甘蓝黑腐病黄单胞菌胞外蛋白酶的致病作用

现代分子生物学实验技术,为研究植物病原菌的致病机理提供了有效的手段。目前已在分子水平上,证实了甘蓝黑腐病黄单胞菌产生的胞外蛋白酶在致病过程中起着重要的作用。本文就此病原菌产生的胞外蛋白酶、胞外蛋白酶基因的克隆分析、胞外蛋白酶突变体的构建以及胞外蛋白酶在致病过程中的作用的研究进展作一概述。     

野油菜黄单胞菌一个假定蛋白激酶基因与多糖合成及致病力相关

已报道的野油菜黄单胞菌8004菌株基因组中,开放阅读框XC3631注释编码一个假定蛋白激酶。通过定点整合突变方法构建了XC3631基因的非极性突变体NK3631,表型分析发现NK3631合成脂多糖和胞外多糖的能力显著下降,致病力极显著减低,但产生胞外水解酶的能力不变。用带有全长XC3631基因序列的pLAFR3对NK3631进行功能互补,其脂多糖合成、胞外多糖产量和致病力均得到恢复。这一研究表明,XC3631基因与野油菜黄单胞菌脂多糖的核心多糖和胞外多糖的合成有关,并在致病中发挥重要作用。     

植物乳杆菌C88胞外多糖生物合成基因的克隆及序列比对

乳酸菌胞外多糖能显著改善发酵乳制品及食品的流变学和质构特性。为进一步了解乳酸菌胞外多糖的生物合成途径及调控机制,本研究对参与植物乳杆菌C88胞外多糖生物合成基因簇的部分序列进行了克隆和鉴定。根据GenBank中己报道植物乳杆菌基因序列的保守区域设计特异性引物,扩增出植物乳杆菌C88生物合成蛋白基因（cps4A）序列,并通过染色体步移方法克隆了植物乳杆菌C88参与胞外多糖合成基因簇的部分序列（4．9kb）。利用生物信息学方法预测基因簇中6个阅读框的结构和功能,结果表明该序列与己报道的乳酸杆菌胞外多糖生物合成基因具有高度的同源性（〉96％）;对各阅读框功能预测分析发现,这6个基因主要编码参与胞外多糖合成中的多糖合成蛋白、糖链长度检测蛋白、UDP-葡萄糖-4-异构酶和糖基转移酶。本研究将为利用基因工程方法调控多糖的合成和产量提供理论依据。     

细菌毒素在病原物致病过程中的作用

植物病原细菌毒素是重要的植物病原毒素,其化学类别,包括胞外多糖、有机酸、肽、糖肽等。不少植物病原菌引起病害与病原菌产生的植物毒素有关,致病毒素是诱发植物病害的一类重要化合物,其主要伤害效果是:作用于质膜,所以原生质体肿胀,电解质渗漏;抑制光合作用的ATP磷酸化的合成,从而引起能量匮乏,导致体内合成的破坏,影响叶绿素的合成;结合原生质膜蛋白,从而导致宿主感染。由此,主要综述了两大类细菌毒素在病原物致病过程中的作用研究进展。     

球孢白僵菌基因组候选效应子的预测与分析

球孢白僵菌(Beauveria bassiana)是一种广泛应用的生防菌株,效应子在其对寄主昆虫侵染过程中发挥着重要作用。为高通量地筛选球孢白僵菌的效应子,本研究根据球孢白僵菌全基因组信息,利用SignalP、TMHMM、TargetP和Protcomp等生物信息学软件和预测程序对球孢白僵菌中10 364个蛋白序列进行分泌蛋白预测。结果表明,共分析到940个分泌蛋白,包含了185个碳水化合物活性酶家族蛋白。其中碳水化合物结合模块(CBM)40个,碳水化合物酯酶(CE)23个,糖苷水解酶(GH)92个,糖基转移酶(GT)4个,多糖裂解酶(PL)2个,辅助功能酶(AA)24个。进一步将上述分泌蛋白与胞外酶数据库进行比对分析、半胱氨酸含量分析、多个串联重复序列分析、病原寄主互作数据库进行比对及剔除大于300个氨基酸序列的蛋白,共获得18个候选效应子,其中11个为功能未知的假定蛋白,其余为胞外蛋白、细胞壁蛋白、几丁质酶蛋白等。本研究结果为进一步探究效应子在病原菌与昆虫寄主中的互作奠定了理论基础,并为其他病原菌效应子的预测及分析提供了技术参考。     

野油菜黄单胞菌XC1892基因与致病力关系的分析

野油菜黄单胞菌野油菜变种(简称Xcc)是十字花科作物黑腐病的病原菌。在前期工作中,从Xcc8004菌株获得1株编号为XC1892的基因的转座子Tn5gusA5突变体,其致病力降低。根据基因组注释,XC1892编码1个DsbE蛋白,参与细胞色素的合成。为评估XC1892的功能,采用自杀质粒pK18mob对XC1892进行诱变,获得非极性突变体1892nk。对突变体的表型分析发现,其致病力及胞外多糖产量分别是野生型的59%和55%。用一段包含XC1892基因的DNA片段对突变体1982nk进行功能互补,其致病力和胞外多糖产量基本得到恢复。这表明XC1892基因与Xcc的致病力和胞外多糖合成产量有关。     

山冈单胞菌ZL261几丁质酶基因的克隆及原核表达

分离自西藏色季拉山海拔4 530 m高山草甸土壤的山冈单胞菌(Collimonas pratensis)ZL261具有很高的几丁质酶活性,为了明确其几丁质酶种类及作用特点,依据已报道的食真菌山冈单胞菌(Collimonas fungivorans)Ter331胞外内切几丁质酶基因chiI相关序列设计引物,利用分段扩增的方法得到了菌株ZL261几丁质酶基因,并对其序列和编码蛋白的氨基酸进行分析。结果表明,全长1 338 bp的chiIQ基因编码的几丁质酶ChiIQ由445个氨基酸组成,分子量为46.04 kDa,等电点为5.67。序列比对和同源性分析发现,该基因与食真菌山冈单胞菌Ter331几丁质酶基因chiI序列相似性最高(92%),其氨基酸序列含有几丁质酶GH18家族高度保守序列SXGG和GXDXDXE,因此,ChiIQ属GH18家族。利用双酶切法构建了ZL261几丁质酶基因原核表达载体,并在大肠杆菌BL21中成功诱导表达。本试验结果为后期研究几丁质酶在山冈单胞菌生防应用中的功能以及生防菌ZL261的开发和应用奠定了基础。     

香蕉根际土壤原生动物群落特征及与枯萎病病原菌的关系

香蕉枯萎病由病原真菌尖孢镰刀菌4号生理小种引起,其根际微生物组是抵御病原菌入侵的关键屏障。根际原生动物群落在根际微生物群落调控以及植物健康方面有不可忽视的作用。利用Illumina MiSeq高通量测序技术分析连作蕉园中植株发病前后根际土壤原生动物群落特征变化以及其与病原菌的相互关系。结果表明：相较于可培养细菌与真菌,原生动物的数量对病原菌的种群数量变化的影响更大;根际原生动物的群落多样性与丰富度都随植株生长而降低,且在发病植株中更低;抽蕾前后的健康与发病植株中根际原生动物群落结构存在显著差异;总体而言,吞噬型原生动物的相对丰度显著高于其他功能类型的原生动物,且在健康与发病植株中差异显著;吞噬型原生动物中与病原菌具有显著相关性的多为丝足虫门;在抽蕾期的健康植株中,丝足虫门的高丰度属中Group-Te与Cercomonas属与病原菌呈显著负相关;在抽蕾期的患病植株中,相对丰度较高的植物致病型原生动物Pythium属与病原菌呈显著正相关。综上所述,根际土壤中原生动物对病原菌的影响较大,在植株生长过程中,原生动物的群落特征发生改变,且在发病与健康植株中存在差异。与病原菌具有显著负相关的吞噬型原生动物,尤其是丝足虫门的部分高丰度属,可能会在防控香蕉枯萎病上产生一定潜力。     

菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因的克隆与生物信息学分析

菌寄生真菌的几丁质酶有很强的降解几丁质能力,在控制植物病害方面起着重要的作用.为克隆和研究菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因(tfchi1),本研究根据真菌几丁质酶基因的保守序列设计扩增基因中间片段的简并引物,采用RT-PCR、3'-RACE及5+-TAIL的方法获得了该基因的DNA和mRNA序列(GenBank:GU361769,GU361770).tfchi1长为2 561 bp,具有6个内含子,长度分别为129、78、68、65、53和59 bp,包含1 194bp的ORF,编码一个由397个氨基酸组成的蛋白.推导的tfchi1氨基酸序列以及蛋白质结构生物信息学分析表明,该蛋白具有典型的几丁质酶催化区保守序列SXGGW和DGXDXDWE,属于糖苷水解酶18家族几丁质酶,与柄篮状菌(Talaromyces stipitatus ATCC 10500)几丁质酶(XP_002480365)氨基酸序列同源性为96％,分子量为43.47kD,等电点为4.97.该蛋白无信号肽序列,有15个潜在的N-糖基化位点,Tfchi1的二级结构以无规卷曲和α-螺旋为蛋白的主要结构元件,三级结构中有(α/β)8的圆桶形结构.tfchi1转化毕赤酵母(Pichia pastoris )GS 115,酵母工程菌可分泌具几丁质酶活性的表达产物,重组蛋白的分子量与理论值相符.结果说明,本研究已从T.flavus中正确克隆了1个糖苷水解酶18家族几丁质酶基因.     

海带渣菌肥作为土壤添加剂对土壤抑制病原真菌活性影响的研究

试验以海带渣固体发酵微生物菌肥作为土壤添加剂为研究对象,以土壤浸出液抑制植物病原真菌寄生曲霉的活性来表示土壤自身抑制植物病原真菌的活性。试验通过Tip-culture定量检测方法对不同处理组的不同时期土壤浸出液抑制病原真菌寄生曲霉活性进行定量的测定。试验表明, 海带渣固体发酵微生物菌肥能显著提高土壤抑制植物病原真菌活性的能力。花生盆栽试验结果表明, 海带渣固体发酵微生物菌肥能极显著地提高土壤抑制病原真菌寄生曲霉的能力,其抑制活性对比未添加的CK对照组的抑菌活性提高了37.67%。海带渣固体发酵微生物菌肥作为土壤添加剂提高了土壤抑制病原真菌活性的能力,可用于特异抑菌土壤的培育。     

Relationship between soil enzyme activities, nutrient cycling and soil fungal communities in a northern hardwood forest

Soil fungi are highly diverse and act as the primary agents of nutrient cycling in forests. These fungal communities are often dominated by mycorrhizal fungi that form mutually beneficial relationships with plant roots and some mycorrhizal fungi produce extracellular and cell-bound enzymes that catalyze the hydrolysis of nitrogen (N)- and phosphorus (P)- containing compounds in soil organic matter. Here we investigated whether the community structure of different types of mycorrhizal fungi (arbuscular and ectomycorrhizal fungi) is correlated with soil chemistry and enzyme activity in a northern hardwood forest and whether these correlations change over the growing season. We quantified these relationships in an experimental paired plot study where white-tailed deer (access or excluded 4.5 yrs) treatment was crossed with garlic mustard (presence or removal 1 yr). We collected soil samples early and late in the growing season and analyzed them for soil chemistry, extracellular enzyme activity and molecular analysis of both arbuscular mycorrhizal (AM) and ectomycorrhizal/saprotrophic fungal communities using terminal restriction fragment length polymorphism (TRFLP). AM fungal communities did not change seasonally but were positively correlated with the activities of urease and leucine aminopeptidase (LAP), enzymes involved in N cycling. The density of garlic mustard was correlated with the presence of specific AM fungal species, while deer exclusion or access had no effect on either fungal community after 4.5 yrs. Ectomycorrhizal/saprotrophic fungal communities changed seasonally and were positively correlated with most soil enzymes, including enzymes involved in carbon (C), N and P cycling, but only during late summer sampling. Our results suggest that fine scale temporal and spatial changes in soil fungal communities may affect soil nutrient and carbon cycling. Although AM fungi are not generally considered capable of producing extracellular enzymes, the correlation between some AM taxa and the activity of N acquisition enzymes suggests that these fungi may play a role in forest understory N cycling.     

The relative importance of host vigor and hormonal response to pathogens in controlling the development of arbuscular mycorrhizal fungi

Plants are routinely colonized by both beneficial and detrimental microorganisms. These two microbial guilds may indirectly interact with each other via their host, either by modifying its vigor, or by altering its hormonal/defense status. Here, we studied indirect interactions between arbuscular mycorrhizal (AM) fungi and three plant pathogens. We show that AM fungal sporulation was only triggered by the least aggressive fungal pathogen, which is known to induce a jasmonate-based hormonal response by the host without affecting its vegetative growth and vigor. Conversely, the most aggressive fungal pathogen considerably reduced host vigor but did not alter AM fungal growth and sporulation. Our results thus suggest that the plant hormonal system is an important component of indirect interactions between AM fungi and plant pathogens.     

Plant pathogen protection by arbuscular mycorrhizas: A role for fungal diversity?

Arbuscular mycorrhizal (AM) fungi can confer protection to host plants against some root pathogens, and several mechanisms for these phenomena have been proposed. If AM fungal taxa vary in the ways that they limit the negative effects of pathogens on host plants, additive and/or synergistic interactions among members of diverse AM fungal assemblages and communities may result in a greater pathogen protection than is currently predicted. However, in a review of the literature on interactions between AM and pathogenic fungi, we found few examples that compared the effectiveness of single- and multi-species AM fungal assemblages. Here, we briefly recount the generally recognized mechanisms of pathogen protection by AM fungi and present evidence, where appropriate, for functional diversity among AM fungal taxa with regard to these mechanisms. We propose that functional complementarity of AM fungal taxa in interactions with pathogens could mimic, or even be the cause of, previously observed relationships between AM fungal diversity and plant productivity.     

Trichoderma–plant–pathogen interactions

Biological control involves the use of beneficial organisms, their genes, and/or products, such as metabolites, that reduce the negative effects of plant pathogens and promote positive responses by the plant. Disease suppression, as mediated by biocontrol agents, is the consequence of the interactions between the plant, pathogens, and the microbial community. Antagonists belonging to the genus Trichoderma are among the most commonly isolated soil fungi. Due to their ability to protect plants and contain pathogen populations under different soil conditions, these fungi have been widely studied and commercially marketed as biopesticides, biofertilizers and soil amendments. Trichoderma spp. also produce numerous biologically active compounds, including cell wall degrading enzymes, and secondary metabolites. Studies of the three-way relationship established with Trichoderma, the plant and the pathogen are aimed at unravelling the mechanisms involved in partner recognition and the cross-talk used to maintain the beneficial association between the fungal antagonist and the plant. Several strategies have been used to identify the molecular factors involved in this complex tripartite interaction including genomics, proteomics and, more recently, metabolomics, in order to enhance our understanding. This review presents recent advances and findings regarding the biocontrol-resulting events that take place during the Trichoderma–plant–pathogen interaction. We focus our attention on the biological aspects of this topic, highlighting the novel findings concerning the role of Trichoderma in disease suppression. A better understanding of these factors is expected to enhance not only the rapid identification of effective strains and their applications but also indicate the potentials for improvement of natural strains of Trichoderma.     

In situ dynamics of soil fungal communities under different genotypes of potato, including a genetically modified cultivar

Fungi are key to the functioning of soil ecosystems, and exhibit a range of interactions with plants. Given their close associations with plants, and importance in ecosystem functioning, soil-borne fungi have been proposed as potential biological indicators of disturbance and useful agents in monitoring strategies, including those following the introduction of genetically modified (GM) crops. Here we report on the impact of potato crop varieties, including a cultivar that was genetically modified for its starch quality, on the community composition of the main phyla of fungi in soils, i.e. Ascomycota, Basidiomycota and Glomeromycota in rhizosphere and bulk soil. Samples were collected at two field sites before sowing, at three growth stages during crop development and after the harvest of the plants, and the effects of field site, plant growth stage and plant cultivar (genotype) on fungal community composition assessed using three phylum-specific T-RFLP profiling strategies and multivariate statistical analysis (NMDS ordinations with ANOSIM test). In addition, fungal biomass, arbuscular mycorrhizal colonization of roots and activities of extracellular fungal enzymes (laccases, Mn-peroxidases and cellulases) involved in degradation of lignocelluloses-rich organic matter were determined. Fungal community compositions, densities and activities were observed to differ significantly between the rhizosphere and bulk soil. The most important factors determining fungal community composition and functioning were plant growth stage for the rhizosphere communities and location and soil properties for the bulk soil communities. The basidiomycetes were the most numerous fungal group in the bulk soils and in the rhizosphere of young plants, with a shift toward greater ascomycete numbers in the rhizosphere at later growth stages. There were no detectable differences between the GM cultivar and its parental cultivar in terms of influence on fungal community structure of function. Fungal community structure and functioning of both GM- and parental cultivars fell within the range of other cultivars at most sampling moments.     

Biochemical quality of organic amendments affects soil fungistasis

Soilborne plant pathogens are among the most important limiting factors for the productivity of agro-ecosystems. Identifying reliable and effective control methods is crucial for efficient biological control. Soil fungistasis is the capability of soils to inhibit the germination and growth of soil-borne fungi in presence of optimal abiotic conditions. The aim of this study is to clarify the relationships between soil amendments with plant residues spanning a wide variety of biochemical quality and soil fungistasis. Microcosms experiments were performed with 42 different plant residues and the effect on soil fungistasis was assessed by using four different fungi (Aspergillus niger, Botrytis cinerea, Pyrenochaeta lycoperici and Trichoderma harzianum). We measured soil respiration and FDA enzymatic activity and compared classic litter proximate chemical analysis with ¹³C-CPMAS NMR spectroscopy. Results showed that quality of organic amendments is a major controlling factor of soil fungistasis. The dramatic relief of soil fungistasis when soil was amended with lignin poor, but labile C rich, substrates gives strong support to the competition-based hypothesis. The positive correlation between soil respiration and fungal growth further supports the competition hypothesis. Finally, ¹³C NMR results showed a relationship between soil fungistasis and the biochemical quality of plant residues, and provided a quantitative assessment of the time required for fungistasis restoration after organic materials application.     

Isolation and identification of antifungal fatty acids from the basidiomycete Gomphus floccosus

Bioautography of extracts of the fruiting bodies of the basidiomycete Gomphus floccosus (Schw.) Singer indicated the presence of fungitoxic compounds in the ethyl acetate fraction against the plant pathogens Colletotrichum fragariae, Colletotrichum gloeosporioides, and Colletotrichum acutatum. Bioassay-guided fractionation of this fraction resulted in the isolation of the bioactive fatty acids (9 S,10 E,12 Z)-9-hydroxy-10,12-octadecadienoic acid (1), (9 E,11 Z)-13-oxo-9,11-octadecadienoic acid (2), and (10 E,12 E)-9-oxo-10,12-octadecadienoic acid (3). These three oxylipins were further evaluated for activity against a greater range of fungal plant pathogens (C. fragariae, C. gloeosporioides, C. acutatum, Botrytis cinerea, Fusarium oxysporum, Phomopsis obscurans, and Phomopsis viticola) in in vitro dose-response studies. Phomopis species were the most sensitive fungi to these compounds. At 120 h of treatment, the IC50 values for compounds 1, 2, and 3 for P. obscurans were 1.0, 4.5, and 23 microM, respectively, as compared to 1.1 microM for the captan positive control.     

Arbuscular mycorrhizal fungal spore-associated bacteria affect mycorrhizal colonization,plant growth and potato pathogens

Arbuscular mycorrhizal (AM) fungi and their bacterial associates are essential living components of the soil microbiota. From a total of 385 bacteria previously isolated from spores of AM fungi (AMB), 10 were selected based on ability to inhibit growth of plant pathogens. Effects of these isolates on AM fungal colonization, plant growth in potato (Solanum tuberosum L.) and inhibition of pathogens was investigated. AM fungal root colonization of potato was 7-fold higher in the presence of the Pseudomonas FWC70 isolate in a greenhouse and was 6–9-fold higher in the presence of the three isolates Pseudomonas FWC70, Stenotrophomonas FWC94 and Arthrobacter FWC110 in an outdoor pot experiment. Several growth traits of potato were stimulated by the Pseudomonas isolates FWC16, FWC30 and FWC70 and by the Stenotrophomonas isolate FWC14. All three Pseudomonas isolates showed inhibition against Erwinia carotovora, Phytophthora infestans and Verticillium dahliae but Stenotrophomonas isolates were variable. Protease(s), siderophores and indole acetic acid were produced by all isolates. Chitinase(s) were produced by all Stenotrophomonas and phosphate-solubilizing activity by all Pseudomonas isolates, the Stenotrophomonas FWC14 isolate and the Arthrobacter FWC110 isolate. We conclude that some AMB are multifunctional and production of extracellular enzymes and bioactive compounds are likely mechanisms for their multifunctional activities. Our results show that some AMB are likely to contribute to the often described ability of AM fungi to inhibit pathogens, acquire mineral nutrients and modify plant root growth.     

Ectomycorrhizal and Saprotrophic Fungal Communities Vary Across mm-Scale Soil Microsites Differing in Phosphatase Activity

To understand nutrient cycling in soils, soil processes and microorganisms need be better characterized. To determine whether specific trophic groups of fungi are associated with soil enzyme activity, we used soil imprinting to guide mm-scale sampling from microsites with high and low phosphatase activities in birch/Douglas-fir stands. Study 1 involved sampling one root window per site at 12 sites of different ages (stands); study 2 was conducted at one of the stem-exclusion stands, at which 5 root windows had been installed. Total fungal and ectomycorrhizal (EM) fungal terminal-restriction fragment length polymorphism (TRFLP) fingerprints differed between high-and low-phosphatase activity microsites at 8 of 12 root windows across 12 sites. Where differences were detected, fewer EM fungi were detected in high-than low-phosphatase activity microsites. Using 5 root windows at one site, next-generation sequencing detected similar fungal communities across microsites, but the ratio of saprotrophic to EM fungal reads was higher in high-phosphatase activity microsites in the two windows that had low EM fungal richness. In windows with differences in fungal communities, both studies indicated that EM fungi were less successful than saprotrophic fungi in colonizing fine-scale, organic matter-rich microsites. Fine-scale sampling linked with in situ detection of enzyme activity revealed relationships between soil fungal communities and phosphatase activity that could not be observed at the scales employed by conventional approaches, thereby contributing to the understanding of fine-scale phosphorus cycling in forest soils.