Combined Toxicity Test of Complex Agents of Metalaxyl and Other Fungicides against Phytophthora parasitica var. nicotianae

[ Objective] To overcome the resistance of Phytophthora parasitica var. nicotianae against metalaxyl and effectively control its damage, new efficient complex agent of metalaxyl was studied and developed. [ Method] The toxieities of nine fungicides against P. parasitica were measured using growth rate method. On this basis, the fungicides with good effects were selected to compound with metalaxyl, and the optimum complex ratio was confirmed. [Result] The toxicity of metalaxyl was the strongest with EC50 of 2. 130 5μg/ml; followed by carbendazim, mancezeb and dimethomorph with EC50 of 2.357 9, 2.639 8 and 2. 778 8 μg/ml. The effect of cyazofamid was the poorest with EC50 of 6. 278 8 μg/ml. The optimum complex ratios of dimethomorph, carbendazim and mancezeb with metalaxyl were 40： 60, 30：70 and 20： 80, and their co-toxicity coefficients were 138.80,124.25 and 115.00, respectively. [ Conclusion] The complex agents had application and promotion value, which could be used to carry out further field trials.

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。

A Preliminary Study on the Resistance to Different Fungicides in Phytophthora parasitica var. nicotianae

[Objective] This study aimed to investigate the resistance to different fungicides in Phytophthora parasitica var. nicotianae. [Method] Under indoor incubation conditions, the resistance to dimethomorph, metalaxyl-mancozeb, propamocarb and ovraclostrobin.dimethomorph in P. parasitica strain isolated from Zhenyuan County in Qiandongnan State was analyzed with colony growth measurement method. [Result] P. parasitica exhibited different levels of sensitivity to four fungicides. To be specific, P. parasitica exhibited the highest resistance to dimethomorph, and ECho reached 1.19 μg/ml. [Conclusion] In Zhenyuan tobacco-growing area, long-term single use of dimethomorph possesses certain resistance risk in prevention and control of black shank disease.

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。

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.

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.

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.

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。

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.

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。

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。

Protaphelinus, a newly recorded genus of Aphelinidae(Hymenoptera: Chalcidoidea) from China

The genus Protaphelinus Mackauer （Hymenoptera： Aphelinidae） is recorded for the first time from China and Protaphelinus nikolskajae （Yasnosh） is redescribed in detail and illustrated by macrophotography. This is the parasitoid specifically associated with the gall-making aphid, Pemphigus borealis Tullgren （Aphidoidea： Pemphyidae） found on the poplar tree （Populus sp.） in Wudalianchi, Heilongjiang Province.

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。

烟草黑胫病菌侵染叶片的动态

对抗烟草黑胫病菌（Phytophthora parasitica var. nicotianae）的烟草品种RBST与红花大金元（红大）叶片进行黑胫菌接种,采用细胞生物学的方法,在不同时间取样、染色、显微观察,揭示黑胫病菌在抗感叶片时的侵染动态。结果表明,接种感病红大叶片16-20 h,黑胫病菌通过气孔侵入红大叶表皮组织,接种48 h后,黑胫病菌长满整个接种小叶盘；而接种抗病RBST叶片24 h后,抗病的RBST叶片才出现极少的坏死斑,随着接种时间的延长,坏死斑区域有少量的扩大,但在接种72 h后,坏死面积只占接种小叶盘面积的很小部分。

六种杀菌剂对茄子绵疫病菌的室内毒力测定

采用菌丝生长抑制法测定了啶酰菌胺、嘧菌酯及氟吗啉等6种杀菌剂对茄子绵疫病菌(Phytophthora parasitica)的室内活性。结果表明,20%氟吗啉可湿性粉剂的抑制效果最好,其次为50%啶酰菌胺水分散粒剂和100 g/L氰霜唑悬浮剂;50%嘧菌酯水分散粒剂随着药量的增加,抑制率增长并不显著,但其EC50不是很高;40%百菌清悬浮剂和722 g/L霜霉威盐酸盐水剂两种药剂对茄子绵疫病菌的防效较低,抑制菌丝生长的效果较弱。