球孢白僵菌对四种十字花科蔬菜害虫的兼控潜力评价

研究了分离自小猿叶甲的一株球孢白僵菌(Beauveria bassiana(SCAU-BB01D))对十字花科蔬菜4种非目标害虫烟粉虱Bemisia tabaci、小菜蛾Plutella xylostella、黄曲条跳甲Phyllotreta striolata和菜缢管蚜Lipaphis erysimi的致病能力。结果表明,在实验室条件下,该菌株对以上4种害虫均有不同程度的侵染力。1×108孢子/mL的白僵菌悬浮液对1,2,3,4龄烟粉虱若虫的侵染率分别为84.88,86.81,55.94和38.78%;对小菜蛾2,3和4龄幼虫的侵染率为67,59和44%;对黄曲条跳甲幼虫和成虫分别为63%和60%;对菜缢管蚜成蚜和若蚜的侵染率分别为44%和35%。通过机率值分析,得出该菌株对烟粉虱1~4龄若虫的LT50分别为4.14,3.78,6.24和7.59 d;对小菜蛾2~4龄幼虫的LT50分别为7.29,8.16和9.82d;对黄曲条跳甲成虫和幼虫的LT50分别为11.22和8.11 d;对菜缢管蚜成蚜和若蚜的LT50分别为11.01和12.15 d。说明该球孢白僵菌菌株对烟粉虱、小菜蛾和黄曲条跳甲3种非目标害虫具有一定的兼控作用,在蔬菜害虫的生物防治中具一定的应用潜力。     

Purification and Characterization of Beauveria bassiana Proteinases

Two chymotrypsin‐like serine proteinases are produced by B. bassiana 278 when grown on different carbon and nitrogen sources. By employing acetone precipitation, gel filtration and ion‐exchange chromatographies, the enzymes were separated from the culture filtrate after propagation of the fungus on medium enriched either with ground larvae of Apis mellifera (Proteinase I) or porcine blood plasma (Proteinase II). The purified enzymes with a molecular mass of approximately 32 kDa hydrolyzed natural protein substrates: casein, hide powder azure (HPA), azocoll and much less elastin Congo Red and collagen. They differ from each other in the optimum pH value, amino acid composition, Michaelis constant and susceptibility to natural chymotrypsin inhibitors. Both proteinases hydrolyze suc‐Ala‐Ala‐Pro‐Phe‐p‐NA with an apparent K_m of 2.03 × 10^—3 M and 1.04 × 10^—4 M, respectively. The turkey ovomukoid (OMTKY) and cathepsin G/chymotrypsin inhibitor inhibit only Proteinase II from the larvae hemolymph of Apis mellifera (AMCI). The association constant of the interaction of this enzyme with AMCI was estimated to be very high (4.11 × 10⁹ M^—1).     

Beta-rhizobia from Mimosa pigra, a newly discovered invasive plant in Taiwan

A total of 191 rhizobial isolates from the root nodules of three geographically separate populations of the invasive plant Mimosa pigra in Taiwan were examined using amplified rDNA restriction analysis, 16S rDNA sequences, protein profiles and ELISA. Of these, 96% were identified as Burkholderia and 4% as Cupriavidus taiwanensis. The symbiosis-essential genes nodA and nifH were present in two strains of Burkholderia (PAS44 and PTK47), and in one of C. taiwanensis (PAS15). All three could nodulate M. pigra. Light and electron microscopy studies with a green fluorescent protein transconjugant variant of strain PAS44 showed the presence of fluorescent bacteroids in M. pigra nodules. These bacteroids expressed the nifH protein, hence this is the first confirmation that Burkholderia is a genuine symbiont of legume nodules. The predominance of Burkholderia in Taiwanese M. pigra suggests that this species may have brought its symbionts from its native South America, rather than entering into association with the Taiwanese Mimosa symbiont C. taiwanensis which so successfully nodulates Mimosa pudica and Mimosa diplotricha.     

防治西花蓟马的病原真菌-白僵菌微菌核的液体培养

微菌核是许多植物病原真菌产生的越冬结构,目前还没有关于白僵菌微菌核的报道。用含不同氮源的介质培养出了2株白僵菌菌株,RSB和SZ21的微菌核。在液体培养条件下,2株菌株不仅能产生典型的芽孢和菌丝,还能产生微菌核。在以玉米粉(RSB和SZ21菌株分别为6.5×102和6.3×102mL-1)为氮源的介质中产生的微菌核浓度高于以大豆粉(RSB和SZ21菌株分别为2.5×102和2.2×102mL-1)为氮源的介质。菌株SZ21产生的芽孢浓度(在以大豆粉和玉米粉为氮源的介质中,分别为17.9×105和7.4×105L-1)高于RSB菌株的芽孢浓度(在以大豆粉和玉米粉为氮源的介质中,分别为14.8×105和6.2×105L-1)。含有硅藻土的微菌核制剂能在真空干燥(<5%湿度)后存活下来,并且对于活力没有显著影响。干燥后的微菌核颗粒在水洋菜培养基上复水和培养后,能够萌发菌丝和产生高浓度的分生孢子。将微菌核颗粒预混到土壤中能引起西花蓟马Frankliniella occidentalis(Pergande)土栖阶段的高死亡率。这是首次报道白僵菌的微菌核的培养,并为用虫生真菌防治土栖昆虫提供了一个新的方法。     

球孢白僵菌对黄瓜代谢产物及西花蓟马取食行为的影响

球孢白僵菌Beauveria bassiana灌根处理黄瓜后,发现西花蓟马Frankliniella occidentalis对叶片危害面积显著低于对照组;为探究产生这一现象的原因,测定黄瓜叶片中防御酶系和次生代谢产物等含量变化,采用直流刺吸电位仪(direct current electrical penetration graph,DC-EPG)分析西花蓟马成虫取食黄瓜叶片的行为.球孢白僵菌对黄瓜灌根后叶片中过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)含量均呈上升趋势,其中CAT活性增加最高,增加率为37.26％;丙二醛MDA的含量呈下降趋势;可溶性蛋白、类黄酮的含量增加.DC-EPG显示西花蓟马在经球孢白僵菌处理的黄瓜上,取食时间和次数均低于对照组.结果表明,黄瓜植株经球孢白僵菌处理后防御酶物质增加,进而导致西花蓟马对叶片危害下降,利于对西花蓟马的防治.     

利用冷冻切片对球孢白僵菌侵染西花蓟马过程的观察

为探索球孢白僵菌对西花蓟马的侵染,研究球孢白僵菌在西花蓟马体表和虫体内的侵染过程,采用冷冻切片与HE染色技术,对被球孢白僵菌侵染的西花蓟马成虫进行组织切片及HE染色。结果表明,接菌4 h,体表粘附的孢子开始萌发,8-12 h芽管继续伸长并出现穿透体壁。18 h虫菌体出现在西花蓟马体内,球孢白僵菌成功侵入西花蓟马体内。之后,西花蓟马体内虫菌体、菌丝明显增多,各组织器官均受到感染,消化道、肌肉组织等发生病变。     

Temperature and moisture requirements for conidial germination of an isolate of Beauveria bassiana,pathogenic to Rhodnius prolixus

The effects of temperature, relative humidity and water activity on germination of conidia of an isolate of Beauveria bassiana (Bals.) Vuill. pathogenic to the triatomine vector of Chagas' disease, Rhodnius prolixus Stål., were investigated in vitro. Germination occurred at temperatures between 15 °C and 35 °C under saturated atmosphere and the optima ranged from 25 °C to 3O °C. At the extreme temperatures tested (15 °C and 35 °C) the germination process was delayed, but germination rates reached more than 95%. Germination of B. bassiana conidia was strongly affected by moisture conditions. The availability of water, in both atmospheric and liquid conditions, caused changes in germination times as well as in germination rates. For example, at 25 °C + O.5 °C, germination took place within 20 h at 95.5% RH, whereas it needed 72 h of incubation at 90% RH. Germination times increased as the water activity declined from 0.96 a_w to 0.92 a_w. Below 0.92 a_w, o germination was observed after a 72 h incubation time.     

中国一新记录种——黑顶小皮伞

<正>最近作者在检查中国科学院昆明植物研究所隐花植物标本馆(HKAS)小皮伞属Marasmius Fr.的标本时,发现了二份具大型子实体的标本,经鉴定它们与采自美国的黑顶小皮伞M.nigrodiscus(Peck)Halling等模式标本(HKAS 39834)在宏观和微观特征上一致,为中国的新记录种。颜色对照采用Kornerup & Wanscher(1978)的颜色手册描述。现描述如下:     

Use of chitin to improve a Beauveria bassiana alginate-pellet formulation

A study was undertaken to evaluate optimum concentrations of chitin in sodium alginate pellet formulations to enhance conidia production. Chitin concentrations tested were 0, 0.5, 1, 2, 3 and 4% (w/v), with (2%, w/v) or without wheat bran. The different chitin-wheat bran pellet combinations were prepared with Beauveria bassiana isolate Qu-B306 at 10⁸ conidia mL^-1. After 21 days of incubation in a humid chamber at 28°C, conidia production was assessed. Improvements up to three times the initial conidia number were achieved using 2% chitin and 2% wheat bran. Higher levels of chitin decreased the number of conidia per pellet. For all chitin concentrations, conidia number increased with the addition of wheat bran (P≤0.05). Contamination by saprophytic fungi was reduced by the incorporation of chitin in the pellet formulation.     

Effect of entomopathogenic fungus, Beauveria bassiana on larvae of three species of mosquitoes

Beauveria bassiana, an entomopathogenic fungus, was evaluated for its potential against second and third instar larvae of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. Conidiospores of this fungus were effective in causing infection leading to mortality of different larval instars. Larvae of Cx. quinquefasciatus were more susceptible to infection than An. stephensi and the second instar larvae of these two species were more susceptible than third instar larvae. Larvae of Ae. aegypti were resistant to infection by B. bassiana.     

Production in vitro of toxic macromolecules by strains of Beauveria bassiana, and purification of a chitosanase-like protein secreted by a melanizing isolate

The production of macromolecular insecticidal toxins in Adamek's medium by two selected strains of Beauveria bassiana was investigated. Filtrates and dialysates of the melanizing strain 618 were toxic when injected into the lepidopteran insect Galleria mellonella. Separation by DEAE chromatography revealed that peaks eluted respectively with 100 and 200 mM NaCl (P 100 and P 200) had an insecticidal activity and induced cuticular blackening. A hydrophilic protein, Bclp, which causes the formation of brownish spots of the integument, was purified from P 200 by means of chromatographic and electrophoretic methods. Bclp exhibited clear sequence homologies with fungal chitosanases of Fusarium solani. It has a molecular mass of 28 kDa, a pHI of 4 and is thermolabile. Injection of Bclp causes the same cytoxic effects and alterations of the cuticule as those observed during mycosis, and may contribute to the virulence of strain 618. Comparatively, the most obvious characteristic of the weakly melanizing strain 101 is the lack of significant toxic activity of its P 200, which does not contain Bclp. However, this strain secretes other insecticidal molecules active on lepidopterans, presently unidentified.     

球孢白僵菌对小猿叶甲的致病力测定

在室内研究了分离自小猿叶甲的一株球孢白僵菌(SCAU-BB 01D)对小猿叶甲的致病力。结果表明,该菌株能感染小猿叶甲的成虫和各龄幼虫,但对不同虫期的致病力存在差异。在105~108孢子/m l的浓度范围内,随着处理浓度的升高,各虫期小猿叶甲的感病死亡率增加,在最高浓度1×108孢子/m l,处理后成虫第14天及1~3龄幼虫第10天的累计死亡率分别为84.7%、94.0%、96.0%和81.0%。用TDM模型对成虫和各龄幼虫的致病力数据进行模拟,所建模型均顺利通过Hosm er-L em eshow拟合异质性检验,表明模型拟合良好,并由模型估计出了该菌株对小猿叶甲各虫期的致死剂量与致死时间。在处理后第10天,成虫和1~3龄幼虫的致死中浓度(LC50)分别为2.68×107、1.07×106、1.63×105孢子/m l和8.31×106孢子/m l,而第14天成虫的LC50为2.38×106孢子/m l。随着浓度的增加,各虫期所需的感病死亡时间缩短,在最高浓度1×108孢子/m l,球孢白僵菌对小猿叶甲成虫及1~3龄幼虫的致死中时(LT50)分别为9.28、4.29、4.40d和5.06 d。综合分析白僵菌对各虫期的致死剂量及致死时间可以看出,不同虫期的小猿叶甲对球孢白僵菌敏感性不同。结果表明该菌株在小猿叶甲生物防治中具有一定的潜力。     

毒鹿花菌——一个发现于中国的毒蘑菇新种

基于形态学和系统发育研究结果,本研究报道在中国华中和西南地区发现的一个隶属于鹿花菌属鹿花菌亚属的有毒新种——毒鹿花菌。该种在形态上与鹿花菌Gyromitra esculenta十分相似,但毒鹿花菌的囊盘被分化较为明显,外表面几乎由一层栅状细胞排列而成,而鹿花菌的囊盘被基本没有分化。此外,毒鹿花菌通常具有更长的菌柄并生于阔叶林地上。为了准确理解鹿花菌的物种概念,本研究指定了一份采自法国的标本（即HMAS 21460）作为该种的附加模式。     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

Consumption of cyanogenic bamboo by a newly discovered species of bamboo lemur

Three species of bamboo-eating lemurs were found to be sympatric in the southeastern rain forests of Madagascar. Sympatric species generally differ in habitat utilization or diet, but these three closely related bamboo lemurs lived in the same habitat and all ate bamboo. Behavioral observation revealed that they did select different parts of the bamboo, and chemical analyses confirmed that there was a difference in the secondary compound content present in those selections. The growing tips of Cephalostachyum ef uiguieri selected by the golden bamboo lemur (Hapalemuraureus) contained 15 mg of cyanide per 100 g fresh weight bamboo while the leaves of C. perrieri selected by the gentle bamboo lemur (H. griseus)and the mature culms of C. cf uiguieri selected by the greater bamboolemur (H. simus) did not contain cyanide. Since each individual golden bamboo lemur ate about 500 g of bamboo per day, they daily ingestedabout 12 times the lethal dose of cyanide. The mechanism by which this small primate avoids the acute and chronic symptoms of cyanide poisioning is unknown.     

Genetic diversity among Brazilian isolates of Beauveria bassiana: comparisons with non-Brazilian isolates and other Beauveria species

Aims:  The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp.
Methods and Results:  The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp.
Conclusion:  Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil.
Significance and Impact of the Study:  This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu . The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil.     

我国入侵害虫西部喙缘蝽的溯源分析

本文对我国外来入侵物种西部喙缘蝽Leptoglossus occidentalis Heidemann, 1910进行了溯源分析,依据对国内种群的观察和标本简要描述了其形态特征和生活习性。通过野外采集国内种群标本,提取扩增COI基因序列,结合原产地和国外其他入侵种群的公共序列,构建西部喙缘蝽COI基因数据集,以中国入境口岸对木材的检疫情况为参考,分析西部喙缘蝽的入侵来源。全世界目前已知10种单倍型,中国存在4种。山东青岛种群有H＿8、H＿9和H＿10三种单倍型,其中H＿8与韩国共享,H＿9和H＿10为中国特有单倍型,青岛种群可能部分来自韩国;山东烟台两个采集点的种群都属于H＿2,可能来自北美东部,与青岛种群的入侵来源不同。以上分析表明,我国西部喙缘蝽种群可能来源于多次独立入侵事件,同时在国内发生了小范围的扩散迁移。本研究首次对我国入侵害虫西部喙缘蝽做了溯源分析,为更好地制定规制措施,开展检疫防治工作提供了科学依据。