细胞壁降解酶在球刺盘孢侵染过程中的作用

为明确球刺盘孢Colletotrichum coccodes产生的细胞壁降解酶(CWDEs)在其侵染马铃薯过程中的作用, 本文对球刺盘孢离体和活体条件下产生的CWDEs种类及活性进行检测, 并通过RT-qPCR测定了球刺盘孢侵染过程中多聚半乳糖醛酸酶(PG)基因的表达情况。通过定性测定发现, 球刺盘孢能够产生果胶酶、纤维素酶和蛋白酶; 定量测定发现, 球刺盘孢可产生羧甲基纤维素酶(Cx)、β-葡萄糖苷酶(β-Glu)、PG和聚甲基半乳糖醛酸酶(PMG)。在活体条件下, 球刺盘孢可在马铃薯茎秆内产生PG、PMG和多聚半乳糖醛酸反式消除酶(PGTE), 且酶活分别于接种后48、72 h和144 h达到最大值, 分别为26.50、77.61 U/mL和0.16 U/mg。分别将羧甲基纤维素钠和果胶诱导后的酶液接种至马铃薯茎秆可引起植物组织浸腐, 与球刺盘孢引起的典型症状相似。除此之外, 球刺盘孢的4个PGs基因均在其侵染后24 h和48 h显著共上调表达。综上所述, 该研究结果表明, 果胶酶在球刺盘孢致病过程中发挥重要作用。     

番茄芝麻斑病原菌产生的细胞壁降解酶种类及其活性变化

番茄芝麻斑点病原菌能够产生一系列细胞壁降解酶,即果胶甲基酯酶(PE)、多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)、多聚半乳糖醛酸反式消除酶(PGTE)、果胶甲基反式消除酶(PMTE)和纤维素酶(Cx)。酶动力学研究表明:产生的各种细胞壁降解酶均有特定的最适反应条件。水解酶类的PG、PMG、Cx最大酶活的pH均为5.0,温度均为50℃;裂解酶类的PGTE和PMTE最大酶活的pH均为9.0,温度均为30℃,与其他病菌产生的细胞壁降解酶的特性基本相同。在活体外,随着培养天数的增加PG、PMG、PGTE、PMGE、Cx的活性都大幅度增加,但所有酶活性都明显低于活体内的酶活性。     

玉米茎腐病菌产生的细胞壁降解酶种类及其活性分析

 玉米茎腐病菌Pythium aphanidermatum、Fusarium graminearum能够产生一系列细胞壁降解酶,即果胶甲基酯酶(PE)、多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)、多聚半乳糖醛酸反式消除酶(PGTE)、果胶甲基反式消除酶(PMTE)和纤维素酶(Cx)。Fusarium graminearum产生的细胞壁降解酶活性明显高于Pythium aphanidermatum。病菌在活体内和活体外产生的细胞壁降解酶活性明显不同。酶动力学研究表明:Fusarium graminearum产生的各种细胞壁降解酶均有特定的最适反应条件。水解酶类的PG、PMG、Cx最大酶活的pH分别为6.0、5.0、6.0,温度分别为50、40、50℃;裂解酶类的PGTE和PMTE最大酶活的pH均为9.0,温度分别为30、20℃,与其它病菌产生的细胞壁降解酶的特性基本相同。     

莲子草假隔链格孢SF．193的细胞壁降解酶研究

采用3,5一二硝基水杨酸法分析了莲子草假隔链格孢NimbyaalternantheraeSF一193菌株在活体内外产生的羧甲基纤维素酶（Cx）、口．葡萄糖苷酶、木聚糖酶、聚甲基半乳糖醛酸酶（PMG）和多聚半乳糖醛酸酶（PG）的活性。结果表明,SF一193可产生cx、伊葡萄糖苷酶、木聚糖酶和PG,且4种细胞壁降解酶在活体外和活体内的活性显著不同。其发酵滤液中以PG活性最高,其次为木聚糖酶、,β-葡萄糖苷酶和Cx;在空心莲子草叶片内以Cx活性最高,其次为β一葡萄糖苷酶、木聚糖酶和PG。在活体内,4种酶活性随接种天数和温度的变化而不同。Cx和卢．葡萄糖苷酶的活性在接种第3d达到最大,木聚糖酶和PG活性则分别在第2d和第5d达到最大;Cx、卢一葡萄糖苷酶和PG活性在30℃时最高,而木聚糖酶活性在35℃时最高。     

酚类物质和代谢对瓜果腐霉菌产生的细胞壁降解酶活性的影响

 本文明确了间苯三酚、邻苯二酚、香草醛对黄瓜苗期猝倒病菌瓜果腐霉菌(Pythium aphanidermatum)生长及其细胞壁降解酶体外合成和活性的影响。结果表明:3种酚类物质中香草醛抑制病菌生长比较明显,随着香草醛浓度提高,病菌菌落直径下降。3种酚类物质均能明显抑制病菌4种果胶酶(PG,PMG,PGTE,PMTE)的合成和活性,3种酚类物质抑制作用因果胶酶种类而异。邻苯二酚和香草醛对PG合成抑制比较明显。间苯三酚和邻苯二酚在各浓度下均完全抑制PMG合成。酚含量20ppm以上时,香草醛完全抑制了PGTE和PMTE合成,间苯三酚完全抑制了PMTE合成。香草醛对PG、PMG和Cx活性均有明显抑制作用。随苗龄增加过氧化物酶活性升高,木质化程度加强,寄主抗果胶酶降解能力也相应增加。寄主细胞壁降解中释放的半乳糖醛酸有刺激过氧化物酶活性的作用,这可能属于一种寄主自卫反应。     

马铃薯早疫病病菌产生细胞壁降解酶的条件研究

为明确马铃薯早疫病病菌产生细胞壁降解酶的条件,采用液体培养基培养病菌。结果表明,病菌在改良的Marcus液体培养基中能产生多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)、纤维二糖水解酶(Cx)和β-葡萄糖苷酶(βG)细胞壁降解酶。其中,果胶酶(PG、PMG)的活性较高,纤维素酶(Cx、βG)的活性较低。病菌产生果胶酶的适宜条件为培养液初始p H为4.0,30℃持续振荡培养4 d,纤维素酶初始p H为5.0,25℃持续振荡6 d。     

增强UV-B辐射对立枯丝核菌抗氧化酶及细胞壁降解酶活性影响研究

 作为太阳光谱的一部分,到达地球表面的UV-B辐射有逐渐增加的趋势,影响地球生态系统。为揭示增强UV-B辐射对立枯丝核菌的影响,本文采用UV-B灯辐照处理病菌菌丝,并对菌丝生长、菌核形成,活性氧稳态和细胞壁降解酶进行分析。结果表明,增强UV-B辐射可导致供试菌株菌丝生长抑制、刺激菌核提早形成。处理菌株O^-·₂和H₂O₂的含量随处理时间的延长呈现不同程度的增加,其中U60处理较对照分别增加132.3%和71.3%;尽管SOD、APX、POD和CAT抗氧化酶活性也随处理时间增加而显著增强,但菌株MDA含量依然显著逐渐增加。同时,立枯丝核菌纤维素酶Cx、FPA的活性以及PG、PMG的活性均随辐射处理时间的延长而下降。此外,UV-B辐射处理菌株对高粱幼苗致病表型减弱。综上所述,增强的UV-B辐射打破立枯丝核菌体内活性氧稳态平衡,致使菌丝生长受到抑制,细胞壁降解酶活性降低,从而影响病菌致病性。     

香蕉枯萎病菌1号和4号生理小种细胞壁降解酶的比较

对香蕉枯萎病菌1号和4号生理小种的细胞壁降解酶进行比较。通过测定4号生理小种在寄主体内细胞壁降解酶的活性发现,能检测到多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)、多聚半乳糖醛酸反式消除酶(PGTE)、果胶甲基反式消除酶(PMTE)和纤维素酶(Cx)的活性。在不同碳源培养条件下,2个生理小种均有以上5种酶的活性,以1%柑桔果胶为碳源时产生的PMG和PG活性明显高于其他几种酶的活性,而以1%CMC为碳源时,所产生的Cx都比其他几种酶的活性高。细胞壁降解酶同工酶电泳后发现,4号生理小种在寄主体内和体外培养时都比1号生理小种多分泌一种PG。2个生理小种在体外培养时分泌的PMG、PGTE和PMTE没有差异。4号生理小种在寄主体内比1号生理小种多分泌一种PMG,却少分泌一种PGTE,2个生理小种在寄主体内的PMTE则没有差异。     

温湿度调控对番茄灰霉病菌产生的细胞壁降解酶的影响

 番茄灰霉病菌在致病过程中能够产生4种细胞壁降解酶,以PMG酶活性最高,其次是β-葡萄糖苷酶和PG酶,Cx最少。灰霉病菌在不同温度下侵染番茄叶片时产生的致病酶活性不同,4种酶在20℃时表现了最高的活性,15℃次之,当温度达到25℃时,各种酶的活性都急剧下降,随着温度的再升高,酶活更低。随着湿度的增高,病菌产生的细胞壁降解酶的活性也增加,当相对湿度达到90%以上时,4种酶的活性也达到最高。温湿度对番茄灰霉病菌产生细胞壁降解酶的影响趋势,与其对发病的影响趋势是一致的。     

细胞壁降解酶在油茶炭疽病菌致病过程中的作用研究

为明确细胞壁降解酶在油茶炭疽病菌致病过程中的作用,本文研究了活体内外炭疽病菌产生的细胞壁降解酶活性及其对叶片的降解情况。结果表明,活体外以羧甲基纤维素钠(CMCNa)为诱导底物,羧甲基纤维素酶(Cx酶)和漆酶活性最高;以柑橘果胶为诱导底物,果胶酶活性最高;以油茶叶为诱导底物,纤维素酶、果胶酶和漆酶可产生较高活力;并且经5种诱导物诱导的酶液对叶片均有降解作用。发病叶片的各部位,以病健交界处细胞壁降解酶活性最高。接种4d后开始发病,其细胞壁降解酶活性迅速增强;6d后滤纸酶(FPA)、β-葡萄糖苷酶和漆酶活性达最大值,分别为4.53、7.44、1.21U/mg;而Cx酶和果胶酶在第8天时酶活性最高,分别为15.79和25.49U/mg;接种10~16d,酶活性比较稳定。上述结果表明,纤维素酶、果胶酶和漆酶在油茶炭疽病菌致病过程中起重要作用。     

Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

The antifungal glycoalkaloid -tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades -tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (>98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, -tomatine-inducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by two-dimensional gel electrophoresis. A protein with a molecular mass of 64kDa accumulated in the -tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading -tomatine to lycotetraose and tomatidine.     

Fusarium oxysporum f.sp. cucurbitacearum n.f. embracing all formae speciales of F. oxysporum attacking Cucurbitaceous crops

Isolates ofFusarium oxysporum from wilted muskmelons, watermelons, cucumbers and from the muskmelon rootstockBenincasa hispida were screened for pathogenicity on seedlings and adult plants of these crops and related species. In seedling tests the isolates were not typically species-specific, contrary to what might be expected as an implication of their characterization as forma specialis. They often attacked species of several genera of plants, but not beyond the family of theCucurbitaceae. In the adult stage, plants were much more exclusively attacked by their corresponding formae speciales, but essential exceptions occurred. Isolates from cucumber were highly pathogenic to muskmelons, in the adulstage even causing more wilt of the latter than of cucumber.Comparing the results of these experiments with data from the literature, it is argued that the proposed f.sp.cucurbitacearum, embracing all formae speciales which specialize on plants within the family of theCucurbitaceae, would best fit in with the present state of knowledge. A proposition is given for equivalence of old and new classifications of isolates.Samenvatting Isolaten vanFusarium oxysporum uit verwelkte meloenen, watermeloenen, komkommers en uit de meloene-onderstamBenincasa hispida werden getoetst op pathogeniteit voor zaailingen en volwassen planten van deze gewassen en verwante soorten. In zaailingtoetsen waren de isolaten weinig soort-specifiek, in tegenstelling tot wat mocht worden afgeleid uit hun karakterisering als forma specialis. Zij tastten vaak soorten uit verschillende geslachten aan, maar niet buiten de grenzen van de familie derCucurbitaceae. in het volwassen stadium waren de interacties veel specifieker en werden planten slechts aangetast door de bijbehorende formae speciales. Enkele essentiële uitzonderingen kwamen echter voor. Isolaten van komkommer waren zeer pathogeen voor meloen. In het volwassen stadium veroorzaakten zij zelfs sterkere verwelking van meloen dan van komkommer.Vergelijking van de resultaten van deze proeven met gegevens uit de literatuur leidt tot de conclusie dat de voorgestelde f.sp.cucurbitacearum, die alle formae speciales met specialisatie opCucurbitaceae omvat, het best overeenkomt met de huidige stand van kennis. Een voorstet wordt gedaan ter vervanging van de oude classificatie van isolaten door corresponderende nieuwe aanduidingen.     

DNA sequence analysis of ribosomal ITS region 1 of Phytophthora vignae f. sp. adzukicola, the pathogen that causes stem rot of adzuki bean

Sequences of the internal transcribed spacer (ITS) region 1 were used to examine the phylogenetic relationships among races of 19 isolates of Phytophthora vignae f. sp. adzukicola and between this forma specialis and three isolates of the closely related P. vignae f. sp. vignae. The ITS 1 sequences were highly conserved (> 98.7% similarity) among representatives of both formae speciales groups. The results of this study indicate that P. vignae is a monophyletic group. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession nos. AB120062–AB120080 and AB120122     

玉米大斑病菌两性交配型菌株的出现频率及其育性分析

玉米大斑病菌属于异宗配合真菌,在自然界中存在A、a交配型菌株和Aa两性交配型菌株,为明确两性菌株的出现频率及其对有性生殖的影响,采用有性态诱导和交配型PCR鉴定的方法,对2011—2014年采集的野生菌株及室内诱导产生的有性杂交后代F1、F2代进行交配型组成鉴定,通过检测两性菌株子囊壳和子囊孢子的发育情况明确其有性生殖能力,结合RT-PCR技术对有性生殖过程中MAT1和MAT2基因的表达情况进行分析。结果表明,野生菌株和有性杂交后代中均存在两性交配型菌株,且出现频率相当,介于2.09%和6.25%之间;Aa两性菌株的育性与杂交时对应菌株的交配型组成相关,两性菌株与A交配型菌株或a交配型菌株杂交均可育,能产生成熟的子囊壳和子囊孢子,但其自交时败育,只产生子囊壳,不产生子囊孢子。两性菌株Aa自交和两性菌株Aa与A交配型或与a交配型菌株杂交时,Aa菌株中MAT1和MAT2基因表达量无明显差异,推测MAT基因在有性生殖产生子囊壳的过程中发挥了重要作用。     

The basis of host recognition in Fusarium oxysporum f. sp. lycopersici

Filtrates from shake-cultures of Fusarium oxysporum f. sp. lycopersici race 1, concentrated to 20% of the original volume, caused cell death in tomato leaf protoplasts from near-isogenic lines corresponding to the compatible cultivar/race reactions of whole plants. Maximum activity was found in late log phase cultures on Czapek-Dox supplemented with 2% casamino acids. Selective toxicity was associated only with the protein fraction of the culture filtrate. LD₅₀ values for susceptible Ace and Moneycross to F. oxysporum f. sp. lycopersici race 1 culture filtrates were 1·92 and 0·36 μg protein ml⁻¹. Corresponding values for cvs Royal Ace and MM161, each containing the I-gene conferring resistance to race 1, were >350. Culture filtrates from F. oxysporum f. sp. lycopersici race 2 gave LD₅₀ values of 2·34 and 2·08 μg protein ml⁻¹ on cvs Ace and Royal Ace, both susceptible to race 2. The LD⁵⁰ of cv. Ace to a non-pathogenic isolate of F. xysporum f. sp. lycopersici was > 350. Culture filtrates from non-host formae of F. oxysporum were 9–149-fold less toxic on cv. Ace. Protoplasts from Pisum sativum, Lactuca sativa, Zea mays, Gossypium barbadense and Solanum melongena, all non-hosts of F. oxysporum f. sp. lycopersici, were 6–175 times less sensitive to F. oxysporum f. sp. lycopersici filtrates than susceptible tomato. The putative toxins lycomarasmin and fusaric acid showed no differential toxicity to I⁺ and I⁻ tomato protoplasts. The results are discussed in the wider context of host-pathogen interaction in which specificity is considered as the recognition of susceptibility by a proteinaceous toxic metabolite of the pathogen. This hypothesis is further extended to include the specificity of F. oxysporum formae and races.     

Development of sequence tagged site markers to identify races of Fusarium oxysporum f. sp. lactucae

By random amplified polymorphic DNA (RAPD) analysis of the representative isolates of each race of Fusarium oxysporum f. sp. lactucae, RAPD fragments of 0.6, 1.6, and 2.9kb were obtained. The 0.6-kb RAPD fragment was common to the representative isolates of all three races. Amplification of the 1.6- and 2.9-kb fragments were unique to the isolates of races 1 and 2, respectively. Sequence tagged site (STS) marker FLA0001, FLA0101, and FLA0201 were generated from the 0.6-, 1.6-, and 2.9-kb RAPD fragments, respectively. Polymerase chain reaction (PCR) analysis showed that FLA0001 was common to all 49 isolates of F. oxysporum f. sp. lactucae. FLA0101 was specifically generated from all 23 isolates of race 1 but not from races 2 or 3. FLA0201 was specifically amplified from all 12 isolates of race 2 but not from races 1 or 3. In two isolates of F. oxysporum f. sp. lactucum, PCR amplified FLA0001 and FLA0101 but not FLA0201. On the other hand, these STS markers were not detected from isolates of five other formae speciales. Because these STS markers were not generated from isolates of other plant pathogenic fungi, bacteria, or plant materials examined in this study, PCR analysis combined with the three STS markers should be a useful means for rapid identification of races of F. oxysporum f. sp. lactucae.     

Gene genealogies support Fusarium oxysporum f. sp. ciceris as a monophyletic group

Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of fusarium wilt of chickpea, consists of two pathotypes (yellowing and wilting) and eight races (races 0, 1B/C, 1A and 2–6) of diverse geographical distribution. Six Foc isolates, one each of races 0, 1B/C, 1A, 4, 5 and 6, representing the two pathotypes and the geographical range of the pathogen, showed identical sequences in introns of the genes for translation elongation factor 1α ( EF1 α), β-tubulin, histone 3, actin and calmodulin. Eleven additional Foc isolates representative of all races, pathotypes and geographical range, and three isolates of F. oxysporum (Fo) nonpathogenic to chickpea were further analysed for sequence variation in the EF1 α gene. All isolates pathogenic to chickpeas shared an identical EF1 α gene sequence, which differed from that shared by the three Fo isolates nonpathogenic to chickpea. EF1 α gene sequences from the 17 Foc isolates and the three Fo isolates were compared with 24 EF1 α gene sequences in GenBank from isolates of 11 formae speciales of F. oxysporum by parsimony analysis. Foc isolates formed a grouping distinct from other formae speciales and nonpathogenic isolates. These results indicate that F. oxysporum f. sp. ciceris is monophyletic.     

Phylogeny of Fusarium oxysporum f. sp. lactucae Inferred from Mitochondrial Small Subunit, Elongation Factor 1-alpha, and Nuclear Ribosomal Intergenic Spacer Sequence Data

ABSTRACT Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathogen recently reported in Arizona. Sequence analysis of the mitochondrial small subunit (mtSSU), translation elongation factor 1-alpha (EF-1alpha) gene, and the nuclear ribosomal DNA intergenic spacer (IGS) region was conducted to resolve relationships among f. sp. lactucae isolates, F. oxysporum isolates from other hosts, and local non-pathogenic isolates. Analysis of mtSSU sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from 13 other F. oxysporum isolates. Analysis of EF-1alpha sequences resulted in moderate resolution, grouping seven formae speciales with the lactucae isolates. Analysis of the IGS region revealed numerous sequence polymorphisms among F. oxysporum formae speciales consisting of insertions, deletions, and single nucleotide transitions and substitutions. Repeat sequence analysis revealed several duplicated subrepeat units that were distributed across much of the region. Based on analysis of the IGS sequence data, lactucae race 1 isolates resolved as a monophyletic group with three other formae speciales of F. oxysporum. In all analyses, lactucae race 2 isolates composed a separate lineage that was phylo-genetically distinct and distantly related to the lactucae race 1 isolates.     

番茄枯萎病菌和颈腐根腐病菌KASP-SNP检测技术的建立

为快速、准确地对番茄枯萎病菌Fusarium oxysporum f. sp. lycopersici(FOL)和番茄颈腐根腐病菌F. oxysporum f. sp. radicis-lycopersici(FORL)进行检测,基于尖孢镰刀菌F. oxysporum多聚半乳糖醛酸外切酶基因pgx4的单核苷酸多态性（single nucleotide polymorphism,SNP）位点,设计FORL、FOL生理小种1(FOL-R1)、2(FOL-R2)和3(FOL-R3)的竞争性等位基因特异性PCR-SNP(kompetitive allele specific PCR-SNP,KASP-SNP)引物,建立番茄颈腐根腐病菌和番茄枯萎病菌KASP-SNP检测技术,并通过与常规PCR比对及ITS与pgx4序列分析对该检测技术的可靠性进行验证。结果显示,在FORL、FOL-R1、FOL-R2和FOL-R3中存在35个变异SNP位点,设计出18对KASP-SNP引物,筛选出FORL＿KASP、FOLrace1＿KASP、FOLrace2＿KASP和FOLrace3＿KASP共4对分型清晰的...     

苦瓜枯萎病菌的鉴定及其遗传多样性分析

 对分离获得的32株苦瓜枯萎病菌菌株进行形态学特征和寄主专化型测定, 结果表明, 测试的苦瓜枯萎病菌株均为尖孢镰刀菌苦瓜专化型 (Fusarium oxysporum f. sp. momordicae), 这些菌株可以侵染苦瓜和瓠瓜幼苗, 但不侵染其他葫芦科瓜类作物。对苦瓜枯萎病菌菌株的rDNA-ITS区 (ITS1、5.8S和ITS2)序列进行扩增测序, 结果显示其序列长度均为456 bp;聚类分析表明测序菌株与镰刀菌属中尖孢镰刀菌不同专化型的菌株聚为一群。利用RAPD标记技术分析苦瓜枯萎病菌的遗传多样性, 结果显示苦瓜枯萎病菌株与其他葫芦科瓜类作物枯萎病菌株间的遗传相似系数范围为0.59~0.99, 当遗传相似系数为0.85时, 供试的48个菌株分成10个类群 (G_1~10)。在RAPD聚类树中所有苦瓜枯萎病菌株聚在一个分支上 (G₁群), 菌株间的遗传相似系数范围为0.92~1.00, 具有较高的遗传相似性, 且菌株的聚群与地理来源存在一定的相关性。