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.

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.

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.

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.

福建省芋疫霉交配型测定及防治药剂筛选

【目的】明确福建省芋疫霉交配型,筛选可用于防治芋疫病的化学药剂。【方法】对2020年采集自福建4个地区的芋疫病样本进行病原菌分离纯化,结合形态特征观察、致病性测定和Ypt1序列同源性分析对病原菌进行鉴定,通过与辣椒疫霉A1、A2交配型菌株对峙培养来测定其交配型,并利用菌丝生长法测定6种杀菌剂对病原菌的室内毒力。【结果】通过形态特征观察、致病性测定和ITS-LSU-Ypt1融合序列同源性分析,明确所分离病原菌均为芋疫霉(Phytophthora colocasiae)。交配型测定发现,所分离的125个芋疫霉菌株中,122个为A2交配型,3个为A1A2交配型。室内药剂筛选结果表明,98%甲霜灵对芋疫霉抑菌效果最好,EC_(50)值为(0.146±0.032)μg·m L^(-1);95%烯酰吗啉、98%氟吡菌胺和94%氰霜唑抑菌效果较好,EC_(50)值介于(0.239±0.011)~(0.713±0.088)μg·m L^(-1);而95%嘧菌酯的抑菌活性最低,EC_(50)值为(23.447±3.666)μg·m L^(-1)。【结论】福建省芋疫霉以A2交配型为优势群体,甲霜灵、烯酰吗啉、氟吡菌胺和氰霜唑可作为芋疫病高效防控的轮换使用药剂。

枯草芽胞杆菌GXHZ16抑制烟草疫霉病菌的表达谱分析

烟草黑胫病由烟草疫霉菌Phytophthora nicotianae侵染引起,是烟草生产上一种分布广泛、为害严重的土传病害。本实验室前期从广西贺州富川烟区健康烟株根际土壤中分离筛选获得一株枯草芽胞杆菌Bacillus subtilis GXHZ16,对峙培养发现该菌株对烟草疫霉菌具有良好的拮抗活性(抑制率65.79%)。为探究菌株GXHZ16对烟草疫霉菌的可能抑制分子机理,本研究分别针对枯草芽胞杆菌GXHZ16与烟草疫霉菌对峙培养36h(处理组)和0h(对照组)的混合样品,采用高通量转录组测序(RNA-Seq)技术结合生物信息学分析对峙培养36 h后枯草芽胞杆菌的基因差异表达谱及所涉及的信号通路变化。结果发现:对峙培养36 h后枯草芽胞杆菌GXHZ16有1607个基因发生了显著差异表达,包括810个上调表达基因和797个下调表达基因;其中筛选获得的显著表达基因特别是上调表达的基因(包括鞭毛装配、细菌趋化性相关基因以及与表面活性素、丰原素和制磷脂菌素合成相关的10个相关基因)可能均涉及枯草芽胞杆菌对烟草疫霉菌的抑制作用。进一步的GO富集分析显示,共有1343个差异表达基因被注释到不同的功能亚类中,涉及差异基因最多的为细胞组分类别中的细胞解剖实体。特别地,KEGG富集分析显示,共有551个差异表达基因归到数百个Pathway中,其中涉及差异表达基因最多的是微生物在不同环境中的代谢途径;其他主要代谢途径有鞭毛装配和淀粉蔗糖代谢等;此外,细菌趋化性通路和单环菌素生物合成通路也高度富集。总之,本研究从转录组水平分析获得了枯草芽胞杆菌(与烟草疫霉对峙培养后)的基因差异表达图谱及相关调控代谢途径;为进一步阐明芽胞杆菌抑制植物病原真菌的生防机制并提高枯草芽胞杆菌的生防效果奠定了基础。

几种中草药提取物对马铃薯晚疫病菌的抑菌活性

中草药因其独特的优势应用于农作物病虫害防控中备受关注,为探究可用于防治马铃薯晚疫病的中草药制剂,助力晚疫病绿色防控,选用无水乙醇、石油醚及乙酸乙酯(体积比为4:3:3)三元溶剂,通过冷浸渍法提取百里香等11种中草药不同部位获得相应制剂。分别采用平板菌丝生长速率法和叶片接种法测定11种中草药制剂对马铃薯晚疫病菌的离体和半活体抑菌活性。百里香和大黄具有较强的抑菌活性,EC_(50)分别为130.19和364.90 mg/kg,优于阳性对照药剂丁子香酚(669.52 mg/kg);防风、齿果酸模、广藿香、蛇床子、野胡萝卜和补骨脂抑菌活性较强,EC_(50)分别为768.28、807.33、874.75、953.52、1519.11和1659.58 mg/kg,低于丁子香酚。可知,百里香、大黄、防风、齿果酸模、广藿香、蛇床子、野胡萝卜和补骨脂对马铃薯晚疫病具有较强的防治效果和潜在应用价值。

烤烟黑胫病抗性评价及生理响应特性分析

本研究旨在揭示不同烤烟品种对烟草黑胫病的抗性机理。通过对抗病性的鉴定,从24个烤烟品种(系)中鉴定出4个高抗品种、8个中抗品种、8个中感品种和4个高感品种。对这些品种在烟草疫霉(Phytophthora parasitica var.nicotianae)侵染后,从中选取2个高抗品种和2个高感品种测定其抗氧化酶系活性、水杨酸含量以及抗病相关基因的表达情况。结果表明,在受到病原菌侵染时,高抗品种的抗氧化酶活性显著高于高感品种,暗示着更强的氧化应激防御能力。此外,高抗品种中的内源水杨酸合成以及抗病相关基因的诱导表达均显著高于高感品种,表明这些品种能够更有效地激活其防御系统以应对烟草黑胫病菌的侵染。本研究结果为理解烤烟对黑胫病的抗性机制提供了重要信息,并为烟草抗病育种提供了理论依据。

2株有效抑制剑麻斑马纹病菌的生防细菌鉴定

剑麻斑马纹病是一种由烟草疫霉(Phytophthoranicotianae)引起的传染性病害,导致剑麻出现叶斑、茎腐和轴腐的症状,对剑麻纤维的产量和质量造成严重影响。目前,剑麻斑马纹病的防治主要采取以农业栽培措施为主,抗病育种和化学药剂防治相结合的综合防治策略,但是农业防治人工成本高,工人操作技术参差不齐;抗病育种难度大,周期长;化学防治受剑麻叶片表面带有蜡质层的影响,存在药液不易吸附的问题,而且大量施药也容易造成病原抗药性提高和环境污染。因此,开发防治剑麻斑马纹病的生防菌资源,探索综合防控新途径显得尤为重要。本研究以烟草疫霉菌为靶标菌,通过五点对峙法筛选,获得2株生防菌PpHyHNCJ2和PpHyHNCJ5,其菌丝生长抑制率分别为85.92%和83.10%。通过对烟草疫霉菌菌丝显微结构进行观察发现,菌株PpHyHNCJ2和PpHyHNCJ5均抑制了烟草疫霉菌菌丝的生长,生防菌对峙的烟草疫霉菌出现菌丝体膨大增粗、分枝畸形、缠绕等现象。基于细胞形态、生理生化反应以及16S rDNA基因序列比对分析,菌株PpHyHNCJ2和PpHyHNCJ5鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa)。进一步研究显示菌株PpHyHNCJ2和PpHyHNCJ5均具有广谱拮抗作用,能够很好地抑制辣椒疫霉菌(Phytophthora capsica)、大豆疫霉菌(P.sojae)、瓜疫霉菌(P.melonis)、棕榈疫霉菌(P.palmivora)、豇豆疫霉菌(P.vignae)、尖孢镰刀菌(Fusarium oxysporum)、黑曲霉菌(Aspergillus niger)、暹罗刺盘孢菌(Colletotrichum siamense)等11种病原菌的生长。此外,菌株PpHyHNCJ2和PpHyHNCJ5具有解有机磷能力、解无机磷能力、解钾能力、固氮能力和产铁能力,可为作物的良好生长提供条件。结果表明,菌株PpHyHNCJ2和PpHyHNCJ5对剑麻斑马纹病的防治具有良好的潜力,可为剑麻斑马纹病的生物防治提供基础材料。

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。

番茄疫霉根腐病菌拮抗细菌HP8-1的鉴定及生物防治潜力

【目的】探究HP8-1在番茄上的生物防治潜力,为番茄疫霉根腐病的生物防治提供优良生物防治资源。【方法】通过形态学观察、生理生化特性检测并结合16S rRNA序列分析鉴定HP8-1菌株,探讨其对植物病原菌的抑菌效果和生物学功能及在番茄根际的定殖能力,并通过盆栽试验测定其对番茄疫霉根腐病的防治效果和对番茄生长的影响。【结果】HP8-1为多粘类芽孢杆菌(Paenibacillus polymyxa),对多种植物病原菌具有抑菌效果,具有解磷、分解蛋白、分解纤维素、产嗜铁素和产吲哚乙酸等生物学功能;HP8-1在番茄根系和根际土壤具有稳定的定殖能力,尤其是在接种疫霉菌的条件下其定殖量趋于增加并显著高于未接种疫霉菌的条件;HP8-1处理对番茄疫霉根腐病具有明显的防治效果,防治效果达到57.41%;同时,HP8-1处理显著促进番茄植株生长、提高叶绿素含量和根系活力。【结论】HP8-1菌株稳定定殖在番茄根系和根际土壤,防治番茄疫霉根腐病,促进番茄生长,是对番茄疫霉根腐病具有生物防治潜力的资源。

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.

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.

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。

对马铃薯晚疫病菌(Phytophthora infestans)有杀菌毒性的中草药的筛选(英文)

将来源于 44科的 88种中草药等植物材料的提取物稀释 5 0 0倍后 ,与马铃薯晚疫病菌 (Phytophthorainfestans)的孢子囊悬浮液混合。有 31种材料的提取物在该浓度下表现了对马铃薯晚疫病菌的游动孢子囊间接萌发的完全抑制作用 ,有 46种提取物也表现了不同程度的抑制作用 ,另有 11种提取物在该浓度下没有表现抑制作用。在抑制菌丝生长作用的试验中 ,植物提取物用黑麦培养基稀释 2 0 0倍 ,然后倒皿 ,在 8.5cm直径的培养皿中央放一马铃薯晚疫病菌菌片 ,16℃下培养 ,15d后测量菌落直径大小。结果表明 ,在供试的 83种提取物中 ,在稀释 2 0 0倍浓度下 ,有 32种提取物表现了对菌丝生长的完全抑制作用 ,2 4种表现了比较强的抑制作用 ,而有 2 7种提取物没有表现对菌丝生长的抑制作用 ,甚至还表现了对菌丝生长的促进作用。同时 ,还有

云南省发现马铃薯晚疫病菌(Phytophthora infestans)A2交配型

将一批分离自楚雄、大理、昆明、曲靖、宣威等马铃薯产区的晚疫病菌在利马豆 Ｖ８ 琼脂培养基上与 Ａ１ 交配型标准菌株进行交配型测定， ４７ 个菌株中出现了１０ 个 Ａ２ 交配型菌株。亲和性菌株交配时产生大量具有典型特征的有性结构。对卵孢子、穿雄生的藏卵器以及雄器的形态特征和大小进行了描述。这是云南马铃薯晚疫病菌中存在 Ａ２ 交配型菌株的首次报道。

影响大豆疫霉菌(Phytophthora sojae)游动孢子产生的条件

间歇性地给菌丝块换水可以诱导菌丝产生孢子囊 ,进而释放游动孢子。挑取 5～ 8块直径为 8mm于利马豆或 V8汁平板上培养 4～ 8d的菌块 ,置于直径 7cm的培养皿内 ,加入蒸馏水正好没过菌块表面 ,每 30 min换水一次 ,换水 4次后加入 15 ml Petri培养液 ,2 5℃、黑暗条件下培养 18～ 2 0 h,可诱发菌块大量产孢。

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.