马铃薯致病疫霉研究进展

马铃薯致病疫霉(Phytophthora infestans)属卵菌纲(Oomycetes)霜霉目(Peronosporales)腐霉科(Pythiaceae)疫霉属(Phytophthora),是马铃薯和番茄晚疫病病原菌。由于晚疫病对马铃薯生产的毁灭性和严重性,对致病疫霉的研究一直是关注的重点。本文首先对病害引起的症状、发生特点及流行规律进行阐述,对有性生殖发生的遗传规律和多种交配型共存的大环境下病原菌群体结构变异特点进行归纳总结。随着2009年致病疫霉基因组测序的完成,本文比对了疫霉属目前已完成测序各个种的基因组学特点,介绍了致病疫霉在效应子克隆方面的研究进展及线粒体基因组研究现状,阐述了功能基因组学的两个重要技术:高密度遗传连锁图谱(high density linkage mapping)和全基因组关联分析(genome-wide association study,GWAS),及其在挖掘致病疫霉重要功能基因上的应用。本文有助于了解致病疫霉研究热点及后续突破方向,可为深入解析致病疫霉的功能基因及致病机制提供参考,对开发马铃薯晚疫病菌药物靶标及预测病害的大规模流行趋势也具有重要意...     

致病疫霉有性生殖诱导及F1代遗传多样性

致病疫霉Phytophthora infestans为马铃薯晚疫病的重要病原菌。通过从昆明市寻甸县采集110P和H-6两株致病疫霉,明确其染色体倍性、交配型、线粒体单倍型、毒性和甲霜灵敏感性,经对峙培养,利用改良的卵孢子萌发方法获得有性生殖F1代群体POP1（60株）,并对POP1进行表型和基因型测定。结果表明：冷冻处理24h为最佳条件,卵孢子萌发率达5.09%±0.15%;POP1的交配型、毒性和甲霜灵敏感性均发生了分离,其中交配型分离比为A1:A2:A1A2:自育型（SF）=16:5:17:22,毒性分离比为抗性（R）:敏感性（S）=11:49,甲霜灵敏感性分离比为抗性（R）:敏感性（S）=2:58;3个表型的分离均偏离孟德尔单基因显性遗传特点。基于8对SSR多态性引物对POP1基因型分析表明,遗传相似系数为0.98时,可将所有菌株分为14个基因型;遗传相似系数为0.95时,可将POP1分为6个分支,其中优势群体为S1,占分离群体的61.67%。关联分析进一步表明,8对SSR所代表的基因型和几个重要表型有显著相关性（R2=0.6667）。本研究建立了高效的致病疫霉卵孢子萌发体系,解析了有性生殖后代群体遗传结构特点,为深入探索致病疫霉的变异规律及病害流行趋势提供了理论基础。     

一个马铃薯种质资源圃致病疫霉群体的分析

由致病疫霉Phytophthora infestans引起的晚疫病是马铃薯生产上最严重的病害之一,认识其群体结构特征,可为晚疫病防控策略的制定以及抗病品种的合理布局提供指导。对2009年采自宁夏一个种植有93个品种(品系)的马铃薯种质资源圃的致病疫霉进行了交配型、致病型和线粒体DNA单倍型分析,结果表明,116个致病疫霉菌株中存在A1、A2和自育型3种交配型,发生频率分别为24.1%、57.8%和18.1%,A2交配型为优势类型;对其中43个菌株的致病型进行测试,检测到两种致病类型:1.2.3.4.5.6.7.8.9.10.11和3.4.10,发生频率分别为95.3%和4.7%,可克服所有11个抗病基因的1.2.3.4.5.6.7.8.9.10.11类型占绝对优势;对62个菌株的线粒体DNA单倍型进行分析,检测到Ia和IIa两种类型,发生频率分别为74.2%和25.8%。综合表型和基因型数据分析发现,该马铃薯种质资源圃中致病疫霉群体致病型单一,但致病型毒力因子高度复合;线粒体DNA分析表明,该马铃薯种质资源圃引入了遗传背景较为复杂的致病疫霉"新"群体。     

辣椒疫霉致病毒素

辣椒疫霉在培养液中振荡培养时可分泌毒素,这种纱能经志辣椒叶片形成水渍状褐腐斑,类似病原菌侵染后形成的症状,Ｐｌｉｃｈ’ｓ和Ｖ８Ｃ２液适于Ｐ．ｃａｐｓｉｃｉ的生长和产毒;生长适宜温度和ＰＨ范围分别为２０－３０℃和ＰＨ６－７,在２５－３０℃、Ｐ５－８的条件下２滤液毒性最强;培养１５天产毒达到最高值,液先后经８５％硫酸铵沉淀,ＤＥ５２、Ｃ几ＳｅｐｈａｄｅｘＧ－７５柱层将毒素纯化。经鉴定该纯毒素为３９．８     

几种真菌发酵液对致病疫霉的抑制作用

测定了８种真菌发酵液在５种不同浓度下对致病疫霉菌丝生长、游动孢子静止、静止胞萌发、附着胞形成和侵入丝形成等不同阶段的影响。结果表明,供试真菌不同浓度的发酵液,对致病疫霉上述各个阶段均有一定程度的抑制作用,并均随发酵液浓度增加,抑制作用逐渐增强,浓度为１００％时,抑制作用均达到最高。其中,立枯丝核菌发酵液的抑制作用最强,浓度为１００％时,对致病疫霉菌丝生长的抑制率达到９０．４％,而静止胞萌发率仅为２．４％,附着胞及侵入丝均未见形成。     

福建省部分地区马铃薯致病疫霉群体遗传多样性分析

致病疫霉(Phytophthora infestans)引起的晚疫病是马铃薯的一种毁灭性病害。有效控制马铃薯晚疫病需要明确致病疫霉的群体遗传结构特征。采用8对SSR引物对采自福建省福州、长乐、漳州2010年分离的95株马铃薯致病疫霉进行遗传多样性分析。结果共检测出21个等位基因和26个基因型。三个地点致病疫霉菌群体间的平均遗传分化系数FST为0.22,在8个位点中有5个位点的等位基因频率分布差异显著。三个群体的观测纯合度小于期望纯合度,观测杂合度大于期望杂合度,以无性生殖为主。结果表明福建群体的遗传多样性高,群体间的存在较高的遗传分化度。     

辣椒疫霉致病毒素

辣椒疫霉(Phytophthora capsici Leonian)在培养液中振荡培养时可分泌毒素,这种毒素能引起辣椒叶片形成水渍状褐腐斑,类似病原菌侵染后形成的症状。Hich’s和V₈培养液适于P capsici的生长和产毒;生长适宜温度和pH范围分别为20～30℃与pH6～7,在25～30℃、pH5～8的条件下培养滤液毒性最强;培养15天产毒达到最高值。滤液先后经85％硫酸铵沉淀、DE52、CM32和SephadexG-75柱层析将毒索纯化。经鉴定该纯毒索为39．8kD酸性糖蛋白。并对80℃以上高温或蛋白酶敏感,而β-D-葡萄糖苷酶处理不影响其毒性,蛋白亚基为毒紊的活性中心。毒素处理寄主叶片引致病理性坏死,这一作用与辣椒品种抗病性有关。     

致病疫霉PiSAK1的生物学功能分析

【背景】致病疫霉是引起世界范围内马铃薯晚疫病的重要病原菌。Stress-activated protein kinases (SAPKs)是一类胁迫激活的mitogen-activated protein kinases (MAPKs),研究表明真菌SAPKs在调控细胞应答外界胁迫等方面有重要作用。致病疫霉中存在一个SAPK,即PiSAK1,其生物学功能并不明确。【目的】探究PiSAK1在致病疫霉生长发育、抵抗外界胁迫及侵染马铃薯过程中发挥的生物学功能。【方法】利用生物信息学手段分析PiSAK1的特性,通过RT-qPCR分析明确致病疫霉PiSAK1在不同发育阶段及侵染马铃薯不同时期的表达量,最后构建PiSAK1沉默、过表达菌株并测定其各项生物学表型。【结果】PiSAK1具有丝裂原活化蛋白激酶典型的Ser/Thr蛋白激酶催化结构域,并且与其他卵菌的SAPKs同属一个进化分支。致病疫霉PiSAK1分别在休止孢阶段、侵染马铃薯48 h时表达量最高,且0.3 mol/L NaCl及3 mmol/L H2O2胁迫刺激0.5 h后PiSAK1的表达量均显著升高。构建PiSAK1沉默、过表达菌株并测...     

恶疫霉致病力和对甲霜灵敏感性的遗传与变异

以分离自黑龙江腐烂苹果的恶疫霉Phytophthora cactorum Schroeter野生型菌株Ap14为亲本,采用菌丝块创伤接种法测定了恶疫霉对苹果的致病力在无性繁殖和有性生殖后代的遗传,结果是连续2代单游动孢子后代在苹果上所致病斑半径分别为22.4-24.1mm和21.8-23.4mm,与亲本所致病斑半径22.5mm无显著差异;而其20个自交后个体所致病斑半径为21.4-25.8mm,与亲本有显著差异。其中2株致病力显著强于亲本,其余与亲本相似,表明恶疫霉对苹果的致病力在无性后代可稳定遗传,而在自交有性后代发生分离,同时在含甲霜灵0.05μg/ml的LBA平板上测定了恶疫霉菌丝生长对甲霜灵的敏感性在无性和有性后代的遗传。结果是恶疫霉对甲霜灵的敏感性在无性单孢后代无显著变异,而在50个自交有性后代中,上述浓度对其菌丝生长的抑制率分布范围为67.3-97.1%,与亲本78.3%有极显著差异。其中3株高于亲本,3株低于亲本,44株与亲本相似,表明恶疫霉对甲霜灵的敏感性同样在无性后代稳定遗传而在有性后代发生变异,上述结果提示,供试恶恶疫霉菌株中上述性状可能由细胞核杂合基因控制。     

马铃薯晚疫病菌DK98-1、DX98-2和DX98-3的生物学特性及AFLP指纹分析

利用黑麦培养基和V8-蔬菜汁培养基研究了马铃薯晚疫病菌Phytophthora infestans特异菌株DK98-1、DX98-2和DX98-3 的生物学特性,发现该菌株与普通菌株相比菌落生长速度慢、孢子囊产生数量少、有性杂交后卵孢子产生量大（2047～75623个/cm²）;利用AFLP分子标记研究这3个菌株的DNA指纹图谱,发现用引物E+CG/M+CC扩增菌株DK98-1、DX98-2和DX98-3后,在330bp处与普通菌株相比各缺失一条谱带,用引物E+AC/M+CT扩增菌株DK98-1、DX98-2和DX98-3后,在370bp处比普通菌株增加1条谱带,说明这3个菌株与普通菌株在遗传上明显不同。同时可以利用上述2对特异性引物,鉴定在自然界的晚疫病菌群体中这类特异菌株的出现频率。     

Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F₁ populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F₁ populations analysed, whereas the RI allele segregated with a 1:1 ratio as expected in five F₁ populations. The mode of inheritance of the R10 allele could not be deduced as only very few F₁ hybrids bearing R10 were obtained. Linkage analysis in two F₁ populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromdsome XI.     

拮抗烟草疫霉的枝穗霉菌株筛选

【背景】烟草疫霉(Phytophthora nicotianae)引起的烟草黑胫病(tobacco black shank)是烤烟生产上重要的土传根茎病害之一,生产上防治困难。【目的】筛选对病原菌具有强拮抗能力的有益微生物菌株是开展生物防治的基础。【方法】采用平板对峙法筛选对烟草疫霉具有拮抗作用的枝穗霉菌株。根据枝穗霉在烟草疫霉菌落上的覆盖程度和产孢量,以及对烟草疫霉菌丝、孢囊梗和孢子囊的缠绕情况,将枝穗霉的拮抗能力划分为强、中等、弱和无4个等级。【结果】供试8种65株枝穗霉中,6株具有强拮抗能力、27株具有中等拮抗能力、22株具有弱拮抗能力、10株无拮抗能力;不同枝穗霉菌株对烟草疫霉的抑制率大小为20.0%-86.7%。【结论】粉红枝穗霉(Clonostachys rosea)菌株7901、11361和亚麻生枝穗霉(C. byssicola)5072、6729、7507及条孢枝穗霉(C. grammicospora)6730对烟草疫霉具有强拮抗能力,这为后续盆栽试验及作用机理研究等提供了种质资源。     

AFLP Linkage Map of the OomycetePhytophthora infestans

Here we present the first comprehensive genetic linkage map of the heterothallic oomycetous plant pathogenPhytophthora infestans.The map is based on polymorphic DNA markers generated by the DNA fingerprinting technique AFLP (Voset al.,1995,Nucleic Acids Res.23:4407–4414). AFLP fingerprints were made from single zoospore progeny and 73 F1 progeny from two field isolates ofP. infestans.The parental isolates appeared to be homokaryotic and diploid, their AFLP patterns were mitotically stable, and segregation ratios in the F1 progeny were largely Mendelian. In addition to 183 AFLP markers, 7 RFLP markers and the mating type locus were mapped. The linkage map comprises 10 major and 7 minor linkage groups covering a total of 827 cM. The major linkage groups are composed of markers derived from both parents, whereas the minor linkage groups contain markers from either the A1 or the A2 mating type parent. Non-Mendelian segregation ratios were found for the mating type locus and for 13 AFLP markers, all of which are located on the same linkage group as the mating type locus.     

Biosynthesis of scopoletin in Hevea brasiliensis leaves inoculated with Phytophthora palmivora

Inoculation of resistant (R) and susceptible (S) Hevea brasiliensisleaves with Phytophthora palmivorainduced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Heveaphytoalexin. The degree of resistance of four clones, as classified by the necrotic lesions, was related to the rapidity and intensity of Scp production. The resistant BPM-24, and marked partially resistant clone PB-235, displayed an early secretion of scopoletin that intensified and lasted longer than the weak partially resistant RRIT251, and susceptible clone RRIM600. The lesion size and amount of Scp after infection were positively correlated to the concentration of spores applied to Hevealeaves. In addition, in leaflets inoculated with high spore concentration, Scp reached the highest level earlier than those with low spore concentration. A fungitoxic effect of Scp on mycelium growth was shown in bioassays; the I₅₀ value tested on Phytophthora palmivora was relatively the same as on Phytophthora botryosa, but much lower than those found on other leaf pathogens of rubber tree, Corynespora cassiicolaand Colletotrichum gloeosporioides. The lesion size and level of Scp upon spore inoculation may be appropriate for classifying Heveaclones according to their R/S with respect to P. palmivora     

Expression of the Phytophthora infestans ipiB and ipi0 genes in planta and in vitro

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.     

马铃薯对晚疫病水平抗性的组织细胞学观察

以马铃薯晚疫病水平抗性品种LBr-12和感病品种费乌瑞它为材料,采用普通光学和电子显微镜技术,系统研究了马铃薯与晚疫病菌(致病疫霉)互作的组织细胞学反应特征。观察结果显示:(1)接种后,水平抗性材料LBr-12出现过敏反应,病菌被限制在侵染点的几个细胞中,菌丝产生较少的分支和吸器。(2)感病品种费乌瑞它被侵染细胞呈蔓延趋势,菌丝产生较多的分支和吸器。(3)电镜观察发现,抗病品种上病菌的胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,包括原生质的电子致密度加深、液泡增多变大、菌丝细胞壁不规则增厚、细胞器排列紊乱及解体、吸器母细胞及吸器形态异常、病菌最终畸形坏死,同时发现抗病品种受病菌侵染时可迅速产生结构防卫反应,形成的细胞壁沉积物使胞壁极度增厚或细胞膜上产生乳突状结构。     

Phytophthora captiosa sp. nov. and P. fallax sp. nov. causing crown dieback of Eucalyptus in New Zealand

A locally severe crown disease of exotic plantation Eucalyptus trees has been recorded periodically in New Zealand since 1986. Symptoms include leaf spots, petiole infection and twig and small branch lesions. Outbreaks of disease are episodic and individual trees may show marked variation in crown symptoms ranging from unaffected to total defoliation. Two previously unknown species of Phytophthora are associated with the disease. These are described and formally designated here as P. captiosa, from Eucalyptus botryoides and E. saligna; and P. fallax, from E. delegatensis, E. fastigata, E. nitens and E. regnans. Both P. captiosa and P. fallax have non-papillate, non-caducous sporangia and both are self-fertile. Phylogenetic analysis on the basis of ITS rDNA sequence data indicates they are closely related to each other but evolutionarily distant from the majority of described Phytophthora taxa. They share a common ancestor with another assemblage of Phytophthora lineages that includes P. insolita, P. macrochlamydospora and P. richardiae. Sporulation of P. captiosa and P. fallax has not been observed in the field. The mode of infection and spread of these non-caducous Phytophthora species in the eucalypt tree canopy remains unknown. This issue, and the possible geographic origins of these two Phytophthora species are discussed.