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。     

温度和氧对节丛孢属食线虫真菌产生捕食性菌网的影响

国外利用食线虫真菌作为一种新的防治动物线虫病的手段近年来已开展了广泛的研究Ｌａｒｓｅｎｅｔａｌ．１９９１;１９９４;１９９６）。国内开展这方面的研究才刚刚起步。捕食线虫真菌对线虫的作用方式主要是通过捕食性菌网从线虫体内吸取营养,导致线虫死亡（Ｇｒｏｎｖｏｌｄｅｔａｌ,１９９６）。迄今尚未见到较为系统的有关影响节丛孢属真菌产生捕食性菌网的研究。本研究对温度和氧对食线虫真菌产生的捕食性菌网数的影响进行初步观察。１　材料和方法１．１供试菌　　共９个菌种,分别由中国农业大学动物医学院和中国农业科学院生物…     

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。     

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。     

食线虫真菌基因组测序和分析研究进展

食线虫真菌作为线虫的天敌,是一种潜在的线虫病害的生防制剂。食线虫真菌通过形成特殊的捕食器官或产生粘性孢子和毒素等方式捕捉和侵染线虫。近年来,随着测序技术的进步和生物信息学的应用,越来越多真菌基因组已经被测定和报道。目前,已经有7种食线虫真菌的基因组被报道,包括捕食线虫真菌寡孢节丛孢(Arthrobotrys oligospora)、线虫卵寄生真菌厚垣普可尼亚菌(Pochonia chlamydosporia)和内寄生真菌明尼苏达被毛孢(Hirsutella minnesotensis)等。本文对食线虫真菌的基因组特点、毒力相关基因家族的扩张、捕食器官形成调控和进化机制进行了系统地总结,对组学时代食线虫真菌研究面临的关键问题进行了评述。     

食真菌线虫对热或铜胁迫下土壤生态功能稳定性的影响

在模拟胁迫条件下(施加 CuSO₄ 的持续胁迫或加热40 ℃的瞬时胁迫),以大麦叶粉短期分解过程代表土壤功能,采用室内培养试验研究了土壤食真菌线虫(Aphelenchus avenae)与微生物的相互作用对土壤生态功能稳定性(抗性和恢复力)的影响.结果表明：无论施加胁迫与否,食真菌线虫的活动都有促进土壤微生物活性的趋势,尤其是在施加铜胁迫后第8 天开始到培养期结束,接种食真菌线虫导致土壤基础呼吸显著增加(P<0.05),但加热胁迫后食真菌线虫对土壤基础呼吸的促进作用仅在第8天有显著差异(P<0.05),反映了食真菌线虫对土壤微生物活性的影响程度与胁迫类型有关.在两种胁迫条件下,接种食真菌线虫对土壤功能的抗性没有影响,但都能促进胁迫条件下土壤功能的恢复.培养后期,两种胁迫条件下接种食真菌线虫处理真菌生物量低于未接种线虫处理,表明胁迫条件下食真菌线虫对真菌的取食可能限制甚至抑制了真菌生长,导致真菌对细菌的竞争压力减少,从而使细菌获得更大的生长优势,间接促进了细菌的生长.     

食线虫真菌资源研究概况

食线虫真菌是指寄生、捕捉、定殖和毒害线虫的一类真菌,这类真菌是自然界中线虫种群控制的重要因子,也是动植物病害生物防治的重要研究材料,具有特殊的研究意义和经济价值。目前全世界共报道700余种食线虫真菌,包括捕食线虫真菌380余种,线虫内寄生真菌120余种,产毒真菌270余种和大量机会真菌。针对丰富的食线虫真菌资源,近年来世界各国尤其是中国科学家对其进行了广泛研究,在捕食线虫真菌资源的分类、系统进化、生态分布、有性无性联系等方面的研究取得了重要进展,在线虫内寄生真菌侵染宿主的方式及产毒真菌的次生代谢产物挖掘等方面也进行了广泛研究,文章综述了以上研究进展并简述了食线虫真菌资源的生物防治应用概况。     

不同水分管理方式对稻田土壤生物学特性的影响

在下辽河平原单季稻地区研究了常规浅湿干灌溉 (CK)、浅湿干灌溉薄膜阻渗 (IC)、湿润灌溉薄膜阻渗 (MC)、淹水灌溉薄膜阻渗 (FC) 4种不同水分管理方式下土壤线虫及土壤微生物量的动态变化。结果表明 ,耙耕前 ,CK、FC处理食细菌线虫数量显著高于MC、IC处理 ;薄膜阻渗在黄熟期显著降低了土壤食细菌线虫的数量 ,在耙耕前显著降低了食真菌线虫数量。潮棕壤稻田食真菌线虫与食细菌线虫相比数量较低。在耙耕前不同水分管理方式下土壤微生物量C显著低于对照。不同水分管理方式在水稻分蘖期、抽穗期对食细菌线虫数量、食真菌线虫数量、微生物量C和微生物量N没有影响。土壤食细菌线虫、食真菌线虫数量与土壤微生物量C、N没有达到显著相关。     

土壤食细菌线虫的原位富集培养方法

采用两种孔径的尼龙网袋（1mm和5μm）,将盆钵供试土壤分成内外两层,内层土壤混合猪粪或稻草,并以不添加猪粪和稻草的供试土壤作为空白;外层直接接入供试土壤,进行培养,以获取土著食细菌线虫大量富集的试验土壤。结果表明：添加基质（猪粪和稻草）显著地促进了土壤线虫的繁殖,大量繁殖的线虫通过1mm网袋迁移至外层未加基质的土壤,而采用5μm网袋则限制了线虫向外层土壤的迁移。添加猪粪的1mm网袋处理经过28d培养后,外层土壤线虫数是空白处理的9．1倍;添加稻草的1mm网袋处理经过35d培养后,外层土壤线虫数是空白处理的5．9倍。添加两种基质的5μm网袋处理,外层土壤线虫数和空白处理差异不大。添加基质主要是促进了食细菌线虫的繁殖,在培养结束时,添加猪粪的1mm网袋处理的食细菌线虫比例达到98．2％,添加稻草的1mm网袋处理的食细菌线虫比例达到90．5％,两个处理食细菌线虫的总数分别是空白处理的14．8倍和8．9倍,并且主要是Protorhabditis sp．线虫的增加。     

节丛孢属丝孢菌对松材线虫和拟松材线虫的捕食

利用节丛孢属捕食线虫真菌的8个种,11个菌株,对松材线虫（Bx）和拟松材线虫（Bm）进行了捕食力的测定。结果显示节丛孢属真菌对这两种线虫的捕食能力既存在种间差异,也存在同种不同菌株间的差异。指状节丛孢的3个菌株Ad－1,Ad－2,Ad－3菌株对两种线虫均表现出较高的捕食率,接川7d后对Bx和Bm的捕食率分别达到了98．08％,91．16％,86．3％和96．28％,90．45％,85．38％;同一菌株对松材线虫和拟松材线虫的捕食没有选择性。此外,通过对松材线虫和拟松材线虫繁殖真菌的筛选实验,肯定了利用拟松材线虫代替松材线虫进行生防菌株筛选的可行性。     

In vitro stress selection of nematophagous fungi for biocontrol of parasitic nematodes in ruminants

Laboratory experiments were designed to select nematophagous fungi that were able to survive in vitro conditions simulating passage through the gastro-intestinal tract of cattle. All of the tests were conducted at 39 degrees C. In a primary stress selection step in diluted rumen fluid, 21 isolates were obtained. Each of the primary stress selected isolates was tested in synthetic saliva, rumen fluid simulating the activity in the rumen, rumen fluid followed by pepsin-hydrochloric acid treatment simulating the additional effect of ruminal and abomasal activity, pepsin-hydrochloric acid solution simulating conditions in the abomasum and finally in a trypsin solution as an example of enzyme activity in the gut. The effect of the rumen fluid alone, or rumen fluid followed by pepsin-hydrochloric acid treatment, were responsible for the reduction in surviving fungal isolates. Only six of thirteen isolates belonging to the genus Arthrobotrys survived while seven of eight isolates of the genus Duddingtonia survived. Fourteen isolates were tested for their predatory capacity in a dung pat bioassay. Fungi of the genera Arthrobotrys and Duddingtonia reduced the development of Ostertagia ostertagi third stage larvae by approximately 75% and 96% respectively compared to the number of larvae that developed from fungus-free control pats.     

Ureolytic bacteria in sheep rumen.

Estimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producting organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophyticus and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Gram-positive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.     

Taxon-specific associations between protozoal and methanogen populations in the rumen and a model rumen system

Methanogen populations in the rumen and in model rumen systems (operated over a 240-h period) were studied using the small subunit (SSU) rRNA phylogenetic framework for group-specific enumerations. Representatives of the family Methanobacteriaceae were the most abundant methanogen population in the rumen, accounting for 89.3% (± 1.02%) of total archaea in the rumen fluid and 99.2% (± 1.8%) in a protozoal fraction of rumen fluid. Their percentage of archaea in the model rumen systems declined from 84% (± 8.5%) to 54% (± 7.8%) after 48 h of operation, correlated with loss of protozoa from these systems. The Methanomicrobiales, encompassed by the families Methanomicrobiaceae, Methanocorpusculaceae, and Methanospirillaceae were the second most abundant population and accounted for 12.1% (± 2.15%) of total SSU rRNA in rumen fluid. Additionally this group was shown to be essentially free living, since only a negligible hybridization signal was detected with the ruminal protozoal fraction. This group constituted a more significant proportion of total archaea in whole rumen fluid, 12.1% (± 2.1%) and model rumen fluid containing no protozoa (26.3 ± 7.7%). In contrast, the Methanosarcinales, generally considered the second most abundant population of rumen methanogens, accounted for only 2.8% (± 0.3%) of total archaeal SSU rRNA in rumen fluid.     

Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows

The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.     

Comparison of growth characteristics of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a defined medium

Anaerobic fungi were isolated from rumen fluid of a domestic sheep (Ovis aries; a ruminant) and from faeces of five non-ruminants: African elephant (Loxodonta africana), black rhinoceros (Diceros bicornis), Indian rhinoceros (Rhinoceros unicornis), Indian elephant (Elephas maximus) and mara (Dolichotis patagonum). The anaerobic fungus isolated from the sheep was a Neocallimastix species and the isolates from non-ruminants were all species similar to Piromyces spp. A defined medium is described which supported growth of all the isolates, and was used to examine growth characteristics of the different strains. For each fungus the lipid phosphate content was determined after growth on cellobiose and the resulting values were used to estimate fungal biomass after growth on solid substrates. The ability of isolates from ruminants and non-ruminants to digest both wheat straw and cellulose was comparable. More than 90% and 60%, respectively, of filter paper cellulose and wheat straw were digested by most strains within 60-78 h. Growth of two fungi, isolated from rumen fluid of a sheep (Neocallimastix strain N1) and from faeces of an Indian rhinoceros (Piromyces strain R1), on cellobiose was studied in detail. Fungal growth yields on cellobiose were 64.1 g (mol substrate)-1 for N1 and 34.2 g mol-1 for R1. The major fermentation products of both strains were formate, lactate, acetate, ethanol and hydrogen.     

Phylogenetic analysis of protozoa in the rumen contents of cow based on the 18S rDNA sequences

AIMS: To examine the diversity of protozoa in the rumen contents of cow. METHODS AND RESULTS: Protozoa that inhabit the rumen were detected by PCR using protozoan-specific primers. Libraries of protozoan rDNA sequences were constructed from rumen fluid, solid tissues and epithelium. Twenty-three clones isolated from rumen fluid fell into two genera identified as Entodinium (69.6% of clones) and Epidinium (31.4% of clones). Of the clones isolated from rumen fluid, a moderate number were unidentifiable (30.4%). CONCLUSIONS: The predominant protozoan genus identified in the whole rumen belonged to the Entodinium group (81.1%). Protozoa were not detected in the rumen epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that rumen fluid and solid tissues contain different protozoan populations that may play specific roles in rumen function. Quantitative PCR techniques and a more specific set of phylogenetic probes that distinguish between protozoan species are needed to determine the significance of newly identified groups and to determine the distribution of identified protozoan clusters in rumen microbial communities.     

Diversity of Phytases in the Rumen

Examples of a new class of phytase related to protein tyrosine phosphatases (PTP) were recently isolated from several anaerobic bacteria from the rumen of cattle. In this study, the diversity of PTP-like phytase gene sequences in the rumen was surveyed by using the polymerase chain reaction (PCR). Two sets of degenerate primers were used to amplify sequences from rumen fluid total community DNA and genomic DNA from nine bacterial isolates. Four novel PTP-like phytase sequences were retrieved from rumen fluid, whereas all nine of the anaerobic bacterial isolates investigated in this work contained PTP-like phytase sequences. One isolate, Selenomonas lacticifex, contained two distinct PTP-like phytase sequences, suggesting that multiple phytate hydrolyzing enzymes are present in this bacterium. The degenerate primer and PCR conditions described here, as well as novel sequences obtained in this study, will provide a valuable resource for future studies on this new class of phytase. The observed diversity of microbial phytases in the rumen may account for the ability of ruminants to derive a significant proportion of their phosphorus requirements from phytate.     

Enumeration and Isolation of Lactate-Utilizing Bacteria from the Rumen of Sheep

A highly specific medium was developed for the enumeration of lactate-utilizing bacteria in the rumen of sheep. This medium, which contained 2.0% lactate, 2.0% Trypticase, 0.2% yeast extract, and volatile fatty acids, hemin, and trace elements in place of rumen fluid, enabled high counts (42 × 10⁷ to 190 × 10⁷/g of ingesta) of lactate-utilizing bacteria to be made with a high degree of specificity (96%). The medium also supported the growth of all species of predominant lactate-utilizing bacteria reported to occur in the rumen and thus is of importance for ecological studies where the incidence and influence of the different species on lactate metabolism under changing conditions in the rumen cannot be predicted. The survival rate of isolates was increased from 60 to 96% by addition to the modified maintenance medium of 40% rumen fluid in place of the volatile fatty acids, hemin, and trace elements used in the counting medium. These results, together with the slow growth of colonies in roll bottles, showed that, although highly selective, the counting medium was not optimal for the types selected.     

Lysis of Viable Rumen Bacteria in Bovine Rumen Fluid

Streptococcus bovis and Butyrivibrio sp. were labeled with thymidine-methyl-(3)H, washed, and resuspended in rumen fluid or rumen fluid fractions obtained from Holstein and Jersey cows fed alfalfa hay once daily. Factors affecting the lytic activity found in untreated rumen fluid were examined. Day to day variation and differences before and after feeding were observed for the same cow. There were also differences between cows on the same day. For a given rumen fluid, the rate of release of label was roughly proportional to the number of labeled cells present over a 100-fold range in concentration. Removal of protozoa largely abolished the lytic action of fresh rumen fluid for S. bovis, but some soluble lytic activity remained. Mixed rumen protozoa added to media containing labeled S. bovis caused label to appear in solution. In a sample of rumen fluid containing 4.3 x 10(4) protozoa/ml 5.2% of the S. bovis population were destroyed by protozoa per hr. The mean rate of destruction for 12 runs on whole rumen fluid was 8.7% per hr with a standard deviation of 6.05. Parallel experiments with Butyrivibrio indicated that soluble lytic factors were more important for this organism. They could be destroyed by autoclaving and were generated when viable rumen bacteria were resuspended in autoclaved rumen fluid. The lysis of S. bovis and Butyrivibrio, at equal cell densities, by mixed rumen protozoa was compared in 30% rumen fluid media, and Butyrivibrio appeared to be more readily lysed than S. bovis.     

Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence

Phylogenetic analysis of archaea in the rumen ecosystem was analysed by PCR of 16S rDNA from the bovine rumen using archaea-specific primers. The libraries were constructed from rumen fluid (AF), rumen solid (AS), and rumen epithelium (AE) from a rumen-fistulated Korean cow (Hanwoo). The 45 AF clones could be divided into three groups and the largest group was affiliated with the Methanomicrobiaceae family (96% of clones). The AF clones contained a high proportion of unidentifiable clones (67%). The 39 AE clones could be divided into two groups and the largest group was also affiliated with the Methanomicrobiaceae family (95% of clones). The AE clones contained a low proportion of unidentifiable clones (5%). The 20 AS clones could be divided into two groups that were affiliated with either the Methanobacteriaceae family (55%) or the Methanomicrobiaceae family (45%). The AS clones contained a moderate proportion of unidentifiable clones (40%). The predominant family of whole rumen archaea was found to belong to the Methanomicrobiaceae (85%). Methanomicrobiaceae were predominant in the rumen epithelium and the rumen fluid while Methanobacteriaceae were predominant in the rumen solid. One clone from the rumen fluid and two clones from the rumen epithelium contained rDNA sequences of Non-Thermophilic-Crenarchaeota (NTC) and Thermophilic-Crenarchaeota (TC), respectively, which have not previously been described from the rumen.