蝙蝠蛾幼虫肠道中的冬虫夏草菌体研究

西藏林芝地区冬虫夏草天然产区野生虫草蝙蝠蛾幼虫的肠道液pH范围为9.69-10.99,体外在此pH下培养冬虫夏草菌长势较差。同时,在该pH的体外模拟中肠液中,虫菌体也会在极短的时间内消亡,而对自然侵染的蝙蝠蛾幼虫的血淋巴、消化道及其“反吐物”（肠液）进行切片和显微镜检却发现,蝙蝠蛾幼虫消化道、呕吐物中均存在与血淋巴中相同的冬虫夏草菌菌体形态,生存状态良好,正常增殖,表明蝙蝠蛾幼虫肠道的生理状态可以维系并支撑冬虫夏草菌菌体某种形态的存在与生长,并证实冬虫夏草菌由口器及消化道入侵蝙蝠蛾幼虫的可能性;肠道虫菌体与血淋巴虫菌体不一定同时存在,表明冬虫夏草菌对蝙蝠蛾幼虫的侵染可能同时存在表皮侵染和肠道侵染两种途径。本文为冬虫夏草菌对蝙蝠蛾幼虫的侵染途径研究提供更加多样化的信息支持。     

蝙蝠蛾幼虫肠道真菌复合体中的多菌种与中国被毛孢分生孢子联合侵染蝙蝠蛾幼虫提高侵染效能

应用真菌特异性引物PCR测序、克隆测序和SNP质谱基因分型法,鉴定2株蝙蝠蛾幼虫活体肠道的真菌分离株(CH1和CH2)中的多种菌物。应用这2株分离株与中国被毛孢分生孢子联合侵染蝙蝠蛾幼虫,观察其协同侵染活性。结果表明这2株分离株呈现中国被毛孢的体外培养生长和显微形态特征。通用引物ITS4/5和蝙蝠蛾拟青霉特异性引物Pp4/5扩增子测序检出蝙蝠蛾拟青霉和中国被毛孢ITS序列。扩增子克隆-测序和SNP基因分型法证实多种转换和颠换点突变基因型在2株分离株中共存。反复固体、液体培养证实蝙蝠蛾拟青霉和中国被毛孢ITS序列共存,并非真菌间DNA片段漂移。应用子囊孢子或中国被毛孢侵染4~5龄蝙蝠蛾幼虫,幼虫染菌率为1.37%~3.52%,幼虫变僵时间为35~50 d。应用2株分离株与中国被毛孢分生孢子联合侵染,幼虫变僵时间为5~8 d,染菌率达55.2%(P0.001),侵染效能提高15~39倍。蝙蝠蛾拟青霉和多种点突变基因型冬虫夏草菌(包括中国被毛孢)在蝙蝠蛾幼虫肠道以天然真菌复合体形式共存;多种菌物组成的侵染源联合侵染蝙蝠蛾幼虫大幅度提高侵染效能,呈现多菌协同侵染的共生关系。     

冬虫夏草寄主昆虫选育及生殖退化研究

选育发育速度快、繁殖能力强、中国被毛孢侵染率和冬虫夏草产出率高的寄主昆虫品系是冬虫夏草培植的基础,同时明确冬虫夏草的寄主昆虫蝙蝠蛾退化机制是保证冬虫夏草培植的条件。本文从冬虫夏草产区采集蝙蝠蛾种群种质资源,分别针对蝙蝠蛾发育速度、繁殖能力、中国被毛孢侵染率、冬虫夏草产出率4个方面通过杂交及四代选育,获得新种群（A1）,该种群对冬虫夏草繁育环境的适应力强,发育整齐度高,繁育一代的种群扩繁倍数从8.2倍提高至26.3倍;对中国被毛孢的敏感性较好,侵染率高,冬虫夏草产出率从原始种群的11.2%提高至18.5%。同时,仿照高原生态环境长期饲养冬虫夏草的寄主昆虫蝙蝠蛾,其子代出现雌雄比失调、发育历期分化、生殖力降低等退化现象。经过研究分析得知卵孵化率下降的主要原因是雄性的生殖力下降。组织切片观察蝙蝠蛾雄性生殖系统,在幼虫后期、蛹、成虫期会出现生精囊减少及畸形等异常情况,推测此为雄性生殖力降低、卵孵化率下降的生理原因。研究结果为高效、稳定地饲养蝙蝠蛾保证冬虫夏草培植具有重要意义。     

冬虫夏草菌在寄主钩蝠蛾幼虫中的潜伏侵染过程研究

冬虫夏草是青藏高原的特色名贵药材,是冬虫夏草菌Ophiocordyceps sinensis寄生于钩蝠蛾幼虫后形成的虫生子囊真菌,其发生机理和侵染途径至今未知。本文利用real-time q PCR实时定量技术检测了寄主蒲氏钩蝠蛾Thitarodes pui幼虫表皮、脂肪体、血淋巴和肠壁等组织中冬虫夏草菌定殖量,分别建立了起始于表皮的侵染路径(R20.678)和起始于肠道的侵染路径(R20.271)两种Holt数学模型,经比较分析后,推论冬虫夏草菌随口腔摄食侵染寄主幼虫的方式具有一定合理性和可行性,揭示冬虫夏草菌可以通过进食途径实现其对钩蝠蛾幼虫的带菌生长、侵染和寄生,从而为深入阐释冬虫夏草菌与寄主钩蝠蛾幼虫相互作用关系提供重要参考。     

冬虫夏草研究的新进展

冬虫夏草是冬虫夏草菌寄生于虫草蝙蝠蛾（Hepialus armoricanus）幼虫体后发育成的真菌子座和充满菌丝的僵死幼虫的复合体,与人参、鹿茸齐名,为我国三大珍贵滋补防病中药材。冬虫草源流于我国,主产于四川、青海、西藏和甘肃等省,从冬虫夏草的基本概况、生物学特性、理化成分、人工培育现状及其应用前景等进行了综述,以期推动冬虫夏草资源的研究和开发利用进程。     

糖类对冬虫夏草菌Ophiocordyceps sinensis芽生孢子产量及毒力的影响

冬虫夏草Chinese cordyceps是食药两用的传统名贵资源。其人工培养需要冬虫夏草菌Ophiocordyceps sinensis的优质芽生孢子。为获得高产量及高感染力芽生孢子,本研究测定不同糖类培养基对冬虫夏草菌芽生孢子产量及毒力（对小金蝠蛾Thitarodes xiaojinensis幼虫的存活率、携菌率和僵虫率）的影响。两个冬虫夏草菌株于含6种糖（葡萄糖、果糖、阿拉伯糖、麦芽糖、蔗糖和海藻糖）的液体培养基中分别培养30、45和60 d,计数芽生孢子产量并将收集的孢子注射感染两个品系的小金蝠蛾6龄幼虫,置于不同温度下观察幼虫的存活率、携菌率及僵虫率。结果显示：不同糖源培养基中芽生孢子的产量差异显著,其中含麦芽糖培养基中芽生孢子产量最高;注射菌株和幼虫品系对被注射幼虫的存活率无显著差异;不同糖源培养基获得的芽生孢子对蝠蛾幼虫的存活率无显著差异,对携菌率和僵虫率产生影响;以含麦芽糖为糖源的培养基、芽生孢子培养时间显著影响被注射幼虫的存活率与僵虫率,其中培养30 d的冬虫夏草菌所注射的幼虫的存活率、僵虫率显著高于60 d的;温度也显著影响被注射幼虫的存活率、僵虫率,10℃的幼虫存活率和僵虫率显著高于14℃、18℃的。研究结果为培养优质冬虫夏草芽生孢子,提高被侵染蝙蝠蛾幼虫僵虫率提供了参考。     

粉棒束孢Isaria farinosa的研发进展

粉棒束孢Isaria farinosa是一种常见的昆虫病原真菌,已被用作一种生物因子防治温带农林害虫、植物病原和线虫。同时,粉棒束孢作为冬虫夏草中的定殖真菌,严重危害冬虫夏草菌Ophiocordyceps sinensis寄主蝙蝠蛾幼虫的饲养,引起了资源昆虫研究者的高度重视和控制。作为生防真菌,粉棒束孢资源的分布和筛选、分子鉴定和遗传多样性、培养、活性成分和药效、分子生物学、害虫生物防治、病害控制、安全性等是学术界的研究重点;作为冬虫夏草寄主昆虫病原菌,学界和产业界一直研究其感染特征、侵染机理和防霉剂筛选等,为这一真菌的高效安全控制提供基础。本文将综述粉棒束孢在利用和控制两大方面的研发进展。     

蝙蝠蛾幼虫感染粉棒束孢后的病症、组织病理变化及侵入途径

蝙蝠蛾幼虫的饲养是冬虫夏草培植的先决条件,粉棒束孢作为蝙蝠蛾幼虫饲养过程中的一种常见和主要致病菌,具有极强的致病性和流行性。结合形态学观察和组织切片技术,本文研究了蝙蝠蛾幼虫感染病原菌粉棒束孢后的症状、主要组织病理变化及粉棒束孢侵染蝙蝠蛾幼虫的途径。结果表明,用浓度为1×10⁵个/mL的粉棒束孢分生孢子悬液处理蝙蝠蛾5龄幼虫,4d后在腹部气门处有黑斑出现,气门变黑,虫体随后瘫痪死亡、体色改变、收缩化僵、菌丝层包裹、形成众多孢梗束等症状。幼虫被粉棒束孢感染后,菌丝体首先利用血淋巴进行增殖,脂肪体、肌肉、消化道、丝腺和马氏管等主要组织器官先后出现不同程度的破坏,虫体体腔被大量菌丝充斥并最终僵化。组织切片观察结果表明,粉棒束孢可以通过气门侵入蝙蝠蛾幼虫体内并造成虫体死亡,菌丝穿透气管进入血淋巴后断裂成许多长短不一的短菌丝段,目前尚未观察到菌丝直接穿透幼虫体壁和从消化道侵入的现象。本研究为蝙蝠蛾幼虫饲养过程中的病害防治提供信息参考。     

冬虫夏草菌丝状菌源增殖培养的研究

冬虫夏草菌是珍稀濒危名贵中药材冬虫夏草的无性型菌种,是侵染蝠蛾幼虫的唯一菌种,其侵染后形成名贵中药材冬虫夏草。冬虫夏草菌在不同营养及条件下生长形态不同,主要有丝状体和菌球体两种生长形态。冬虫夏草菌丝状菌体能够侵染蝠蛾幼虫,有可能作为一种新的接种体被广泛应用;但丝状菌体对营养要求苛刻、生长缓慢、菌丝体得率低的特点,阻碍了冬虫夏草菌丝状菌体作为侵染蝠蛾幼虫的接种体的开发及应用,从而阻碍了冬虫夏草人工培殖产业化的进程。为了提高冬虫夏草菌丝状菌体的生物表达量,本文对营养及培养条件进行了优化研究,得出了最佳条件为:葡萄糖质量浓度40 g/L、酵母粉质量浓度65 g/L、培养温度17℃、MgSO_4质量浓度3 g/L、KH_2PO_4质量浓度1.5 g/L、培养时间12 d,按最优培养条件,冬虫夏草菌丝状菌体干重得率15.67 g/L,为下一步新的接种体的制备提供菌源保障。     

冬虫夏草线状菌体制剂对蝠蛾幼虫感染的研究

为明确冬虫夏草线状菌体制剂的侵染力,进行了冬虫夏草线状菌体制剂对蝠蛾幼虫的感染试验。在相同条件下对蝠蛾幼虫进行人工接种,并对感染温度、菌剂用量和虫龄进行了优化。结果表明冬虫夏草线状菌体具有侵染蝠蛾幼虫的能力,优化后的感染条件为:菌体剂量为5 g(即5个标准剂量)、幼虫虫龄为3龄、培养温度为14℃。该结果为蝠蛾幼虫新型接种体的制备与开发提供了依据。     

The medicinal fungus Cordyceps militaris: research and development

Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a highly valued medicinal fungus. This entomopathogen has a limited distribution, has been overharvested in the wild, and its stromata have not been artificially cultivated. Another entomopathogenic fungus, Cordyceps militaris (commonly known as orange caterpillar fungus), has chemical capacities similar to those of O. sinensis, but unlike O. sinensis, its stromata can be easily cultivated. Consequently, C. militaris is being studied as an alternative to O. sinensis, and the large-scale production of stromata is receiving substantial attention. Significant research has been conducted on the genetic resources, nutritional and environmental requirements, mating behavior, and biochemical and pharmacological properties of C. militaris. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biology. The current paper reviews the biological aspects of the fungus including host range, mating system, cytology and genetics, insect- and non-insect nutritional requirements, environmental influence on stroma development, and commercial development.     

Phylogeography and evolution of a fungal–insect association on the Tibetan Plateau

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high‐frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis–ghost moth parasitoidism on the Tibetan Plateau.     

People,money, and protected areas: the collection of the caterpillar mushroom <Emphasis Type="Italic">Ophiocordyceps sinensis</Emphasis> in the <Emphasis Type="Italic">Baima Xueshan</Emphasis> Nature Reserve,Southwest China

The caterpillar mushroom Ophiocordyceps sinensis (syn. Cordyceps sinensis) is among the most valuable mushrooms in the world, and plays a major role for the local economies in its distribution area on the Tibetan Plateau and adjacent regions. Large proportions of its habitat fall into protected areas, and best practice of sustainable harvest is under discussion, considering both, O. sinensis as a valuable income source for rural poor and protection of its populations and habitat. This study aims for a detailed analysis of O. sinensis collection in a nature reserve in Southwest China. We found that harvesting is unevenly distributed among households and villages, with households who have access to the resource but lack adequate alternatives for income generation such as rewarding wage labor, fertile agricultural fields or harvest of other high value products being most involved. Although collection is de jure forbidden, authorities of the nature reserve apply adaptive management strategies for sustainable resource use. This includes the allocation of collection areas to communities based on their traditional land use strategies and the control of harvesters from outside, triggering self-policing of the resource by the local people. The strategies applied provide a promising model also for other protected areas where the caterpillar mushroom is collected.     

一株产虫草素的冬虫夏草内生真菌 Bionectria ochroleuca OS17

虫草素是一种核苷类似物,具有多种药理作用。利用虫草素产生菌发酵的方式获得虫草素,可能是产生虫草素的一种新方法。为筛选能产虫草素的内生真菌,从中国传统的中药冬虫夏草子实体中共分离得到42株真菌,经PDB液体培养后,采用高效液相色谱（HPLC）鉴定其代谢产物。结果表明：菌株OS17的代谢产物中含有虫草素,且虫草素产量为40.16 mg/L。基于形态学特征和ITS序列分析,菌株OS17被鉴定为淡色生赤壳菌(Bionectria ochroleuca)。这是首次从冬虫夏草中分离得到能产虫草素的生赤壳属,表明了内生真菌具有产生多种生物活性物质的潜力。     

On the reliability of fungal materials used in studies on <Emphasis Type="Italic">Ophiocordyceps sinensis</Emphasis>

Ophiocordyceps sinensis (≡Cordyceps sinensis) is one of the best known traditional Chinese medicines, with great benefits to human health and huge economic value. The reliability of fungal materials used in studies of the species is particularly important because contradictory results have been found in various studies in the past decades. Examination of fungal materials specified in reports on O. sinensis showed great variation in both sources and culture conditions of living strains. To test the reliability of the materials used, experiments were carried out to study the effect of culture conditions on the growth of living strains of O. sinensis by using six reliable strains representing the major production regions of the fungus on the Tibetan Plateau. The results showed that O. sinensis is a slow-growing fungus at comparatively low temperature, and that temperature and growth period are crucial factors which can be verified by experiment. Analyses of fungal materials used in 152 papers on O. sinensis from PubMed since 1998 showed that 41 papers lacked detailed information on the fungal materials; 26 used natural products, 11 used artificially cultivated fruit bodies, and 80 used fermentation products from living strains. Of the latter category (using fermentation products), 64 of the papers were found to use unreliable (45) or uncertain (19) strains for fermentation products based on the temperature and growth period for O. sinensis strains verified in this study. Apart from the natural products of O. sinensis, which require scientific identification, a total of at least 116 papers (over three-quarters) used unreliable, uncertain or unspecified materials, including so-called cultivated fruit bodies which were apparently from other species. The reliability of materials or living strains used in studies on O. sinensis is discussed in this paper, and suggestions are made for use of reliable fungal materials in further studies of this fungus.     

Impact of Climate Change on Potential Distribution of Chinese Caterpillar Fungus (Ophiocordyceps sinensis) in Nepal Himalaya

Climate change has already impacted ecosystems and species and substantial impacts of climate change in the future are expected. Species distribution modeling is widely used to map the current potential distribution of species as well as to model the impact of future climate change on distribution of species. Mapping current distribution is useful for conservation planning and understanding the change in distribution impacted by climate change is important for mitigation of future biodiversity losses. However, the current distribution of Chinese caterpillar fungus, a flagship species of the Himalaya with very high economic value, is unknown. Nor do we know the potential changes in suitable habitat of Chinese caterpillar fungus caused by future climate change. We used MaxEnt modeling to predict current distribution and changes in the future distributions of Chinese caterpillar fungus in three future climate change trajectories based on representative concentration pathways (RCPs: RCP 2.6, RCP 4.5, and RCP 6.0) in three different time periods (2030, 2050, and 2070) using species occurrence points, bioclimatic variables, and altitude. About 6.02% (8,989 km²) area of the Nepal Himalaya is suitable for Chinese caterpillar fungus habitat. Our model showed that across all future climate change trajectories over three different time periods, the area of predicted suitable habitat of Chinese caterpillar fungus would expand, with 0.11–4.87% expansion over current suitable habitat. Depending upon the representative concentration pathways, we observed both increase and decrease in average elevation of the suitable habitat range of the species.     

Steps towards sustainable harvest of Ophiocordyceps sinensis in Bhutan

The insect-pathogenic fungus Ophiocordyceps sinensis (better known as Cordyceps sinensis) is harvested over much of the Himalayan plateau as a highly prized remedy in traditional Oriental medicine. Over the past 10 years its financial value has increased dramatically, with collectors paid as much as US $12,500 kg⁻¹ for top-quality material. This is causing significant distortion to local economies, and there is widespread concern that the current rate of collection is unsustainable. This paper introduces the fungus and its insect hosts, documents some of the biological and social constraints to achieving sustainability, describes the socioeconomic climate within which harvest and sale occurs in Bhutan, and details the measures put in place by the Royal Government of Bhutan to promote wise management of this valuable natural resource.

基于MaxEnt模型的中国沙棘潜在适宜分布区分析

中国沙棘主要分布于我国华北、西北、西南等地森林—草原过渡地带,是我国北方地区退耕还林、生态修复等工程的重要造林树种,对维持干旱、半干旱地区的生态环境稳定具有重要意义。探讨限制中国沙棘分布的主导气候因子,模拟其潜在适宜分布区,以期为中国沙棘在林业生态工程和生态经济林建设中的合理种植和推广提供理论依据。基于中国沙棘自然分布的328个地理样点,利用最大熵(MaxEnt)模型对中国沙棘的潜在分布区的主导气候因子进行分析,并预测中国沙棘的潜在分布范围。结果表明,基于气候变量的MaxEnt模型训练集和测试集受试者工作特征曲线下面积(AUC值)分别为0.962±0.001和0.949±0.001,均大于0.9,表明MaxEnt模型对中国沙棘潜在分布区的预测具有极高的准确度,可信度好。基于环境变量贡献率和刀切法的结果表明年降雨情况、生长季的水热状况、最干季降雨和最冷月最低温等是限制中国沙棘分布的主要气候因素,其中年降雨是限制中国沙棘分布的主导气候因子。通过模拟得到现代中国沙棘潜在地理分布的总适生区面积为165.1万km~2;其中高适生区和中适生区面积共93.3万km~2,主要集中分布于河北西部、北部,...     

Investigations into the natural infection rate of <Emphasis Type="Italic">Haemaphysalis qinghaiensis</Emphasis> with Piroplasma using a nested PCR

Here we describe the natural infection rate in China of Haemaphysalis qinghaiensis with four Piroplasma species, namely Theileria uilenbergi, T. luwenshuni, T. sinensis and Babesia motasi. Specifically, a nested PCR was designed based on 18S ribosomal RNA genes and its specificity and sensitivity were established. The result showed that 62 flat adult field H. qinghaiensis ticks (27 females and 35 males) out of 136 (55 females and 81 males) were infected by one or more parasites. All 62 (45.6%) were infected with T. uilenbergi; nine (five males and four females; 6.6%) were infected with T. luwenshuni; two (1.5%) females were infected with T. sinensis; and one (0.7%) male was infected with B. motasi. Twelve (19.4%) were infected with more than one pathogen. There was no significant difference in infection rate between males and females. The high figure 45.6% Theileria infection rate indicates the serious prevalence of theileriosis; while the presence of T. sinensis and B. motasi implies the potential existence of the corresponding diseases in the area studied.