜育期为8~17天,发病率可达13.0%,病情流行基本呈线性上升扩散规律,开花期是油菜菌核病的主要流行期,其病情流行呈Logistic函数模型运动轨迹。     

河套灌区向日葵菌核病发生的气象条件分析

向日葵是巴彦淖尔市的主要经济作物,占全市作物总播面积的30%以上,而菌核病制约着向日葵产业的健康发展。根据气象指标,提前判断向日葵菌核病出现早晚、发生程度的轻重,对于指导生产与科学防治至关重要。根据河套灌区向日葵菌核病中心病株出现期、始盛期与同期气象资料的相关性分析,研究其与气象条件的关系,确立向日葵菌核病发生的农业气象指标。结果表明,确定的13个指标中,有7个是4月下旬以前的指标。而这些指标大都直接、间接影响到土壤湿度,通过土壤湿度变化影响向日葵菌核病发生的早晚、发生程度的高低。6月份是关键时段,6月大风每增加1天,菌核病中心病株出现期和始盛期分别提前12.1天和2.5天,6月中旬平均最低气温每升高1℃,菌核病始盛期提前1天出现,6月中旬平均最高气温每升高1℃,菌核病发生程度提高0.09级。根据菌核病发生期和发生程度平均状况,可确定农业气象指标。为降低菌核病孢子存活的土壤条件,变秋灌为春灌,使菌核病存活的条件降低,待播种前进行灌溉,灌后播种,降低菌核病发生程度。加强农田防护林带建设力度,减缓大风对作物的伤害,推迟菌核病发生期。     

10种杀菌剂对瓜叶菊菌核病菌的室内生物活性测定

为了筛选得到对瓜叶菊菌核病菌室内抑制活性较高的杀菌剂,采用菌丝生长速率法测定了10种化学杀菌剂对瓜叶菊菌核病菌(Sclerotinia sp.)的抑制活性。结果表明,瓜叶菊菌核病菌对保护性有机硫杀菌剂敏感性较高,其中福美双对病菌的EC50值为0.8742 μg/mL,EC90为19.4750 μg/mL,而代森锰锌对病菌的抑制活性低于福美双。对于现代选择性杀菌剂,菌核病菌对咪鲜胺、苯醚甲环唑、腐霉利、异菌脲、戊唑醇、腈菌唑、三唑酮和嘧菌酯的敏感性依次下降,其中三唑类杀菌剂咪鲜胺对病菌的抑制活性最高,EC50达0.0418 μg/mL,EC90为0.3138 μg/mL,二甲酰亚胺类杀菌剂的抑菌活性以腐霉利最高,EC50达0.2113 μg/mL,EC90为0.5889 μg/mL,甲氧基丙烯酸酯类杀菌剂嘧菌酯对病菌的抑制活性最低,其中EC50为7.6224 μg/mL,EC90为59.5022 μg/mL。因此,咪鲜胺和腐霉利为瓜叶菊菌核病室内生物活性较高的杀菌剂,可以推荐供生产上使用,提倡保护性杀菌剂和内吸选择性杀菌剂复配使用,以提高病害的防控效果。     

安徽省油菜菌核病气象等级预报方法研究

为满足现代农业气象业务服务中病虫害发生发展气象等级预报需求,利用安徽省江淮、沿江和皖南3个区域代表站1991—2010年油菜菌核病观测数据和气象资料,通过相关分析,确定影响菌核病发生发展的主要影响时段和气象因子,再通过归一化处理、加权等方法得到影响油菜菌核病发生发展的综合气象条件指数。以综合气象条件指数为自变量,以油菜菌核病加权平均病株率为因变量,采用曲线回归方法,建立不同区域代表站油菜菌核病发生发展气象等级预报模型。模型拟合结果表明：江淮、沿江和皖南地区拟合准确率分别为80%、70%和75%;利用模型对2011—2013年发生情况进行预测,结果显示：江淮地区代表站全部正确,沿江和皖南地区代表站均为2年正确,一年与实际发生情况误差一个等级。建立的模型基本上能满足油菜菌核病气象等级预报业务服务的需求。     

三重PCR检测黄瓜炭疽病菌、菌核病菌和细菌性萎蔫病菌

 本试验建立一种可同时检测黄瓜炭疽病(Colletotrichum orbiculare)、黄瓜菌核病(Sclerotinia sclerotiorum(Lib.)de Bary)和黄瓜细菌性萎蔫病(Erwinia tracheiphila)等黄瓜主要病害病原菌的三重PCR检测体系。采用正交试验设计方法, 对三重PCR的影响因素分析研究, 进行退火温度优化, 并以3个引物组、Taq DNA聚合酶、dNTP和Mg²⁺ 共6因素3水平进行多重PCR体系优化, 成功建立了适合黄瓜主要病害的三重PCR最佳检测体系, 即25 μL的反应体系中含有0.24 μmol·L^-1 CY1/CY2;0.72 μmol·L^-1 SSFWD/SSREV;0.336 μmol·L^-1 ET-P1/ ET-P2;1 U Taq聚合酶;0.15 mmol·L^-1 dNTP;1 mmol·L^-1 MgCl₂, 最适退火温度为63℃。该方法能够快速从田间黄瓜发病植株和根围土壤中将黄瓜炭疽病菌、黄瓜菌核病菌和黄瓜细菌性萎蔫病菌检测出来, 灵敏度可以达到10 pg·μL^-1。