捕食性真菌Duddingtonia flagrans基因组DNA的提取及基因组survey分析

采用透析膜培养法结合DNA柱式提取法进行基因组DNA提取,同时利用第2代高通量测序技术,分析了捕食性真菌Duddingtonia flagrans基因组概况。结果显示:透析膜培养结合DNA柱式提取法所提取的基因组DNA质量及浓度均能达到测序标准;捕食性真菌Duddingtonia flagrans基因组约为37.6 Mb,GC含量为44.95%,基因组杂合度较低。结果表明:透析膜培养法结合柱式法提取其基因组DNA的方法简便可行,为丝状真菌DNA提取,提供了新的思路;基因组survey分析为捕食性真菌Duddingtonia flagrans全基因组精细图谱的绘制奠定了基础。     

捕食线虫性真菌Duddingtonia flagrans冻干制剂临床模式

本试验选用耐高温高压的菌种袋作为玉米粒培养基的培养容器,对Duddingtonia flagrans厚壁孢子进行批量培养,然后经孢子洗脱液将孢子洗脱后,将其制备成冻干制剂,并在内蒙古地区的呼和浩特市和林格尔、包头市萨拉齐、呼伦贝尔西旗等地养殖场共165头绵羊中进行了临床应用研究。通过设立不同的试验组和对照组,使用常用驱虫药伊维菌素进行驱虫试验,同时配合口服捕食线虫性真菌Duddingtonia flagrans厚壁孢子,在不同时间进行动物直肠采粪,对粪便进行第三期幼虫培养,然后检测比较不同组别粪便中感染性幼虫的数量,评价捕食线虫性真菌临床应用模式效果。结果显示,将伊维菌素与D.flagrans冻干制剂联合应用于绵羊的寄生性线虫病防治,可使粪便中幼虫数量降低100%,效果优于单独用药组。结果表明,捕食线虫性真菌Duddingtonia flagrans冻干生物制剂与驱虫药物联合使用的临床应用模式,可以取得较好的家畜线虫病临床防控效果,值得进一步在生产实践中进行深入研究和推广。     

不同培养条件对捕食性真菌Duddingtonia flagrans生长的影响

为了研究不同生长条件对捕食线虫性真菌Duddingtoniaflagrans生长的影响,本研究将带有D.flagrans菌丝的琼脂块分别接种于不同碳氮比及不同pH值(4.0~8.5)的CMA固体培养基中,在不同温度下(10℃~45℃)进行培养,每天定时观察菌株的发育情况,并测量菌丝生长长度。结果表明,D.flagrans生长最适pH值范围为6.5~7.0,其中以pH 7.0中生长最快;最适温度为35℃;最适碳氮比范围为5∶1~10∶1,其中在碳氮比为5∶1时,生长率最高。菌丝呈辐射状生长,纵横交错,无色,分隔,生长后期有缠绕圈和厚壁孢子的形成。上述结果为进一步研究D.flagrans的生长特性及捕食作用条件奠定了基础。     

捕食线虫性真菌Duddingtonia flagrans冻干制剂的杀虫效果

本试验通过在含有0.05%琼脂粉的沙氏葡萄糖肉汤培养基中培养Duddingtonia flagrans(D.flagrans)再转接到玉米粒培养基的方法,对D.flagrans厚壁孢子进行培养,之后将其洗脱并制备成冻干制剂,然后对冻干制剂的萌发率和杀虫率进行观察。结果显示,D.flagrans冻干制剂厚壁孢子在接种玉米粉琼脂培养基(CMA)培养后5d萌发率达到峰值78.3%;经口饲喂绵羊D.flagrans冻干厚壁孢子剂量为1×106个/kg时,幼虫平均减少率为81.4%。结果表明,D.flagrans冻干制剂使用方便,复苏和杀虫效果较好,其在生物防制家畜胃肠道线虫病方面有很大的应用前景。     

不同培养基及诱导物对捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用的影响

为研究捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用,使用不同营养成分组成的液体培养基及诱导物,对Duddingtonia flagrans进行培养和诱导,测定其发酵液中的蛋白质含量、蛋白酶活性、磷酸酶活性和杀虫作用,进而确定Duddingtonia flagrans产生胞外蛋白质和杀虫作用最适的培养基与诱导物。结果显示,不同培养基及诱导物对Duddingtonia flagrans的代谢过程会产生明显影响,导致其分泌的蛋白质种类、浓度以及蛋白酶和磷酸酶的活性显著不同;Duddingtonia flagrans发酵液不仅具有杀线虫活性,而且有杀蝇蛆作用。上述结果为进一步研究捕食线虫性真菌的杀虫机理提供了重要参考依据。     

相对湿度和培养时间对食线虫真菌Duddingtonia?flagrans产孢量的影响

本研究旨在评估食线虫真菌Duddingtonia flagrans当地分离株(SDH035)在生长发育的过程中培养时间和环境中的相对湿度对其产孢量的影响。利用实验室已保存的当地分离的SDH035分离株,用液固双相法分别在35%和85%的相对湿度下分别培养7 d、14 d、21 d、28 d、35 d、42 d、49 d、56 d和62 d,用0.05%(w)的灭菌Twain-80将生长在谷粒表面的菌物洗脱下来,用双层纱布过滤,获得含有厚垣孢子的菌悬液,用血细胞计数版对其孢子进行计数,最后推算出每克谷粒产生的厚垣孢子数量。结果表明,随着培养时间的延长,该菌株的产孢量逐渐升高,当培养时间为7～14 d时,产孢量缓慢上升,此后产孢量急速增加,在培养28 d后该菌株的产孢量趋于平缓,培养至49 d时其产孢量达到高峰。分离株在两种相对湿度、不同培养时间段的产孢量。在相同的培养时间下,85%的相对湿度所获得的产孢量显著高于35%的相对湿度。培养时间和湿度条件均影响厚垣孢子的产量。     

大肠杆菌O78原生质体制备与再生的研究

以耐药标记的鸭大肠杆菌ZB078(Ery<'r>,Chl<'s>)为亲本茵株,在DNase Ⅰ始终存在的条件下,采用溶茵酶-EDTA法制备原生质体,然后在高渗琼脂平板上再生.通过优化溶菌酶-EDTA的工作浓度与作用时间、原生质体不同的处理方式和再生培养基中蔗糖浓度等条件.摸索出ZB078以0.2g/L溶菌酶作用20 rnjn后补加EDTA至终浓度为0.01 mmol/L继续作用16 min为最佳试验条件,并成功获得了91.94%的原生质体制备率和37.29%的再生率,为下一步进行融合试验提供了依据.     

糙皮侧耳原生质体制备与再生条件的研究

以糙皮侧耳为材料,研究酶的组成、酶解时间、酶解温度、菌龄和渗透压稳定剂对糙皮侧耳原生质体制备率和再生率的影响.结果表明:静置培养7d的糙皮侧耳菌丝体,以0.6 mol/L甘露醇溶液为渗稳剂,2％溶壁酶+2％纤维素酶+1％蜗牛酶酶体系30℃酶解3h,原生质体制备率为415万个/mL;培养9d的菌丝体以0.6 mol/L硫酸镁溶液作为渗稳剂,2％溶壁酶+2％纤维素酶+2％蜗牛酶,28℃酶解4h,原生质体最佳再生率为2.86％.     

捕食动物线虫性真菌相关研究进展

畜禽线虫病是由线形动物门中多种线虫引起的一类寄生虫病,给养殖业带来了严重的经济损失.长久以来,采用化学药物驱虫进行防治产生的耐药性、环境污染及动物源性食品药物残留等问题愈发严重,迫切需要寻找能替代化学药物驱虫的防治手段.利用捕食线虫性真菌进行动物寄生性线虫的生物防治对建立可持续发展战略和无公害农业具有重要意义.通过综述...     

捕食动物线虫性真菌凝集素的研究进展

凝集素是广泛存在于生物体内的一类糖蛋白,对生物体有着重要作用,近年来凝集素的研究取得了重要成果。笔者主要对捕食动物线虫性真菌凝集素的生物学特性、功能、作用机制及其在分子生物学方面的研究进展作了较详细的阐述。     

Capability of the nematode-trapping fungus Duddingtonia flagrans to reduce infective larvae of gastrointestinal nematodes in goat feces in the southeastern United States: dose titration and dose time interval studies

Infection with gastrointestinal nematodes, particularly Haemonchus contortus, is a major constraint to goat production in the southeastern United States. Non-anthelmintic control alternatives are needed due to increasing resistance of these nematodes to available anthelmintics. Two studies were completed in Central Georgia in August 1999, and April–May 2000, using Spanish does naturally infected with Haemonchus contortus, Trichostongylus colubriformis, and Cooperia spp. to evaluate effectiveness of nematode-trapping fungi as a biological control agent. In the first experiment, five levels of Duddingtonia flagrans spores were mixed with a complete diet and fed once daily to the does (three per treatment) in metabolism crates. The treatment concentrations were (1) 5×10⁵, (2) 2.5×10⁵, (3) 10⁵, and (4) 5×10⁴ spores per kilogram body weight (BW), and (5) no spores. Fungal spores were fed for the first 7 days of the 14-day trial, and fecal samples were collected daily from individual animals for analysis of fecal egg count and establishment of fecal cultures. Efficacy of the fungus at reducing development of infective larvae (L3) in the fecal cultures was evaluated. The mean reduction in L3 from day 2 of the treatment period until the day after treatment stopped (days 2–8) was 93.6, 80.2, 84.1, and 60.8% for animals given the highest to lowest spore doses, respectively. Within 3–6 days after termination of fungal spore feedings, reduction in L3 development was no longer apparent in any of the treated animals. In a second experiment, effectiveness of 2.5×10⁵ spores of D. flagrans per kilogram BW fed to does every day, every second day, and every third day was evaluated. Reduction in L3 development by daily feeding was less in the second experiment than in the first experiment. Daily fungal spore feeding provided more consistent larval reduction than intermittant feeding (every second or third day). When fed daily under controlled conditions, D. flagrans was effective in significantly reducing development of L3 and appears to be an effective tool for biocontrol of parasitic nematodes in goats.     

嗜线虫真菌Duddingtonia flagrans减少绵羊粪便中线虫感染性幼虫剂量测定

嗜线虫真菌Duddingtonia flagrans是目前发现的一种最具有动物胃肠道线虫病生物防治应用潜力的真菌。为了解该菌捕杀绵羊粪便中感染性幼虫效果与剂量的关系,为今后该制剂应用和质量检验标准的制定提供依据,用不同剂量的厚垣孢子,分别以不经消化道直接加入感染羊粪便,或作添加剂通过饲喂进入消化道后采集直肠粪便,经培养检测感染性幼虫数量的变化。结果表明,嗜线虫真菌Duddingtonia flagrans具有良好的捕杀胃肠道线虫感染性幼虫的生物学特性。以制剂厚垣孢子4&#215;103/g剂量加入感染羊粪便,或以每天5&#215;105/kg体质量的剂量饲喂绵羊,可使粪便培养物中线虫感染性幼虫数减少83.6%~87.5%。     

Effect of biological control through the daily application of spores of Duddingtonia flagrans in lambs kept under an evasive grazing system in the Netherlands

In 2004, an experiment was carried out to evaluate the effect of biological control through feeding spores of Duddingtonia flagrans on parasitic gastroenteritis in lambs, kept under an evasive grazing system. In total 66 lambs were used. Forty naturally infected 3-month old ram lambs were weaned in mid June, and divided into four groups of 10 lambs. On 21 June, G1–G4 were moved to four separate virtually clean plots, they were moved after 4 and 8 weeks to similar plots, and housed after 12 weeks to be necropsied 16 days later. The other 26 lambs had been raised helminth-free, and were used as pairs of tracer lambs. All but one of these pairs, were grazed during the last 2 weeks on each plot. The remaining pair (TA) was grazed during the last 2 weeks on pasture (30 August to 13 September) on the plot that had been grazed by G3 between 19 July and 16 August, to study inhibited development in Haemonchus contortus. All lambs were fed 200 g of concentrates daily throughout the whole period, and those of G1 and G2 were also fed 500,000 spores of D. flagrans/kg bodyweight daily.

The faecal cultures demonstrated a high reduction in yield as a result of fungal application. However, no differences between groups were seen in weight gain, faecal egg counts, pasture larval counts, worm counts and tracer worm counts. H. contortus was the dominant species, and it is obvious that the moves at 4-week intervals prevented the development of severe haemonchosis. This is in particular demonstrated by the much higher worm counts in the two TA tracer lambs grazed. Nevertheless, increases to high faecal egg counts 3 weeks after the first and second moves, indicated acquisition of infection before these moves and at least subclinical haemonchosis. This was supported with the worm counts of lambs and tracer lambs. A higher proportion of inhibited early L4 than in other tracers and than in the permanent lambs were found in the pair of TA tracer lambs. This indicates that moves to new pastures in late summer and autumn delays the onset of inhibition.     

Predacious activity of the nematode-trapping fungus Duddingtonia flagrans against cyathostome larvae in faeces after passage through the gastrointestinal tract of horses

This study was undertaken to examine the potential of the nematode-trapping microfungus Duddingtonia flagrans to survive passage through the gastrointestinal tract of horses and subsequently to destroy free-living stages of cyathostomes in faecal cultures. Three different oral dose levels were tested, two horses being used for each level. Faeces were collected twice daily and the number of parasite eggs per gram of faeces were determined. The numbers of infective third stage larvae which developed in faecal cultures were determined after the cultures had been incubated for 2 weeks at 24°C. Results showed a positive relationship between dose level and reduction in the number of infective larvae. Fungi were recovered in faeces at times which corresponded to high larval reduction.     

Soil Nematode Populations Beneath Faecal Pats from Grazing Cattle Treated with the Ivermectin Sustained-release Bolus or Fed the Nematophagous Fungus Duddingtonia flagrans to Control Nematode Parasites

The size and composition of the nematode assemblage in soil under faecal pats derived from young cattle treated or untreated with either ivermectin sustained-release boluses, or the nematophagous fungus Duddingtonia flagrans, were studied in each of three years. Soil samples taken 4, 6, 8 and 10 weeks after four deposition dates in 1998 showed significant temporal effects in many taxa and treatment effects in a few genera. In 2000, soil samples taken 10 weeks after deposition in July, August and September showed treatment effects in the plant-associated Tylenchus and Cephalenchus, and the bacterial-feeding Cephalobus 1 and Cephalobus 2 taxa. However, overall it was found that the nematode assemblages were similar below all three types of pat, and the assemblages varied with the season of deposition. D. flagrans, the novel biological control agent being tested against the free-living stages of nematode parasites of cattle, had no detectable impact on the size or the structure of the soil nematode communities under the faecal pats.     

Efficacy of the nematode-trapping fungus Duddingtonia flagrans against three species of gastro-intestinal nematodes in laboratory faecal cultures from sheep and goats

The ability of the nematode-killing fungus Duddingtonia flagrans to reduce number of infective larvae of three species of gastro-intestinal parasitic nematodes developing in dung was investigated in both goats and sheep. Groups of lambs and kids (12-20 weeks old) were given mono-specific infections of Haemonchus contortus, Ostertagia (Teladorsagia) circumcincta or Trichostrongylus colubriformis. Following patency of the infections (t1) faecal samples were collected for determination of faecal nematode egg count (FEC) and culture of parasite larvae. Groups of animals were then dosed on 2 consecutive days with one of the two dose rates of the fungus (250,000 or 500,000 spores/kg liveweight). One (t2) and 5 (t3) days after the second dose of fungus samples were again collected for FEC and culture. The number of larvae recovered from the faecal cultures at t1 and t3 were used as controls to assess the efficacy of the experimental treatment at t2. Average efficacy was 78% with group means ranging from 40 to 93%. Dose rate of fungus appeared to influence efficacy against O. circumcincta but not against H. contortus or T. colubriformis. Overall, there were no differences in the efficacy of the fungus against any of the parasite species or in either host animal. The results of this trial indicate the potential use of this fungus as a broad spectrum anti-parasite agent for use in both goats and sheep.     

Absence of obvious short-term impact of the nematode-trapping fungus Duddingtonia flagrans on survival and growth of the earthworm Aporrectodea longa

The nematode-trapping fungus Duddingtonia flagrans may be used in biological control of parasitic nematode larvae in faeces of domestic host animals after feeding the hosts with fungal chlamydospores. In this experiment a possible undesirable fungal impact on earthworms, of the species Aporrectodea longa, was investigated. As earthworms eat animal faeces, D. flagrans may come into contact with earthworms both in their alimentary tract and on their body surface. However during the experimental period of 20 days, when earthworms were living in soil and eating cattle faeces that were heavily infested with viable chlamydospores of D. flagrans there were no indications of internal or external mycosis among the earthworms.