金龟子绿僵菌选择性培养基的筛选

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适宜金龟子绿僵菌产孢的发酵培养基及其制备和使用方法[发明专利]

(10)申请公布号(43)申请公布日 (21)申请号 201510131011.8(22)申请日 2015.03.25C12N 3/00(2006.01)C12R 1/645(2006.01)(71)申请人河南科技大学地址471000 河南省洛阳市涧西区西苑路48号(72)发明人侯颖徐建强林晓民刘圣明李晓艳(74)专利代理机构洛阳公信知识产权事务所(普通合伙) 41120代理人罗民健(54)发明名称适宜金龟子绿僵菌产孢的发酵培养基及其制备和使用方法(57)摘要适宜金龟子绿僵菌产孢的发酵培养基及其制备和使用方法，涉及一种培养基；所述培养基，包括种子培养基和固体发酵培养基。

所述种子培养基包括固体种子培养基和液体种子培养基；固体种子培养基中含有马铃薯汁1000mL、葡萄糖、KH 2PO 4、MgSO 4和琼脂，余量为水；液体种子培养基中含有蛋白胨、蔗糖、K 2HPO 4、MgSO 4和NaCl，余量为水；所述固体发酵培养基的成分为：麦麸、玉米糁和蒸馏水;其中麦麸和玉米糁的质量比为1:1，蒸馏水的质量为麦麸和玉米糁总质量55-60%。

本发明适宜金龟子绿僵菌产孢的发酵培养基适于金龟子绿僵菌的生长，每克发酵原料可产生1×109以上个孢子；制备的孢子粉萌发率可达95%。

(51)Int.Cl.(19)中华人民共和国国家知识产权局(12)发明专利申请权利要求书1页说明书7页附图1页(10)申请公布号CN 104651294 A (43)申请公布日2015.05.27C N 104651294A1.适宜金龟子绿僵菌产孢的发酵培养基，其特征在于：包括种子培养基和固体发酵培养基；所述种子培养基包括固体种子培养基和液体种子培养基；每升固体种子培养基中含有质量浓度为20%马铃薯汁1000mL、葡萄糖20g、KH2PO43g、MgSO41.5g和琼脂 20g，余量为水；每升液体种子培养基中，含有10 g蛋白胨、10 g蔗糖、0.3 g K2HPO4、0.3 g MgSO4和0.3g NaCl，余量为水；所述固体发酵培养基的成分为：麦麸、玉米糁和蒸馏水；其中麦麸和玉米糁的质量比为1:1，蒸馏水的质量为麦麸和玉米糁总质量55~60%。

对斜纹夜蛾高效绿僵菌的筛选

对斜纹夜蛾高效绿僵菌的筛选马丽娟;滕忠才;臧欢;刘廷辉;李瑞军;郭巍;巩兰菊【摘要】对从土壤中分离到的金龟子绿僵菌中生长性状较好的6个菌株,通过测定对黄粉虫幼虫的僵虫率和致死中时,选出3株致病效果好的菌株M7-9、M7-(16-18)、M7-(64-65),再采用点滴法接种对斜纹夜蛾进行毒力测定.结果表明,不同菌株对斜纹夜蛾的毒力与菌株种类有密切关系,3株菌株对斜纹夜蛾的LT50分别为2.70、3.07、5.79d.以此3株菌进行抗高温及抗紫外线试验,菌株M7-9和M7-(16-18)对高温和紫外线都具有较好的抗性,且M7-9对紫外线的抗性强于M7-(16-18),两菌株均具有进一步研究的价值.%Six strains of Metarhizium anisopliae with good growth traits, isolated from soil in Baoding area, were screened against Tenebrio molitor Linnaeus by bioassay. Among them, M7-9, M7-(16-18)and M7-(64-65)strains, which showed higher virulence than other isolates, were selected to test their pathogenicity against Spodoptera li-tura(Fabricius). The LT50 of strain M7-9, M7-(16-18) and M7-(64-65) against S. litura were 2.70, 3.07 and 5. 79 d, respectively. The 3 strains were then treated with the ultraviolet ray and heat. The results demonstrated that the resistance of M7-9 and M7-( 16-18) to high temperature and ultraviolet ray was higher, and the resistance of M7-9 to the ultraviolet ray was higher than that of M7-(16-18). These results suggested that strains M7-9 and M7-(16-18) have better exploitation and application value in the future.【期刊名称】《植物保护》【年(卷),期】2012(038)005【总页数】6页(P78-83)【关键词】金龟子绿僵菌;斜纹夜蛾;生物测定;筛选【作者】马丽娟;滕忠才;臧欢;刘廷辉;李瑞军;郭巍;巩兰菊【作者单位】河北农业大学植物保护学院,河北省农作物病虫害生物防治工程技术研究中心,保定071001;保定学院生化系,保定071000;河北农业大学植物保护学院,河北省农作物病虫害生物防治工程技术研究中心,保定071001;河北农业大学植物保护学院,河北省农作物病虫害生物防治工程技术研究中心,保定071001;河北农业大学植物保护学院,河北省农作物病虫害生物防治工程技术研究中心,保定071001;河北农业大学植物保护学院,河北省农作物病虫害生物防治工程技术研究中心,保定071001;河北省曲周县农牧局,曲周057250【正文语种】中文【中图分类】S476.12斜纹夜蛾［Spodopteralitura （Fabricius）］属鳞翅目（Lepidoptera）夜蛾科（Noctuidae），是一种世界性分布的重要农业害虫，食性广，繁殖力强，发生为害严重［1］。

海南椰心叶甲病原菌金龟子绿僵菌的分离、鉴定及其生防潜力

海南椰心叶甲病原菌金龟子绿僵菌的分离、鉴定及其生防潜力詹儒林;覃伟权;宋妍;张世清;H H HO;许天委;黄俊生【摘要】椰心叶甲[Brontispa longissima(Gestro)]是椰子的重要害虫,近年来,该虫在海南岛发生普遍,椰子受害严重.由于椰心叶甲受到自然界中某些致病微生物的侵袭,在受害的椰子树心叶上常可发现椰心叶甲僵虫,并发现大部分僵虫表面长出了霉菌,本研究的目的在于从椰心叶甲僵虫表面的霉菌中分离出绿僵菌,并对分离菌株进行鉴定和致病性测定.从僵虫表面刮下孢子或菌丝体,置于绿僵菌选择性培养基(DOA)上培养,挑出真菌菌落,经纯化后,进行生物学特性、菌落生长速率及产孢量的测定,并从PPDA、OMA、V8A和PDA中筛选菌落生长及产孢最适培养基,同时对所分离的菌株进行对椰心叶甲的致病性测定.结果表明,所有分离菌株均鉴定为金龟子绿僵菌[Metarhizium anisopliae(Metschnikoff)],PPDA是菌落生长及产孢的最适培养基,大多数菌株对椰心叶甲有较强的致病力.选取强毒菌株MA4在田间进行防治效果的初步测定,结果表明,该菌株能显著降低椰心叶甲成虫的虫口密度.这些金龟子绿僵菌菌株是首次从海南的椰心叶甲僵虫中分离到的昆虫病原真菌,该菌对海南的椰心叶甲具有很好的生防潜能.%Coconut hispid beetles, Brontispa longissima (Gestro) (Coleoptera:Chrysomelidae) have been prevalent all over Hainan Island in China, and millions of coconut palms were attacked by this pest. The objective of this work was to isolate and identify strains of entomopathogenic fungi from natural infections of B. longissima collected from coconut palms.Conidia or mycelia collected from the surface of beetle cadavers were cultured and the colonies were screened on dodine oatmeal agar medium (DOA). The colony morphology, mycelia growth rate and conidial production were further studied on various agar media: PPDA,OMA, V8A and PDA. In order to confirm whether these isolates were pathogenic to B.longissima, their virulence to the beetles were also tested in laboratory. The results showed two different kinds of strains MA and MB were all identified as Metarhizium anisopliae(Metschnikoff) base on the macromorphological and micromorphological characteristics, and PPDA was the optimal culture medium for vegetative growth and conidial production. Several high virulent strains were screened in vitro, and application on controlling coconut hispid beetles was carried out preliminarily in vivo. The results showed a significant reduction in adult population was caused by the isolate. It was proved that M.anisopliae isolated from the natural infected cadavers of B. longissima have great potential as a means of biocontrol against this serious pest.【期刊名称】《生态学报》【年(卷),期】2007(027)004【总页数】7页(P1558-1564)【关键词】金龟子绿僵菌;分离;鉴定;生物防治;椰心叶甲【作者】詹儒林;覃伟权;宋妍;张世清;H H HO;许天委;黄俊生【作者单位】热带作物生物技术国家重点实验室,海口,571101;中国热带农业科学院环境与植物保护研究所,儋州,571737;中国热带农业科学院南亚热带作物研究所,湛江,524091;中国热带农业科学院椰子研究所,文昌,571201;热带作物生物技术国家重点实验室,海口,571101;中国热带农业科学院环境与植物保护研究所,儋州,571737;热带作物生物技术国家重点实验室,海口,571101;中国热带农业科学院环境与植物保护研究所,儋州,571737;Department of Biology,State University of New York,New Palz,USA,12561;热带作物生物技术国家重点实验室,海口,571101;中国热带农业科学院环境与植物保护研究所,儋州,571737;热带作物生物技术国家重点实验室,海口,571101;中国热带农业科学院环境与植物保护研究所,儋州,571737【正文语种】中文【中图分类】Q16Brontispa longissima (Gestro) was originally described from the Am Islands (Maluku Province) [1].It was native to Indonesia,and also to Papua New Guinea,including the Bismarck Archipelago.It has now spread widely in Australia,Pacific Islands and Asia such asSingapore,Vietnam,Nauru,Thailand and Maldives[1].From 2002,this beetle has spread to Hainan Island.Because of the absence of natural antagonists,it has become a serious and devastating pest on the island.At the beginning,it was prevalent in several important coconut-growing regions such as Haikou,Wenchang and Sanya.In recent years,it has broken out to cover 17 counties and cities,almost 2 million coconut palms were attacked and resulting in heavy losses.Thus,emergency operations were carried out assisted by local governments.It has been proved effectively by introduction and enhancement of parasitoids-Tetrastichus brontispae,Asecodes hispinarum and spraying of entomopathogenic fungi[1].Chemical control was also recommended by using relatively safe pesticides to eliminate the pest before biological control.The entomopathogenic fungus,Metarhizium has been proved effectively in controlling more than 200 species of insect pests[2].However,there are no reports about Metarhizium strains isolated from other insect pests whether can be used to control B.longissima effectively in Hainan.The exploratory surveys for parasitoids in the original home of this pest and the isolation of highly virulent strains have been suggested.In this study,a number of host cadavers of B.longissima were found on the tree,and mycelia and conidia were collected from the cuticle surfaces.Fungi were isolated on the selective medium,and their identification,virulence to B.longissima in vitro and effects of application in vivo were further tested.1.1 Collection of natural infectious and healthy beetlesCoconut hispid beetles ( B.longissima ) were collected from seriously infected regions including Haikou and Wenchang of Hainan Island.Foliar buds of coconut palms infected with beetles were cut down and a number of larvae and adults were picked out.Cadavers were collected and transferred in sterilized Petri dishes,and the healthy ones were transferred into glass bottles for further rearing.Cadavers were used to isolate entomopathogenic fungi,and the living insects were used to perform pathogenic tests for isolated fungus.1.2 Preparation of culture mediaFour different media were prepared:PDA (potato dextrose agar),PPDA ( PDA with 2% peptone),V8A (10% V8 vegetable juice agar) [3],and OMA ( oatmeal agar) medium.DOA medium was prepared as the selective medium by adding selective elements for screening entomopathogenicfungi.The selective elements of DOA medium mainly contained Dodine200μg/ml,Penicillin 100μg/ml,Streptomycin 50μg/ml [4],Carbendazim0.8μg/ml and Metalaxyl 0.5μg/ml.1.3 Isolation and identification of Metarhizium anisopliaeCadavers of coconut hispid beetles including larvae and adults were collected from Wenchang and Haikou.Mycelia or conidia were isolated with sterile needles from cuticle of cadavers and transferred into a 1.5 ml Eppendorf tube containing sterile distilled water.Conidial suspension(5×104 conidia/ml) was prepared and 100μl of it were transferred and incubated on a Petri dish containing 15ml DOA medium at temperature of (28±2)℃.Three days later,daily checks were performed up to the tenth day.Discs of the fungal colonies appearing on each Petri dish was then transferred to test tubes containing PPDA medium,and incubated under the same conditions described above.Purification of every strain was carried out under an inverted Olympus microscope.A single conidium on the surface of water agar medium was picked up with a sterile capillary tube and transferred onto PPDA medium for obtaining pure culture.Identification of the isolates were performed at a species level according to the papers or books such as published by Pu zhelong[2],Hawksworth[5],Roddam et al[6],and Zimmerman[7].The strains were identified by evaluation of the macromorphological characteristics of the colonies ,such as diameter,color,mycelia texture and other fearures,as well as the micromorphological characteristics,such as the aspects ofhypha,conidiophore,conidial fructification and conidium,which were detected under biological microscope.1.4 Tests on biological characters of isolated strains1.4.1 Tests on colony growth rate and conidia production on different culture mediumAll strains were cultured in Petri dishes containing PDA,PPDA,V8A and OMA medium.After five days of incubation,the daily colony diameter was measured by crisscross method up to the tenth day.The average of the increases in colony diameter per day was considered as the growth rate. Conidia were harvested from 15 days old cultures by surface scraping.Homogenous spore suspensions were made by placing harvested spores in 20 ml sterile distilled water in glass bottles containing 0.1% Tween80,and then were agitated on a vortex for 30 Sec.Spore concentrations were determined using a haemocytometer.The procedure was replicated three times for each strain.1.4.2 Tests on colony macromorphological aspectsAll isolates were cultured on the optimal medium (PPDA) according to above results.After 4 days,daily checks of different colony color and mycelia texture were carried out and documented by photography.The sizes of 50 conidia of each strain were measured under a Leica microscope and average values were compared for all strains.1.5 Tests on virulence to B.longissima and preliminary applying on adults in vivoAll isolates were cultured and maintained on PPDA medium.Conidia were harvested and its suspensions (1×108conidia ml-1 ) were prepared.Twentyadults or larvae of B.longissima reared for 1 week were sprayed with 3 ml conidial suspension respectively and then placed into glass bottles and fed by fresh coconut leaf buds (replaced once every 3 days).The glass bottle was capped with a plastic lid with several tiny punctures to allow aeration for insects living and incubated at ambient temperatures of 25-30℃.Each isolate was repeated 3 times on adults or larvae.The control was only sprayed with 3 ml of sterile water containing 0.1% Tween-80.After rearing for 9 days,dead beetles was put down and mortalities were calculated for every strain.All dead beetles were transferred to Petri dishes in humid ambient to see whether have mycosis growth on the surface. Preliminary effect tests of application on adults in vivo were carried out in Sanjiang farm of Haikou,where coconut palms were infected seriously by B.longissima.The conidial suspension were prepared by mixing 80% plant oil and 20% paraffine,and adjusted to the conidia concentratio n of ≥1×109 conidia ml-1.The suspensions (20ml per-tree) were brushed with brush on the surface of bud leaves of 10 coconut palms two times after an interval of 20 days.The trials were repricated three times in the same experiment area.Controls were also brushed only by 80% plant oil and 20% paraffine.2.1 Strains isolationFrom a number of cadavers (Fig.1),21 filamentous fungal colonies were isolated on DOA medium from different Petri dish (Fig.2).The isolate from different Petri dish was regarded as a strain,respectively.All of these strains were purified and transferred to PPDA medium for further identification.As a result,all isolates were identified as Metarhizium anisopliae,and no anyother species such as Beauveria spp or others were detected. According to the macromorphological characteristics,we found there were two distinct strains based on the differences in colony morphology and acervuli,and were thus recorded as MA(13 strains) and MB(8 strains) isolates.After 5 days of incubation,The colony of all MA strains on PPDA medium were almost entirely covered with acervuli but few acervuli in all MB strains under the same conditions( Fig.3).However,there was no difference in conidial shape between the two kinds of isolates:cylindrical with obtuse ends ,slightly narrowing in the center( Fig.4),and the conidial width(2.5-3μm) and length (5-8μm)as well as the structure of conceptacles were no different from those described by Pu Zhelong.2.2 Screening of optimal culture medium for isolates with colony growth and conidia productionThe results showed PPDA was the optimal culture medium for colony growth and conidia production for both MA and MB strains.The results were summarized in Fig.5 (only show testing results of MA4 and MB4,the others were as same as the two strains).Colony growth rate on PPDA media was faster than the others,especially during 1 to 3days.Otherwise,there was no significantly difference in colony growth rate on other media.The average conidial production of MA4(others were as the same as th is strain) strain was 2.62×109 conidia ml-1 on PPDA media,which was significantly more than 2.68×108 conidia ml-1 onOMA ,1.96×108 conidia ml-1 on PDA and 1.85×108 conidia ml-1onV8.However,the last three media showed no significant differences inconidial production(Fig.6).2.3 Pathogenicity to B.longissima and application effects in vivoAll strains both MA(13 strains) and MB(8 strains) were showed pathogenic to B.longissima according to the testing results.The mortality ranged from 52% to 100% either in adults or larvae,showing significantly higher than the control.Isolates of MA2,MA3,MA4,MA8,MA9,MB2,MB4,MB5,MB6 and MB7,were high pathogenic to B.longissima (Table 1) among of all strains.In moist condition,conidia and mycelia were observed on the surface of cadavers and identified as the same fungi species used in the initial inoculation.The mortality of the control was only 20% and 30% in adults and larvae,respectively,and few mycelia growth were found on cadavers.The results showed that the strains isolated from natural occurring endomopathogenic fungi in B.longissima were pathogenic to B.longissima and most of strains were highly virulent to the beetles.In this study,the high virulent strain of MA4 were employed to apply on controlling B.longissima in vivo.The averages of adult beetles population taken before application varied from 55.2 to 19.0 after 90 days and to 11.8 after 120 days,and cadavers with mycosis were found at more than 90% trees,while few changes occurred at controls,which the adult beetles population density taken varied from 45.2 to 48.0 and to 46.7 at the same time detection (Fig.7),and few cadavers were found.From the cadavers of B.longissima collected from Haikou and Wenchang of Hainan,21 filamentous fungi were isolated and identified as Metarhizium anisopliae.PPDA was the optimal culture medium for colonygrowth and conidia production among PPDA,PDA,OMA and V8A.M.anisopliae has already been isolated naturally from soils[6,8,9] and insects[10～12] in different regions of the world.And M.anisopliae had ever been applied to against the coconut leaf hispid [13,14].However,it was the first time that strains of M.anisopliae were isolated from B.longissima in Hainan Island of China.Our studies proved that these isolates were pathogenic to B.longissima based on the symptoms after inoculation of the beetles with these isolates.In pathogenic tests,the mortality in treatments was significantly higher than the control.In addition,fungal outgrowth was found on the surface of dead beetles in humid condition,but not in the control.A high pathogenic strain of MA4 were employed to apply in vivo,with which caused significantly reduction in the adult population.The results showed the great potential of M.anisopliae as a means of biological against the coconut leaf hispid.Recently,the effective strategies on B.longissima control were performed in Hainan,involved rearing of natural antagonists of T.brontispae,A.hispinarum and spraying insecticides.As an important part of integrated pest management (IPM) strategy,M.anisopliae could be widely used due to few harmful side effects caused by it.M.anisopliae can be considered as promising for biocontrol on coconut hispid beetles by reducing the pest population and lessening the dependence on chemical control.*通讯作者Correspondingauthor.E-mail:***************【相关文献】[1]Singh S P,Rethinam P.Coconut hispid beetle Brontispa longissima (Gestro) (Coleoptera:Chrysomelidae).Cord,2004,Vol.20,1～20.[2]Pu zhelong.Insect pathology.Guangzhou:Guangdong Science&Technology,Press,1994.8,360.[3]Ho H H,Ann P J,Chang H S.The Genus Phytophthora in Taiwan.Taipei.Published by Institute of Botany,Academia Sinica,1995,1～86.[4]Chase A R,Osborme L S,Ferguson V M.Selective isolation of the entomopathogenic fungi,Beauveria bassiana and Metarhizium anisopliae from an artificial potting medium.Flo Entol,1986,69:285～292.[5]Hawksworth D L.Mycologist's handbook.England:CAB Press,1971.[6]Roddam L F,Rath A C.Isolation and characterization of Metarhizium anisopliae and Beauveria bassiana from Subantartic Macquarie Island.J Invertebr Pathol,1997,69:285～288.[7]Zimmerman G.The entomopathogenic fungus Metarhizium anisopliae and its potential as a biological agent.Pesticide Science,1993,37:375～379.[8]Tarasco E,Bievre C de,Papierok B,et al.Occurrence of entomopathogenic fungi in soils in Southern Italy.Entomologica,Bari,1997,31:157～166.[9]Tigano-Milani M S,Faria M R de,Martins I,et al.Ocorr ncia natural de Beauveria bassiana (Bals) Vuill,Metarhizium anisopliae (Metsch) Sorok.e Paecilomyces sp.em solos de diferentes regi es do Brasil.An Soc Entomol Brasil,1993,22:391～393.[10]Hern ndez-Crespo P,Santiago-lvarez C.Entomopathogenic fungi associated with natural populations of the Moroccan Locust Dociostaurus maroccanus (Thunberg) (Orthoptera:Gomphocerinae) and other Acridoidea in Spain.Biocontrol Science and Technology,1997,7:357～363.[11]Poprawiski T J,Yule W N.Incidence of fungi in natural populations of Phyllophaga spp.and susceptibility of Phyllophaga anxia (Le Conte) (COL.Scarabalidae) to Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina).J Appl Ent,1991,112:359～365. [12]Samsinakova A.Beauveria globulifera (Speg) Pic Jako Parasit Klistete Ixodes ricinus.L Zoologie List,1956,6:329 330.[13]Marshall K J,Vargo A M ,Fatuesi S .Application of a New Strain of Metarhizium anisopliae (FungiImperfecti) as a Means of Biological Control Against the Coconut Leaf Hispid,Brontispa Longissima (Coleoptera:Hispidae) in Samoa.Research Extension Series College of Tropical Agriculture and Human Resourse,University of Hawaii,Cooperative Extension Service (USA),1991,11:137～140.[14]Wikardi E A.Protects for controlling coconut hispids with Metarhizium sp.[Plesis pareichei,and Brontispa longissima].Pemberitaan Lembaga TanamanInd.Bogor:Lembaga,1982.8(44):35～38.。

金龟子绿僵菌的液固双相发酵研究

河南农业科学，２０１３，４２（２）：９３－９７
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金龟子绿僵菌的液固双相发酵研究
许天委，张世清，黄俊生。
的优点，先生产大量的菌丝或芽生孢子，缩短了菌
子产量和菌丝生长量，试验设计３个重复，以筛选最
优的液体发酵培养基。
１．２．２固体培养基的筛选将菌块移接到最优的
ｓｈａｌｌｏｗｔｒａｙｆｅｒｍｅｎｔａｔｉｏｎ，ｗｈｅｎｓｏｌｉｄｃｕｌｔｕｒｅｍｅｄｉｕｍＦ（ｒｉｃｅ３００ｇ，ｂｒｏｋｅｎｒｉｃｅ６００ｇ，Ｈ２Ｏ５０９，６，
Ａｂｓｔｒａｃｔ：Ｍｅｔａｒｈｉｚｉｕｍａｎｉｓｏｐｌｉａｅｉｓａｎｉｍｐｏｒｔａｎｔｐｅｓｔ — ｐａｔｈｏｇｅｎｉｃｆｕｎｇｕｓ．Ｔｈｉｓｅｘｐｅｒｉｍｅｎｔｃａｒ－
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生孢子产量和菌丝生长量均最高，分别达到４．３ ×１Ｏ。个／Ｌ、５．９９ｇ／Ｌ。固体发酵时，浅盘发酵方法优

金龟子绿僵菌选择性培养基的筛选

维普资讯
一
７一６
江苏农业科学
２００７年第５期
金龟子绿僵菌选择性培养基的筛选
程子路，詹儒林，许天委，宋妍，郭立佳张世清黄俊生，，
（．中国热带农业科学院环境与植物保护研究所，１海南儋州５１３；．中国热带农业科学院南亚热带作物研究所，７７７２广东湛江５４９）２０１
２１１选择性培养基上的成菌落数．．
对绿僵菌在
选择性培养基上形成的目标菌落数量进行方差分
析，发现Ｉ号培养基与 Ⅵ号培养基差异不显著，与其
余培养基差异显著。 Ⅱ号培养基与Ⅶ号培养基差异
不显著，与其余培养基差异显著。 Ⅲ号培养基除与
按表１配制各种培养基，ＰＤ以ＰＡ培养基作为
对照。各培养基灭菌后倒入直径为９ｃ的培养皿ｍ中，制成平板备用。
１４方法．
数量的多少，影响其防治持效性的关键。土壤是是
一
个相对复杂的环境，病原菌受到诸多因素的影响，
Ⅷ 号培养基差异不显著外，与其他培养基差异都显
维普资讯
程子路等：金龟子绿僵菌选择性培养基的筛选
Ⅱ Ⅲ Ⅳ Ｖ Ⅵ Ⅶ ｍ
一７一７
燕麦片３，５ｇ琼脂５ｇ结晶紫１０ｇｍ，，０／ｌ多菌灵０８／ｌ．ｇｍ，青霉素１ｇｍ，００／ｌ硫酸链霉素１０／ｌ１０ｌ０ｇｍ，０ｍ水０

高毒力杀蝗绿僵菌的紫外线诱变选育

高毒力杀蝗绿僵菌的紫外线诱变选育程辉彩1,敦冬梅2,张丽萍1,张根伟1,董超1,崔冠慧1(1.河北省生物研究所,河北石家庄050081;2.石家庄职业技术学院,河北石家庄050081)摘要:以金龟子绿僵菌M 105复壮菌株M 1053为出发菌株,通过紫外线诱变选育,获得P r1酶活是出发菌株1.36倍的诱变株M 105-52。

经测定,诱变株的遗传性能稳定,紫外线抗性和杀蝗毒力均有显著提高。

关键词:杀蝗绿僵菌;紫外线;诱变选育中图分类号:S433 2 文献标志码:A 文章编号:1002-1302(2008)03-0106-02收稿日期:2007-10-08基金项目:河北省科技攻关项目(编号:05820121D )。

作者简介:程辉彩(1974 ),女,河北栾城人,硕士,助理研究员,主要从事微生物农药研究。

Te:l (0311)83014879;E -m ai:l hu icai ch eng @163.co m 。

蝗灾危害范围广泛,与水灾、旱灾并称为严重威胁我国农牧业生产、影响人民生活的三大自然灾害。

长期以来,我国防治蝗灾仍然以化学农药为主,如有机磷类、菊酯类及其复配制剂等,化学农药在蝗灾的控制中发挥了重要作用,但同时也给环境安全带来了较大隐患。

绿僵菌(M etarhiz i u m )是一种有效的蝗虫生防制剂,为蝗虫体内寄生病原真菌,对蝗蝻和成虫均有效。

作为专用性生物杀虫剂,绿僵菌对昆虫天敌无害,无二次中毒,不污染环境,可持续有效控制蝗虫种群在经济受害水平以下[1-6]。

紫外线诱变是一种使用时间长、效果好、设备简单、所需费用少、突变随机性强、可在短时间内获得大量突变体的常用诱变选育方法。

本试验以研究室保存的M 105菌株虫体复壮后进行紫外线诱变,以选育高毒力杀蝗绿僵菌菌株,为制备田间具有高防效菌剂奠定基础。

1 材料与方法1.1 试验材料1.1.1 菌种金龟子绿僵菌(M etarhizium anisop li ae )M 105,为本研究室保存。

不同来源金龟子绿僵菌菌株生物学特性

不同来源金龟子绿僵菌菌株生物学特性苏文晶【摘要】选择不同地理、寄主来源的15株金龟子绿僵菌(Metarhizium ankopliae)菌株,对各菌株菌落形态、生长速度、产孢量等生物学性状进行比较.结果表明:(1)根据菌丝质地和颜色、产孢能力、孢子颜色等将15株菌株分为6类,其中A、B、C类菌株分别有3、3、6株,D、E、F各有1株.(2)不同金龟子绿僵菌菌株的生长速度在初期(10 d以内)差异显著,MaQZ-01、MaJGZ-01和MaFZ-0,菌株10 d时菌落直径均超过6 cm,显著快于其他菌株;到生长后期(15 d),由于进入了产孢阶段,除Ma-20、MaJGSTR-01菌落直径较小外,其他菌株差异不显著.(3)不同菌株产孢量差异显著,MaQZ-01、MaZPTR-04和MaSHTR-05菌株产孢量显著高于其他菌株,分别达到0.147×109、0.145×109和0.137×109个·cm-2.综合生长速度与产孢量两项指标,MaQZ-01菌株具备生长迅速、产孢量大等优良生产性能,可作为优良菌株进一步在害虫防治中加以利用.【期刊名称】《亚热带农业研究》【年(卷),期】2015(011)002【总页数】5页(P123-127)【关键词】金龟子绿僵菌;生物学特性;菌落形态;生长速度;产孢量【作者】苏文晶【作者单位】泉州市森林病虫防治检疫站,福建泉州362000【正文语种】中文【中图分类】S476.12Key words:Metarhiziumanisopliae; biological characteristics; colonial morphology; growth speed; sporulation绿僵菌[Metarhizium (Metsch.) Sorokin]是虫生真菌的重要类群之一，寄主范围广，能寄生8个目30个科约200种昆虫、螨类及线虫[1]。

金龟子绿僵菌发酵培养基响应面优化-精品文档资料

金龟子绿僵菌发酵培养基响应面优化金龟子绿僵菌（Metarhizium anisopliae）是一类重要的昆虫病原真菌，它作为生物防治制剂具有环境安全性好、昆虫不易产生抗性等特点。

此外，绿僵菌的寄主广泛性和广谱性使得其成为一种研究昆虫病原真菌侵染寄主过程的重要模式生物[1-2]。

目前，植物病害在全球范围内每年造成高达数千亿美元的损失，而其中又以昆虫病害最为严重。

金龟子绿僵菌具有寄生范围广、无残毒、后效期长等特点，已成为国内外杀菌、除虫的一线药物之一[3-4]。

然而，在金龟子绿僵菌发酵生产过程中，菌体生物量低，生产成本高，发酵周期长等问题限制了其推广应用[5-7]；因此，对金龟子绿僵菌发酵培养基优化及条件探索具有重大意义。

本研究以金龟子绿僵菌NJYHWG 3820为试验菌株，应用响应面法系统考察了影响金龟子绿僵菌生物量的培养基成分，得到了最佳培养基配方，从而提高了生物量。

1 材料与方法1.1 菌种金龟子绿僵菌：Metarhizium anisopliae NJYHWG 3820，由笔者课题组保藏。

1.2 培养基斜面培养基：马铃薯200 g/L，蔗糖20 g/L，琼脂15～20 g/L，自然pH值。

种子培养基：蔗糖10 g/L，酵母膏10 g/L，KH2PO4 5 g/L，MgSO4 2 g/L，pH值6.5。

发酵培养基：蔗糖10 g/L，酵母膏10 g/L，KH2PO4 5 g/L，MgSO4 2 g/L，pH值6.5。

1.3 主要试剂和仪器蔗糖、葡萄糖、麦芽糖、乳糖、木糖、甘油、可溶性淀粉、蛋白胨、酵母膏、牛肉浸膏、甘氨酸、琼脂粉，由国药集团化学试剂XX公司提供；FeSO4?7H2O、MgSO4?7H2O、NaCl、K2HPO4?3H2O、CaCl2、NaNO3、CuSO4、CaCO3，由广东汕头市西陇化工厂提供；马铃薯，市售。

电热恒温培养箱，上海跃进医疗器械厂；灭菌锅，华粤行仪器XX公司；医用净化工作台，苏州净化设备厂；鼓风电热恒温干燥箱，上海试验仪器厂XX公司；台式高速离心机，德国艾本德股份公司；电子天平，瑞士梅特勒-托利多仪器XX公司；基础分析型纯水机，青岛富勒姆科技XX公司。

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金龟子绿僵菌选择性培养基的筛选
作者：程子路，詹儒林，许天委，宋妍，郭立佳，张世清，黄俊生
作者单位：程子路,许天委,宋妍,郭立佳,张世清,黄俊生(中国热带农业科学院环境与植物保护研究所,海南儋州,571737)，詹儒林(中国热带农业科学院环境与植物保护研究所,海南儋州
,571737;中国热带农业科学院南亚热带作物研究所,广东湛江,524091)
刊名：
江苏农业科学
英文刊名：JIANGSU AGRICULTURAL SCIENCES
年，卷(期)：2007(5)
被引用次数：2次
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2.宋漳.景云.蔡和谦应用绿僵菌防治马尾松毛虫初探 1997(02)
3.江英成绿僵菌和白僵菌侵染马尾松毛虫试验比较[期刊论文]-浙江林学院学报 2000(04)
4.Weiner J E B M.Kennedy C Growth and variability in crowded and uncrowded populations of dwarf marigold(tagetes patula) 1990
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8.张跃西.钟章成亚热带次生常绿阔叶林乔木优势种邻体干扰竞争模型的研究 1997
9.张跃西.钟章成亚热带次生常绿阔叶林优势种间的竞争效应与竞争反应[期刊论文]-应用与环境生物学报
2003(04)
10.史红霞.胡明龙球孢白僵菌选择性培养基的筛选和检测应用[期刊论文]-浙江林学院学报 2002(02)
11.王滨.樊美珍球孢白僵菌选择性培养基的筛选[期刊论文]-安徽农业大学学报 2000(01)
12.Veen K H.Ferron P A selective medium for the isolation of Beauveria tenella and of Metarhizium anisopliae 1966
13.樊美珍.李增智绿僵菌在土壤中的延续及控制桃小食心虫的潜力 1996(01)
1.孔琼.袁盛勇.郭亚力.张宏瑞.田学军.李河球孢白僵菌MZ041016菌株及其与化学农药混配对禾谷缢管蚜的毒力测定[期刊论文]-江苏农业科学 2008(4)
2.程辉彩.敦冬梅.张丽萍.张根伟.董超.崔冠慧高毒力杀蝗绿僵菌的紫外线诱变选育[期刊论文]-江苏农业科学2008(3)
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