食线虫真菌致病相关丝氨酸蛋白酶的研究进展

食线虫真菌是一类土壤微生物,作为线虫的天敌,它们对于维持线虫在土壤生态环境中的种群动态平衡发挥着十分重要的作用。食线虫真菌通过形成特殊的捕食器官或产生毒素等方式来捕捉和杀死线虫。丝氨酸蛋白酶是食线虫真菌侵染线虫的重要毒力因子,近年来,研究人员对不同食线虫真菌来源的致病相关丝氨酸蛋白酶进行了大量的研究,尤其在丝氨酸蛋白酶的晶体结构和分子进化方面的研究取得了较大的进展。本文对食线虫真菌致病相关丝氨酸蛋白酶的生物化学性质和功能进行了系统的总结,对丝氨酸蛋白酶的晶体结构、催化机制及分子进化等最新的进展进行了评述。     

中国食线虫真菌的种类

对我国24个省市,涉及40余种植物的300多个土样的调查,分离出食线虫真菌近千个分离物,共鉴定出食线虫真菌29属57种,包括捕食线虫真菌4属25种,其中Arthrobotrys arthrobotryoides, A. brochopaga, A. cladodes var. macroides, A. microspora,A. musiformis, A. straminicola, Monacrosporium ellipsosporinm, M. thaumasium, M. pravicollis,Stylopage grandis, Dactylella ramiformis, Monacrosporium qiuanum, Monacrosporium sinense等10种为国内新记录。内寄生真菌4属4个种、其中Catenaria angullulae, Harposporiumarcuatum, Nematoctonus leiosporus为国内新记录。定殖于大豆孢囊线虫真菌22属25种,其中Catenaria auxiliaris, Cylindrocarpon heteronema, C. willkommii, Fusariella bizzozeriana,Nematophthora sp., Phoma eupyrena, Phoma leveilei,Pithomyces sp,Trichocladium opacum.Verticillium chlamydosporium等10属种为国内新记录,其中Nematophthora, Fusariella,Trichocladium三属为国内新记录属,Rhizoctonia sp., Stachybotrys elegans, F.bizzozeriana,C.heteronema, C.willkommii等为首次发现定殖于大豆孢囊线虫孢囊上。对所鉴定的真菌进行了描述和讨论并制作了检索表。     

食真菌线虫与真菌的相互作用及其对土壤氮素矿化的影响

采用悉生培养微缩体系,探讨了食真菌线虫(燕麦真滑刃线虫)与两种真菌(真菌Ⅰ：外皮毛霉和真菌Ⅱ：丛梗孢科的一种)间的相互作用及其对土壤氮素矿化的影响．结果表明,燕麦真滑刃线虫在取食两种真菌时表现为在真菌Ⅱ上的生长优于真菌Ⅰ,两个处理的线虫数达到显著差异．食真菌线虫对真菌的取食活动促进了真菌的增殖：接种真菌Ⅱ加线虫处理中真菌Ⅱ的数量是仅接种真菌Ⅱ处理的2．5～3．5倍,增幅在整个培养期基本稳定;而接种真菌Ⅰ加线虫处理中真菌Ⅰ的数量在培养前期(10d)是仅接种真菌Ⅰ处理的1．1～2．0倍。之后增幅达5．0～5．7倍．线虫和真菌的生长及增殖基本保持同步．食真菌线虫与真菌的相互作用显著提高了土壤铵态氮和矿质态氮含量,促进了土壤氮的矿化,其中线虫与真菌Ⅰ的相互作用对提高矿质态氮含量的贡献显著大于线虫与真菌Ⅱ的相互作用。     

土壤抑菌作用对食线虫真菌及其制剂的影响

通过我国东北及黄淮海地区主要大豆种植区１１种土壤对４株定殖于大豆胞囊线虫真菌淡紫拟青霉（ＰａｅｃｉｌｏｍｙｃｅｓＬｉｌａｃｉｎｕｓ）Ｍ－１４,厚垣孢子轮枝菌（Ｖｅｒｔｉｃｉｌｌｉｕｍｃｈｌａｍｙｄｏｓｐｏｒｉｕｍ）ＪＮ－１１,ＳＨ－２６和ＢＱＬ－１孢子的抑制作用及其影响因子的研究,表明土壤元菌作用普遍存在,但依土壤条件,菌种类型而异,其中以安徽宿到,济南农科院,北京,黑龙江宝泉岭农场的土壤抑菌作用     

食线虫真菌28S rDNA PCR扩增片段的RFLPs分析

本文对16个食线虫真菌(节丛孢属、隔指孢属和单顶孢属)菌株和3个其他相关丝孢菌(顶辐孢属和单端孢属)菌株的28S rDNA 片段进行了扩增, 并用限制性内切酶Sau3A I, Msp I, Rsa I and Hae III对PCR产物进行了消化。采用UPGMA法对 PCR 产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA 的ITS区间、5.8S和18S的序列分析结果一致。     

食线虫真菌侵染性胞外酶和生防应用

食线虫真菌作为重要的植物寄生线虫的生物防治资源,深入了解它们的侵染方式、毒力因子是了解食线虫真菌侵染的分子机理和开发高效、稳定的生物杀线虫制剂的关键。目前的研究表明,食线虫真菌能分泌具有降解线虫体壁或线虫卵壳的胞外酶,它们在食线虫真菌侵染线虫的过程中起着非常重要的作用。对这些侵染性胞外水解酶的深入研究将促进人们对食线虫真菌的侵染过程和侵染机制的了解以及高效生防制剂的开发。综述了近年来食线虫真菌侵染性胞外酶的研究概况,对食线虫真菌胞外丝氨酸蛋白酶进行同源性分析,对以后食线虫真菌侵染性胞外酶的研究和高效生防制剂开发进行了评述。     

根结线虫天敌真菌及其高效菌株筛选

本文报道捕食和寄生南方根结线虫(Meloidogyne incognita)和爪哇根结线虫(M.javanica)的天敌真菌6属14种。其中Arthrobotrys candida A.cladodes var.macroides,A.musiformis,Monacrosporium cionopagum,M.doedycoides,Drechmeria coniospora,Harposporium crassum,H.lilliputanum,Myzocytium humicola和Verticillum coccospora为我国新纪录种。室内测定结果表明:GACA 1B(Arthrobotrys oligospora)、GACM 102(Monacrosporium doedycoides)菌株对南方根结线虫和爪哇根结线虫均具极高的捕食率,接种线虫7天后,捕食率达98%以上,接种20天后,捕食率均达100%。     

Ecology of nematophagous fungi: vertical distribution in a deciduous woodland

Summary The distribution of nematophagous fungi in soil collected from a deciduous woodland is compared to various biotic and abiotic soil factors. The microfungi are isolated at all depths down to a maximum of 35 cm. Predators forming constricting rings, adhesive branches and adhesive knobs are restricted to the upper litter and humus layers. The net forming predators and endoparasites are isolated at all depths, although they are significantly more abundant in the lower mineral rich soils. A much greater species diversity of nematophagous fungi is recorded in the upper organic zones.Preliminary soil analysis indicates thatCephalosporium balanoides is independent of all soil variables, while predators able to form traps spontaneously are restricted to the organic soils which are rich in nematodes. Non-spontaneous trap forming predators, which are excellent saprophytes, are isolated from the deeper soils which are low in nutrients. The ecological significance of these results is discussed.     

The living strategy of nematophagous fungi

The infection structures, trophism, and ecological character of nematophagous fungi are reviewed in this article on the basis of data extracted from the literature and the most recent experiments conducted in this area. Traditionally, nematophagous fungi are classified into four groups according to their modes of attacking nematodes: nematode-trapping fungi using adhesive or mechanical hyphal traps, endoparasitic fungi using their spores, eggparasitic fungi invading nematode eggs or females with their hyphal tips, and toxin-producing fungi immobilizing nematodes before invasion. In the present review, we focus on the first two groups. The living strategies of these nematophagous fungi depend on the diversity of their infection structures, such as different traps and spore types, which determine the modes of infecting nematodes. The diversity of trophic modes of nematophagous fungi is an important prerequisite for fungal survival and activity in soil. The abundance and activity of Hirsutella rhossiliensis and H. minnesotensis, representatives of endoparasites and potential biocontrol agents against nematodes, are highly dependent on environmental factors. Comprehensive understanding of the survival and activity of nematophagous fungi in soil is fundamental for the exploitation of these fungi as successful biocontrol agents.     

Suppression of nematophagous fungi by enchytraeid worms: a field exclosure experiment

The feeding biology of Enchytraeus crypticus and other enchytraeids is poorly understood as is their effect on nematophagous fungi. Because enchytraeids had been associated with nematophagous fungi in the field and had suppressed these fungi in soil microcosms, we tested the hypothesis that exclusion of enchytraeids, largely E. crypticus, would improve establishment of certain nematophagous fungi in field plots. The fungi, Hirsutella rhossiliensis and Monacrosporium gephyropagum, are being studied as potential control agents of plant-parasitic nematodes and were formulated as hyphae in alginate pellets. The pellets were mixed into soil without enchytraeids and placed in cages (PVC pipe, 80 cm³ volume) with fine (20 μm) or coarse (480 μm) mesh; cages were buried 15 cm deep in field plots and then recovered after 6–52 days. When fine mesh was used, enchytraeids were excluded and the fungi increased to large numbers. When coarse mesh was used, enchytraeid numbers in cages increased rapidly and the fungi did poorly. Although mesh also affected other potential fungivores, including collembolans and large dorylaimid nematodes, we suspect that enchytraeids were more important because large numbers were consistently found in cages with coarse mesh soon after the cages were placed in soil. Organisms smaller than enchytraeids (bacteria, fungi, and protozoa) also appeared to be important because the fungi did better in heat-treated soil than in non-heat-treated soil, regardless of mesh size. The rapid increase in enchytraeid numbers in cages with hyphal pellets and coarse mesh was probably caused by movement of enchytraeids toward the pellets with hyphae: increase in enchytraeid numbers was minimal when movement into cages was blocked (or when cages contained pellets without hyphae). Overall, the data were consistent with the hypothesis that enchytraeids, or other meso- or macrofauna, contributed to suppression of nematophagous fungi in our field plots. Received: 22 April 1997 / Accepted: 16 June 1997     

Predatory activity and passage of six nematophagous fungi species in gastrointestinal tract of trichostrongylide-infected sheep*

With the development of anthelmintic resistance of parasitic nematodes, screening the potential of predatory fungi candidates by in vitro and in vivo assay is necessary for biocontrol of parasitic nematodes in sheep. Fifteen native isolates of fungi species of six predators were evaluated through in vitro and in vivo experiment to evaluate the capacity of passing through the gastrointestinal tract of sheep. The results of the in vitro experiment showed that the reduction percentages of the third-stage larvae (L3) of trichostrongylides in faeces were 97.02–98.49% for five isolates of Arthrobotrys (Monacrosporium) sinense; 83.47–99.12% for three isolates of Arthrobotrys oviformis; 80.00–97.41% for four isolates of Arthrobotrys conoides; and 99.18%, 77.56%, and 75.72% for one isolate of Arthrobotrys microscaphoides, Arthrobotrys salina, and Dactylellina leptospora, respectively. In the in vivo assay, the results exhibited that the larval recoveries in faeces were significantly decreased (p?D. leptospora. After fungal treatment (within 24?h) of one isolate of A. salina, although the efficacy against L3 was 43.83%, tested fungus was detected in the faeces. The remaining isolates, except one isolate of D. leptospora and one isolate of A. conoides, were positive for either L3 reduction or fungal survival in faeces. The present study showed that nematophagous fungi could survive in the passage through the alimentary tract of sheep and should be potential candidates as a feed supplement.     

锈菌类真菌基因组结构分析研究进展

锈菌种群庞大,可以引起许多重要经济作物和林木病害,严重威胁全球粮食和林业生产安全。全基因组分析为锈菌基因功能研究、毒性变异研究及锈菌演化规律研究提供了重要基础,为制定锈病有效防控策略和创制抗锈新材料提供理论依据。本文综述了目前锈菌全基因组分析领域的进展,对锈菌的基因组结构、基因组成、基因组变异等特征进行了归纳分析,对基因组变异与其专性寄生特性的关系、基因组变异对其毒性变异的可能影响等进行了阐述。基因组学将为最终揭示锈菌生活史复杂性和毒性高度变异性的根本成因提供有力工具。     

Decoding the genome beyond sequencing: the new phase of genomic research

While our understanding of gene-based biology has greatly improved, it is clear that the function of the genome and most diseases cannot be fully explained by genes and other regulatory elements. Genes and the genome represent distinct levels of genetic organization with their own coding systems; Genes code parts like protein and RNA, but the genome codes the structure of genetic networks, which are defined by the whole set of genes, chromosomes and their topological interactions within a cell. Accordingly, the genetic code of DNA offers limited understanding of genome functions. In this perspective, we introduce the genome theory which calls for the departure of gene-centric genomic research. To make this transition for the next phase of genomic research, it is essential to acknowledge the importance of new genome-based biological concepts and to establish new technology platforms to decode the genome beyond sequencing.     

Culture medium characteristics on the predatory activity of the nematophagous fungus Duddingtonia flagrans

The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L₃ in the media supplemented with casein and pH 7.0.     

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.     

Ovicidal effect of nematophagous fungi on Taenia taeniaeformis eggs

This work evaluated the ovicidal effect of the nematophagous fungi Monacrosporium sinense (SF53), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) on Taenia taeniaeformis eggs in laboratory conditions. T. taeniaeformis eggs were plated on 2% water-agar with the grown isolates and control without fungus and examined at seven and fourteen days post-inoculation. At the end of the experiment, P. chlamydosporia showed ovicidal activity (P < 0.01) on T. taeniaeformis eggs unlike the other two species, mainly for internal egg colonization with percentage results of 32.2–54.0% at 7th and 14th day, respectively. The other fungi only showed lytic effect without morphological damage to eggshell. Results demonstrated that P. chlamydosporia was in vitro effective against Taenia taeniaeformis eggs unlike the other fungi. In this way, the use of P. chlamydosporia is suggested as a potential biological control agent for eggs of this cestode.