Vacuolar processing enzyme positively modulates plant resistance and cell death in response to Phytophthora parasitica infection

Oomycete, particularly Phytophthora species, causes the most devastating crop diseases, such as potato late blight,and threatens the sustainable crop production worldwide. Our previous studies identified Resistance to Phytophthora parasitica 1(RTP1) as a negative regulator of Arabidopsis resistance to multiple biotrophic pathogens and RTP1 lossof-function plants displayed rapid cell death and reactive oxygen species(ROS) production during early colonization of P. parasitica. In this study, we aim to decipher the mechanism of RTP1-mediated cell death, and identify a member of vaculoar processing enzymes(VPEs), γVPE, playing a role in rtp1-mediated resistance to P. parasitica and cell death occurrence. Our results showed up-regulation of the expression of γVPE as well as increased VPE/caspase 1-like protease activity in P. parasitica-infected rtp1 mutant plants. Besides, we found that the VPE/caspase 1-like protease activity was required for the cell death occurrence in Arabidopsis plants during the infection of P. parasitica as well as rtp1-mediated resistance to P. parasitica. Further pathogenicity assays on either Arabidopsis γvpe mutant plants or leaves of Nicotiana benthamiana with transient overexpression of γVPE demonstrated γVPE could positively affect plant resistance to P. parasitica. Together, our studies suggest that γVPE might function as an important regulator of plant defense and cell death occurrence in response to P. parasitica infection, and VPE/caspase 1-like protease activity is required for rtp1-mediated resistance to P. parasitica.

柑桔脚腐病菌Phytophthora parasitica杀菌剂的室内筛选

柑桔脚腐病是为害柑桔主干基部的病害。采用菌丝生长速率法对柑桔脚腐病菌寄生疫霉Phytophthoraparasitica进行了21种农药的室内毒力测定。结果表明，各药剂对寄生疫霉菌丝的生长均有抑制作用，并且其抑制率与药剂浓度呈正相关。其中，毒力作用最强的是50％烯酰吗啉水分散粒剂，EC50为0．2598μg／mL；其次是60％唑醚·代森联水分散剂、2％春雷霉素水乳剂、70％丙森锌可湿性粉剂、10％多抗霉素B可湿性粉剂、50％多·锰锌可湿性粉剂和50％醚菌酯水分散粒剂，EC5。在1．0～7．7μg／mL之间；再次是65％代森锌可湿性粉剂、10％井冈霉素水乳剂、10％苯醚甲环唑水分散粒剂、40％腈菌唑水分散粒剂、43％戊唑醇悬浮剂、25％丙环唑乳油、3％中生菌素可湿性粉剂、68％碱式硫酸铜水分散粒剂、40％氟硅唑乳油和12．5％烯唑醇可湿性粉剂，EC50在18．0～69．0μg／mL之间；较差的是50％敌克松可湿性粉剂、50％异茵脲可湿性粉剂、70％甲基硫菌灵可湿性粉剂和50％甲基硫菌灵悬浮剂，EC50在329．0μg／mL以上，尤其甲基硫茵灵70％可湿性粉剂和50％悬浮剂的EC50分别高达979．1665和1713．8330μg／mL，不宜推荐继续用于柑桔脚腐病的防治。

Phytochemical and anti-bacterial activity of epidermal glands extract of Christella parasitica (L.) H. Lev.

Objective:To study the morphology,biochemistry and bioactivity of the epidermal glands of the glandular morphotype of Christella parasitica(C.parasitica)(L.) H.Lev.Methods: Morphological studies on epidermal glands were earned out by using light microscope and scanning electron microscope.To prepare the extract,the shade-dried fronds of glandular morphotype were soaked in acetone.For antibacterial studies paper disc method was followed by using various pathogenic bacteria.Results:Detailed micromorphological,phytochemical and bioactivity studies on a medicinal fern C.parasitica(L.) H.Lev.showed its inlraspecific variation in antibacterial activity.The presence or absence of the epidermal glands was the key factor for antibacterial activity in the morphovariants of this species.The epidermal glands were orange-coloured,stalked and elongated ones of about 84.2μm×45μm,and distributed on the undersurface of cosla,coslules and veins in croziers,young and mature leaves.Frequency of glands varied from 15/cm on costa in mature leaves to 140/cm on costules in croziers.The acetone extract of the glands showed antibacterial activities and also toxic effect against mosquito larvae and tadpoles of frog.Preliminary phytochemical analysis and HPLC studies of the gland extract showed the presence of various kinds of terpenoids,alkaloids,tannins,saponins and flavonoids in it.Conclusions:The present study shows that epidermal glands of the glandular morphotype of C. parasitica(L.) H.Lev.have several bioactive compounds and such rare moiphovariant should be conserved in nature.The next step is to isolate the pure compounds and to screen the bioactivity of individual compounds of the epidermal glands.

中国不同地区烟草寄生疫霉 (Phytophthora parasitica var.nicotianae)菌株的随即多态性DNA扩增分析(英文)

来自中国云南、辽宁、山东 3省的烟草寄生疫霉 (Phytophthoraparasiticavar.nicotianae)菌株的致病性已被划分为 3种致病类型组 ,即强致病性组、中致病性组和弱致病性组。上述省是中国的烟草主产区 ,选自云南省的 15个烟草寄生疫霉菌株、山东省的 13个烟草寄生疫霉菌株和辽宁省的 2 0个烟草寄生疫霉菌株 ,选择 3个烟草栽培品种在温室内进行接种试验测定不同菌株的致病性分化。提取受试菌株的DNA ,利用PCR技术对受试菌株的模板DNA进行随机多态性扩增分析 ,对扩增DNA片段谱带借助于UPGMA分析法构建遗传树 ,结果表明 ,受试菌株被划分为4个遗传聚类组 ,每个遗传聚类组内包括不同的烟草寄生疫霉致病性菌株 ,而且来自于不同烟区相同的致病性菌株和每种致病性的不同菌株皆不属于同一个遗传聚类组内。结果表明RAPD -PCR的遗传标记分析结果与不同致病性组的划分未有明显的区别。因此 ,随机多态性DNA图谱的相同与不同不能当作区分来自不同烟区的烟草寄生疫霉的致病性分化的分子检测的工具。

A Comparison of the Growth and Asexual Reproduction by Cryphonectria parasitica Isolates Infected with Hypoviruses CHV3-County Line, CHV1-Euro7, and CHV1-Ep713

The impact of chestnut blight, caused by the fungal pathogen Cryphonectria parasitica, has diminished in Europe due to a natural biological control caused by hypovirus infection. Hypovirulence-mediated biological control has been far less successful in North America meriting further evaluation of field isolates that have the ability to produce non-lethal cankers, generate hypovirulent inoculum, and exhibit a greater ecological fitness in forest systems. In this study, Cryphonectria hypoviruses (CHV) CHV3-County Line, CHV1-Euro7, and CHV1-Ep713 were evaluated in five different isolates of C. parasitica. One hundred and eighty cankers representing each treatment combination were initiated on American chestnut sprouts in the Monongahela National Forest, West Virginia, USA. The size of cankers, the persistence of hypovirulent (HV) isolates, stroma production, and hypovirus transmission to conidia were assessed four and 12 months after canker expansion. CHV3-County Line infected isolates produced significantly smaller cankers than the isolates infected with either CHV1-type. With regard to CHV1-Euro7 isolates, the fungal genome appeared to contribute to the differences in canker size. After four months, HV isolates harboring either CHV1-type (30%) were retrieved at a significantly higher rate than isolates containing CHV3-County Line (14%). After 12 months, the HV recovery was similar among the three hypoviruses indicating smaller cankers will maintain their HV status after one year. Very few stroma were produced after one year in the field from HV isolates. In vitro, CHV3-County Line (49%) had a significantly lower rate of hypovirus transmission to conidia when compared to CHV1-Euro7 (87%) and CHV1-Ep713 (80%). Significant differences existed among the five different isolates indicating HV transmission is dependent on the fungal genome. This research provided additional evidence that each hypovirus interacts with its host differently and certain isolate/hypovirus combinations have better biological control potential than others.

Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica

Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation,affecting both yield and quality.Thus,rapid and accurate detection of P.parasitica plays an important role in disease management.The aim of this study was to develop a simple diagnostic method to detect P.parasitica infection by combining recombinase polymerase amplification and lateral flow strips(LF-RPA).To establish the LF-RPA assay of P.parasitica,the primers and probe designed based on the Ypt1 gene were tested for specificity to P.parasitica,which showed no cross-reactivity with DNAs of other related oomycete species.The LF-RPA assay detected the amount of genomic DNA of P.parasitica which was as low as 1 pg.To make the LF-RPA assay useful in low-resource settings,four simplified DNA extraction methods were compared,after which the LF-RPA assay was applied,with no specialized equipment,to analyze a diverse range of citrus tissues by using a simplified PEG-NaOH method for DNA extraction.This method was successful in detecting P.parasitica in infected plant samples within 30 min.Combining the LF-RPA assay and a simplified DNA extraction method could be a potential detection test for P.parasitica,especially in areas with limited resources.

烟草黑胫病菌(Phytophthora parasitica var. nicotianae)生理小种鉴定技术研究

1981—1985年从全国8个主产烤烟省30个地、县共采集和分离到烟草黑胫病菌(Phytophthora parasitica var.nicotianae)57个分离物,用游动孢子悬浮液接种根及茎基部,试验表明这种接种方法效果为好。用此方法将各分离物接种在常用的Burley_(21)、Burley_(37)、N.nesophila、N.stocktonii等4个鉴别寄主上进行了鉴定试验。通过鉴定,采自全国57个分离物中有44个属于生理小种Ⅰ号,占77.2%;有12个属于生理小种0号,占21.1%,还有个别分离物有待进一步鉴定。

拟氨基多糖抑菌剂K1、K2对烟草黑胫病原菌(Phytophthora parasitica var．nicotianae)的抑制效果研究

采用生长速率法测定了拟氨基多糖类抑菌剂K1、K2对烟草黑胫病原菌(Phytophthora parasitica var.nicotianae)的抑菌效果。结果表明:K1、K2对烟草黑胫病原菌均有显著的抑制作用,且抑制效果随K1、K2浓度的增大而增强;20 mL/L的K1、K2对烟草黑胫病原菌的抑制率分别为50.58%、84.68%,EC50分别为26.15、4.87 mL/L。

寄生疫霉Phytophthora parasitica侵染对岩溪晚芦果皮挥发性物质的影响

以人工接种寄生疫霉Phytophthora parasitica的晚熟柑桔品种岩溪晚芦的果皮为材料,采用顶空固相微萃取-气相色谱-质谱联用技术测定果皮挥发性物质,研究寄生疫霉病菌侵染对柑桔挥发性物质的影响。结果表明,所检测到的挥发性物质主要成分由烯萜类、醇类、酯类、醛类、芳香族化合物及少量的其他物质组成。健康果皮共检测到45种物质,发病果皮检测到79种,两者共有物质36种。在感病后未检测出而在健康果皮中存在的物质9种,其含量占健康果皮挥发性物质含量的12.80%。感病后特有的挥发性物质有43种,其含量占感病果皮挥发性物质含量的15.05%。与健康果皮相比,感病后挥发性物质总含量增加了56.24%,其中含量增加最多的是烯萜类物质,增加值为7.704μg/g。

一种适用于高分辨质谱分析的高效丝状真菌囊泡提取及蛋白制样方法

使用液氮冷冻研磨破碎真菌菌丝细胞,结合醋酸钠沉淀和超速离心纯化,获得的囊泡样品进一步使用超滤法浓缩和过滤,扫描电镜观察和蛋白电泳分析,实验结果显示所制备的真菌囊泡完整性好,蛋白纯度高,未见明显杂质污染,符合后续实验要求。进一步使用串联质谱标签技术联用高分辨生物质谱仪对真菌囊泡蛋白组进行分析,在提取的低毒病毒侵染后的真菌囊泡中成功鉴定到病毒蛋白的存在。实验方法的建立和优化,为丝状真菌囊泡提取、囊泡蛋白组学和病毒宿主相互作用研究提供了方法学保障。

Variations in Disease Resistance of Chestnut Cultivars against Chestnut Blight Fungus,Cryphonectria parasitica

Chestnut blight caused by Cryphonectria parasitica is one of the major chestnut diseases occurred on stems and branches. Oriental chestnut has more resistance than American one. Twenty-five cultivars of chestnut treeincluding various cultivars such as Tsukuba, Okkwang, Rihei, Ginyose, Eunsan, etc. were tested with a rapid, simple and reliable method. The cultivar Banseki is one of the most resistant cultivars tested and has the necrotic area of 0.40 cm2. However, the necrotic areas of the cultivars of American chestnut, Eunsan, Kwangjujoyul and Eaton, ranges from 3.35 to 2.61 cm2 and these cultivars are relatively susceptible to this pathogen. Many pycnidia of this pathogen were observed on the inner and outer bark tissues after incubating for 3 weeks.

烟草疫霉拮抗放线菌的生防潜力评价

由寄生疫霉烟草致病变种Phytophthora parasitica var.nicotianae引起的烟草黑胫病是连作烟田的重要土传病害之一,重发年份常给烟农造成巨大的经济损失。本研究通过稀释分离法从贵州省遵义市烟草黑胫病重病区的健康烟草根际土壤中共分离获得179株放线菌。通过平板对峙培养法筛选得到15株对烟草黑胫病菌菌丝生长有明显抑制作用的菌株。测定了这15个菌株代谢液对烟草疫霉菌丝生长的抑制作用,其中H-3菌株代谢液对烟草疫霉菌丝生长抑制率最高,达到85.68%。研究了H-3代谢液对烟草疫霉游动孢子萌发的抑制作用。结果显示培养6、12和24 h对游动孢子萌发的抑制率分别为72.75%、65.20%和63.45%。结合形态特征及16S rDNA序列分析,初步鉴定H-3菌株属于链霉菌属Streptomyces sp.。盆栽防效和田间防效试验结果表明,施用H-3对烟草黑胫病具有一定的防效,当添加H-3固体发酵物22.5 kg/hm2时田间防效最好,防效可达70.42%。通过荧光定量PCR技术检测了烟草根际土壤中烟草疫霉的量,结果发现,随着H-3用量的增加,根际土壤中烟草疫霉的拷贝数呈下降趋势,说明H-3处理后,能有效降低烟草根际土壤中烟草疫霉的菌量。

基于非靶向代谢组学的烟草镰刀菌根腐病和黑胫病拮抗链霉菌及其代谢产物的鉴定

针对烟草两种土传病害病原菌—镰刀根腐病菌Fusarium oxysporum和黑胫病菌Phytophthora parasitica,本实验室前期从烟草根际土壤中筛选得到1株具有良好生防作用的链霉菌,结合形态学、生理生化特性及分子生物学分析,将其鉴定为淀粉酶产色链霉菌Streptomyces diastatochromogenes。通过对该生防菌发酵液进行色谱-质谱联用(LC/MS)非靶向代谢组学分析,确定其重要代谢产物为茴香霉素。利用高效液相色谱对产物进行验证及含量测定,表明每克粗提物中含有茴香霉素423.98μg。菌丝生长抑制试验结果表明,茴香霉素对镰刀根腐病菌和黑胫病菌的EC_(50)值分别为1556.36和40.74μg/m L。本研究为烟草镰刀菌根腐病和黑胫病的生物防治提供了新的方法,并为其代谢产物—茴香霉素的进一步利用提供了有价值的理论基础。

根部内生真菌—寄生疫霉—拟南芥互作模式体系的构建与鉴定

为构建根部内生真菌—寄生疫霉—拟南芥三方互作模式研究体系,以影响马铃薯晚疫病害发生的潜在内生真菌雪球微座孢(Microdochium bolleyi)为例,利用WGA-Alexa Fluor■488染色和带有GFP标记的寄生疫霉(Phytophthora parasitica)转化菌株,通过荧光显微镜观察分析M.bolleyi侵染拟南芥根部以及M.bolleyi与寄生疫霉和拟南芥根部三方互作的细胞学特征;利用皿内平板拮抗测试初步分析内生真菌M.bolleyi对致病疫霉(Phytophthora infestans)的抑制效果,并进一步利用皿内拟南芥与真菌M.bolleyi和寄生疫霉三方互作体系,鉴定分析真菌M.bolleyi对植物与寄生疫霉亲和互作的影响。结果表明,真菌M.bolleyi可成功侵染并定殖于根组织,而未引发植物细胞坏死现象,说明M.bolleyi具有典型的内生真菌特征;相比于对照预处理,真菌M.bolleyi预处理能够抑制寄生疫霉在拟南芥根部的定殖和扩展,初步揭示真菌M.bolleyi在植物与疫霉菌互作中的影响作用;真菌M.bolleyi不仅对致病疫霉的皿内抑制率可达63.55%,还能够显著减轻拟南芥根部对寄生疫霉的感病性。可见,所建立的根部内生真菌—寄生疫霉—拟南芥三方互作模式体系不仅为快速鉴定有益真菌影响植物抗病抗逆生物学功能提供了技术体系,也为深入解析植物根部与不同微生物之间的互作机制奠定基础。

烟草黑胫病生防功能菌筛选及其应用效果

以野生络石Trachelospermum jasminoides为试验材料,采用组织平板法从其植株体内分离培养出31株内生细菌活体纯培养物,以烟草黑胫病原Phytophthora parasitica var.nicotianae为指示菌株筛选出的HHH12具有明显的抑菌作用,抑菌圈直径达到24 mm,初步定性为具有生防活性菌株。HHH12的多相鉴定结果为假单孢菌属Pseudomonas菌株,暂定名Pseudomonas sp.HHH12。盆栽试验证实,HHH12菌悬液的灌根+叶面喷雾混合处理对烟草黑胫病不同发病阶段均表现出比叶面喷雾和灌根单一处理有更好的应用效果,药后14 d时的防治效果达到72.86%。菌株HHH12具有良好的开发应用前景。

抗水霉菌菌株的分离鉴定与应用

水霉病(saprolegniasis)是危害淡水养殖鱼类的重要疾病,其相关防治策略尤其是环境友好的绿色生物防治制剂研制与应用是近年来科研与养殖一线的关注点。本研究通过分子扩增、测序以及生化鉴定等方法,从患水霉病的异育银鲫中分离鉴定了一株寄生水霉(Saprolegnia parasitica)JY1,并从正常淡水养殖环境中分离获得一株荧光假单胞菌(Pseudomonas fluorescens)JD;2个菌株与寄生水霉菌株SP/OM/UK/H4/2021和荧光假单胞菌菌株L321的序列相似度分别为99.85%和100%。荧光假单胞菌JD与寄生水霉JY1的体外对峙试验结果显示,JD可在长满寄生水霉的马铃薯葡萄糖琼脂(PDA)平板上缓慢而持续抑杀JY1,直至JY1菌丝全部降解;菌株JD和JY1在液体沙氏培养基中共培养时则显示,共培养的寄生水霉JY1出现菌丝管壁破损,菌丝折断、脱落,孢子囊壁破损,菌丝和孢子囊内孢子逸出等现象,最后大量菌丝降解、死亡。结果表明,荧光假单胞菌菌株JD或其分泌成分具有开发成防治淡水鱼水霉病制剂的潜力。

烟草黑胫病菌的分离及培养性状研究

烟草黑胫病是烟草(Nicotiana tabacum)的一种毁灭性病害。试验采集云南省昭通市的烟草黑胫病病株,采用组织分离法对病原菌进行分离、纯化,对其培养性状进行研究。结果表明,燕麦培养基适合烟草黑胫病菌(Phytophthora parasitica)的培养,菌丝生长旺盛;烟草黑胫病菌菌丝粗细不均匀,无隔膜,具有膨大体;孢子囊顶生,具脱落性,具1个或2个乳突。这些培养性状与前人研究结果相似,表明烟草黑胫病菌的培养性状较为稳定。

烟草黑胫病抗性鉴定方法比较及其相关性分析

为明确烟草黑胫病(Phytophthora parasitica var.nicotianae Tucker)抗性鉴定方法,采用粗毒素浸种法、粗毒素浸根法、孢子悬浮液浸根法及田间病圃接种法,研究烟草革新3号(抗病品种,R)、G28(R)、金星6007(中抗品种,MR)、小黄金1025(感病品种,S)、红花大金元(S)等对烟草黑胫病的抗性,并对其抗性鉴定效果进行相关性分析。结果表明,孢子悬浮液浸根法的鉴定结果与粗毒素浸种法的鉴定结果呈显著负相关,与田间病圃接种法的鉴定结果呈显著正相关。粗毒素浸种法不受季节限制,可以对烟草材料黑胫病的抗性进行大批量筛选。粗毒素浸根法和田间病圃接种法有一定的缺陷,可以作为粗毒素浸种法和孢子悬浮液浸根法的辅助鉴定方法。在整个抗性鉴定过程中,孢子悬浮液浸根法可观测到烟株病情指数和发病率的动态变化,较其他3种方法更能准确、合理地评价品种抗性。

芽孢杆菌1205和烟草疫霉处理后对烟苗防御酶的测定

分别将烟苗接种芽孢杆菌(Bacillus subtilis)1205和烟草黑胫病菌病原菌烟草疫霉(Phytophthora parasitica Gzufp-9)在接种后1、3、5、7、9、11 d测定烟苗过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)3种与植物抗病密切相关的酶活性变化。结果表明,接种芽孢杆菌1 205后,PAL、PPO活性在5 d时达到最高值,明显高于对照和接种烟草疫霉,POD活性在接种1 d时即达到最高值。而接种烟草疫霉后PAL、PPO活性均在3 d时达到最高值,POD活性在接种1 d时达到最高值。

栗疫病菌弱毒性菌株的筛选及其dsRNA的转化稳定性

为获取栗疫病生物防治的基础信息,本研究根据菌株在PDA培养基上培养7天后的菌丝生长速度、分生孢子形成能力等培养特性,从韩国国立山林科学院树木病理研究室保藏的60个栗疫病菌菌株中筛选了2个弱致病力菌株,进行dsRNA检测、弱致病力菌株和强致病力菌株间的细胞融合试验。结果表明:2个弱致病力菌株(KCP-135和KCP-136)中均检测到了dsRNA,弱毒性菌株KCP-22和其他19个强毒性菌株之间的菌落形成明显的隔离带并沿着隔离带产生分生孢子,没有明显的细胞融合现象,而弱毒性菌株KCP-22和强毒性菌株KCP-9之间的菌落则呈现了显著的细胞融合现象,而且其细胞融合菌株的培养特性和转化dsRNA数量均发生了变异。