【作者】 黄志宏；

【导师】 冯明光；

【作者基本信息】 浙江大学 ， 微生物学， 2008， 博士

【摘要】 蚜虫是全球性害虫，自然死亡因子很多，尤其侵染蚜虫的病原真菌（主要包括虫霉目真菌）多达30余种，由于蚜虫具有群集和迁飞特性以及其主要病原真菌特殊的流行学特征，成为昆虫流行病学研究的经典对象之一。本研究首次在中缅接壤的低纬度高原地区开展了蚜虫病原真菌资源的调查，通过大量空中诱捕和单头饲养有翅蚜而建立了不同组别迁入有翅蚜（感病、寄生、健康）的定殖能力、传病能力和生殖概率模型，首次研究揭示了努利虫疠霉的侵染生物学和流行学特征，主要结果分述如下：云南低纬度高原地区空中迁飞性有翅蚜传带的病原真菌和寄生蜂为了普查中缅接壤的低纬度高原地区的蚜虫病原真菌种类，2006年1-12月在云南勐海县一茶村设点，在一建筑物顶楼平台建立黄色防雨布上放盆载甘蓝苗的诱蚜区，整年不间断从空中诱获萝卜蚜（Lipaphis erysimi）、桃蚜（Myzus persicae）及甘蓝蚜（Brevicoryne brassicae）的有翅蚜共计3553头，分别带回室内在18-22℃和12L：12D的条件下单头饲养观察7-14天。其中，19．2％的有翅蚜（683头）在单头饲养期间发病死亡，病因为9种虫霉目真菌和1种丝状真菌，均为寄主专化性或非专化性蚜虫病原真菌。在发病死亡的有翅蚜中，77．8％系由虫霉目真菌感染，优势种为新蚜虫疠霉[Pandora neoaphidis（占42．7％）]和根虫瘟霉[Zoophthora radicans（14．5％）]，其余按发生频率依次为努利虫疠霉[P.nouryi（～5％）]、弗雷生新接霉[Neozygites fresenii（3％）]、普朗肯虫霉[Entomophthoraplanchoniana（2．3％）]、三种耳霉[Conidiobiolus spp．（1．9％）]及蚜虫瘟霉[Z.aphidis（个别）]。属于丝状真菌的蜡蚧菌（Lecanicillium lecanii）多出现在雨季诱获的萝卜蚜有翅蚜中，占总感染数的22．2％。另有占总观察数2．8％的有翅蚜（99头）被寄生蜂寄生变成僵蚜，从中羽化出的寄生蜂包括烟蚜茧蜂（Aphidius gifuensis）、菜小脉蚜茧蜂（Diaeretella rapae）、麦蚜茧蜂（Ephedrus plagiator）和苹果绵蚜蚜小蜂（Aphelinus mali），它们在被寄生的有翅蚜中分别占44．4％、46．5％、2．0％和7．1％。旱季诱获较多的甘蓝蚜有翅蚜，感病和寄生的频率都很低。迁入带病和寄生有翅蚜的定殖潜能空中诱获的带菌有翅蚜在单头饲养期间，前3天发病死亡的占83％，很少死于第6-7两天，平均潜伏期为2．30±1．26天。被寄生蜂寄生的有翅蚜在单头饲养期间平均存活4．89±1．94天（仅个别超过7天），显著长于感病有翅蚜的存活时间，僵蚜经5．6±1．3天的发育后羽化为寄生蜂。从空中诱捕的萝卜蚜、桃蚜、甘蓝蚜有翅蚜，在单头饲养期所产后代的蚜群随定殖天数而增大，但不同组别之间差异很大。在感病有翅蚜组中，萝卜蚜后代蚜群从定殖后第1天的平均2．0±2．0头（n=559）增长到第14天的35．4±51．3头（n=349），桃蚜从平均2．1±2．0头（n=119）增长到72．1±76．5头（n=78），甘蓝蚜从平均4．4±3．7头（n=5）增长到6．3±2．5头（n=5）。相同定殖时间内，寄生组萝卜蚜后代蚜群的平均数量分别从1．7±1．6头（n=58）增长到86．6±73．7头（n=32），桃蚜从1．3±1．4头（n=39）增长到99．5±124．3头（n=22），甘蓝蚜从0．7±1．1头（n=3）增长到23．5±33．2头（n=3）。健康组后代蚜群的平均数量明显多于感病组和寄生组，萝卜蚜从2．6±2．0头（n=959）增长到65．3±125．8头（n=381），桃蚜从2．8±1．8头（n=605）增长到180．3±114．4头（n=378），甘蓝蚜从2．6±2．7头（n=260）增长到69．7±64．5头（n=62）。在感病有翅蚜的后代蚜群中均发生了接触传染，萝卜蚜的后代蚜群占73．4％，桃蚜占58．6％，甘蓝蚜占60％。感病组、寄生组和健康组有翅蚜的后代蚜群随定殖天数变化的增长趋势很好地拟合逻辑斯蒂模型（r²≥0．98），后代蚜群的增长潜能依次是健康组>寄生组>感病组。萝卜蚜、桃蚜和甘蓝蚜三种感病有翅蚜的后代因接触传染的发病死亡率随定殖时间增长的趋势符合Gompertz模型（r²≥0．92），拟合萝卜蚜和桃蚜后代的最大发病率分别为44．1％和27．0％，与第14天的最大观察值很相近。对不同组别有翅蚜定殖后6天内产m头若蚜的累积概率P（m≤N）通过拟合逻辑斯蒂方程而建立生殖力概率模型（r²≥0．99），结果显示，健康组更多若蚜的概率远高于感病组或寄生组；而感病组和寄生组相对于健康组而言，产较少若蚜数的概率更高。拟合的模型显示，空中诱捕的有翅蚜无论是被病原真菌侵染还是被寄生蜂寄生，均能在迁入后成功定殖并建立后代蚜群，而且随有翅蚜带入的病害能通过接触传染的方式就地扩散。努利虫疠霉的侵染生物学与流行学特征蚜虫病原性虫霉的休眠孢子形成是认识其季节性流行和宿存机制的关键，但对这一生物过程的认识十分缺乏。为了探索休眠孢子形成的可能机制，用从云南低纬度低区空诱带病有翅蚜中分离到的蚜科专化性努利虫疠霉菌株，在既适于病原菌侵染也适于寄主繁衍的条件下(（20±2℃，12L：12D）与桃蚜进行互作，观察其侵染生物和流行学特征。将甘蓝叶片上的桃蚜用分离菌株产生的分生孢子进行“孢子浴”接种的有重复生物测定（接种剂量即侵染体密度为7．7-70．2个孢子／mm2），致死中浓度(LC₅₀)从接种后第4天的6．7个孢子／mm²)下降为第7天的0．9个孢子／mm²，该菌对桃蚜表现出很强的毒力。所获蚜尸产休眠孢子（azygospores）或分生孢子，或两类孢子同时产，且产休眠孢子的蚜尸比例(P_CFRS)有随侵染体密度（C）增大而长高的趋势。继而在扩大接种剂量范围和增加剂量处理数的延伸试验中，C扩大为0．3-1759．8个孢子／mm²，处理数增至58个，各剂量下平均处理33（22-42）头蚜虫。所获观察数据的模拟分析结果十分令人鼓舞。P_CFRS对C的依赖关系很好地拟合逻辑斯蒂模型（r²=0．975），拟合的最大P_CFRS为0．6774，同240-1760个孢子／mm²范围内的26个高接种剂量下的P_CFRS平均值（0．674±0．124）几乎相等。当C不超过16．2个／mm²时，仅有0％-3．7％的蚜尸体内有休眠孢子形成。在介于28-240个／mm²的孢子密度范围内，P_CFRS随孢子密度的增加而快速上升，而后便不再随孢子密度的增加而变化，即稳定在最高水平附近。这一结果首次揭示了努利虫疠霉在寄主体内的休眠孢子形成依赖于侵染体密度的定量关系。我们由此提出新的生物学假说，在病原真菌与寄主蚜虫互作系统中或许存在某类生化信号物质，病原菌能从群体水平上“感受”这类信号物质而对即将发生的寄主密度变化作出及时反应，或产生分生孢子继续侵染周围还存在的健康寄主，或形成休眠孢子就地渡过缺乏寄主的季节。综上所述，本研究首次查清了中缅接壤低纬度高原地区的蚜虫病原真菌资源，尤其努利虫疠霉完整生活史的发现、菌株分离及其对蚜虫毒力的测定结果，丰富了虫疠霉属既有的科学认识；通过在低纬度高原地区大量空中诱捕和单头饲养有翅蚜的结果分析，建立了感病、寄生、健康有翅蚜迁入后的定殖能力、传病能力和生殖概率模型，有力佐证了寄主迁飞既传播蚜虫病原真菌又传播寄生蜂的观点，丰富了蚜虫迁飞生物学的科学内涵；首次揭示了努利虫疠霉产休眠孢子的蚜尸比例与感染寄主的侵染体密度之间的依赖关系，并由此提出了新的生物学假说。更多还原

【Abstract】 Aphids are global pests of crops and economic plants and are attacked by varieties of natural mortality agents, which include over 30 species of aphid-pathogenic fungi （mainly Entomophthorales）. Aphid mycoses are considered one of classic pathogen-host interactions due to the well-known biology of aphid dispersal flight and dense colonies and the special epizootic features of main pathogens. This study sought to survey for the first time the resources of aphid-pathogenic fungi that occur in the low-latitude plateau of China-Burma border, to examine the potential of pathogen-infected, wasp-parasitized and healthy host alates in colonization, fecundity and mycosis transmission after immigration, and explore for the first time the infective and epizootiological features of obligate aphid pathogen Pandora nouryi found in the plateau. The results are given as follows.Pathogenic fungi and parasitoids of aphids present in air captures of migratory alates. To survey fungal pathogens and parasitoids of aphids in the low-latitude plateau of Yunnan, southwest China, 3553 migratory alates of Brevicoryne brassicae, Lipaphis erysimi, Myzus persicae were attracted to a yellow-plus-plant trap from air during a full-year period and individually reared on cabbage leaves for 7-14 days at 18-22℃and L:D 12:12. Among the trapped alates, 19.2% survived averagely for 2.3 （1-7） days before killed by 11 species of aphid-pathogenic fungi. Another 2.8% were mummified by the wasps Aphidius gifuensis, Diaeretella rapae, Ephedrus plagiator and Aphelinus mali after survival of 4.9 （3-13） days. Most of the mycosed alates （77.8%） were attributed to Entomophthorales predominated by Pandora neoaphidis （42.7%） and Zoophthora radicans （14.5%）, followed by P. nouryi, Neozygites fresenii, Conidiobiolus spp., Entomophthora planchoniana and Z. aphidis at decreasing frequency. A mitosporic fungus, Lecanicillium lecanii, was found frequently in L. erysimi alates trapped in wet season. However, the B. brassicae alates captured in dry season were infected or parasitized very occasionally. Overall, the fecundity of the infected or parasitized alates before death warranted successful colonization on plants, though greatly reduced, and was well shown by the fitted probability for a given fecundity per capita and the increasing mean size of their progeny colonies. Contagious transmission of the alate-borne mycosis observed in most of the colonies caused high progeny mortalities within 14 days. The results highlight for the first time the diversity of aphid pathogens as well as the spread of both pathogens and parasitoids with host dispersal flight in the low-latitude plateau.Colonization potential of infected and parasitized alates trapped from air. Among the infected alates trapped from air, 83% became mycosed during colonization after trapping and a very few survived on day 6 or 7 with a mean （±SD） latent period of 2.30±1.26 days. All the parasitized alates averagely survived 4.89±1.94 days （with a few surviving for >7 days） before being mummified; wasp adults emerged from their mummies after developing for 5.6±1.3 days.The sizes of progeny colonies from the trapped alates generally increased over days after colonization but differed among the different groups. Overall mean （±SD） sizes of the colonies （no. nymphs per capita） from the infected alates increased from 2.0±2.0 （n=559） on day 1 to 35.4±51.3 （n=349） on day 14 for L.erysimi, 2.1±2.0 （n=119） to 72.1±76.5 （n=78） for M. persicae, and 4.4±3.7 （n=5） to 6.3±12.5 （n=5） for B. brassicae, respectively. The same observations from the parasitized alates of the three aphid species increased separately from 1.7±1.6 （n=58） to 86.6±73.7 （n=32）, 1.3±1.40 （n=39） to 99.5±124.3 （n=22） and 0.7±1.1 （n=3） to 23.5±33.2 （n=3）. In contrast, the progeny colonies of the alates free of both fungal infection and parasitism were conspicuously larger during the 14-day colonization, increasing from 2.6±2.0 （n=959） to 165.3±125.8 （n=381） for L. erysimi, 2.8±1.8 （n=605） to 180.3±114.4 （n=378） for M. persicae and 2.6±2.7 （n=260） to 69.7±64.5 （n=62） for B. brassicae.The fecundity of the infected or parasitized alates before death warranted successful colonization on plants, though greatly reduced, as was well shown by the fitted cumulative probability [P（m≤N）] for the alates producing≤m nymphs per capita and the increasing mean size of their progeny colonies. The relationship between m and P（m≤N） fit very well to a modified logistic equation for a given group of the alates or an aphid species （r²≥0.988, P<0.001）. Based on the fitted equations, healthy alates from air captures were highly capable of producing more nymphs than those infected or parasitized.Infective and epizootiological features of P. nouryi. Resting spore formation of some aphid-pathogenic Entomophthorales is important for the seasonal pattern of their prevalence and survival but this process is poorly understood. To explore the possible mechanism involved in the process, P. nouryi （obligate aphid pathogen） interacted with green peach aphid M. persicae on cabbage leaves under favorable conditions. Host nymphs showered with primary conidia of an isolate (LC₅₀: 0.9-6.7 conidia mm^-2 4-7 days post-shower) from air captures in the low-latitude plateau produced resting spores （azygospores）, primary conidia or both spore types. Surprisingly, the proportion of mycosed cadavers forming resting spores (P_CFRS) increased sharply within the concentrations （C） of 28-240 conidia mm^-2, retained high levels at 240-1760, but was zero or extremely low at 0.3-16. The P_CFRS-C relationship fit well the logistic equation P_CFRS=0.6774/[1+exp（3.1229-0.0270C）] （r²=0.975）. This clarified for the first time the dependence of in vivo resting spore formation of P. nouryi upon the concentration of infective inocula. A hypothesis is thus proposed that some sort of biochemical signals may exist in the host-pathogen interaction so that the fungal pathogen perceives the signals for prompt response to forthcoming host-density changes by either producing conidia for infecting available hosts or forming resting spores for surviving host absence in situ.In summary, 10 species of fungal pathogens were identified for the first time from the alates of three aphid species air-trapped in the low-latitude plateau with the discovery of full infection cycle of P. nouryi enriching our knowledge about Pandora species. Observations and modeling analysis have confirmed that both infected and parasitized alates were able to independently establish progeny colonies after immigration despite their reduced fecundity in comparison with that of healthy alates. Particularly, the alalte-borne mycoses were successfully transmitted to most of the progeny colonies. This further supports a hypothesis that both aphid pathogens and parasitoids may spread with host dispersal flight and provides deep insights into the biology of host flight. A new hypothesis for possible mechanisms involved in resting spore formation of Entomophthorales is proposed based on the first finding of the dependence of P. nouryi resting spore formation on the concentration of its primary conidia as inocula.更多还原