防治苦瓜枯萎病的拮抗放线菌分离筛选及鉴定

以苦瓜枯萎病菌为靶标菌,通过对峙培养试验和发酵滤液抑菌试验对分离自苦瓜根际土壤的放线菌进行筛选。候选菌株0250具有广谱抗真菌活性,根据培养特征、生理生化特性以及与同源性相近的菌株进行平均核苷酸一致性分析,被鉴定为Streptomyces rhizosphaericus,并评估了该菌株在温室和田间对苦瓜的促生长和防治枯萎病效果。结果表明: 链霉菌菌株0250对苦瓜枯萎病菌的平板抑制率为69.2%,对17种植物病原真菌的平板抑制率达64.3%~85.6%;该菌株的菌悬液处理能促进盆栽和田间苦瓜植株根、茎生长发育,提升产量,对苦瓜枯萎病的防病效果分别为66.9%和61.5%。预先用菌株0250菌悬液处理土壤再接种病原菌,对土壤尖镰孢菌数量抑制率达62.1%,显著提高了苦瓜幼苗苯丙氨酸解氨酶、过氧化物酶和β-1,3-葡聚糖酶活性以及根系活力。总之,菌株0250是一株对苦瓜枯萎病具有巨大生防潜力的放线菌资源。     

白僵菌防治菜青虫的研究

通过应用6株白僵菌不同菌株对菜青虫幼虫进行室内和田间防治试验比较,结果表明,不同菌株间的毒力存在显著差异,以筛选自菜青虫僵虫尸上的Bpr3菌株毒力最强,用浓度为1×108 mL-1的孢子液对菜青虫幼虫进行室内和田间感染,室内幼虫校正死亡率达100%,LT50为2.9806 d,田间防治效果达96.6%,经大面积示范应用,取得了显著的防治效果.     

杀松材线虫海洋放线菌HT-8的鉴定及培养条件的研究（英文）

松树萎蔫病(Pine wilt disease,PWD)是由松材线虫(Bursaphelenchus xylophilus)引起的一种毁灭性疾病,该病对世界上许多国家和地区的针叶林资源造成严重破坏。为了寻找具有高杀松材线虫活性的海洋放线菌,对分离自海洋环境中的放线菌菌株进行杀线虫活性测定。选用稀释涂布平板法对采自青岛近海海域的样品进行放线菌分离,采用浸渍法测定菌株的杀松材线虫活性,并对所筛选出的高杀线虫活性菌株的形态学、培养特征和16S rD NA序列进行测定,运用单因素试验测定菌株产生杀线虫活性物质的最适培养条件。结果显示,从采集的样品中共分离到28株放线菌,经杀线虫活性筛选得到1株具有高杀线虫活性的放线菌HT-8。该菌株分离自海沙,其培养上清液处理松材线虫30 h时,线虫的校正死亡率高达88.30%。根据形态学、培养特征和16S rD NA序列测定及其系统发育分析结果,将菌株HT-8鉴定为Streptomyces termitum。单因素试验结果表明,该菌株产生杀线虫活性物质的最适培养条件为接种体菌龄48 h,接种量6%,培养基海水浓度100%,初始pH 7.5,培养温度25℃,培养时间7 d。该研究为海洋微生物资源的开发及其杀松材线虫天然活性物质的利用提供理论依据。     

绿僵菌大孢变种的生物学特征及其对蛴螬的毒力研究

以报道较多的蛴螬病原真菌 布氏白僵菌、绿僵菌小孢变种作参照菌,系统研究了新分离鉴定的绿僵菌大孢变种的生物学特性,测定了其在偏低的自然气温下对蛴螬的致病能力.结果表明,绿僵菌大孢变种与参照菌的培养要求接近,最适宜的温度为25 ℃.3种供试的培养基中,PPDA为最适宜的培养基,其次为SDAY.在偏低的温度环境下绿僵菌大孢变种对蛴螬的毒杀能力强于布氏白僵菌,土壤和喷雾处理后累计僵虫率分别达88.23%和76.47%,有很好的田间应用前景.     

絮凝剂产生菌培养条件的研究及在废水处理中的应用

通过菌种富集、分离、纯化,从含有大量微生物菌群的土壤和活性污泥中筛选到一株对高岭土悬浊液具有较高絮凝活性的絮凝剂产生菌,将其命名为B23.通过研究该菌株在不同培养时间的生长情况和发酵液的絮凝活性,从而得出发酵液絮凝活性与菌体生长量呈正相关.通过对该菌株培养条件的研究表明,在培养时间为24h、培养温度为30℃、培养基初始pH值为8.0,以葡萄糖为碳源、(NH4)2SO4为氮源时,发酵液絮凝活性最强.用B23菌株所产絮凝剂处理废水后,废水中CODCr的去除率为62.48%,SS的去除率为84.47%,表明该菌株有良好的实际应用前景.     

松材线虫拮抗细菌的杀线活性及其发酵培养特性

旨为筛选具有高效杀松材线虫活性的拮抗细菌。采用浸测法对3株芽孢杆菌进行杀线活性测定,并对高效杀线菌株发酵滤液处理线虫后的虫体进行形态观察及该菌株培养条件和杀线物质稳定性测定。结果表明3株细菌发酵滤液处理松材线虫(Bursaphelenchus xylophilus)48 h后,线虫校正死亡率均达100%,且松材线虫在蜡样芽孢杆菌(Bacillus cereus)JK-XZ3发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率分别达99.55%和88.32%,明显高于短小芽孢杆菌(B. pumilus)HR10和瓦雷兹芽孢杆菌(B. velezensis)YH-20;该JK-XZ3菌株发酵滤液处理过的线虫虫体断裂,内含物外溢,体壁消解;该菌株在30℃培养4 d的发酵滤液杀线活性最高,且松材线虫在发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率均达100%;松材线虫在40℃、60℃、80℃和100℃处理后的发酵滤液8倍稀释液中处理48 h后,其校正死亡率均在84%以上;在pH为8-10的发酵滤液8倍稀释液处理48 h后,其校正死亡率均在88%以上;说明该菌株发酵滤液具有耐高温和耐碱特性。蜡样芽孢杆菌JK-XZ3是一株具有开发和应用潜力的高毒力杀线菌株。     

二化螟幼虫病原真菌的分离纯化及其对烟粉虱和褐飞虱的毒力研究

通过对罹病二化螟Chilo suppressalis (Walker)幼虫病原真菌的分离纯化,筛选出5种不同真菌,探究不同营养对不同真菌生物活性的影响,并测定其对烟粉虱Bemisia tabaci (Gennadius)和褐飞虱Nilaparvata lugens (Stal)成虫和若虫的室内毒力。选择毒力最强的菌株,通过生物型的初步鉴定,明确高毒力菌株的种类。结果表明：所筛选的5种真菌对两种害虫均具有较高的毒力,其中菌株A26和Bbr14对烟粉虱和褐飞虱具有较强的毒力,在处理后第9 d,菌株A26对烟粉虱成虫和若虫的累计校正死亡率均最高分别达84.69%、77.62%,感染率为91.17%、88.24%,LT50为4.31 d、4.87 d;处理后第10 d对褐飞虱成虫和若虫的累计校正死亡率达87.54%、73.18%,感染率为90.13%、87.13%,LT50为4.27d、5.14 d,而菌株Bbr14的效果仅次于A26。鉴定的结果显示菌株A26为曲霉,菌株Bbr14为球孢白僵菌,曲霉的安全性有待研究,球孢白僵菌的应用技术已较成熟,有望能在防治这两类重要害虫中发挥作用。     

一株寄生油松毛虫的白僵菌毒素化学成分

[目的]从一株寄生于油松毛虫(Dentrolimus tabulaeformis Tsai et Liu)越冬幼虫上的高毒力卵孢白僵菌(Beauveria tenella)的发酵液中,分离纯化出对油松毛虫幼虫具有杀虫活性的毒素物质.[方法]对这株白僵菌进行了液体培养,用乙酸乙酯对发酵液进行萃取,然后对粗提物进行了硅胶色谱分离,利用GC/MS对第6组物质进行化学成分分析.[结果]其发酵液的乙酸乙酯粗提物对油松毛虫具有杀虫活性(校正死亡率42.52%)对粗提物进行了硅胶色谱分离,共得到6组物质,其中第6组物质对油松毛虫的校正致死率达80.26%.利用GC/MS对第6组物质进行化学成分分析,得知其包括17个组分,其中相对含量大于10%的有:2-哌啶酮占14.02%,2.香豆满酮占47.10%,六氢化-吡咯环[1,2-a]吡嗪-1,4-二酮占21.05%.用其中的2-哌啶酮和2-香豆满酮分别作杀虫试验,油松毛虫的校正死亡率分别为83.32%和91.61%.[结论]2-哌啶酮和2-香豆满酮是该白僵菌菌株的代谢毒素物质.     

深海链霉菌选择性分离及活性菌株16S rRNA聚类分析

选择性分离深海链霉菌,检测其抗肿瘤细胞,抗金黄色葡萄球(Staphylococcus aureus ATCC 51650),抗PTP1B和抗caspase-3活性,对活性菌株做16s rRNA聚类分析.用8种选择性培养基分离培养,检测活性和构建活性菌株16S rRNA聚类分析图.共分离到90株链霉菌,其中在RH培养基上分离到的菌株数量和类群最多,占38.9%,次之为GS1和OA培养基,而M5培养基未分离到菌株;共筛选到活性菌株44株,有抗肿瘤活性14株,抗金黄色葡萄球菌22株,抗PTP1B活性19株和抗caspase-3活性2株,其中16株至少有两种活性;16S rRNA聚类分析结果表明44株活性菌株分布在4个clade,分别是S.coelicolor,S.pactum,S.stramineus和S.restomycificus clade,以S.coelicolor和S.restomycificus clade为主,分别占66.0%和11.1%.在常温,常压下可分离培养到大量高活性和活性多相样性的深海链霉菌,KH培养基最适用于分离深海链霉菌.     

贵州兴义喀斯特洞穴土可培养细菌多样性及其产蛋白酶、淀粉酶活性筛选

【目的】进一步了解贵州喀斯特洞穴土可培养细菌的物种多样性组成及其产蛋白酶、淀粉酶生物活性能力。【方法】选取11种分离培养基,利用稀释直接涂布平板法对贵州黔西南兴义市喀斯特地区白碗窑镇魔家大溶洞洞内土壤进行可培养细菌分离;利用两种鉴定培养基对相关细菌进行生物活性判定。【结果】根据16S rRNA基因序列的系统进化分析,将分离得到的217株细菌分别归类到24个属的63个不同种类,其中红球菌属(Rhodococcus)和链霉菌属(Streptomyces)为该洞内土壤可培养细菌的优势菌群,分别占24.42%和21.66%。大多数菌株与已知典型菌株的16S rRNA基因序列相似性为97.90%-99.99%,其中至少有4株菌株(D3T01、D911、D961和D502)为潜在的新分类单元。对217株细菌进行蛋白酶和淀粉酶活性筛选,其中具有蛋白酶或淀粉酶活性的99株,占分离菌株的45.62%,分别属于18个属的38个不同种;同时具有蛋白酶和淀粉酶活性的36株,占具有酶活性菌株的36.36%,占分离菌株的16.59%。【结论】贵州兴义喀斯特洞穴土中存在丰富多样的细菌类群,且蕴藏着一定数量的潜在新物种资源;此外功能酶菌株在喀斯特洞穴土壤中大量存在,为工业应用奠定了资源基础,极具进一步发掘和研究的价值。     

Comparison of application methods for suppressing the pecan weevil (Coleoptera: Curculionidae) with Beauveria bassiana under field conditions

The pecan weevil, Curculio caryae (Horn), is a key pest of pecans. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin is pathogenic to C. caryae. One approach to managing C. caryae may be application of B. bassiana directed toward adult weevils as they emerge from the soil to attack nuts in the tree canopy. Our objective was to compare different application methods for suppression of C. caryae adults. Treatments included direct application of B. bassiana (GHA strain) to soil under the tree canopy, soil application followed by cultivation, soil application in conjunction with a cover crop (Sudan grass), direct application to the tree trunk, and application to the trunk with an UV radiation-protecting adjuvant. The study was conducted in a pecan orchard in Byron, GA, in 2005 and 2006. Naturally emerging C. caryae adults, caught after crawling to the trunk, were transported to the laboratory to determine percentage mortality and signs of mycosis. When averaged over the 15-d sampling period, weevil mortality and signs of mycosis were greater in all treatments than in the nontreated control in 2005 and 2006; >75% average mortality was observed with the trunk application both years and in the trunk application with UV protection in 2005. Results indicated trunk applications can produce superior efficacy relative to ground application, particularly if the ground application is followed by cultivation. Efficacy in the cover crop treatment, however, did not differ from other application approaches. Future research should focus on elucidating the causes for treatment differences we observed and the extent to which B. bassiana-induced C. caryae mortality reduces crop damage.     

Persistence and efficacy of Beauveria brongniartii strains applied as biocontrol agents against Melolontha melolontha in the Valley of Aosta (northwest Italy)

AIMS: To monitor and select genetically characterized strains of Beauveria brongniartii to be used as microbiological control agents against Melolontha melolontha in different climatic conditions of the Valley of Aosta (northwest Italy). METHODS AND RESULTS: Molecular random amplified polymorphic DNA markers allowed monitoring of five B. brongniartii strains (C2, F, K2, N3 and W2) in field trials. Ten sites were chosen at Joven?an, Saint-Pierre and Quart areas, where a mixture of the five strains colonizing rye kernels was applied to the soil of each M. melolontha infested site. Growth, persistence and virulence on M. melolontha larvae of five fungal strains were evaluated in two subsequent 24-month studies. Beauveria brongniartii grew best at the Joven?an sites. Not only did strain F persist better than the other strains in most soil samples but it was also the most virulent strain. Strain F was isolated the most frequently from infected M. melolontha larvae recovered from the test sites. A general decrease in the larvae rate was detected in the test field soil. CONCLUSIONS: Strain F of B. brongniartii was better than other strains in growth, persistence and virulence against M. melolontha larvae in the test site soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Results obtained from preliminary field trials support the use of strain F as a biological control agent against M. melolontha in the Valley of Aosta even if further targeted studies are still necessary.     

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.)

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.     

Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus

The potential of a strain of Beauveria bassiana (Ascomycota: Clavicipitaceae) obtained from a naturally infected Rhynchophorus ferrugineus (Coleoptera: Curculionidae) pupa as a biological control agent against this weevil was evaluated both in the laboratory and in semi-field assays. Laboratory results indicate that this strain of B. bassiana can infect eggs, larvae and adults of R. ferrugineus (LC₅₀ from 6.3 × 10⁷ to 3.0 × 10⁹ conidia per ml). However, mortality was not the only indicator of treatment efficacy because adults of either sex inoculated with the fungus efficiently transmitted the disease to untreated adults of the opposite sex, with male-to-female and female-to-male rates of transmission of 55% and 60%, respectively. In addition, treatment with B. bassiana significantly reduced fecundity (up to 62.6%) and egg hatching (32.8%) in pairing combinations with fungus-challenged males, females or both sexes. Likewise, 30-35% increase in larval mortality was observed in larvae obtained from eggs from fungus-challenged females or from untreated females coupled with inoculated males, resulting in an overall 78% progeny reduction. Semi-field preventive assays on potted 5-year old Phoenix canariensis palms, with efficacies up to 85.7%, confirmed the potential of this strain as a biological control agent against R. ferrugineus.     

Laboratory and greenhouse evaluation of a new entomopathogenic strain of Beauveria bassiana for control of the onion thrips Thrips tabaci

Abstract

The onion thrips Thrips tabaci is one of the most important pests of greenhouse and open-field broccoli, onion and other crops. However, the current strategy of using synthetic pesticides for its control is inadequate and unsustainable, leading to a growing interest in novel and effective biological control alternatives such as entomopathogenic fungi. Among 20 isolates of Beauveria bassiana tested for virulence against T. tabaci in laboratory bioassays, we found strain SZ-26 as the most potent, causing 83–100% mortality in adults at 1×10⁷ mL^?1conidia after 4–7 days. Further experiments in greenhouses showed the strain SZ-26 significantly lowered the numbers of adult and larval stages.     

Importance of direct spray and spray residue contact for infection of Trichoplusia ni larvae by field applications of Beauveria bassiana

Commercial formulations and unformulated conidia of Beauveria bassiana strain GHA were applied to field-grown plants and artificially infested with Trichoplusia ni (Hübner) larvae to compare the relative insecticidal activity resulting from direct spray contact with insecticidal activity due to contact with dry spray residue. In general, applications to cabbage, Brassica oleracea L., resulted in nearly equal mortalities when comparing insects exposed to direct spray contact with those exposed by spray residue, suggesting a potential benefit by improving formulations to extend residual activity. For applications to beans, Phaseolus vulgaris L., direct spray contact provided significant insect mortality, but mortality due to residual contact was generally not different than the untreated control. In contrast to the differences observed for larvae exposed in the field, larvae exposed in laboratory bioassays to leaf disks collected from the same treated cabbage and bean plants (residual contact exposure) resulted in nearly identical mortalities. Field applications of Beauveria showed rapid loss of activity, expressed as a loss of conidia viability and loss of insecticidal activity during the first 8 h after application. Evidence of significant mortality by residual contact and the rapid loss of insecticidal activity with field exposure support additional research to improve formulations to extend the residual activity of fungal biopesticides.     

黄绿绿僵菌Mf82菌株对不同虫态褐飞虱的毒力

对水稻重要害虫褐飞虱Nilaparvata lugens目前市场上尚无一种理想的微生物杀虫剂。昆虫病原真菌具有从体壁侵入的能力因而对刺吸性害虫的防治具有优势。为此, 本研究选用不同原寄主和来源地的3种昆虫病原真菌（金龟子绿僵菌Metarhizium anisopliae、 黄绿绿僵菌Metarhizium flavoviride和球孢白僵菌Beauveria bassiana）的12个不同菌株, 以1 100孢子/mm²孢子悬浮液进行室内毒力测定。结果表明： 在参试的不同菌种12个菌株中, 黄绿绿僵菌Mf82菌株对褐飞虱成虫致病力最高, 10 d累计校正死亡率为83.5%, 致死中时（LT₅₀）为4.6 d。其不同浓度孢子液对褐飞虱3个发育阶段有不同程度的致病力, 毒力大小顺序为成虫>高龄若虫>低龄若虫。黄绿绿僵菌孢子液对各处理稻株褐飞虱产卵痕部位、 卵粒均有侵染作用, 10 d侵染率分别为66.7%和51.2%, 卵龄越低, 侵染效果越好, 卵龄为0.5 d时侵染率最高。本研究表明黄绿绿僵菌Mf82菌株对褐飞虱成虫、 若虫和卵均有较强的致病性, 是一株极具应用潜力的生防真菌。     

Effects on Diversity of Soil Fungal Community and Fate of an Artificially Applied Beauveria bassiana Strain Assessed Through 454 Pyrosequencing

The entomopathogenic fungus Beauveria bassiana is widely used as a biological control agent (BCA) for insect pest control, with fungal propagules being either incorporated into the potting media or soil or sprayed directly onto the foliage or soil. To gain a better understanding of entomopathogenic fungal ecology when applied as a BCA to the soil environment, a case study using tag-encoded 454 pyrosequencing of fungal ITS sequences was performed to assess the fate and potential effect of an artificially applied B. bassiana strain on the diversity of soil fungal communities in an agricultural field in India. Results show that the overall fungal diversity was not influenced by application of B. bassiana during the 7 weeks of investigation. Strain-specific microsatellite markers indicated both an establishment of the applied B. bassiana strain in the treated plot and its spread to the neighboring nontreated control plot. These results might be important for proper risk assessment of entomopathogenic fungi-based BCAs.     

白僵菌Bbt1菌株与化学农药防治荔枝蝽效果比较

应用白僵菌Bbt1菌株和常用化学农药90％晶体敌百虫、2．5％溴氰菊酯、5％顺反氯氰菊酯等进行室内毒力测定和田间防治荔枝蝽试验。结果表明,荔枝蝽已对敌百虫高度不敏感,药效下降,产生抗药性;5％顺反氯氰菊酯乳油、2．5％溴氰菊酯乳油虽有较好防效,但污染环境、残留大,不宜使用;Bbt1尽管初期见效慢,但7—15d效果达到高峰（达90％以上）,其粉剂兑水100倍液喷雾防治,室内和田间15d防效分别达94．5％和93．0％,具有很好的防治效果,且属无公害农药,可以代替化学农药使用。