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.

GmSKP1,a Novel S-phase Kinase-associated Protein 1 in Glycine max,Enhancing Resistance Against Phytophthora sojae Infection

Phytophthora root and stem rot of soybean caused by Phytophthora sojae(P.sojae)is a devastating disease that affects soybean[Glycine max(L.)Merr.]all over the world.S-phase kinase-associated protein 1(SKP1)proteins are key members of the SKP1/Cullin/F-box protein(SCF)ubiquitin ligase complex and play diverse roles in plant biology.However,the role of SKP1 in soybean against the phytopathogenic oomycete P.sojae remains unclear.In this study,a novel member of the soybean SKP1 gene family,GmSKP1 which was significantly induced by P.sojae,was reported.The expression of GmSKP1 was simultaneously induced by methyl jasmonate(MeJA),salicylic acid(SA)and ethylene(ET),which might suggest an important role for GmSKP1 of plant in responses to hormone treatments.Functional analysis using GmSKP1 overexpression lines showed that GmSKP1 enhanced resistance to P.sojae in transgenic soybean plants.Further analyses showed that GmSKP1 interacted with a homeodomain-leucine zipper protein transcription factor(GmHDL56)and a WRKY transcription factor(GmWRKY31),which could positively regulate responses to P.sojae in soybean.Importantly,several pathogenesis-related(PR)genes were constitutively activated,including GmPR1a,GmPR2,GmPR3,GmPR4,GmPR5a and GmPR10,in GmSKP1-OE soybean plants.Taken together,these results suggested that GmSKP1 enhanced resistance to P.sojae in soybean,possibly by activating the defense-related PR genes.

Antibacterial Activity and Mechanisms of Ethanol Extracts from Scutellaria baicalensis Georgi and Magnolia officinalis against Phytophthora nicotianae

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced.At present,the control of P.nicotianae mainly depends on chemical methods,with considerable environmental and health issues.We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi(SBG)and Magnolia officinalis(MO).On mycelial growth,sporangium formation,and zoospore release of P.nicotianae.Both extracts inhibited the growth of P.nicotianae,with mycelial growth inhibition rates of 88.92%and 93.92%,respectively,at 40 mg/mL,and EC50 values of 5.39 and 5.74 mg/mL,respectively.The underlying mechanisms were the inhibition of sporangium formation,the reduction of zoospore number,and the destruction of the mycelium structure.At an SBG extract concentration of 16.17 mg/mL,the inhibition rates for sporangia and zoospores were 98.66%and 99.39%,respectively.At an MO extract concentration of 2.87 mg/mL,the production of sporangia and zoospores was completely inhibited.The hyphae treated with the two plant extracts showed different degrees of deformation and damage.Hyphae treated with SBG extract showed adhesion and local swelling,whereas treatment with MO extract resulted in broken hyphae.Mixture of the extracts resulted in a good synergistic effect.

Analysis of Population Genetic Change to Single Oospore Strains of Phytophthora infestans

The occurrence of sexual reproduction accelerates the population genetic variation of Phytophthora infestans and makes it more difficult to control.The systematic analysis of the differentiation of phenotype(mating type and metalaxyl sensitivity)and genotype(mtDNA haplotype and SSR genotype)of 65 single oospore strains of P.infestans was carried out in this article.Five test strains were isolated from Heilongjiang Province and Mongolia Autonomous Region.The experiment results showed that the isolation ratio of metalaxyl resistance(MR:HR)of single oospore strains produced through the cross of medium resistance and high resistant parents was 18:13;the isolation ratio of the metalaxyl resistance(S:MR:HR)of single oospore strains produced through the cross of sensitive and high resistant parents was 4:12:7.The progenies of single oospore strains produced through self-fertility parents with medium resistance were all of the medium resistance.The mating types A1:A2 was greater than 1:1 in single oospore strains of the progenies,which did not conform to the Mendel's law of inheritance.All single oospore strains of the progenies inherited mitochondrial DNA fragments from only one parent.Sexual recombination of single oospore strains was verified by using two pairs of SSR primers(Pi4B and Pi4G).At the locus of Pi4B and Pi4G in the cross of KS-37 and KS-25,the separation frequencies of allele were 19:12 and 14:17,respectively.They produced two new genotype strains.This study could provide a basis for formulating disease control strategies.

Primed Expression of Defense-Related Genes by Streptomyces cameroonensis-Based Bioformulation (SCaB) on Cocoa Seedlings in a Nursery Challenged with Phytophthora megakarya

A Streptomyces cameroonensis based bioformulation (SCaB) has been developed and shown to be stable and effective in controlling the early proliferation of P. megakarya and promoting the growth of cocoa seedlings in nursery. This study was carried out to explore the molecular mechanisms associated with the interaction of SCaB, cocoa seedlings, and the pathogen during the early stages of seedling growth in the nursery. For this purpose, seedling treatment with 10% W/W SCaB under greenhouse conditions evaluated SCaB’s capacity to stimulate the defense mechanisms in cocoa. Agronomic growth parameters and the level of induction of defense-associated compounds were analyzed. Real-time (rt) PCR was used to assess the level of expression of defense genes. Here, we showed that the application of SCaB as a seedling treatment enhanced the growth of cocoa seedlings in the nursery by an average of 15.6% after 30 days of growth and led to an average reduction in disease severity of 64% when challenged with P. megakarya. The latter led to an increased synthesis of total phenolic compounds, flavonoids, chitinases, peroxidases, and β-1,3-glucanases and an induced up-regulation of TcChiB, TcGlu-1, TcPer-1, and TcMYBPA genes. This research provides a basis for the optimization of beneficial microorganisms as a viable alternative to chemical fungicides used in disease suppression.

影响大豆疫霉菌Phytophthora sojae卵孢子生活力和萌发的因素

本研究的目的是寻找一种能够促使大豆疫霉菌卵孢子在短时间内大量萌发的方法。结果表明,卵孢子菌龄、预处理温度、化学物质及其浓度在某种程度上均影响大豆疫霉菌卵孢子的生活力。胡萝卜琼脂平板上24℃密封培养条件下,30d菌龄的卵孢子中只有10.56%处于萌动状态,但经0.4%的KMnO4处理20min或35℃处理5d后,萌动率分别提高到58.49%和83.55%,但并不萌发。以3mL大豆感病品种Sloan的根系分泌物为培养液,26℃黑暗或光照培养7d即可获得80%以上的卵孢子萌发率。

Cloning of Potato POTHR-1 Gene and Its Expression in Response to Infection by Phytophthora infestans and Other Abiotic Stimuli

A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which we had cloned using suppression subtractive hybridization (SSH) technique. The potato POTHR-1 gene encodes a protein of 225 amino acids, which shares 81% identity with tobacco hin1 gene-enoded protein (harpin-induced protein). Southern blot revealed that there are two to three copies of POTHR-1 in potato genome. The POTHR-1 gene expression in potato leaves showed that its transcripts accumulated remarkably in leaves after 36 h inoculation with P. infestans. Mechanical wounding and jasmonic acid (JA) could induce the POTHR-1 gene expression and osmotic stress just induce a slight accumulation of POTHR-1 gene mRNA, while salicylic acid (SA) had no detectable function on the induction accumulation of POTHR-1 gene transcripts. The potato POTHR-1 gene may preferentially associate with hypersensitive response (HR) or biotic cell death during interaction between host and pathogen.

Phylogenetic Analysis of the Sequences of rDNA Internal Transcribed Spacer (ITS) of Phytophthora sojae

The internal transcribed spacer （ITS） region （ITS1, ITS2 and 5.8S rDNA） of the nuclear ribosomal DNA （nrDNA） was amplified via the PCR method in seventeen different isolates of Phytophthora sojae using the common primers of the ITS of fungi. Around 800 bp- 1,000 bp fragments were obtained based on the DL2000 marker and the sequences of the PCR products were tested. Taking isolate USA as outgroup, the phylogenetic tree was constructed by means of maximum parsimony analysis, and the genetic evolution among isolates was analyzed. The results showed that there is a great difference between the base constitution of ITS 1 and ITS2 among various isolates. The seventeen isolates are classified into three groups, and the isolates from the same region belong to the same group, which shows the variation in geography.

Screening of Antagonistic Bacteria against Phytophthora infestans and Its Inhibition Effect

[ Objective ] The paper was to screen bacterial strain with significant antagonistic effect against Phytophthora infestans, so as to provide basis for further development and utilization of antagonistic bacteria to inhibit P. infestans and control potato late bright. [ Method] Plate dual culture and filter paper method were used to determine the inhibition effect of strains in vivo, fermentation broth and bacterial liquid of 61 strains against P. infestans and the resistance-induction effect of SR13-2 strain. [ Result] The inhibition rate of 24 strains among 61 tested strains against mycelial growth of P. infestans was greater than 60%, and the inhibi- tion effect of HT-6 strain was the strongest with the inhibition rate of 89.92%. However, fermentation broth of all tested strains had no significant inhibition effect against P. infestans, while the inhibition effect of bacterial liquid of most strains was significantly higher than strain in vivo; the inhibition effect of $34-1 strain was the strongest with inhibition rate of 91.50%. The bacterial liquid of SR13-2 strain was found to have significant resistance-induction effect with protective rate of 60%. [ Conclusion] The inhibition effect of strains in vivo and fermentation broth of antagonistic strains S34-1 and SR13-2 had no relationship with each other, while bacterial liquid had great application potential in controlling potato late bright.

不同浓度臭氧水对致病疫霉菌Phytophthora infestans孢子萌发抑制作用初报

采用不同浓度臭氧水对致病疫霉菌Phytophthora infestans(Mont.)de Bary孢子囊萌发进行了试验,结果表明:臭氧水处理对孢子囊萌发的影响与臭氧水浓度密切相关,以9 mg/kg臭氧水处理1 min可以抑制95%以上孢子囊萌发,以18 mg/kg臭氧水处理1 min则100%抑制孢子囊萌发。无论在高浓度还是在低浓度条件下,处理时间1 min或2 min对孢子囊萌发没有产生显著影响,杀菌作用几乎是瞬间完成的。臭氧水处理孢子囊后导致孢子囊形态发生变化,这种变化随时间延长变得更加明显。鉴于高浓度臭氧水对植物本身不产生毒害作用,因此,高浓度臭氧水在控制晚疫病方面显示出广阔的应用前景。

Screening and Identification of Antagonistic Strain against Phytophthora sojae

[ Objective] The paper was to screen the antagonistic strain against Phytophthora sojae with biocontrol potential, and provide basis for searching control measures and designing new control strategies against P. sojae. [ Method] The rhizosphere soil of soybean was collected from three different places in Heilongjiang Province, and various soil microorganisms were isolated. Dual culture method was used to screen the microorganism with antagonistic effect against P. sojae. On this basis, the growth inhibition rate of the microorganism with stronger antagonistic effect against P. sojae was determined, and its control effect against P. sojae was also measured. [ Result] A strain of bacterium with relatively good antagonistic effect was isolated from soil, and named as strain B048. Dual test showed that the growth inhibition rate of antagonistic bacterium 11048 against P. sojac reached 97.5%. Antagonistic endurance tests showed that the width of inhibition zone was still 20.0 mm after dual culture with P. sojac for21 d. In potting experiment, the control effect of B048 against P. sojae was 100%. The antagonistic bacterium was primarily identified to be Bacillus pumilus through morphology and 16S rDNA sequence analysis. [Condusion] The antagonistic bacterium B048 had good prospect to be developed as the biocontrol bacterium against P. sojae.

Improvement of Polyacrylamide Gel Electrophorus in Phytophthora infeatans SSR Marker

[Objective] The paper was to improve polyacrylamide gel electrophorus in Phytophthora infeatans SSR Marker.[Method] With the disease sample of P.infeatans collected from Guyuan in Ningxia Province in 2009 as test material,its DNA was extracted and amplified with PCR,and its products were carried out polyacrylamide gel electrophoresis.[Result] 12% polyacrylamide gel electrophoresis was used to detect primers D13,G11 and PI02,and 8% polyacrylamide gel electrophoresis was used to detect primers PI4B,PI63,SSR4,SSR8 and SSR11,then 0.1% silver nitrate was used to stain,and an ideal electrophoresis and staining effect was obtained.[Conclusion] The electrophoresis and staining method suitable for P.infeatans SSR Marker established in the study had the characteristics of high sensitivity,simple operation and clear bands,which was an effective,simple and quick detection method.

Distribution and Virulence Diversity of Phytophthora sojae in China

By investigating occurrence of Phytophthora root rot in fields and isolating P.sojae fromdiseased plants and soils, the distribution of P.sojae in China was surveyed. In addition tonortheast region, P.sojae existed in Huanghe-Huaihe basin and Yangtze basin too. Eighty- threeisolates of P.sojae isolated from different areas were identified on virulence using 13differential soybean cultivars, abundant virulence diversity was found in P.sojae. The greaterdiversity in virulence of P.sojae was in isolates from soil than from plants. And the greatestvirulence diversity of P.sojae was found in Yangtze basin.

烟草黑胫病原菌(Phytophthora parasitica Var·nicotianae)生理生化特性的研究

本文主要介绍了用人工合成培养基测定烟草黑胫病原菌的致死温度、最适生长的pH值和能够生长的pH值范围的方法及结果。对黄河等“适宜于马铃薯晚疫病菌的一种液体合成培养基”,在配方和实验方法上做了一些改进。通过实验,进一步证实了该菌在营养生长过程中钙素的重要性和三羧酸循环(TCA)中有机酸的重要性;该菌的营养生长并不需要较复杂的营养条件。

Effect of Different Culture Conditions on Sporulation Quantity of Phytophthora capsici in Capsicum annuum L. var. dactylus M. in Xunhua

[Objective] The paper was to explore the effect of different culture conditions on sporulation quantity of Phytophthora capsici in Capsicum annuum L.var.dactylus M.in Xunhua.[Method] The effects of light hours,pH,medium and temperature on sporulation quantity during isolation and culture process of P.capsici were studied.[Result] The sporulation quantity of P.capsici under the conditions of 24 h/day light,pH 7.0,potato medium（PSA） and 30 ℃ was the largest,and pH,basal medium and temperature had greater impact on sporulation quantity.[Conclusion]The study laid foundation for the study on natural incidence condition of C.annuum in Xunhua.

Resistance Evaluation of Soybean Germplasm from Huanghuai Region to Phytophthora Root Rot

The aim of the study was to establish a set of differential strains and to identify soybean resistant genes to Phytophthora root rot and then to apply those strains for analysis of the resistant genes Rps1a,Rps1c,and Rps1k that soybean cultivars or lines may carry.Virulence formula of 125 Phytophthora sojae isolates were determined using the hypocotyls inoculation technique,the strains,which includ 6 isolates with different virulence formulas,were applied to identify the resistance of 55 soybean cultivars or lines and resistant genes were analyzed using the gene postulating procedure.Eighteen reaction types occurred in 55 cultivars or lines and results of gene postulation indicated that 2 cultivars or lines probably carried gene Rps1c and no cultivar may carry genes Rps1a or Rps1k.A few of soybean cultivars or lines from Huanghuai Region carry Rps genes Rps1a,Rps1c and Rps1k and tend to infect by P.sojae,so resistant cultivars or lines need to be bred and popularized actively.

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.

大豆疫霉菌(Phytophthora sojae)增强型绿色荧光蛋白遗传转化载体的构建

载体的构建是建立遗传转化体系的基础。以真核表达载体pcDNA3.1(-)/hygro为基本骨架,构建大豆疫霉菌遗传转化载体,通过限制性内切酶酶切、去磷酸化、连接等基因重组技术,将增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)基因和来自莴苣霜霉菌(Bremia lactucae)的启动子(ham34)、终止子重组到真核表达载体pcDNA3.1(-)/hygro中,经大肠杆菌转化后对转化子进行了酶切验证,为大豆疫霉菌遗传转化体系的建立提供载体。

Phytophthora α1性信息素C(9)～C(16)片段的合成

以甾体皂甙元氧化降解废弃物（R）-4-甲基-5-羟基戊酸甲酯为原料，经包括酯的格氏加成反应、烯烃的臭氧化反应和环氧化合物的亲核加成等六步反应，以60．5％的总收率合成得到了Phytophthora α1性信息素的C（9）-C（16）片段4。

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。