球孢白僵菌固态罐发酵产孢培养的研究

建立了一套两升圆柱体玻璃发酵罐的固态通风培养装置及通风系统。以球孢白僵菌Bb371为生产菌,培养基220g(82％麸皮 4．5％黄豆粉 4．5％玉米粉 9％稻壳)作为生产原料,接种30ml,23℃培养温度下,分阶段调节通风量和空气湿度,培养5d,产孢量可达到116亿／g干培养基。     

分阶段调控关键因素提高白僵菌固态发酵产孢量

采用分阶段调控关键因素策略,对白僵菌固态发酵过程进行优化.优化后最佳发酵模式为:初始含水量为65％,发酵至48 h将含水量调为68％;发酵至20 h和40h分别适当翻料;0～48h(适应期)发酵温度为25℃,48 ～108 h(快速生长期)发酵温度为24℃,108 ～168 h(稳定期)发酵温度为28℃;同时,48 ～108 h按通气比1∶2通气,0～48 h和108 ～168 h不通气.采用上述关键因素调控策略后,白僵菌固态发酵产孢量达18亿孢子/g(干培养基),与优化前相比产孢量提高约360％,效果显著.     

布氏白僵菌微菌核的诱导培养及条件优化

菌核是一些丝状菌的休眠结构,具有很强的抗逆性和稳定性。近年来发现有些虫生真菌在特定的营养条件下能形成微菌核(MS)。以适合用于地下害虫防治的布氏白僵菌为对象,研究观察了该菌摇瓶培养微菌核形成的过程,通过单因子实验和部分因子实验确定培养液的碳源A、碳氮比B和接种量C为影响MS产量的显著因子,而装液量和转速是不显著因子;通过最陡爬坡实验和中心组合实验得出MS摇瓶培养参数的最优二次多项式回归模型,确定在A=30.66g/L,B=7.59:1,C=1.03%时,MS产量理论最大值为最高(8.24×103MS/mL),MS实验产量在8.00–8.25×103MS/mL,优化的结果产量比初始试验时的最大值提高21%–25%。研究结果为探索使用布氏白僵菌菌核防治地下害虫奠定了基础。     

球孢白僵菌菌株间差异性的研究

本文对不同来源的4个球孢白僵菌菌株进行了培养观察。在培养基量、接种量相同;培养条件一致的情况下,比较它们之间的产孢量。并通过生物检测,计算出对菜青虫的致死中时。结果表明,菌株间在培养性状、产孢量及毒力诸方面均存在差异。     

防治稻水象甲优效球孢白僵菌YS03菌株产孢培养基优化

采用单因素试验和响应面分析对球孢白僵菌YS03菌株进行产孢培养基的配方改良。单因素试验筛选出对产孢量影响较大的3种原料为花生、麦麸和玉米粉,进而根据Box-Behnken的中心组合原理进行三因素三水平的实验设计,最终得出使产孢量最大的配方为花生∶麦麸∶玉米粉=2.6∶3.5∶3.9。经验证,预测值与验证试验平均值接近,且新配方的产孢量是PDA培养基产孢量的1.92倍。     

利用RAPD-PCR检测三种白僵菌及球孢白僵菌种内变异

采用PCR-RAPD技术研究了球孢白僵菌2株野生型多孢株及其各1株单孢分离株的 RAPD扩增片段的多态性,比较了彼此间的相似率。结果显示,相似率在63.6～99.3％之间变化很大,表明各分离子间的基因组DNA已发生变化。球孢白僵菌与另一种白僵菌布氏白僵菌之间的相似率在64.9％～80.9％之间,与粘孢白僵菌之间的相似率在50.0～61.9％之间。     

对蜱致病性球孢白僵菌培养条件的优化

对前期筛选得到的具有生防潜力的杀蜱真菌Beauveria bassiana AT17菌株进行相关生物学性状的研究,建立一套规范的实验室培养的技术和方法,同时对其液固双相发酵技术进行优化,在于提供优质高效的生防材料,为其真菌制剂的规模化生产提供实践和理论基础。通过采用单因素筛选方案对其最适基础培养基、温度、pH值、碳源、氮源、微量元素及液固双相发酵配方进行研究,发现该菌株在PDA、PPDA、PDAY、SDAY、SMAY 5种培养基上均能较好生长,在PDA上生长最快,在PDAY上产孢量最大。温度对菌丝的生长和产孢影响显著,25°C菌丝生长最快,且产孢量最大。B.bassiana AT17菌株在pH 4-10范围内均可生长,在偏碱性环境内生长最快,在pH 5-6的偏酸性环境内产孢量最大。综合评价真菌各项指标后,葡萄糖和酵母粉为菌丝生长和产孢的最佳碳源和氮源,固体物料麦麸+玉米粉+米粉与基础培养液3/4 SDAY按2:1均匀混合后,添加0.05%K+可作为菌株固体发酵的最佳物料配方组合。     

长蠕孢菌产孢条件的研究

对番茄褐斑病原菌—长蠕孢菌产孢条件进行了研究,结果表明燕麦片琼胶培养基、Czapek培养基和PDA＋番茄叶片能促进产孢子,V8汁、PSA和番茄汁培养基抑制产孢;碳源果糖明显促进产孢,甘露醇抑制产孢子;氮源氯化铵促进产孢,蛋白胨和硫酸铵抑制产孢;光照和紫外线照射对长蠕孢菌产孢有明显促进作用,特别是紫外线照射60～80min时产孢量达到最大;偏低温或偏高温以及微碱性环境能促进长蠕孢菌产孢,温度为15℃或30℃,pH8～9时最有利于产孢。     

分光光度法测定白僵菌孢子粉的含孢量

研究了用分光光度计法测定白僵菌孢悬液的含孢量。首先用筛选出的适宜稀释液SF1将孢子粉作不同浓度的稀释,使用分光光度计在470nm波长下测出孢悬液的吸光度值（x）,再用血球计数板测出对应合孢量（y）,得到回归方程y＝10^{0.8895x＋6.5869}（r＝0．9678）。经检验,方程回归显著,并且不存在菌株间的差异。孢悬液的适宜稀释浓度范围为1500—2000倍。     

卵孢白僵菌及添加杀虫助剂对两种地老虎的生物活性测定

【目的】确定卵孢白僵菌Beauveria brongniartii菌株NEAU30503对八字地老虎Xestia c-nigrum(Linnaeus)和小地老虎Agrotis ypsilon(Rottemberg)的杀虫活性。【方法】采用浸叶法测定NEAU30503对地老虎低龄幼虫的生物活性,土壤处理法测定对高龄幼虫的杀虫活性。【结果】NEAU30503对5日龄八字地老虎幼虫第15天毒力回归方程为y=0.6568x-0.1636(r=0.9846),其LC_(50)、LC_(80)分别为7.28×10~7孢子/m L、139.14×10~7孢子/m L;对5龄八字地老虎幼虫毒力回归方程为y=1.0929x-3.2893(r=0.9801),其LC_(50)、LC_(80)分别为3.85×10~7孢子/L、22.65×10~7孢子/L。未死幼虫化蛹后仍有部分蛹死于白僵菌感染,并能明显降低成虫羽化率。白僵菌与Bt、茶皂素、以及亚致死剂量的高效氯氢菊酯和阿维菌素混用能明显提高其杀虫活性和杀虫速度。【结论】NEAU30503对地老虎具有较高的杀虫活性,土壤处理防治高龄幼虫效果好,喷雾处理防治低龄幼虫加入少量的高效氯氢菊酯或茶皂素效果明显。本研究为开展白僵菌田间防治地老虎提供了科学依据。     

Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii

The commercial use of entomopathogenic fungi and their products as mycoinsecticides necessitates their registration. Worldwide, several registration guidelines are available, however, most of them focus on similar or even the same safety issues. With respect to the two entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. and Beauveria brongniartii (Sacc.) Petch, many commercial products have been developed, and numerous papers on different biological, environmental, toxicological and other safety aspects have been published during the past 30-40 years. The aim of the present review is to summarise these data. The following safety issues are presented: (1) identity of Beauveria spp.; (2) biological properties of Beauveria spp. (history, natural occurrence and geographical distribution, host range, mode of action, production of metabolites/toxins, effect of environmental factors); (3) analytical methods to determine and quantify residues; (4) fate and behaviour in the environment (mobility and persistence in air, water and soil); (5) effects on non-target organisms (non-target microorganisms, plants, soil organisms, aquatic organisms, predators, parasitoids, honey bees, earth worms and nontarget arthropods); (6) effects on vertebrates (fish, amphibia, reptiles and birds); and (7) effects on mammals and human health. Based on the present knowledge it is concluded that both Beauveria species are considered to be safe.     

Evaluation of Beauveria brongniartii and its Metabolite Oosporein Regarding Phytotoxicity on Seed Potatoes

The phytotoxic potential of Beauveria brongniartii and its main secondary metabolite oosporein were evaluated against seed potatoes ( Solanum tuberosum ) both in vitro and in situ . In a tray test, potatoes were inoculated using conidiospores and grown either in garden soil or in B. brongniartii -enriched soil. The only significant effect of the treatment was the fact that the weight of the roots was different compared with the controls. The weight of haulm and tubers was unaffected by the treatment with B. brongniartii . No oosporein was detected in the potatoes, although the density of B. brongniartii in the soil was forty times higher than recommended for the usage of this biological control agent (BCA). This indicates that oosporein does not enter the food chain via potato tubers. The effect of BCA on seed potatoes was also tested in situ . No B. brongniartii was detected in the potato field before the application of BCA. Three months after application, an average of 3 ×10 4 colony forming units per gram soil (dry weight) were recorded. Although Melolontha melolontha larvae damaged 60% of the potatoes, only 3% of the tissue samples were infected with B. brongniartii . The results obtained in this study suggest that B. brongniartii and its secondary metabolite oosporein pose no risks to potato plants, tubers, or to their consumers.     

Differential Susceptibility of Two Melolontha Populations to Infections by the Fungus Beauveria brongniartii

The susceptibility of second-instar larvae of the European cockchafer, Melolontha melolontha L., originating from two different geographical locations was investigated with eight isolates of the insect pathogenic fungus Beauveria brongniartii (Sacc.) Petch. 77-94% of larvae from northern Italy (Auer, South Tyrolia) succumbed to mycosis with an average survival time (AST) of 22.9-40.1 days. Infections in larvae from south-western Switzerland (Bramois, Valais) were 28-72% and the ASTs varied between 34.7-56.4 days. Differences in susceptibility between the two host populations may be explained by the historical ages of the two populations and the presence of B. brongniartii resulting in a coevolution of tolerance between the host and pathogen. The Italian population is occasionally infected by B. brongniartii; in comparison, the Swiss population has existed for at least 50 years and regular infections by the pathogen are observed. Coevolution between B. brongniartii and M. melolontha from Switzerland may explain the apparent resistance of the host towards this pathogen in laboratory assays.     

提高蛋白激发子产量的培养基及发酵条件的优化

为了进一步提高极细链格孢菌产蛋白激发子的产量,通过单因子和多因子试验与分析,筛选优化了适于极细链格孢菌产生蛋白激发子的培养基和培养条件,并检测了发酵过程中pH、还原糖、氨基氮和菌丝量变化以及与蛋白激发子产量的关系。结果表明,土豆淀粉和黄豆粉对蛋白激发子产量影响最大,其次是蛋白胨和无机盐。优化的发酵培养基主要成分（g／L）：碳源I 15、葡萄糖5、玉米淀粉5、土豆淀粉20、谷氨酸10、氮源I5、黄豆粉10、硫酸铵5。确定了优化的培养条件,调整培养基起始pH为7．0～7．5,将18h菌龄的种子培养液按10％接种量接种到装液量为75mL的500mL摇瓶中,在温度（28±1）℃、摇床转速180r／min下培养可获得理想的蛋白产量。在优化的培养基和培养条件下,发酵12～48h该菌进入对数生长期,48h进入稳定生长期,60h菌丝扣蛋白激发子产量达最高。蛋白产量与菌体生物量呈正相关,当还原糖、总糖量消耗到最低水平时,菌丝产量和蛋白激发子产量达最高。优化的培养基菌丝干重收率迭3．9g／100mL,蛋白激发子产量达到5．17g／L,比普通的土豆液体培养基提高近4倍。     

Distribution of insect pathogenic soil fungi in Switzerland with special reference to Beauveria brongniartii and Metharhizium anisopliae

Between 1998 and 2000 soil samples from 82fields in north, east, central and south westof Switzerland were analysed for presence ofinsect pathogenic soil fungi using a selectivemedium and the Galleria bait method. Thedetection rates with either method were verysimilar. 96% of the fields distributed overall examined regions contained Metarhiziumanisopliae. The presence of Beauveriabrongniartii was limited to sites colonised byits host, Melolontha melolontha. It wasalso present at a site where M.melolontha disappeared about 40 years ago. Onthe other hand, B. brongniartii was notfound in four fields containing M.melolontha populations. Beauveriabassiana, Paecilomyces fumosoroseus and Conidiobolus sp. were other entomopathogenicfungi isolated during this survey. Differencesin the presence of M. anisopliae betweenarable fields and adjacent meadows as well asbetween orchards and meadows are discussed.     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

Nucleotide excision repair and photoreactivation in the entomopathogenic fungi Beauveria bassiana, Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus and Verticillium lecanii

AIMS: To compare the DNA repair capabilities of the entomopathogenic fungus (EPF) bassiana to the EPF Beauveria brongniartii, Beauveria nivea, Metarhizium anisopliae, Paecilomyces farinosus, Verticillium lecanii, and the fungi Aspergillus niger and Neurospora crassa. METHODS AND RESULTS: Germination of B. bassiana conidiospores following ultraviolet (UV) irradiation was used to show that nucleotide excision repair and photoreactivation decrease the post-UV germination delay. These two modes of repair were characterized and compared between the aforementioned EPF, A. niger and N. crassa using a physiological assay where per cent survival post-UV irradiation was scored as colony forming units. CONCLUSIONS: The results showed B. bassiana and M. anisopliae are the most UV-tolerant EPF. The DNA repair capabilities indicated that EPF do not have all DNA repair options available to fungi, such as A. niger and N. crassa. SIGNIFICANCE AND IMPACT OF THE STUDY: A key factor detrimental to the survival of EPF in agro-ecosystems is UV light from solar radiation. The EPF literature pertaining to UV irradiation is varied with respect to methodology, UV source, and dose, which prevented comparisons. Here we have characterized the fungi by a standard method and established the repair capabilities of EPF under optimal conditions.