球孢白僵菌释放后在森林系统中定殖及持续控虫作用的证据

在皖南马尾松林中使用球孢白僵菌无纺布菌条接种式放菌防治松褐天牛。在放菌前进行球孢白僵菌本底调查,在放菌后半年内采集该菌感染的各种僵虫,得到87株分离物。利用28SrDNA-PCRⅠ型内含子标记技术,测定放菌后释放菌株的回收率。结果表明,在87株球孢白僵菌中有66株与释放菌株的核型相同,占75.86%。半年中平均回收率为81.7%(75%-90.9%之间),这表明释放菌株已在该生态系中定殖,对松褐天牛种群发挥持续控制作用。在放菌前后球孢白僵菌种群结构有所变化,释放菌株有取代土著菌株优势地位的趋向。     

松褐天牛球孢白僵菌高毒力航天诱变菌株的筛选

【目的】松褐天牛Monochamus alternatus是我国松材线虫病Bursaphelenchus xylophilus的主要媒介昆虫。为了更好地开发利用松褐天牛病原微生物球孢白僵菌Beauveria bassiana, 本研究通过航天搭载诱变及室内筛选, 获得球孢白僵菌高毒力诱变菌株。【方法】将经神舟八号飞船航天搭载诱变后的孢子稀释液涂布在PDA平板上培养,获得单菌落菌株,进而筛选获得高毒力诱变菌株。观察所获9个航天诱变菌株的菌落形态、菌落生长速度、产孢量、孢子萌发率及抗高温胁迫能力等生物学特性, 在此基础上筛选出生物学性状优良的菌株B159, B252和B305, 并进一步对松褐天牛4龄幼虫进行生物测定。再通过撒菌粉和注射菌液方法, 检验B252和B305对松木段内松褐天牛幼虫的杀虫效果。【结果】球孢白僵菌航天诱变菌株的生物学特性与野生型菌株cfcc81357存在分化。9个航天诱变菌株的菌落形态发生了不同程度的改变,6个菌落生长速率出现负向变异,仅诱变菌株B159, B252和B305能产生分生孢子。航天诱变菌株B252和B305在浓度为1.0×107 cfu/mL时对松褐天牛4龄幼虫的校正死亡率均为100%, 半致死中时(LT₅₀)分别为8.08和8.56 d, 明显优于野生型菌株, 显示出对松褐天牛的极强毒力。使用撒菌粉和孢子液体注射方法, 诱变菌株B252和B305对松木段内松褐天牛幼虫死亡率比野生型菌株高。【结论】诱变菌株B252和B305可能是优良菌株, 对生物防治松褐天牛方面有潜在的实用价值。     

应用球孢白僵菌菌条持续控制松褐天牛效果的分子评价

采用Ⅰ型内含子技术对应用球孢白僵菌无纺布菌条持续控制松褐天牛的防治效果进行了分子生物学评价.结果表明: 采用常规手段评价防治效果,两个处理区的防治效果分别为19.5%和10.8%; 而采用分子手段进行评价,防治效果分别为14.2%和11%.证明常规评价以死亡率或侵染率来计算微生物防治效果,难以排除当地微生物区系中的土著菌株所造成的侵染死亡,而采用分子生物学手段进行防治效果评价可从一定程度上消除土著菌株的干扰,较准确地反映人工放菌的防治效果.     

粗放经营茶园土壤昆虫病原真菌种群多样性及时间生态位

本文系统调查了粗放经营状态下黄山毛峰茶园根际土壤昆虫病原真菌的种群多样性及其时间生态位水平.于2012年8月至2013年6月,在安徽省黄山市汤口镇选择粗放经营茶园采集土壤样品140份,经稀释平板法分离共获得昆虫病原真菌1041株.结果表明： 昆虫病原真菌种群多样性较为丰富,经鉴定分为6属13种.其中,淡紫紫孢菌、球孢白僵菌和金龟子绿僵菌为优势种群,其菌株数分别为309、255和101株,占总数的29.7%、24.5%和9.7%.茶园土壤中淡紫紫孢菌的时间生态位宽度最大,球孢白僵菌最小.昆虫病原真菌之间以球孢白僵菌和布氏白僵菌的时间生态位重叠值最大,为1.965;而爪哇棒束孢和球孢白僵菌的时间生态位重叠值最小,仅为0.374.     

球孢白僵菌种群在松林中的寄主转移及遗传多样性对松毛虫持续控制的影响

通过林间接种式放菌及其后一周年的野外调查,从林间采集到30种寄主昆虫及从土壤、落叶和气流中分离到119株球孢白僵菌.酯酶同工酶分析表明,它们属于32个不同酯酶型,呈现出丰富的遗传多样性.释放菌株所属的酯酶型02包括从11种昆虫上分离出的18个菌株,表明林间释放的菌株已成功地在不同寄主昆虫种群中宿存下来,并以常发的地方病状态存在于松毛虫及松灰象甲等12种昆虫种群之中;当林间目标寄主缺乏时,其它寄主可将食物链维系下去.其它酯酶型分别包括1～23个菌株.一周年内的寄主转移动态结果表明,球孢白僵菌在松林生态系统中不同寄主间可转移寄生.每个酯酶型中的菌株对松毛虫的毒力相差很大.表明球孢白僵菌在松林中的延续和扩散流行不是1条路线,每个酯酶型至少代表食物网上的1条支链.有些环节的寄主连接了不同的酯酶型,使松林中食物网变得十分复杂.另外,从土壤、枯枝落叶层、林冠层和空气中分离到的球孢白僵菌分属于不同的酯酶型,表明松林中还存在着复杂的腐生食物链,有利于松毛虫及其它害虫的持续控制.     

球孢白僵菌种群在松林中的寄主转移及遗传多样性对松毛虫持续控制的影响

通过林间接种式放菌及其后一周年的野外调查,从林间采集到30种寄主昆虫及从土壤、落叶和气流中分离到119株球孢白僵菌．酯酶同工酶分析表明,它们属于32个不同酯酶型,呈现出丰富的遗传多样性．释放菌株所属的酯酶型02包括从11种昆虫上分离出的18个菌株,表明林间释放的菌株已成功地在不同寄主昆虫种群中宿存下来,并以常发的地方病状态存在于松毛虫及松灰象甲等12种昆虫种群之中;当林间目标寄主缺乏时,其它寄主可将食物链维系下去．其它酯酶型分别包括1～23个菌株．一周年内的寄主转移动态结果表明,球孢白僵菌在松林生态系统中不同寄主间可转移寄生．每个酯酶型中的菌株对松毛虫的毒力相差很大,表明球孢白僵菌在松林中的延续和扩散流行不是1条路线,每个酯酶型至少代表食物网上的1条支链．有些环节的寄主连接了不同的酯酶型,使松林中食物网变得十分复杂,另外。从土壤、枯枝落叶层、林冠层和空气中分离到的球孢白僵菌分属于不同的酯酶型,表明松林中还存在着复杂的腐生食物链,有利于松毛虫及其它害虫的持续控制。     

孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛幼虫的致病力的影响

【目的】球孢白僵菌Beauveria bassiana是一种被广泛用于害虫生物防治的生防菌。本研究探讨了孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛Monochamus alternatus 幼虫的致病力的影响,旨在为松墨天牛的生物防治提供科学基础。【方法】分别用0.5% 吐温-80(CK)以及1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸和1×10⁹孢子/mL的球孢白僵菌孢悬液接种松墨天牛4龄幼虫,统计接种后15 d内幼虫死亡率和染菌率。同时用最佳浓度(1×10⁹孢子/mL)的球孢白僵菌孢悬液接种体型大小分别为100~150, 200~220, 300~320, 400~420, 500~520和600~650 mg/头的松墨天牛幼虫,测定接种后20 d内幼虫的死亡率和染菌率。【结果】接种1×10⁵~1×10⁹孢子/mL的球孢白僵菌后,松墨天牛4龄幼虫起初活动自如,后在头部出现烧灼状伤并且体色逐渐变红,最后周身长满菌丝。不同浓度下,随球孢白僵菌孢子浓度升高,松墨天牛4龄幼虫校正死亡率和校正染菌率增加。接种1×10⁶~1×10⁹孢子/mL球孢白僵菌孢悬液15 d的松墨天牛4龄幼虫累计校正死亡率均可达到100%,1×10⁷, 1×10⁸和1×10⁹孢子/mL浓度下,松墨天牛达到100%校正死亡率所需时间最少。接种0 (CK), 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸和1×10⁹孢子/mL球孢白僵菌孢悬液的松墨天牛4龄幼虫校正染菌率在第15天分别为0, 20.00%, 86.67%, 90.00%, 96.67%和100.00%,表现为白僵菌孢子浓度越高,染菌率越高。1×10⁹孢子/mL的接种浓度下,松墨天牛幼虫个体越大,天牛幼虫死亡率和染菌率越高。表现在第20天时,体型大小为100~150, 200~220, 300~320, 400~420, 500~520和600~650 mg/头的幼虫的死亡率分别为76.67%, 76.67%, 66.67%, 93.33%, 100.00%和100.00%,染菌率分别为60.00%, 63.33%, 60.00%, 86.67%, 96.67%和100.00%。【结论】球孢白僵菌悬浮液浓度对松墨天牛幼虫的死亡和侵染有显著影响,表现为随孢子浓度的增加而增加;同时,松墨天牛幼虫个体越大,死亡率和染菌率越高。研究结果对开展利用球孢白僵菌防治松墨天牛具重要借鉴和指导意义。     

孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛幼虫的致病力的影响

【目的】球孢白僵菌Beauveria bassiana是一种被广泛用于害虫生物防治的生防菌。本研究探讨了孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛Monochamus alternatus幼虫的致病力的影响,旨在为松墨天牛的生物防治提供科学基础。【方法】分别用0.5%吐温-80(CK)以及1×10~5, 1×10~6, 1×10~7, 1×10~8和1×10~9孢子/mL的球孢白僵菌孢悬液接种松墨天牛4龄幼虫,统计接种后15 d内幼虫死亡率和染菌率。同时用最佳浓度(1×10~9孢子/mL)的球孢白僵菌孢悬液接种体型大小分别为100～150, 200～220, 300～320, 400～420, 500～520和600～650 mg/头的松墨天牛幼虫,测定接种后20 d内幼虫的死亡率和染菌率。【结果】接种1×10~5～1×10~9孢子/mL的球孢白僵菌后,松墨天牛4龄幼虫起初活动自如,后在头部出现烧灼状伤并且体色逐渐变红,最后周身长满菌丝。不同浓度下,随球孢白僵菌孢子浓度升高,松墨天牛4龄幼虫校正死亡率和校正染菌率增加。接种1×10~6～1×10~9孢子/mL球孢白僵菌孢悬液15 d的松墨天牛4龄幼虫累计校正死亡率均可达到100%,1×10~7, 1×10~8和1×10~9孢子/mL浓度下,松墨天牛达到100%校正死亡率所需时间最少。接种0 (CK), 1×10~5, 1×10~6, 1×10~7, 1×10~8和1×10~9孢子/mL球孢白僵菌孢悬液的松墨天牛4龄幼虫校正染菌率在第15天分别为0, 20.00%, 86.67%, 90.00%, 96.67%和100.00%,表现为白僵菌孢子浓度越高,染菌率越高。1×10~9孢子/mL的接种浓度下,松墨天牛幼虫个体越大,天牛幼虫死亡率和染菌率越高。表现在第20天时,体型大小为100～150, 200～220, 300～320, 400～420, 500～520和600～650 mg/头的幼虫的死亡率分别为76.67%, 76.67%, 66.67%, 93.33%, 100.00%和100.00%,染菌率分别为60.00%, 63.33%, 60.00%, 86.67%, 96.67%和100.00%。【结论】球孢白僵菌悬浮液浓度对松墨天牛幼虫的死亡和侵染有显著影响,表现为随孢子浓度的增加而增加;同时,松墨天牛幼虫个体越大,死亡率和染菌率越高。研究结果对开展利用球孢白僵菌防治松墨天牛具重要借鉴和指导意义。     

感染松墨天牛的金龟子绿僵菌菌株的初步筛选

松墨天牛是松材线虫病的主要媒介昆虫,本文对分离自松墨天牛的6株金龟子绿僵菌,以及从光肩星天牛、大蜡螟、伊藤厚丝角叶蜂和土蝽上分离的各1个菌株,共10个菌株的产孢情况、孢子萌发率进行研究;在此基础上选出金龟子绿僵菌1291、1349和2049三个菌株及球孢白僵菌F-263菌株,采用成虫跗节接种法和幼虫浸渍法进行室内松墨天牛及大蜡螟的毒力测定比较。结果表明,供试绿僵菌菌株对松墨天牛幼虫在接种15天后的感染僵虫率为76.9％～93.1％(1×10⁷孢子/mL);成虫在接种20天后的僵虫率亦达57.9％～75.0％(6.5×10⁵～3.4×10⁶孢子/成虫);2049菌株表现尤其突出;对应的球孢白僵菌F-263对幼虫和成虫的僵虫率分别是96.3％(1×10⁷孢子/mL)和55％(9.7×10⁵孢子/成虫)。但这3个金龟子绿僵菌菌株对大蜡螟的毒力较低,存在较明显的寄主专化性。这3个菌株今后在防治松墨天牛方面具有较大开发应用价值。     

栎旋木柄天牛高毒力球孢白僵菌菌株的筛选

栎旋木柄天牛Aphrodisium sauteri是近年来在安徽黄山风景区大面积暴发的危险性林业有害生物之一,其发生危害已经严重影响了风景区自然与生态景观。本研究选择分离自不同天牛成虫的8个球孢白僵菌Beauveria bassiana菌株,依据菌落形态、菌株生长速度、产孢量、萌发率、抗旱力和产胞外蛋白酶水平等生物学性状进行初步筛选。在此基础上,筛选出生物学性状优良菌株Bb202、Bb646和Bb1898,并进一步对栎旋木柄天牛幼虫进行生物测定。基于此种筛选模式的菌株筛选方法筛选出了具有菌落生长速度快、萌发率高、产孢和抗旱能力强、胞外蛋白酶产酶水平高及毒力强的球孢白僵菌Bb202菌株。该菌株对栎旋木柄天牛的校正死亡率达92.8%,侵染率达93.3%,在浓度为1×10⁷/mL时的半致死中时仅为5.86天,显示出对目标天牛的极强毒力,在栎旋木柄天牛的生物防治中将有重要的应用价值。     

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer,Monochamus alternatus

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.     

Management of pine wilt disease vectoring Monochamus alternatus adults using spray and soil application of Metarhizium anisopliae JEF isolates

Chemical control is widely used to control the Japanese pine sawyer beetle, Monochamus alternatus, but strong chemical regulations require an environmentally sound management strategy. In this work, we investigated the use of entomopathogenic fungi and their application as a means of practical pest management. Thirty-two diverse species of fungal isolates were assayed against adult pine sawyer beetles using a contact method under laboratory conditions, and four isolates showed over 70% virulence consequently. These isolates, two each of Beauveria bassiana and Metarhizium anisopliae were sprayed on the adult beetles at 1 × 10⁷ conidia/ml in plastic containers, respectively. The M. anisopliae-treated adult beetles showed 67% mortality. M. anisopliae isolates JEF-197 and JEF-279 demonstrated dosage-dependent insecticidal activity. Following the laboratory experiments, semi-field trials were conducted in young pine trees under high (RH 94%) and low (RH 35%) humidity conditions. In the high humidity conditions, most of the adult beetles stayed on the top of the branches. When the two M. anisopliae isolates were sprayed on the beetles, they showed ca. 50–70% insecticidal activity 11 days after application. In contrast, in low humidity conditions, the adult beetles tried to move off the branches and onto the soil. When the beetles reached the JEF-197 and JEF-279-treated soil, we measured >90% insecticidal activity. This work suggests that M. anisopliae was the most virulent entomopathogenic fungus against adult Japanese pine sawyer beetles, and this forest insect could be ecologically controlled by the spray and soil application of the M. anisopliae isolates.     

Application of harmonic radar to analyze dispersal behavior of the Japanese pine sawyer beetle,Monochamus alternatus (Coleoptera: Cerambycidae)

Monochamus alternatus (Hope) is a severe wood‐boring pest in coniferous forests and a major vector of pine wilt disease in East Asia. Harmonic radar is a powerful tool for studying the dispersal behavior of insects and it could be applied to control pine wilt disease. In this study, we validated the application of harmonic radar for analyzing the dispersal behavior of M. alternatus beetles in a field environment. We determined the wing capacities of the beetles and the effects of electronic tagging and marking on their movement, flight ability, survivorship, and food consumption in the laboratory to confirm the suitability of this technique. The detection rate and recovery rate for beetles were analyzed separately using radar on caged pine stands and in the field environment. The results showed that the minimum wing capacity of the Japanese pine sawyer was 24.9 mg, which was seven times the weight of the electronic tag (3.5 mg). Marking and tagging the beetles had no significant adverse effects on their movement, flight capacity, food consumption, and survivorship. The detection rate using the radar system and recovery rate based on visual observations of the beetles in caged pines were both 95.6%. However, in the field environment, the detection and recovery rates were only 55.6% and 37.8% after one week, respectively, and 33.3% and 7.8% after two weeks. Harmonic radar is a promising technique for studying the dispersal behavior of the Japanese pine sawyer, but its performance is not satisfactory and major improvements are required for both the radar system and electronic tags.     

Pathogenicity of the pinewood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings

Pathogenicity of the pine wood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings was tested with inoculation experiments under nursery conditions. Water suspensions of nematodes (mixed stages cultured on Botrytis cinerea or dispersal fourth-stage juveniles (DJ4) extracted from the adult Japanese pine sawyer, Monochamus alternatus) were injected into the stems of 2- and 3-year-old Japanese larch and Japanese black pine, Pinus thunbergii, seedlings growing in a nursery. In another treatment, Japanese pine sawyer adults holding DJ4 were released under a net that covered the upper half of the seedlings. Regardless of nematode inoculation method, Japanese larch seedlings were as susceptible as Japanese black pine seedlings to B. xylophilus under nursery conditions. The rate of disease development was similar on larch and pine seedlings. Nematode population densities were lower in the stems of dead larch seedlings than in the stems of dead pine seedlings. Histopathological observations revealed that the distribution of nematodes in the stems of dead larch seedlings was mostly limited to the cortex, phloem and cambial zone. Traumatic resin canal formation was one of the most characteristic symptoms in larch seedlings which was dissimilar to that in pine seedlings.     

Maturation feeding and transmission of Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) by Monochamus alternatus (Coleoptera: Cerambycidae) inoculated with Beauveria bassiana (Deuteromycotina: Hyphomycetes)

We examined the amount of maturation feeding and transmission of pinewood nematodes, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Nematoda: Parasitaphelenchidae), to healthy pine (Pinus spp.) trees by pine sawyer Monochamus alternatus Hope (Coleoptera: Cerambycidae) adults infected with Beauveria bassiana (Balsamo) Vuill. (Deuteromycotina: Hyphomycetes). Inoculated beetles fed less than noninoculated beetles, probably because feeding by inoculated beetles began to decrease at about 4 d postinoculation and inoculated beetles ceased to feed for several days before their death. In inoculated beetles carrying >1,000 nematodes, some beetles died before nematode departure. The remaining heavily nematode-infested beetles lived until the beginning of nematode departure, but they had stopped feeding, preventing the nematodes from entering pine twigs. We suggest that microbial control of pine sawyer adults by B. bassiana may be effective in preventing transmission of pine wilt disease to healthy pine trees.     

A Taxonomie Note on Sclerodermas harmandi,Ectoparasite of Stem and Wood Boring Insect Larvae (Hymenoptera: Chrysidoidea'-Bethylidae) in South Korea

As a result of natural enemy survey against the Japanese pine sawyer (Monochamus alternates (Hope 1842)), the major vector of pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhrer 1934)), many samples of the ectoparasitic wasp, Sclerodermas harmandi (Buysson 1903) were collected nationwide in Korea from June to October in 2005. It is redescribed with the figures and biométrie data of apterous and alate females.     

Mating behavior of Pine Sawyer,Monochamus saltuarius Gebler (Coleoptera: Cerambycidae)

Mating behavior of the pine sawyer, Monochamus saltuarius Gebier (Cerambycidae: Lamiinae) was observed on pine trees in an outdoor net cage kept in one healthy pine tree and a cluster of pinelogs (cut over one month) to attract the beetles. When sawyer adult pairs released into net cage, they all moved to the pinelogs. And then the male was staying motionless with his antenna outstretched while the female was actively moving in his vicinity. The mating behavioral reactions were followed by the next steps: The first phase of courtship was initiated by the female approach toward the male. Second, male dashed, contacted antenna and mounted female's back. Third, female carried the male on her back and walked around and then male began to lick. Fourth, male inserted his penis into the female's genitalia when female stopped walking. While a long pairbond, the mating pair repeated copulations, lastly, when mating is over, male and female stayed separately. The mating began on sunset and resting of both male and female began at the temperature below about 20 °C. Substantially straightforward mate searching by female indicates the presence of a male sex pheromone.     

绿僵菌M189菌株特异性SCAR标记的建立及田间监测应用

为了监测生防真菌绿僵菌菌株在田间释放后的回收,有必要建立菌株DNA分子标记,以此将应用的菌株与其他菌株或田间的土著分离株鉴别开来。作者采用16条随机引物扩增了51株绿僵菌菌株的基因组DNA,得到81个多态性位点。其中M189菌株多态性位点30个,分析得到1个特异性位点,并将该位点的DNA片段测序后转化成为特异性SCAR标记。检验确定了该标记的敏感性,可以从供试的51株菌株中准确鉴定出目的菌株M189。并用该标记检测了从田间回收的3个分离株,确定其中1个与应用菌株M189一致。     

Effect of Monochamus carolinensis on the Life History of the Pinewood Nematode,Bursaphelenchus xylophilus

The development of Bursaphelenchus xylophilus in pine wood infested with and free of Monochamus carolinensis was investigated. Formation of third-stage dispersal juveniles occurred in the presence and absence of pine sawyer beetles. The proportion of third-stage dispersal juveniles in the total nematode population was negatively correlated with moisture content of the wood. Formation of nematode dauer juveniles was dependent on the presence of the pine sawyer beetle. Dauer juveniles were present in 3 of 315 wood samples taken from non-beetle-infested Scots pine bolts and 81 of 311 samples taken from beetle-infested bolts. Nematode densities were greater in wood samples taken adjacent to insect larvae, pupae, and teneral adults compared with samples taken from areas void of insect activity. Nematodes recovered from beetle larvae, pupae, and teneral adults were mostly fourth-stage dauer juveniles, although some third-stage dispersal juveniles were also recovered. Dauer juvenile density was highest on teneral adult beetles.