芒果炭疽病菌抗药性基因tub2的克隆及其转化金龟子绿僵菌的初步研究
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摘要
芒果炭疽病(Colletotrichum gloeosporioides Penz.)是芒果重要的病害。多菌灵、甲基托布津、苯菌灵、特克多等苯并咪唑类杀菌剂(benzimidazoles)对该病害具有良好的防治效果。但此类杀菌剂是单作用位点的内吸性杀菌剂,若不合理使用,容易使病菌产生抗药性。
从我国芒果主产区采集炭疽病菌300多个菌株,对多菌灵的敏感性测定结果表明,芒果炭疽病菌在我国热区芒果上已产生了高水平的抗药性,有些菌株在多菌灵500μg/mL的PDA培养基上仍可正常生长,无法测定其EC50和EC90值,确认为质量性状抗性,且无性繁殖12代后,其抗性依然不变。对抗性菌株的性质测定结果表明,病菌的抗药性突变并不影响其各项生物学功能和寄生适合度。
研究表明,大部分病原菌对多菌灵的抗性突变都与?-微管蛋白基因的单碱基突变有关,尤其发生在?-微管蛋白(tub2)第198位上的氨基酸突变是抗药性形成的主要原因。本研究对芒果炭疽病菌抗、感菌株两条?-微管蛋白基因tub1和tub2的全序列分别进行克隆和测序,对其序列分别进行比对,发现抗药性菌株中两条β-微管蛋白基因都发生了突变,其中发生在tub1中的氨基酸突变与抗药性的发生没有相关性。而在抗性菌株的tub2中,发现第198位氨基酸的突变具有一定的规律性,即在所有的抗性菌株中在此位点都突变为丙氨酸(Ala-A),而在敏感菌株中均为谷氨酸(Glu-E),推断tub2中第198位氨基酸的突变与病菌抗药性的形成有紧密的相关性。通过等位特异PCR和酶切的方法检测发现在其它抗药性菌株中也存在同样的现象,因此,认为tub2基因第198位氨基酸的突变是芒果炭疽病菌抗药性形成的主要原因。至此,我们基本探明了芒果炭疽病菌抗药性形成的分子机制,同时,也从中克隆了抗药性基因,突变了的tub2基因即为多菌灵抗性基因,此基因的克隆为绿僵菌的遗传转化打下了重要的基础。
绿僵菌(Metarhizium.spp)是一种虫生真菌,能寄生8个目30科约200种昆虫、螨类及线虫。它致病力强、效果好,对人、畜、作物及害虫天敌无毒害,是当今虫生真菌研究的主要对象之一。我们也从海南的椰心叶甲中分离到了21个绿僵菌菌株,根据菌落、分生孢子及产孢结构的特征,初步鉴定为金龟子绿僵菌,并根据菌落的不同特征,把这些菌株分成了MA和MB两类。为了对这些菌株的分类作进一步的验证和定位,对MA和MB类菌株rDNA的ITS1-5.8S-ITS2分别进行了克隆和测序,使用MEGA3.1软件构建了绿僵菌属种或变种的系统发育树。结果显示,所构建的最大简约树和邻接树均将MA和MB菌株聚在金龟子绿僵菌小孢变种分支中,再对照其形态特征,最终将这些菌株鉴定为金龟子绿僵菌小孢变种(Metarhizium anisopliae var. anisopliae)。经测定,这些菌株对椰心叶甲的成虫和幼虫均有较强的致病力,致死率最高均可达100%。其中MA4菌株被用于田间的防治,取得了明显的成效,说明这些菌株对椰心叶甲具有很好的生防潜力。
尽管绿缰菌对多种害虫有杀灭效果,但环境条件对绿僵菌的影响也较大,如温度、湿度、紫外光、杀菌剂、杀虫剂等。由于绿僵菌在应用时为分生孢子活菌制剂,因此,杀真菌剂对其有致命的影响。经测定,绿僵菌在多菌灵5μg/mL下便受完全抑制,而多菌灵在田间实际施用浓度为500-1000μg/mL,只要两者同时施用,绿僵菌将难以发挥其对害虫应有的毒力,因此,使绿僵菌获得对多菌灵的抗性至关重要。由于苯并咪唑类杀菌剂低毒、内吸性强、杀菌普广等特点,使其得到了广泛应用,此类药剂施用后,都会产生共同的衍生物多菌灵或它的乙基同系物乙基多菌灵,是与病菌相互作用的最后产物。因此,研究对多菌灵的抗性是研究绿僵菌对此类杀菌剂抗性的关键,也是绿僵菌抗药性改良的主要目标之一。
要获得绿僵菌抗药性菌株,可采取下述途径:一是自然筛选,即从田间收集大量的菌株然后进行筛选。目前,在海南受病菌自然感染的椰心叶甲中只分离到几十个菌株,经测定,这些菌株对多菌灵都较敏感。当然,菌株数量越大,成功筛选的几率也越高,但要从海南岛受害的几百万株椰子树中搜集椰心叶甲僵虫,并从中分离出更多菌株的工作量很大,而且也不一定就能筛选到抗药性菌株,因此,自然筛选的方法难度较大。二是抗性诱变,通过紫外诱变获得了对多菌灵的抗性菌株,在600μg/mL的条件下仍可缓慢生长,抗性提高120倍以上,但这些诱变体在紫外光的再次照射后均发生了回复突变。因此,若把这些菌株用于田间的防治,由于太阳紫外线的照射,也可能表现出同样的不稳定性。因此,用紫外光诱变获得的抗性菌株不适于田间的推广应用。三是本项目采用的遗传转化方法,利用芒果炭疽病菌多菌灵抗性基因(tub2),构建合适的载体,以根癌土壤杆菌介导的方法,把该基因导入到绿僵菌中,使之也获得对多菌灵的抗性。
在载体构建中,以tub2基因、质粒pCAMBIA1300和pBHt2(含TrpC)为基础,真菌强启动子TrpC与tub2以套叠PCR的方法相连,其连接产物为TrpC-tub2,同时, pCAMBIA1300以Bst XI/XhoI酶切去除CaMV35S-hph获得线性大片段,然后以此线性大片段与TrpC-tub2相连。由于tub2序列内含有Bst XI和Xho I等酶切位点,无法在TrpC-tub2的两端设计合适的粘端直接与pCAMBIA1300线性大片段相连。因此,TrpC-tub2与pCAMBIA1300大片段的连接只能先补平后再以平端连接,然后以酶切法鉴定其插入方向的正确性。载体成功构建后转化根癌土壤杆菌GV3103,即用于绿僵菌的遗传转化。在这个载体中,多菌灵抗性基因既作为转化的目的基因,同时也为筛选标记基因。
对绿僵菌遗传转化体系进行了多处理比较,获得了优化的转化体系:多菌灵浓度最低为4.0μg/mL;头孢霉素(Cef)的最佳使用浓度为250μg/mL;溶解绿僵菌的最佳溶剂为0.01%的Triton X-100;绿僵菌分生孢子和根癌土壤杆菌浓度分别以1×106～1×108conidia/mL和0.60～0.75OD600为宜、共培时间为2-3d、IM固体培养基含AS200μmol/L;AS处理根癌土壤杆菌最佳浓度为450μmol/L、最佳时间为6h。
对转化子进行了进一步的培养、分析,发现了一些菌落形态、颜色突变的转化子,另有致病力降低的转化子,这些现象说明了T-DNA可能已插入到绿僵菌功能基因区中。对这些转化子进行了PCR鉴定,并对其PCR产物进行克隆和测序,验证和确认了包括TrpC-tub2序列的T-DNA已插入到绿僵菌的基因组中。
转化子对多菌灵的敏感性测定结果表明,抗性只比原来提高2.8倍,虽然无性繁殖12代后仍可维持原有的抗性,但在多菌灵10μg/mL中已完全不能生长,说明了导入的tub2基因并没有得到理想的表达,其原因有待于进一步研究。
总体而言,本项目基本探明了芒果炭疽病菌抗药性形成的分子机理,克隆了多菌灵抗性基因tub2,并成功构建了以多菌灵为筛选标记的真菌转化载体、初步探索了以根癌土壤杆菌为介导的金龟子绿僵菌的遗传转化,为下一步的研究打下了重要基础。
Mango anthracnose is a serious disease caused by Colletotrichum gloeosporioides which is prevalent in tropical regions of China. The disease can be well controlled by benzimidazoles fungicides such as carbendazim, thiophanate-methyl, benomyl, thiabendazole, etc. However, single-site mutation occurred commonly leading to resistance to the fungicide, especially when the fungicide was applied indiscriminately.
During the period 2003 to 2006 more than 300 isolates of C. gloeosporioides from mango-growing regions in China were tested for resistance to carbendazim. The results showed that resistance to the fungicide had developed in the field. The resistant isolates were able to grow normally on PDA medium with carbendazim at a high level of 500μg/mL.The resistance to carbendazim was stable and inherited unchanged through 12 consecutive generations on carbendazim-free PDA medium. There was no apparent correlation between biological characteristics of the fungal isolates with carbendazim-resistance .
Numerous studies showed that single base-pair mutation of ?-tubulin gene especially at 198 amino acid of the tub2 was closely correlated with carbendazim-resistance. The genes of tub1 and tub2 of resistant(ZR46,ZR43,ZR51) and sensitive(ZS19,ZS29) strains were cloned, sequenced and aligned. The results showed a few base-pair mutations in tub1 and tub2 genes. No definite correlation between mutation and fungicide resistance could be detected in tub1 gene from resistant isolates. However, for tub2 gene, mutation of the amino acid in codon 198 resulted in the formation of Ala-A in all resistant isolates and Glu-A in all sensitive isolates. The mutation of amino acid 198 was detected by allele-specific PCR and enzyme assays and similar results were obtained in other resistant isolates. The results strongly suggested that amino acid change in codon 198 of tub2 played a leading role in conferring carbendazim-resistance to mango anthracnose in South China. Furthermore, the resistant gene of tub2 was cloned and used to construct fungicide-resistant strains of Metarhizium anisopliae, an entomopathogenic fungus.
Species of Metarhizium have been proved effective in controlling more than 200 species of insect pests, and was widely used due to its harmless effect on human, animals, plants and other insects. In our study, 21 strains of Metarhizium were isolated from more than 200 cadavers of the host, Brontispa longissima Gestro adults and lavae in Wenchang and Haikou of Hainan. These isolates were identified initially as M. anisopliae based on the colony color, mycelial texture, and the characteristics of hyphae, conidiophores, conidia and conidial fructification. Two different groups of isolates (MA and MB) could be distinguished only based on their colony characters. The ITS1-5.8S-ITS2 of rDNA from MA and MB strains were cloned and sequenced for identification at the species level. The maximum parsimony tree and neighbor-joining tree were established using the software of MEGA3.1.The results showed that both MA and MB strains should be assigned to Metarhizium anisopliae var. anisopliae. These strains were highly pathogenic to adults and larvae of coconut hispid beetles.
However, there were some environmental factors diminishing the effectiveness of Metarhizium in the field, such as temperature, UV light, insecticides and especially fungicides. So it was essential to construct fungicide-resistant strains of the fungus.
The sensitivity of all 21 isolates of M. anisoplieae var. anisopliae to the fungicide, carbendazim was tested. The results showed that these isolates were highly sensitive (MIC<5μg/mL) to carbendazim. It would not be practical trying to screen for resistant strains from the field because of the scarcity of cadavers of hispid beetles. Only a few were collected from millions of thousands of coconut palms on Hainan island.
Alteratively, UV irradiation was considered a good method to improve the resistance of the isolates. In our study several carbendazim-resistant stains were obtained by UV- induced mutation. Some strains could grow slowly on PDA agar medium even with a high concentration of carbendazim (600μg/mL) .Thus the resistant level had increased 120 times when compared with the parental strains. However, reverse-mutation occurred when these“resistant strains”were illuminated again with UV light. So these strains with unstable resistance could not be used in the field due to the presence of UV in the sunlight.
Another approach was to transform the sensitive strains of Metarhizium with the transfer of a carbendazim-resistant gene(tub2) mediated by a bacterium, Agrobacterium tumefaciens. The binary vector pTUB2 was constructed based on the vector pCAMBIA1300, promotor TrpC cloned from vector pBHt2 and carbendazim-resistant gene tub2 cloned from resistant isolates of Colletotrichum gloeosporioides. TrpC-tub2 cassette was amplified by Gene Splicing by Overlap Extension(gene SOEing) of PCR. The hph gene cassette of pCAMBIA1300 was deleted by Bst XI/Xho I digestion,and its large line fragment was ligated with TrpC-tub2 into vector of pTUB2 using the blunt-end because it contained Bst XI and Xho I in the tub2 sequence.An enzyme assay was employed to detect the ligation direction. Then the pTUB2 was mobilized into A. tumefaciens (strain GV3103).
In our study, a highly efficient and stable transformation system of Metarhizium was established by optimizing the A. tumefaciens-mediated procedure. Firstly, the effects of conidial concentration on transformation efficiency was tested, using a 2 day co-cultivation in the presence of 200μmol/L AS. Secondly, A. tumefaciens incubated for 6h in broth IM medium with 450μmol/L AS before co-cultivation resulted in highest transformation efficiency. Thirdly, A. tumefaciens (OD600:0.60-0.75) broth culture and Metarhizium spore suspension (1×10~6~-1×10~8conidial/mL) were mixed in equal volumes, and 0.01% Triton X-100 was added to dissolve the conidia. Fourthly, maximum efficiency was observed with the carbendazim at 4μg/mL. The transformation efficiency decreased with the increase of the concentration of carbendazim and there was no transformation when the fungicide used was at 10μg/mL.
More than 200 transformants were compared with the parental strains. Differences in the colony color, production and morphology of the spores as well as decreased virulence were found. It is quite feasible that T-DNA was inserted into the functional DNA region of Metarhizium. The transformation was detected by PCR and the fragments were sequenced. The results proved that the tub2 cassette gene had been inserted into the genome of Metarhizium. Thus, the transformation of Metarhizium by the insertion of T-DNA could be readily performed on a large scale using the optimized procedure.
The sensitivity of the transformants to carbendazim was tested. The results showed that the MIC was <10μg/mL and the resistant level increased only 2.8 times when compared with the parental strains. Nevertheless, the acquired resistance was stable and inherited unaltered through 12 consecutive generations on PDA medium. It is very likely that tub2 gene expressed only a low-level resistance after being inserted into the genome of Metarhizium. However, with the transformation vector established successfully and the transformation system optimized, a library of T-DNA mediated mutants can be readily obtained, providing a firm foundation for the investigation of fungicide-resistance and development in M. anisopliae.
从我国芒果主产区采集炭疽病菌300多个菌株,对多菌灵的敏感性测定结果表明,芒果炭疽病菌在我国热区芒果上已产生了高水平的抗药性,有些菌株在多菌灵500μg/mL的PDA培养基上仍可正常生长,无法测定其EC50和EC90值,确认为质量性状抗性,且无性繁殖12代后,其抗性依然不变。对抗性菌株的性质测定结果表明,病菌的抗药性突变并不影响其各项生物学功能和寄生适合度。
研究表明,大部分病原菌对多菌灵的抗性突变都与?-微管蛋白基因的单碱基突变有关,尤其发生在?-微管蛋白(tub2)第198位上的氨基酸突变是抗药性形成的主要原因。本研究对芒果炭疽病菌抗、感菌株两条?-微管蛋白基因tub1和tub2的全序列分别进行克隆和测序,对其序列分别进行比对,发现抗药性菌株中两条β-微管蛋白基因都发生了突变,其中发生在tub1中的氨基酸突变与抗药性的发生没有相关性。而在抗性菌株的tub2中,发现第198位氨基酸的突变具有一定的规律性,即在所有的抗性菌株中在此位点都突变为丙氨酸(Ala-A),而在敏感菌株中均为谷氨酸(Glu-E),推断tub2中第198位氨基酸的突变与病菌抗药性的形成有紧密的相关性。通过等位特异PCR和酶切的方法检测发现在其它抗药性菌株中也存在同样的现象,因此,认为tub2基因第198位氨基酸的突变是芒果炭疽病菌抗药性形成的主要原因。至此,我们基本探明了芒果炭疽病菌抗药性形成的分子机制,同时,也从中克隆了抗药性基因,突变了的tub2基因即为多菌灵抗性基因,此基因的克隆为绿僵菌的遗传转化打下了重要的基础。
绿僵菌(Metarhizium.spp)是一种虫生真菌,能寄生8个目30科约200种昆虫、螨类及线虫。它致病力强、效果好,对人、畜、作物及害虫天敌无毒害,是当今虫生真菌研究的主要对象之一。我们也从海南的椰心叶甲中分离到了21个绿僵菌菌株,根据菌落、分生孢子及产孢结构的特征,初步鉴定为金龟子绿僵菌,并根据菌落的不同特征,把这些菌株分成了MA和MB两类。为了对这些菌株的分类作进一步的验证和定位,对MA和MB类菌株rDNA的ITS1-5.8S-ITS2分别进行了克隆和测序,使用MEGA3.1软件构建了绿僵菌属种或变种的系统发育树。结果显示,所构建的最大简约树和邻接树均将MA和MB菌株聚在金龟子绿僵菌小孢变种分支中,再对照其形态特征,最终将这些菌株鉴定为金龟子绿僵菌小孢变种(Metarhizium anisopliae var. anisopliae)。经测定,这些菌株对椰心叶甲的成虫和幼虫均有较强的致病力,致死率最高均可达100%。其中MA4菌株被用于田间的防治,取得了明显的成效,说明这些菌株对椰心叶甲具有很好的生防潜力。
尽管绿缰菌对多种害虫有杀灭效果,但环境条件对绿僵菌的影响也较大,如温度、湿度、紫外光、杀菌剂、杀虫剂等。由于绿僵菌在应用时为分生孢子活菌制剂,因此,杀真菌剂对其有致命的影响。经测定,绿僵菌在多菌灵5μg/mL下便受完全抑制,而多菌灵在田间实际施用浓度为500-1000μg/mL,只要两者同时施用,绿僵菌将难以发挥其对害虫应有的毒力,因此,使绿僵菌获得对多菌灵的抗性至关重要。由于苯并咪唑类杀菌剂低毒、内吸性强、杀菌普广等特点,使其得到了广泛应用,此类药剂施用后,都会产生共同的衍生物多菌灵或它的乙基同系物乙基多菌灵,是与病菌相互作用的最后产物。因此,研究对多菌灵的抗性是研究绿僵菌对此类杀菌剂抗性的关键,也是绿僵菌抗药性改良的主要目标之一。
要获得绿僵菌抗药性菌株,可采取下述途径:一是自然筛选,即从田间收集大量的菌株然后进行筛选。目前,在海南受病菌自然感染的椰心叶甲中只分离到几十个菌株,经测定,这些菌株对多菌灵都较敏感。当然,菌株数量越大,成功筛选的几率也越高,但要从海南岛受害的几百万株椰子树中搜集椰心叶甲僵虫,并从中分离出更多菌株的工作量很大,而且也不一定就能筛选到抗药性菌株,因此,自然筛选的方法难度较大。二是抗性诱变,通过紫外诱变获得了对多菌灵的抗性菌株,在600μg/mL的条件下仍可缓慢生长,抗性提高120倍以上,但这些诱变体在紫外光的再次照射后均发生了回复突变。因此,若把这些菌株用于田间的防治,由于太阳紫外线的照射,也可能表现出同样的不稳定性。因此,用紫外光诱变获得的抗性菌株不适于田间的推广应用。三是本项目采用的遗传转化方法,利用芒果炭疽病菌多菌灵抗性基因(tub2),构建合适的载体,以根癌土壤杆菌介导的方法,把该基因导入到绿僵菌中,使之也获得对多菌灵的抗性。
在载体构建中,以tub2基因、质粒pCAMBIA1300和pBHt2(含TrpC)为基础,真菌强启动子TrpC与tub2以套叠PCR的方法相连,其连接产物为TrpC-tub2,同时, pCAMBIA1300以Bst XI/XhoI酶切去除CaMV35S-hph获得线性大片段,然后以此线性大片段与TrpC-tub2相连。由于tub2序列内含有Bst XI和Xho I等酶切位点,无法在TrpC-tub2的两端设计合适的粘端直接与pCAMBIA1300线性大片段相连。因此,TrpC-tub2与pCAMBIA1300大片段的连接只能先补平后再以平端连接,然后以酶切法鉴定其插入方向的正确性。载体成功构建后转化根癌土壤杆菌GV3103,即用于绿僵菌的遗传转化。在这个载体中,多菌灵抗性基因既作为转化的目的基因,同时也为筛选标记基因。
对绿僵菌遗传转化体系进行了多处理比较,获得了优化的转化体系:多菌灵浓度最低为4.0μg/mL;头孢霉素(Cef)的最佳使用浓度为250μg/mL;溶解绿僵菌的最佳溶剂为0.01%的Triton X-100;绿僵菌分生孢子和根癌土壤杆菌浓度分别以1×106～1×108conidia/mL和0.60～0.75OD600为宜、共培时间为2-3d、IM固体培养基含AS200μmol/L;AS处理根癌土壤杆菌最佳浓度为450μmol/L、最佳时间为6h。
对转化子进行了进一步的培养、分析,发现了一些菌落形态、颜色突变的转化子,另有致病力降低的转化子,这些现象说明了T-DNA可能已插入到绿僵菌功能基因区中。对这些转化子进行了PCR鉴定,并对其PCR产物进行克隆和测序,验证和确认了包括TrpC-tub2序列的T-DNA已插入到绿僵菌的基因组中。
转化子对多菌灵的敏感性测定结果表明,抗性只比原来提高2.8倍,虽然无性繁殖12代后仍可维持原有的抗性,但在多菌灵10μg/mL中已完全不能生长,说明了导入的tub2基因并没有得到理想的表达,其原因有待于进一步研究。
总体而言,本项目基本探明了芒果炭疽病菌抗药性形成的分子机理,克隆了多菌灵抗性基因tub2,并成功构建了以多菌灵为筛选标记的真菌转化载体、初步探索了以根癌土壤杆菌为介导的金龟子绿僵菌的遗传转化,为下一步的研究打下了重要基础。
Mango anthracnose is a serious disease caused by Colletotrichum gloeosporioides which is prevalent in tropical regions of China. The disease can be well controlled by benzimidazoles fungicides such as carbendazim, thiophanate-methyl, benomyl, thiabendazole, etc. However, single-site mutation occurred commonly leading to resistance to the fungicide, especially when the fungicide was applied indiscriminately.
During the period 2003 to 2006 more than 300 isolates of C. gloeosporioides from mango-growing regions in China were tested for resistance to carbendazim. The results showed that resistance to the fungicide had developed in the field. The resistant isolates were able to grow normally on PDA medium with carbendazim at a high level of 500μg/mL.The resistance to carbendazim was stable and inherited unchanged through 12 consecutive generations on carbendazim-free PDA medium. There was no apparent correlation between biological characteristics of the fungal isolates with carbendazim-resistance .
Numerous studies showed that single base-pair mutation of ?-tubulin gene especially at 198 amino acid of the tub2 was closely correlated with carbendazim-resistance. The genes of tub1 and tub2 of resistant(ZR46,ZR43,ZR51) and sensitive(ZS19,ZS29) strains were cloned, sequenced and aligned. The results showed a few base-pair mutations in tub1 and tub2 genes. No definite correlation between mutation and fungicide resistance could be detected in tub1 gene from resistant isolates. However, for tub2 gene, mutation of the amino acid in codon 198 resulted in the formation of Ala-A in all resistant isolates and Glu-A in all sensitive isolates. The mutation of amino acid 198 was detected by allele-specific PCR and enzyme assays and similar results were obtained in other resistant isolates. The results strongly suggested that amino acid change in codon 198 of tub2 played a leading role in conferring carbendazim-resistance to mango anthracnose in South China. Furthermore, the resistant gene of tub2 was cloned and used to construct fungicide-resistant strains of Metarhizium anisopliae, an entomopathogenic fungus.
Species of Metarhizium have been proved effective in controlling more than 200 species of insect pests, and was widely used due to its harmless effect on human, animals, plants and other insects. In our study, 21 strains of Metarhizium were isolated from more than 200 cadavers of the host, Brontispa longissima Gestro adults and lavae in Wenchang and Haikou of Hainan. These isolates were identified initially as M. anisopliae based on the colony color, mycelial texture, and the characteristics of hyphae, conidiophores, conidia and conidial fructification. Two different groups of isolates (MA and MB) could be distinguished only based on their colony characters. The ITS1-5.8S-ITS2 of rDNA from MA and MB strains were cloned and sequenced for identification at the species level. The maximum parsimony tree and neighbor-joining tree were established using the software of MEGA3.1.The results showed that both MA and MB strains should be assigned to Metarhizium anisopliae var. anisopliae. These strains were highly pathogenic to adults and larvae of coconut hispid beetles.
However, there were some environmental factors diminishing the effectiveness of Metarhizium in the field, such as temperature, UV light, insecticides and especially fungicides. So it was essential to construct fungicide-resistant strains of the fungus.
The sensitivity of all 21 isolates of M. anisoplieae var. anisopliae to the fungicide, carbendazim was tested. The results showed that these isolates were highly sensitive (MIC<5μg/mL) to carbendazim. It would not be practical trying to screen for resistant strains from the field because of the scarcity of cadavers of hispid beetles. Only a few were collected from millions of thousands of coconut palms on Hainan island.
Alteratively, UV irradiation was considered a good method to improve the resistance of the isolates. In our study several carbendazim-resistant stains were obtained by UV- induced mutation. Some strains could grow slowly on PDA agar medium even with a high concentration of carbendazim (600μg/mL) .Thus the resistant level had increased 120 times when compared with the parental strains. However, reverse-mutation occurred when these“resistant strains”were illuminated again with UV light. So these strains with unstable resistance could not be used in the field due to the presence of UV in the sunlight.
Another approach was to transform the sensitive strains of Metarhizium with the transfer of a carbendazim-resistant gene(tub2) mediated by a bacterium, Agrobacterium tumefaciens. The binary vector pTUB2 was constructed based on the vector pCAMBIA1300, promotor TrpC cloned from vector pBHt2 and carbendazim-resistant gene tub2 cloned from resistant isolates of Colletotrichum gloeosporioides. TrpC-tub2 cassette was amplified by Gene Splicing by Overlap Extension(gene SOEing) of PCR. The hph gene cassette of pCAMBIA1300 was deleted by Bst XI/Xho I digestion,and its large line fragment was ligated with TrpC-tub2 into vector of pTUB2 using the blunt-end because it contained Bst XI and Xho I in the tub2 sequence.An enzyme assay was employed to detect the ligation direction. Then the pTUB2 was mobilized into A. tumefaciens (strain GV3103).
In our study, a highly efficient and stable transformation system of Metarhizium was established by optimizing the A. tumefaciens-mediated procedure. Firstly, the effects of conidial concentration on transformation efficiency was tested, using a 2 day co-cultivation in the presence of 200μmol/L AS. Secondly, A. tumefaciens incubated for 6h in broth IM medium with 450μmol/L AS before co-cultivation resulted in highest transformation efficiency. Thirdly, A. tumefaciens (OD600:0.60-0.75) broth culture and Metarhizium spore suspension (1×10~6~-1×10~8conidial/mL) were mixed in equal volumes, and 0.01% Triton X-100 was added to dissolve the conidia. Fourthly, maximum efficiency was observed with the carbendazim at 4μg/mL. The transformation efficiency decreased with the increase of the concentration of carbendazim and there was no transformation when the fungicide used was at 10μg/mL.
More than 200 transformants were compared with the parental strains. Differences in the colony color, production and morphology of the spores as well as decreased virulence were found. It is quite feasible that T-DNA was inserted into the functional DNA region of Metarhizium. The transformation was detected by PCR and the fragments were sequenced. The results proved that the tub2 cassette gene had been inserted into the genome of Metarhizium. Thus, the transformation of Metarhizium by the insertion of T-DNA could be readily performed on a large scale using the optimized procedure.
The sensitivity of the transformants to carbendazim was tested. The results showed that the MIC was <10μg/mL and the resistant level increased only 2.8 times when compared with the parental strains. Nevertheless, the acquired resistance was stable and inherited unaltered through 12 consecutive generations on PDA medium. It is very likely that tub2 gene expressed only a low-level resistance after being inserted into the genome of Metarhizium. However, with the transformation vector established successfully and the transformation system optimized, a library of T-DNA mediated mutants can be readily obtained, providing a firm foundation for the investigation of fungicide-resistance and development in M. anisopliae.
引文
Bollen G J ,Scholten G. Acquired resistance to benomyl and some other systemic fungicides in a strain of Botrytis cinerea in cyclamen.Netherlands journal of plant pathology,1971,77:83-90.
Butters J A, Hollomon D W. Resistance to benzimidazole can be caused by changes in β-tubulin isoforms. Pesticide Science, 1999,55: 501~503
Beraha L, Garber E D. A genetic study of resistance to thiabendazole and sodium o-phenylphenate in Penicillium italicum by the Parasexual cycle .bot.Gaz. 1980, 141:204~209
Borck M K. Studies on the mode of action of benomyl in Neurospora crassa.Ph.D Thesis,Lousiana State University,1973,171
Buhr T L,Dickman M B. Isolation and characterization of a ?-tubulin-encoding gene from Colletotrichum gloeosporioides f.sp. aeschynomene.Gene.1993 124:121～125
Buhr T L ,Dickman M B. Isolation, characterization, and expression of a second ? -tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene. Appl. Environ. Microbiol. 1994,60:4155–4159.
Burland T G, Schedl T, Gull K et al. Genetic analysis of resistance to benzimidazoles in Physarum:differential expression of β-tubulin genes.Genetics,1984,108:123～141.
Cleveland D W, Sullivan K F. Molecular biology and genetics of tubulin. Ann. Rev. Biochem.,1985,54:331-365.
Campbell A G, Sinclair D P. Control of facial eczemain lambs by use of fungicides. Proc. Ruakura Farmers’ Conf. Week.N.Z.,1968.3-12.
Churchill A C L, Ciuffetti L M, Hansen D R et al. Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids.Curr Genet,1990,17:25～31.
De Brabander M J,Van de Veire R M L,Aerts F E M,Borgers M,Janssen P A J. The effects of methyl[5-(2-thienyl-carbonyl)-1H-benzimidazol-2-yl]-carbamate (R17934;
NSC238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro.Cancer Res., 1976, 36: 1011 -1018.
Davidse L C. Benzimidazole fungicides: mechanism of action and biological impact. Annual Review of Phytopathology,1986,24:43～65.
Davidse L C,Flach W. Differential binging of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans.J.Cell.Biol.,1977,72:174-193.
Davidse L C. Biological aspects of benzimidazole fungicides-action and resistance. In: Lyr H eds. Modern selective fungicides-properties, applications, mechanisms of action. London, Longman Group UK,1987:245～257.
Davidse L C, Flach W. Interaction of thiabendazole with fungal tubulin.Biochim. Biophys Acla, 1978,543; 82~90.
Davidse L C . Metabolic conversion of methyl-2-yl carbamate (MBC) in Aspergillus nidulans . Pestic . Biochem. Physiol. 1976,6: 538~546.
Delp C J. Coping with resistance to plant disease control agents. Plant Disease, 1980, 64: 651~657.
Delen N,Yildiz M. Fungicide resistance of some fungal pathogens isolated from greenhouses in Turkey. Journal of Turkish Phytopathology, 1982,11(1-2):33～40.
de Lapeyre de Bellaire L ,Dubois C .Distribution of thiabendazole -resistant Colletotrichum musae isolates from Guadeloupc banana plantations.Plant Disease, 1997,81:1378～1383.
Dumontet C, Sikic B L. Mechnisms of action and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death.J Clin Oncol, 1991, 7(3): 1061~1070.
Daunderer C, Graf R O. Molecular analysis of the cytosolic dictyostelinm gamma-tubulin complex. Eur J Cell Biol, 2002,81(40):175~184.
Elad Y, Yunis H, Katan T. Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel. Plant Patholoty,1992,41:41～46.
Faretra F, Pollastro K S, Ditonno A P. New natural variants of Botryotinia fuckeliana(Botrytis cinerea) couling benzimidazole-resistance to insensitivity toward the N-phenylcarbamate diethofencarb.Phytopathol. Mediterr, 1989, 28:98~104.
Fujimura M,Kamakura T,Yanmaguchi I.Action mechanism of diethofencarb to a benzimidazole-resistant mutant in Neurospora crassa.Journal of Pesticide Science,1992,17: 237～242.
Faretra F ,Pollastro S. Genetic basis of resistance to benzimidazole and dicarboximide fungicides in Botryotinia fuckeliana(Botrytis cinerea).Mycol.Res,1991,95(8): 943~951.
Fling M E,Kopf J, Tamarkin A,Gorman J A,Smith H A,Koltin Y.Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate.Mol.Gen.Genet,1991,227:318-329.
Fujimura M,Kanakura Tmand Yamaguchi,I,Action mechanism of diethofencarb to abenzimidazole-resistant mutant in Neurospora crassa,J .Pesti.Sci.1992,17.237. Fujomura M ,Mode od acrion of deethofencarb to benzimidazole-resistance strains in Neuropora crassa.J.Pesticide Sci.,1994,19:333~334.
Hastie A C.Benlate-induced instability of Aspergillus diploids.Nature,1970,226:771.
Hoebeke R J,Van Nyen G ,De Brabander M.Interaction of oncodazole(R17934),a new antitumoral drug,with rat brain tubulin.Biochem.biophys. Res.Commun., 1976, 69:319-324.
Hoebeke R J,Van Nyen G,De Brabander M.Interaction of oncodazole(R17934),a new antitumoral drug,with rat brain tubulin. Biochem. Biophys. Res. Commun., 1976,69:319-324.
Howard R J,Aist J R.Effects of MBC on hyphal tip organization,growth and mitosis of Fusarium acuminatum and their antagonism by D2O. Protoplasma, 1977, 92:195～210.
Howard R J,Aist J R.Cytoplasmic tubules and fungal morphogenesis:ultrastructural effect of methyl benzimidazole-2-yl carbamate determined by freeze substitution of hyphalo tip cells.Journal of Cell Biology,1980,87:55～64.
Heath I B.The effect of nocodazole on the growth and ultrastructure of the fungus.Saprolegnia ferax:Evidence against a simple node of action.In:Cappuccinelli P,Morris N R,ed.Microtubules in Microorganisms.New York:Dekker,1982.275-311.
Hayashi K.PCR-SSCP:一种检测突变的方法。国外医学卫生学分册,1994,21(4):204-205.
Ishii H,Davidse L C.Decreased binding of carbendazim to cellular protein from venturia nashicola and its involvement in benzimidazole resistance. Priceeding of the 1986
British Grop protection Conference-Pests and Disease, 1986. 576~573.
Ishii H, van Raak M. Inheritance of increased sensitivity to N-phenylcarbamates in benzimidazole-resistant Veuturia nashicola. Phytopathology, 1988, 78: 695~698.
Ishii H,Fukaya M,Faretra F et al.Stability of benzimidazole resistance and diethofencarb sensitivity of Botrytis cinerea.Procceedings of the 10th International Botrytis Symposim,1992,232～237.
Itsyn N, Wigler M. Cloning the differences between two coplex genomes. Science,1993,259(5097):946~951.
Ishii H.Monitoring of fungicides resistance in fungi:biological to biotechnological approaches.In:CRC Hand book of Pest Management in Agriculture.Boca Raton:CRC Press.1995,679-684.
Jung M K.Amino aid alterstions in the benA gene of Aspergitlus nidulans that confer benomyl resistance.Cell Motility and Cytoskeleton, 1992, 22:170~174.
Jung M K, Oakly B R.ldentification of an amino acid substitution in benA, β-tubulin gene of Aspergillus nidulans that confets thiabendazole resistance and benomyl supersensity. Cell Motility and the Cytoskeleton.1990,17(2):87~94.
Jones A L,Shabi E, Ehret G R.Genetics of negatively correlated cross-resistance to aN-phenylcarbamate in benomyl-resistant Venturia inaegualis. Canadian Journal of Plant Patholohy, 1987,9: 195~199.
Josepovits G, Gasztonyi M, Mikite G. Negative cross-resistance to N-phenylanilines in benzimidazole-resistant straina of Botrytis cinerea, Venturia nashicola and Venturia inaegualis. Pesticide Science, 1992, 35: 237~242.
J.C. DODD.R.BUGANTE,I.KOOMEN.P.JEFFRIES et al., Pre-and post-harvest control of mango anthracnose in the Philippines.Plant Pathology, 1991, 40: 576～583.
Jung S,Arndt K M,Muller K M.Selectively infective phage(SIP) technology:scope and limitations.J Immunol methods.1999,231(1-2):93-104.
Kǖnkel W.Antimitotische Aktivitet von Methylbenzimidazol02-yl carbamat (MBC). I.Licht-,elektronenmikroskopische and physiologische Untersuchungen an keimenden Konidiěn von Aspergullus nidulans. Z. Allg. Mikrobiol., 1980,20: 113-120.
Kendall S,Hollomon D W ,lshii H, Heaney S P. Characterisation of benzimidazole resistant. strains of Rhynchosporium secalis. Pesticide Science, 1993, 40:175-181.
Koenraadt H ,Somerville S C, Jones A L.Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi.Phytopathology, 1992,82: 1384~1354.
Koenraadt H,Jones A L.Resistance to binomyl conferred by mutation in codon 198 or 200 of the beta-tubulin gene of Neurospora crassa and sensitivity to diethofencarb conferred by codon 198.Phytopathlogy, 1993,83(8):850～853.
Koenrraadt H,Jones A L.The use of allele-specific oligonucleotide probes to characterize resistance to benomyl in field strains of Venturia inaequalis. Phytopathology, 1992,82: 1354-1359.
Kawchuk L M,Hutchison L J, Verhaeghe C A ,Lynch D R,Bains P S ,Holley J D .lsolation of the β-tubulin gene and characterization of thiabendazole resistance in Gibberella pulicaris.Can J.Pathol, 2002, 24: 233~238.
Kato T. Negative cross-resistance activity of MDPC and diethofencarb against benzimidazole-resistant fungi In: Fungicede resistance in north America, Delp C J ed. Minnesota: APS Press, 1988.40.
Kato T, Suzuki D, Takahashi J, Kamoshita K. Negatively correlated cross-resistance between benzimidazole fungicides and methyl N-(3,4-dichlorophenyl)-carbamate. J Pesticide Scienc, 1984, 9: 485~495.
Kiyoshi Sato, Gen Tamura,Takashi Tsuchiya, et.al Analysis of genetic and epigenetic alterations of he PTEN gene in gastric cancet. Virchows Archiv,2002, 440(2), 160~165.
Kay G J,Cooke L R.A PCR-based method to characterize and identify benzimidazole resistance in Helminthosporium solani.FEMS Microbiology letters,1997,152: 371-378.
Kavallaris M, Bukhart C A, Horwitz S B. Antiisense oligonucleotides to class β-tubulin sensitize drug-resistant cells to Taxol.Bri J Cancet,1999,80(7):1020~1025.
Khodjakov A, Riedet C L.The sudden recruitment of γ-tubulin to the centrosome at the onset of mitosis and its dynamic exchang throughout the cell cycle does not require microtubues. J Cell Biol,1999146(3):585~596.
Locke T.Current incidence in the UK of fungicide resistance in pathogens of cereals.proceedings 1986 British Crop protection ,1986.781-786.
Leroux P,Gredt M.Effects du barbane, chloryelAme, du chlorpropham et du prophams sur diverses souches de Botrytis cinerea Pers.Et de Penicillium expansum Link sensibles ou resistantes au carbendazime et au thiabendazole.C R A cad.Sci.Paris,Ser.1979, 289.691~693.
Luduena R F,Woodward D O.α-and β-tubulin;separation and partial sequece analysis.Annal of the New York Academy of Science,1975,253:272～283.
Luck J E,Gillings M R.Rapid identification of benomyl resistant strains of Botrytis cinerea using the polymerase chain reaction.Mycological Research,1995,99(12):1483-1488.
Linhartova I, Novotna B, Sulimenko V et al.,Gamma-tubulin in chicken crythrocytes:changes in localizatiln during cell differerntion and characterization of cytoplasmic complexes.Dev Dyn,2002,223(2)229~240.
Leguy R, Mellki R, Pantaloni D, et al. Monomeric γ-tubulin nucleates microtubules.J Biol Chem, 2000,275(29):21975~21980.
Molnar A.Hornok L,Miklo’s P. The high level of benomyl tolerance in Fusarium oxysporum is determined by the synergistic interaction of two genes .Experimental Mycology, 1985.9(3):326~333.
May G S,Tsang M L-S,Smith H.Aspergillus nidulans β-tubulin genes are unusually divergent.Gene,1987,55:231～243.
Martin L A ,Fox R T V,Baldwin B C.Rapid method for detection of MBC resistance in fungi:Immunological approach.Proceedings of 10th Inernational Reinardsbrunn Symposium,1992.
Martin L A,Fox R T V,Baldwin B C .Use of polymerase chain reaction for the diagnosis of MBC resistance in Botrytis cinerea.In:1992 Brighton Crop Protection Conference-Pests and Diseases.Surry:BCPC publications, 1992, 207-214.
McKay G J,Cooke L R.A PCR-based to characterize and identify benzimidazole reisistance in Helminthoporium solani.FEMS Microbiology Letters, 1997, 152:371-378.
Menendez M, Rivas G, Diaz J F, et al. Control of the structural stability of the tubulin dimmer by ong high affinity bound magnesium ion at nucleotide N-site.J Biol. Chem, 1998,273(1):167~176.
Moritz M,Agard D A. γ-tubulin complexes and microtubule nucleation.Curr Opin Stru Biol,2001,11(2):174~181.
Murfphy S M,PREble A M, Patel U K, et al.GCP6:Two new members of the human γ-tubulin complex.Mol Biol Cell,2001,12(11):3340~3352.
Nakata A,Snao S, Hashimoto S,Hayakawa K, Nishikawa H, Yasuda Y. Negatively correlated cross-resistant to N-Phylformamidoximes in benzimidazole-resistant phytopathogenic fungi. Annals of the Phytopathological Society of Japan, 1987, 53: 659~662.
Neff N F,Thomas J J,Grisafi P et al.Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo.Cell,1983,33:211～219.
Nogales E,Wolf S G,Downing K H.Structure of the α β tubulin dimmer by electron crystallography.Nature,1998,391(6663):199～203.
Nogales E,Whittaker M, Milligan R A et al.,Hingh-resolution model of microtubule.Cell, 1999,96(1):79~88.
Osmani,S A and OaKley,B R .Cell cycle and tubulin mutations in filamentous fungi.In: Bennet,J.W. and Lasure,L.L.,(Eds),More Gene Manipulations in Fungi.Academic Press,NY,1991,107～125.
Orbach M J ,Porro E B ,Yanofsky C.Cloning and characterizationof the gene for β-tubulin froma benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.Mol.Cell.Biol,1986,6:2452-2461.
Orita M,Suzuki Y,Sekiya T.Rapid and sensitive detection of point mutations and DNA polymorphisms using polymerase chain reaction.Genomics,1989,5:874-879.
Oakley B R. An abundance of tubulins.Tren Cell Biol,2000,10(12):537~542.
Oakley C E, Oakley B R. ldentification of r-tubulin , a new member of the tubulin superfamily encoded by mipaA gene of Aspergillus nidulans. Nature, 1989,338(6217):662~664.
Oakley B R. Г-tubulin, Curr Top Dev Biol,2000,49(1):27~54.
Oakley B R. An abundance of tubulons. Tren Cell Biol,2000,10(12):537~542.
Panaccione,D G .McKiernan,M and Hanau,R M .Colletotrichum graminicola transformed with homologous and heterologous benomyl-resistant genes retains expected pathogenicity to corn.Mol.Plant-Microbe Interact. 1988,I,113～120.
Rosenberger D A, Meyer F W. Negatively correlated cross-resistance to diphenylamine in benomyl-resistant Penicillium expansum. Phytopathol, 1985. 75: 74~79.
Pereira G,Grueneberg U, Knop M et al., lnteraction of the yeast γ-tubulin complex binging protein Spc72p with Karl p is essential for microtubule function during karyogamy. EMBO J,1999,18(15):4180`4196.
Rose V L.Herodima formation and tubulin function in Saccharomyces cereviside.PhD Thesis,Messachusetts institute of Technology,1999.
Ruiz F, Krzywicha A, Klotz C, et al. The SM19 gene,required for duplication of basal bodies in Paramecium, encodes a novel tubulin, -tubulin. Curr boil,2000,10(22): 1451~1454.
Schroeder W T ,Provvidenti R.Resistance to benomyl in powdery mildew of cucurbits. Plant disease Reporter,1969,53:271-275.
Shabi E, Katan T, Marton K. lnheritance of resistance to benomyl in isolates of Venturia inaegualis from lsrael. Plant Pathology, 1983,32:207~211.
Stover,R H .Extranuclear inherited tolerance to benomyl in Mycosphaerella fijiensis var.difformis, Trans.Br. Mycol.Soc, 1977,68(1):122.
Sheir-Neiss G,Lai M H,Morris N R.Identification of a gene for β-tubulin in Aspergillus nidulans.Cell,1978,15:639～647.
Spalding D H .Resistance of mango pathogens to fungicides used to control postharvest diseases.Plant Disease,1982,66:1185～1186.
Sekiya T,Orita M, Lwahana H, et al. An efficient method of detecting nucleotide sequence polymorphisms of human genome by gel electrophoresis as single-strand conformation polymorphisms.J Cell. Biochem, 1989,Sppl. 304.
Susan K D. The tubulin fraternity:alpha to eta. Curr Opin Cell Biol,2001,13(1):49~54. Schiebel E. γ-tubulin complexes:binding to the centrosome, regulatiln and microtubule nucleation.Curr boil,2000,12(1):113~118.
Takeuchi T. Studies of the epidemiology of fungicide resistant gray mold strains.Special Bulletion of the Chiba-Ken Agricultural Experiment Station, 1987.14:1~75.
Thomas J H,Neff N F,Botstein D.Isolation and characterization of mutation in the β-tubulin gene of Saccharomyces cerevisiae.Genetics. 1985,112:715～734.
Van Tuyl J M.Genetics of fungicide resistance.Mededelingen Landbouwhogeschool, Wageningen:1977,77(2):236.
Vonk J W,Sijpesteijin A K. Methyl benzimidazole-2-ylcarbamate,the fungitoxic principle of thiophanate-methyl. Pesticide Science,1971,2:160-164.
Wicks T. Tolerance of the apple scab fungus to benzimidzole fungocides. Plant Disease Relorer, 1974,58; 886~889.
Wilson L,Bryan J.Biochemical and pharmacological properties of microtubules. Advances in Cell and Molecular Biology,1974,4:21～72.
Wheeler I E,Kendall S I,Butters J.Using allete-specific digonucleotide probes tocharacterize benzimidazole resistance in Rhynchosporium secalis.Pestic Sci,1995,43: 201-209.
Wiese C, Zheng Y. γ-tubulin complexes and their interaction With microtubule-organizing centers.Curr Opim Stru Biol,1999,9(2):250~259.
Wiese C .Zheng Y. A new function for the gamma-tubulin ring complex as a microtubule minus-end cap. Nat Cell Biol,2000,10(12):537~542.
Yanden O,Katan T.Mutations leading to substiutions at a mino acids 198 and 200 of beta0tubulin that correlate with benomyl-resistance pheno-types of field strains of Botrytis cinerea.Phytopathology, 1993,83(12):1478～1483.
Yank, Dickman M B. Isolation of a β-tubulin gene from Fusarium moniliforme that confers cold sensitive benomyl resistance. Applied and Environmental Microbiology, 1996, 62: 3053～3056.
Zhao Z, Huang X, Li N et al., Direct cloning of cell differential expression genes with full-lrngth by a new strategy based on the multiple rounds of”long distance” polymerase chain and magnetic based mediated subtraction.J Biotechnol,1999,73(1):35~41.
Zheng Y,Wong M L, Alberts B M et al., Nucleation of microtubule assembly by aγ-tubulin-containing ring complex.Nature,1995,378(6557):78583.
Zhou J Shu H B, Joshi H C.Regulatiln of tubulin synthesis and cell cycle progression in mammalian cell by gamma-tubulin-mediated microtubule nucleation. J Cell Biochem, 2002,84(3):472~483.
韩贻仁主编。分子细胞生物学(第二版)。北京:科学出版社,2002
黄朝豪 主编.热带作物病理学. 中国农业出版社, 1997, 95～96.
金冬雁等译.分子克隆实验指南[第二版]. 北京:科学出版社,1995
陆悦健,周明国,叶钟音,王建新.抗苯并咪唑的小麦赤霉病菌β-tubulin 基因序列分析与特性研究.植物病理学报,2000,30(1):30-34.
刘波等.南京农业大学学报,1993,16(3):50～54.
刘勇等.中国甜菜,1992,(1):27～33.
刘波,苗荣,Hollmon D W.大麦云纹斑病菌中抗多菌灵乙霉威株系及其抗药性对致病性作用分子分析.植物病理学报,1996,26(3):211～215.
陆家云主编.植物病害诊断.北京:中国农业出版社,1997.
戚佩坤.广东果树真菌病害志. 北京:中国农业出版社,2000.
魏景超.真菌鉴定手册.上海:上海科学技术出版社,1979.
王关林,方宏筠. 植物基因工程原理与技术. 北京:科学出版社,1998.
许志刚主编.普通植物病理学.北京:中国农业出版社,1997.
肖倩纯,余卓桐,陈绵香.芒果炭疽病化学防治研究.热带作物学报, 1999,(3):25-30.
杨谦.植物病原菌抗药性分子生物学. 北京:科学出版社, 2003.9.
易克贤,黄俊生,刘国道等.中国拄花草炭疽病原菌遗传多态性的 RAPD 分析.微生物学报,2003,43(3):379-387.
周明国,叶钟音.植物病原菌对苯并咪唑类及相关杀菌剂的抗药性. 植物保护,1987,(2):31～33.
周明国,叶钟音,刘经芬.杀菌剂抗药性研究进展.南京农业大学学报, 1994, 17(3): 33~41.
周明国,王建新。禾谷镰孢霉对多菌灵的敏感性基线及抗药性菌株生物学性质的研究。植物
赵善欢.植物化学保护.北京,中国农业出版社,2002.
Amsellem Z. Long term dry preservation of active mycelia of two mycoherbicidal organisms.Crop protection. 1999,18:643-649.
ANG B T, YEN D F. Studies on the entomogenous fungus, Metarhizium anisoplise Sorokin[J]. Memorirs of the College of Agriculture National Taiwan University.1972,13(2):31～46.
Armstrong J L ,Harris D L. Biased DNA integration in Colletotrichum gloesporioides f. sp. aeschynomene transformants with benomyl resistance. Phytopathology. [St. Paul, Minn. : American Phytopathological Society] ,v. 1993,83 (3) p. 328-332.
Bagga S, Hu G,Screen S E,St Leger R J.Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene, 2004,Vol.324, p. 159-169.
Bidochka M J,McDonald M A,StLeger R J et al.,Differentiation of species and strains of entomopathogenic fungi by random amplication of polymorphic DNA (RAPD).CurrGenet,1994,25:107~113.
Bridge P D,William M A J, Prior C et al.,Morphological, biochemical and molecular characteristics of Metarhizium anisopliae and M.flaroviride.Gen Microbiol, 1993, 239: 1163~1169.
Bogo M. Henning Vainstein M. Lima Aragao F.J. Rech E. Schrank A. 1996.High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae. [Journal Article; Summary] FEMS Microbiology Letters. 142(1):123-127.
Burland T G,Schedl T,Gull K. 1984.Genetic analysis of resistance to benzimidazoles in Physarum:differential expression of β-tubulin genes. Genetics, 108: 123-141.
Butters J A, Hollomon D W. 1999. Resistance to benzimidazole can be caused by changes in beta-tubulin isoforms. Pesticide Science. 55: 4, 501-503. 6 ref.
Bundock P,Hooykaas P J J.1996.Integration of Agrobacterium tumefaciens T-DNA in the saccharamyes cerevisiae genome by illegitimate recombination. Proc. Natl. Acad.sci.USA,93:15272-15275.
Bundock P,Mroczek K,Winkler A A,Steensma H Y.Hooykaas P J J.1999.T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromydes lactis.Mol.Gen.Genet,261:115-121.
Bridge PD, Prior C, Sagbohan J, Lomer CJ, Carey M, Buddie A. Molecular characterization of isolates of Metarhizium from locusts and grasshoppers. Biodiversity and Conservation.,1997,6: 177–189.
Cobb B D,Clarkon J M. Detection of molecular variation in the insect pathogenic fungus Metarhizium using RAPD-PCR.FEMS Microbiol Lett,1993, 112: 319~324.
Cantone F A, Vandenberg J D. Use of the green fluorescent protein for investigations of Paecilomyces fumosoroseus in insect hosts. Journal of Invertebrate Pathology, 1999,74:193-197.
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.
Charnley A K. Entomopathogenic fungi and their role in pest control. In:The Mycota IV Environmental and microbial Relationships, Wicklow/Soderstrom (Eds.), 1997,pp 185-201.
Curran J, Driver F, Ballard J W O, Milner R J.Phylogeny of Metarhizium: sequence analysis of the internally transcribed and 5.8s region of the ribosomal DNA repeat. Mycological Research .1994,9: 547-552.
Driver F, Milner R J, Trueman W H. A taxonomic revision Metarhizium of based on a phylogenetic analysis of rDNA sequence data.Mycol Res,2000,104:134~150
Dickman, M. B. 1988. Whole cell transformation of the alfalfa fungal pathogen Colletotrichum trifolii. Curr. Genet. 14:243-246.
De-Groot MJA,Bundock P,Hooykaas PJJ,Beijersbergen AGM,1998.Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nature Biotechnol,16:839-842.
Douthwaite B, Langewald J, Harris J, Development and Commercialization of the Green Muscle Biopesticide. IITA,Cotonou, Benin,2000.
Daoust,R.A.Effect on formulation on the Viability of Metarhizium anisopliae conidia. J. Invertebr.Pathol. 1983,41:151-160.
Fegan M, Manner J M,MacLean D J. Random amplication of polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var.anisopliae.J Gen Microbiol,1993,139:2075~2081.
Fungaro M H P, Vieira M L C, Pizziranikleiner A A et al.Diversity among soiland insect isolates of Metarhizium anisopliae var. anisopliae detected by RAPD.Lett Appl Microbiol,1996,22:389~392.
Fernandez-Larrea J., and U. Stahl. Transformation of Podospora anserina with a dominant resistance gene. Curr.Genet. 1989,16:57-60.
Fincham J R S. Transformation in fungi. Microbiology Review, 1989,Mar,148-170.
Goettel M S, Leger R J S, Bhairi S,Jung M K, Oakley B R,Roberts D W, Staples R C. Pathogenicity and growth of Metarhizium anisopliae stably transformed to benomyl resistance. Current Genetics. [Berlin, W. Ger. : Springer International], 1990,17(2):129-132.
Goldman G H, Temmermann W, Jacobs D, Contreras R, Montagu M, van Herrera Estrella A. A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate. Molecular and General Genetics,1993,240(1): 73-80.
Gouka R J, Gerk C, Hooykaas P J J, Wouter V C T, de Groot M J A. Transformation of Aspergillus awamori by Agrobacterium tuemfaciens-mediated homologous recombination.Nature Biotechnology, 1999,17:598-601.
Goettel M S, Hajek A E, Siegel J P, Evans H C. Safety of Fungal biocontrol agents.In:Fungi as biocontrol agents,Butt T M,Jackson C, Magan N(eds),CAB International, 2001,PP347-375.
Glare T R, Milner R J, Beaton C D, Variation in Metarhizium, a genus of fungal pathogens attacking Orthoptera: is phialide morphology a useful criterion? Journal of Orthoptera Research . 1996,5: 19–27.
GUO H L, YE B L,YUE Y Y, Three new species of metarhizium. Acta Mycologica Sinicanisopliae, 1986,5(3):177-184.
Haenel H. Preliminary field tests on the use of Metarhizium anisopliae for the control Nasutitermes exitiosus (Isoptera: Termitidae). Bull. Entomol.Res. 1983, 73: 305-314.
Henson J M, Blake N K, Pilgeram A L. Transformation of Gaeumannomyces graminis to benomyl resistance.Curr. Genet,1988,14:113-117.
Ho H H, Ann P J, Chang H S.The Genus Phytophthora in Taiwan.Taipei.Published by Institute of Botany,Academia Sinica,1～86, 1995.
Hawksworth DL. Mycologist’s handbook. England: CAB Press, 1971.
Hernández-Crespo P, Santiago-álvarez 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.
Inglis P W, Aragao F J L, Frazao A H, Magalhaes B P,Valadares-Inglis M C. Biolistic co-Transformation of Metarhizium anisopliae var. acridum strain CG423 with green fluorescent protein and resistance to glufosinate ammonium. FEMS Microbiology Letters. Elsevier Science B.V., Amsterdam, Netherlands: 2000.191(2):249-254.
John E.Hamer;William E,Timberlake. Functional Organization of the Aspergillus nidulans trpC Promoter.MOLLECULAR AND CELLULAR BIOLOGY, July 1987,7(7): 2352-2359.
Koenradt H,Jones A L. Resistance to binomyl conferred by mutation in codon 198 or 200 of the beta-tubulin gene of Neurospora crassa and sensitivity to diethofencarb conferred by codon 198.Phytopathlogy, 1993,83(8):850～853.
Kachroo P ,Potnis A,Chattoo B B. Transformation of the rice blast fungus Magnaporthe grisea to benomy resistance. World Journal of Microbiology & Biotechnology. 1997,13(2):185-187.
Koenraadt H,Sonerville S C,Jones A L. 1992.Characterization of mutations in the beta-tubulin gene of benomy resistant fields strains of Venturia inaepualisans other plant pathogenic fungi.Phytopathology, 82(11):1348～1354.
Leal S C M, Bertioli DJ , Butt T M, et al. Amplification and restriction endonuclease digestion of the Pr1 gene for the detection and charactization of Metarhizium strains.Mycol Res,1997,101:257~265.
Leal S C M, Bertioli D J, Butt T M et al., Characterization of isolates of the entomopathogenic fungus Metarhizium anisopliae by RAPD-PCR.Mycol Res, 1994, 98:1077~1081.
Lomer C J, Bateman R P, Johnson D L, Langewald J, Thomas M, Biological control of locusts and grasshoppers. Annual Review of Entomology . 2001,46: 667–702.
LIU A Y,.A new record of the genus Metarhizium. Acta Mycologica Sinicanisopliae 1988.7(3): 191-192.
Mavridou A, Types M A. Intraspecific polymorphism in Metarhizium anisopliae var.anisopliae revealed by analysis of rDNA gene complex and mtDNA RFLPs.Mycol Res,1998,102(10):1233~1241.
Mullins E D,Chen X,Romaine P,Raina R,Geiser D M,Kang S. Agrobecterium -mediated transformation of Fusarium osysporum:and efficient tool for insertional mutagenesis and gene transfer.Phytopathology, 2001,91:173-180.
Marcel J A. et al..Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology Volume 16 September 1998.
Marshall K J,Vargo A M ,Fatuesi S .Application of a New Strain of Metarhizium anisopliae (Fungi Imperfecti) 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.
Milner R J. Mycoinsecticides:making the most of microbes. Australian Microbiologist . 1998,19: 11-12.
Milner R J, Hunter D M, Recent developments in the use of fungi as biopesticides against locusts and grasshoppers in Australi anisopliae.Journal of Orthoptera Research 2001,10: 271–276.
Milner R J, Samson P R, Bullard G K.FI 1045: A Profile of a Commercially Useful Isolate of Metarhizium anisopliae var. anisopliae Biocontrol Science and Technology, 2002,12(1):43-58.
Milner R J, Lozano L B, Driver F, Hunter D. A comparative study of two Mexican isolates with an Australian isolate of Metarhizium anisopliae var. acrider – strain characterisation, temperature profile and virulence for wingless grasshopper, Phaulacridium vittatum. BioControl 2003,48: 335–348.
Magalhaes B P, De Faria M R, Lecoq M, Schmidt F G V, Silva J B T, Frazao H S, Balanc A G, Foucart A.The use of Metarhizium anisopliae var. acridum against the grasshopper Rhammatocerus schistocercoides in Brazil. Journal of Orthoptera Research , 2001,10: 199–202.
Neff N F,Thomas J J,Grisafi P.Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo.Cell, 1983,33:211-219.
NINA OSSANNAt ,SUE MISCHKE. Genetic Transformation of the Biocontrol Fungus Gliocladium virens to Benomyl Resistance. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1990. p. 3052-3056.
Nigel Dunn-Coleman , Huaming Wang. Agrobacterium T-DNA: A silver bullet for filamentous fungi? Nature Biotechnology, 1998,16:817-818.
Orbach M J , Porro E B, Yanofsky C. Cloning and characterization of the gene for β-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.Mol.Cell.Biol. 1986,6:2452-2461.
O'Donnell K. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris).Curr Genet. , 1992,22(3):213-220.
Peter W. Inglis, Myrian S. Tigano and M. Cléria Valadares-Inglis.Transformation of the entomopathogenic fungi, Paecilomyces fumosoroseus and Paecilomyces lilacinus (deuteromycotina: hyphomycetes) to benomyl resistence. Genet. Mol. Biol. 1999,vol.22n.1S?o Paulo Mar.
Panaccione D G, McKiernana M, Hanau R M. Colletotrichum graminicola transformed with homologous and heterologous benomyl-resistance genes retains expected pathogenicity to corn. Mol. Plant-Microbe Interact. 1988,1:113-120.
Picknett T M ,Saunders G. Transformation of Penicillium chrysogenum with selection for increased resistance to benomyl. FEMS Microbiol. Lett. 1989,10:165-168.
Punt P J, Oliver R P, Dingemanse M A,Pouwel P H,van den Hondel C A M J J. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli.Gene, 1987,56:117-124.
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.
Rombach M C, Humber R A ,Roberts D W. Metarhizium flavoviride var.minusvar. nov.,a pathogenic of plant and leaf hoppers on rice Philippine sand Solomon Islands.Mycotaxon,1986,27:87~92.
Rath A C,Carr C J,Graham B R. Characterization of Metarhizium anisopliae strains by carbohydrate utilization(AP150CH).J Invert Pathol,1995,65:152~161.
Rkotonirainy M S,Dutertre M,Cariou M L et al., Phylogenetic relationships with in the genera Metarhizium based on 28S rRNA sequences and isozyme comparison.MycolRes,1994,98:225~230.
Rupesh Thakur,Hu S S. Development of Transformation system for entomopathogenic fungus Metarhizium anisopliae (ENT 12) based on cnx-gene. Indian Journal of Microbiology, 2003,43(3):187-192.
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.
Singh S P ,Rethinam P . Coconut hispid beetle Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae). Cord, 2004,Vol.20,p.1-20.
Sandhu S S,Kinghorn J R,Rajak R C, Unkles S E. Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.Indian Journal of Experimental Biology, 2001,39(7): 650-653.
Steven E,Screen,Gang Hu,Raymond J, St.Leger. Transformants of Metarhizium anisopliae sf.anisopliae Overexpressing Chitinase from Metarhizium anisopliae sf.acridum Show Early Induction of Native Chitinase but Are Not Altered in Pathogenicity to Manduca Sexta. Journal of Invertebrate Pathology,2001, 78,260-266.
St Leger R J, May B, Allee L L, et al,. Genetic differencesinal lozymesand information of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae.J Inverterb Pathol,1992,60:89~101.
St.Leger R J,Shimizu S,Joshi L,Bidochka M J ,Roberts D W. Co-Transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants. FEMS Microbiology Letters. [Amsterdam, The Netherlands : Elsevier Science B.V], 1995, 131(3):289-294.
St.Leger R J,Screen S E. Prospects for strain improvement of fungal pathogens of insects and weeds.In:fungi as biocontrol agent. CABI international, 2001,pp219-237.
Souza Dias E , Vieira de Queiroz M , Gomes Cardoso P , Goncalves de Barros E , Fernandes Araujo E. Transformation of Penicillium expansum with a heterologous gene which confers resisance to benomyl. World Journal of Microbiology & Biotechnology. 1999,15, (4): 513-514.
SEIP E R,WOLOSHUK C P,PAYNE G A ,CURTIS S E .Isolation and sequence Analysis of a β-Tubulin Gene from Aspergillus flavus and Its Use as a Selectable Marker.APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec.1990, p.3686-3692.
Susumu Shimizu,Motoko Yamaji,2002.Pathogenicity of Entomopathogenic Fungi to the Termite, Reticulitermes speratus .Japanese Journal of Applied Entomology and Zoology.46(2):89-91.
Tulloch M.Thegenus Metarhizium.Trans Brit Mycol Soc,1976,66:407~411.
Tzean S S,Hsieh L S,Chen J L et al., Nomuraea cylindrosporacomb nov.Mycologia, 1993, 85:514~519.
Thomas J H,Neff N F,Botstein D.Isolation and characterization of mutation in the β-tubulin gene of Saccharomyces cerevisiae.Genetics. 1985,112:715-734.
Thompson C J,Movva N R,Tizard R,Crameri R,Davies J E,Lauwereys M,Botterman J. Characterization of the herbicide-resisitance gene bar from streptomydes hygroscopicus. EMBO J, 1987,6:2519-2523.
Tarasco E, Bievre C de, Papierok B, Poliseno M, Triggiani O. Occurrence of entomopathogenic fungi in soils in Southern Italy. Entomologica, Bari 1997; 31: 157–166.
Tigano-Milani M S, Faria M R de, Martins I, et al. Ocorrência 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.
Ulhoa C J , Vainstein M H, Peberdy J F.Transformation of Trichoderma species with dominant selectable markers. Curr. Genet.1992,21: 23-26.
Valadares-Inglis M C, Inglis P W. Transformation of the entomopathogenic fungus, Metarhizium flavoviride strain CG423 to benomyl resistance. FEMS Microbiology Letters. 1997. 155: 2, 199-202.
Weiguo Fang,Yan Pei,Michael J Bidochka. Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens.Canadian Journal of Microbiology; Jul 2006; 52(623-626).
Williams J G K,Kubelik A R,Livak K J et al., DNA polymorphism samplified by arbitary primers are useful as genetic markers.Nucl Acids Res,1990,18: 6531~6535.
Wikardi E A. Protects for controlling coconut hispids with Metarhizium sp. [Plesispareichei, and Brontispa longissima]. Pemberitaan Lembaga Tanaman Ind. Bogor: Lembaga,1982, 8(44):35～38.
Yanden O,Katan T.Mutations leading to substiutions at a mino acids 198 and 200 of beta-tubulin that correlate with benomyl-resistance pheno-types of field strains of Botrytis cinerea. Phytopathology, 1993,83(12):1478～1483.
ZHAN Ru-lin, HUANG Jun-sheng. Cloning and Detecting of a High Carbendazim- Resistant Gene From Colletotrichum gloeosporioides of Mango in South China. African Journal of Biotechnology Vol. 6 (2), pp. 143-147, 18 January, 2007. Zimmerman G. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biological agent. Pesticide Science 1993; 37:375–379.
迟玉杰,杨谦,杜金哲.tub2 基因鉴定及其在毛壳菌中的转化.北京林业大学学报, 2003, 25 (4): 30-34.
陈国相.微生物及其在农业上的应用.河北人民出版社,石家庄,1980,130.
陈祝安,黄基荣.不同来源绿僵菌对云斑金龟蛴螬致病力评价.微生物学通报,1997,24(2):81～83.
丁中,刘峰,慕立义.紫外光诱导哈茨木霉产生腐霉利抗性菌株的研究.中国生物防治, 2002,18(2):75～78.
丁立孝,李文泽,宋子红,徐丽娟,梅汝鸿.双抗性标记花生内生菌的诱变与筛选.莱阳农学院学报,1997,14(4):235～239.
董辉,苏红田,高松.绿僵菌对蝗虫及其捕食性天敌的影响.中国生物防治,2005,21(1):60～62.
方卫国,张永军,肖月华,杨星勇,范艳华,侯磊,罗小英,裴炎. 2002a.根癌土壤杆菌介导球孢白僵菌的遗传转化.首届中国青年学者微生物遗传学学术研讨会论文摘要集.
方卫国,张永军,杨星勇,裴炎.2002b.根癌土壤杆菌介导真菌遗传转化的研究进展.中国生物工程杂志,22(5):41-45.
郭好礼,叶柏龄,岳莹玉等.绿僵菌属的三个新种.真菌学报,1986,5(3):177~184.
郭超,樊美趁.中国虫生真菌的研究与应用.1993,3:14-20.
贾春生.昆虫病原真菌对荔枝蝽的致病性.吉林林业科技,34(3): 32-36.
黄原.分子系统学原理、方法及应用.北京:中国农业出版社,1998
黄大昉主编,农业微生物基因工程.科学出版社,2001.
胡景江,樊美珍.绿僵菌免疫特性的研究.安徽农业大学学报,1996,23(3):433~437
李梅,杨谦,宋金柱.多菌灵抗性基因在球毛壳菌中的转化.高技术通讯,2002(2):43-45.
李维,张义正.1999.以潮毒素 B 磷酸转移酶基因为遗传标记的启动子探针载体的构建.四川大学学报(自然科学版),36(4):736-740.
李宝笃,沈崇尧.植物病原真菌对杀菌剂的抗性对策.植物病理学报,1994,24(3):193-196.
李红霞,陆悦健,王建新,周明国.四种不同植物病原真菌与多菌灵抗药性相关基因突变的比较.南京农业大学学报,2002,24(3):41-44.
李梅,杨谦,宋金柱.多菌灵抗性基因在球毛壳菌中的转化.高技术通讯,2002(2):43-45.
李维,张义正.1999.以潮毒素 B 磷酸转移酶基因为遗传标记的启动子探针载体的构建.四川大学学报(自然科学版),36(4):736-740.
刘波,苗荣,Hollmon D W.大麦云纹斑病菌中抗多菌灵乙霉威株系及其抗药性对致病性作用分子分析.植物病理学报,1996,26(3):211～215.
刘爱英,梁宗琦,曹蕾.双型孢绿僵菌的分离鉴定研究.贵州农学院学报,1989(2):27~31.
刘兆伟,郭好礼,KurtzmanCP.由部分核糖体 RNA序列确定的绿僵菌属种系统发育关系.真菌学报,1994,13(2):139~151.
刘少华,陆金萍,朱瑞良,戴富明,2005. 一种快速简便的植物病原真菌基因组 DNA 提取方法. 植物病理学报 35 (4): 362-365
刘爱英,1988.绿僵菌属的新记录种.真菌学报.7(3):191-192.
刘萍萍,闫艳春.微生物农药研究进展.山东农业科学,2005(2):78～80.
梁宗琦,刘爱英,刘杰麟.虫草一新种及其绿僵菌无性型.真菌学报, 1991, 10(4): 257~262.
雷仲仁,问锦曾.绿僵菌治蝗研究进展.植物保护,2004,30(4):14-17.
卢辉,张礼生,张泽华,沈有孝,吴坤宏,龙瑞军.绿僵菌侵染椰心叶甲的部位及侵染过程的电镜观察. 植物检疫,2005,19(4):201-204.
林华峰,李世广,张磊,王萍莉,周义文.绿僵菌大孢变种的生物学特征及其对蛴螬的毒力研究应用生态学报. 2006,17(2): 351~335.
樊美珍,郭超.金龟子绿僵菌对几种林木害虫的致病性及防治应用试验. 生物防治通报, 1987,4(1):29-32.
李贵芳,高贤华.绿僵菌 Metarhizium SP.防治天麻双叉犀金龟及其应用研究.贵州农学院学报,1994, 13(1):103-104.
农向群,吴正铠,包建中.金龟子绿僵菌原生质体形成和再生的研究.微生物学通报,1990,17(2):76-80.
蒲蛰龙.应用昆虫的病原真菌防治害虫 . 生物防治通报, 1985,1(1):27-31.
蒲蛰龙主编.昆虫病理学.广东科技出版社,1992.
蒲蛰龙,李增智.昆虫真菌学. 安徽科技出版社,1996,P93、378.
轻工部甘蔗糖业科学研究所.绿僵菌(Metarhizium anisopliae)防治黑色蔗龟(Alissonotum spp.)试验报告. 1978.
邱式邦.蝗虫生物防治的新进展.植保参考,1992,6:13-14.
任志红,徐平,王富强,苏彩云,丰玫玫,张佳,戴梦,韦春玲,穆岷,贺建功.产黄青霉工业生
产菌种基因报告系统的构建及启动子效率的评价.菌物学报,2005,24(3):376～384.
孙乃恩,孙东旭,朱德煦编著.分子遗传学.南京大学出版社,2002 年,P161～165.
孙晓君,朱婷婷,秦智,蒋忠平,魏金芝,张艳,薛滨泰.紫外诱变获得耐高浓度丙烯腈菌株的研究. 哈尔滨工业大学学报,2005,37(10):1376～1393.
王音,雷仲仁,张青文.绿僵菌侵染小菜蛾体表过程的显微观察.昆虫学报,2005, 48(2):188-193.
王四宝,黄勇平,张心团,刘云鹏,樊美珍,李增智.松褐天牛成虫高毒力病原真菌筛选及林间感染试验. 中国森林病虫,2004 年,23(6):13-16.
王万立,王勇,刘耕春,郝永娟,刘春燕,陈静.天津市茄子灰霉病菌的抗药性检测.华北农学报,1998,13(4):107～111.
王芳,徐秉良,曹奎荣.深绿木霉紫外光诱导耐低温突变菌株的研究.甘肃农业大学学报, 2005,40(4):507～511.
武觐文.侵染金龟子的几种真菌. 北京林学院学报 1982,(3):88-93.
熊道雅.绿僵菌防治甘薯华叶虫(Colasposoma metallicum).植物保护,1981,7:36.
杨洪俊,宗兆锋,郭小芳.紫外线诱导提高重寄生放线菌PR和F46活性的研究.西北农业学报,2005,14(3):22～25A.
杨谦,赵小岩.多菌灵抗性基因在木霉菌中的转化方法.科学通报,1998,43(22): 2423-2426.
赵善欢.植物化学保护.北京:中国农业出版社,2002,118-154.
赵俊生,郭素萍,武慧贞.利用绿僵菌防治鳞翅目害虫研究.山西农业科学2000,28(2):3～6.
张永军,方卫国,裴颖,裴炎.两株球孢白僵菌 T-DNA 插入突变体的生物学特性.首届中
国青年学者微生物遗传学学术研讨会论文摘要集, 2002 年.
张承来,欧晓明.植物病原物对杀菌剂的抗药性机制概述.湖南化工,2000,30(5):7-10.
张道海,王未名,陆文华,等.生防真菌的分子生物学研究.中国虫生真菌研究与应用.第 4卷.北京:中国农业科技出版社,1997.32~36.
周明国,叶钟音.杀菌剂抗性研究进展.南京农业大学学报.1994,17(3):33-41.
周明国,叶钟音.植物病原菌对苯并咪唑类及相关杀菌剂的抗药性.植物保护,1987,(2):31～33.
周燚,王中康,喻子牛主编.微生物农药研发与应用.化学工业出版社,北京,2006,56-70.
詹儒林,李伟,郑服丛.芒果炭疽病菌对多菌灵的抗药性.植物保护学报,2005(1): 71-75.
詹儒林,许天委,张世清,黄俊生.紫外光的照射与绿缰菌对多菌灵敏感性变化的关系.热带作物学报, 2006,26(4):91-94.
Butters J A, Hollomon D W. Resistance to benzimidazole can be caused by changes in β-tubulin isoforms. Pesticide Science, 1999,55: 501~503
Beraha L, Garber E D. A genetic study of resistance to thiabendazole and sodium o-phenylphenate in Penicillium italicum by the Parasexual cycle .bot.Gaz. 1980, 141:204~209
Borck M K. Studies on the mode of action of benomyl in Neurospora crassa.Ph.D Thesis,Lousiana State University,1973,171
Buhr T L,Dickman M B. Isolation and characterization of a ?-tubulin-encoding gene from Colletotrichum gloeosporioides f.sp. aeschynomene.Gene.1993 124:121～125
Buhr T L ,Dickman M B. Isolation, characterization, and expression of a second ? -tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene. Appl. Environ. Microbiol. 1994,60:4155–4159.
Burland T G, Schedl T, Gull K et al. Genetic analysis of resistance to benzimidazoles in Physarum:differential expression of β-tubulin genes.Genetics,1984,108:123～141.
Cleveland D W, Sullivan K F. Molecular biology and genetics of tubulin. Ann. Rev. Biochem.,1985,54:331-365.
Campbell A G, Sinclair D P. Control of facial eczemain lambs by use of fungicides. Proc. Ruakura Farmers’ Conf. Week.N.Z.,1968.3-12.
Churchill A C L, Ciuffetti L M, Hansen D R et al. Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids.Curr Genet,1990,17:25～31.
De Brabander M J,Van de Veire R M L,Aerts F E M,Borgers M,Janssen P A J. The effects of methyl[5-(2-thienyl-carbonyl)-1H-benzimidazol-2-yl]-carbamate (R17934;
NSC238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro.Cancer Res., 1976, 36: 1011 -1018.
Davidse L C. Benzimidazole fungicides: mechanism of action and biological impact. Annual Review of Phytopathology,1986,24:43～65.
Davidse L C,Flach W. Differential binging of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans.J.Cell.Biol.,1977,72:174-193.
Davidse L C. Biological aspects of benzimidazole fungicides-action and resistance. In: Lyr H eds. Modern selective fungicides-properties, applications, mechanisms of action. London, Longman Group UK,1987:245～257.
Davidse L C, Flach W. Interaction of thiabendazole with fungal tubulin.Biochim. Biophys Acla, 1978,543; 82~90.
Davidse L C . Metabolic conversion of methyl-2-yl carbamate (MBC) in Aspergillus nidulans . Pestic . Biochem. Physiol. 1976,6: 538~546.
Delp C J. Coping with resistance to plant disease control agents. Plant Disease, 1980, 64: 651~657.
Delen N,Yildiz M. Fungicide resistance of some fungal pathogens isolated from greenhouses in Turkey. Journal of Turkish Phytopathology, 1982,11(1-2):33～40.
de Lapeyre de Bellaire L ,Dubois C .Distribution of thiabendazole -resistant Colletotrichum musae isolates from Guadeloupc banana plantations.Plant Disease, 1997,81:1378～1383.
Dumontet C, Sikic B L. Mechnisms of action and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death.J Clin Oncol, 1991, 7(3): 1061~1070.
Daunderer C, Graf R O. Molecular analysis of the cytosolic dictyostelinm gamma-tubulin complex. Eur J Cell Biol, 2002,81(40):175~184.
Elad Y, Yunis H, Katan T. Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel. Plant Patholoty,1992,41:41～46.
Faretra F, Pollastro K S, Ditonno A P. New natural variants of Botryotinia fuckeliana(Botrytis cinerea) couling benzimidazole-resistance to insensitivity toward the N-phenylcarbamate diethofencarb.Phytopathol. Mediterr, 1989, 28:98~104.
Fujimura M,Kamakura T,Yanmaguchi I.Action mechanism of diethofencarb to a benzimidazole-resistant mutant in Neurospora crassa.Journal of Pesticide Science,1992,17: 237～242.
Faretra F ,Pollastro S. Genetic basis of resistance to benzimidazole and dicarboximide fungicides in Botryotinia fuckeliana(Botrytis cinerea).Mycol.Res,1991,95(8): 943~951.
Fling M E,Kopf J, Tamarkin A,Gorman J A,Smith H A,Koltin Y.Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate.Mol.Gen.Genet,1991,227:318-329.
Fujimura M,Kanakura Tmand Yamaguchi,I,Action mechanism of diethofencarb to abenzimidazole-resistant mutant in Neurospora crassa,J .Pesti.Sci.1992,17.237. Fujomura M ,Mode od acrion of deethofencarb to benzimidazole-resistance strains in Neuropora crassa.J.Pesticide Sci.,1994,19:333~334.
Hastie A C.Benlate-induced instability of Aspergillus diploids.Nature,1970,226:771.
Hoebeke R J,Van Nyen G ,De Brabander M.Interaction of oncodazole(R17934),a new antitumoral drug,with rat brain tubulin.Biochem.biophys. Res.Commun., 1976, 69:319-324.
Hoebeke R J,Van Nyen G,De Brabander M.Interaction of oncodazole(R17934),a new antitumoral drug,with rat brain tubulin. Biochem. Biophys. Res. Commun., 1976,69:319-324.
Howard R J,Aist J R.Effects of MBC on hyphal tip organization,growth and mitosis of Fusarium acuminatum and their antagonism by D2O. Protoplasma, 1977, 92:195～210.
Howard R J,Aist J R.Cytoplasmic tubules and fungal morphogenesis:ultrastructural effect of methyl benzimidazole-2-yl carbamate determined by freeze substitution of hyphalo tip cells.Journal of Cell Biology,1980,87:55～64.
Heath I B.The effect of nocodazole on the growth and ultrastructure of the fungus.Saprolegnia ferax:Evidence against a simple node of action.In:Cappuccinelli P,Morris N R,ed.Microtubules in Microorganisms.New York:Dekker,1982.275-311.
Hayashi K.PCR-SSCP:一种检测突变的方法。国外医学卫生学分册,1994,21(4):204-205.
Ishii H,Davidse L C.Decreased binding of carbendazim to cellular protein from venturia nashicola and its involvement in benzimidazole resistance. Priceeding of the 1986
British Grop protection Conference-Pests and Disease, 1986. 576~573.
Ishii H, van Raak M. Inheritance of increased sensitivity to N-phenylcarbamates in benzimidazole-resistant Veuturia nashicola. Phytopathology, 1988, 78: 695~698.
Ishii H,Fukaya M,Faretra F et al.Stability of benzimidazole resistance and diethofencarb sensitivity of Botrytis cinerea.Procceedings of the 10th International Botrytis Symposim,1992,232～237.
Itsyn N, Wigler M. Cloning the differences between two coplex genomes. Science,1993,259(5097):946~951.
Ishii H.Monitoring of fungicides resistance in fungi:biological to biotechnological approaches.In:CRC Hand book of Pest Management in Agriculture.Boca Raton:CRC Press.1995,679-684.
Jung M K.Amino aid alterstions in the benA gene of Aspergitlus nidulans that confer benomyl resistance.Cell Motility and Cytoskeleton, 1992, 22:170~174.
Jung M K, Oakly B R.ldentification of an amino acid substitution in benA, β-tubulin gene of Aspergillus nidulans that confets thiabendazole resistance and benomyl supersensity. Cell Motility and the Cytoskeleton.1990,17(2):87~94.
Jones A L,Shabi E, Ehret G R.Genetics of negatively correlated cross-resistance to aN-phenylcarbamate in benomyl-resistant Venturia inaegualis. Canadian Journal of Plant Patholohy, 1987,9: 195~199.
Josepovits G, Gasztonyi M, Mikite G. Negative cross-resistance to N-phenylanilines in benzimidazole-resistant straina of Botrytis cinerea, Venturia nashicola and Venturia inaegualis. Pesticide Science, 1992, 35: 237~242.
J.C. DODD.R.BUGANTE,I.KOOMEN.P.JEFFRIES et al., Pre-and post-harvest control of mango anthracnose in the Philippines.Plant Pathology, 1991, 40: 576～583.
Jung S,Arndt K M,Muller K M.Selectively infective phage(SIP) technology:scope and limitations.J Immunol methods.1999,231(1-2):93-104.
Kǖnkel W.Antimitotische Aktivitet von Methylbenzimidazol02-yl carbamat (MBC). I.Licht-,elektronenmikroskopische and physiologische Untersuchungen an keimenden Konidiěn von Aspergullus nidulans. Z. Allg. Mikrobiol., 1980,20: 113-120.
Kendall S,Hollomon D W ,lshii H, Heaney S P. Characterisation of benzimidazole resistant. strains of Rhynchosporium secalis. Pesticide Science, 1993, 40:175-181.
Koenraadt H ,Somerville S C, Jones A L.Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi.Phytopathology, 1992,82: 1384~1354.
Koenraadt H,Jones A L.Resistance to binomyl conferred by mutation in codon 198 or 200 of the beta-tubulin gene of Neurospora crassa and sensitivity to diethofencarb conferred by codon 198.Phytopathlogy, 1993,83(8):850～853.
Koenrraadt H,Jones A L.The use of allele-specific oligonucleotide probes to characterize resistance to benomyl in field strains of Venturia inaequalis. Phytopathology, 1992,82: 1354-1359.
Kawchuk L M,Hutchison L J, Verhaeghe C A ,Lynch D R,Bains P S ,Holley J D .lsolation of the β-tubulin gene and characterization of thiabendazole resistance in Gibberella pulicaris.Can J.Pathol, 2002, 24: 233~238.
Kato T. Negative cross-resistance activity of MDPC and diethofencarb against benzimidazole-resistant fungi In: Fungicede resistance in north America, Delp C J ed. Minnesota: APS Press, 1988.40.
Kato T, Suzuki D, Takahashi J, Kamoshita K. Negatively correlated cross-resistance between benzimidazole fungicides and methyl N-(3,4-dichlorophenyl)-carbamate. J Pesticide Scienc, 1984, 9: 485~495.
Kiyoshi Sato, Gen Tamura,Takashi Tsuchiya, et.al Analysis of genetic and epigenetic alterations of he PTEN gene in gastric cancet. Virchows Archiv,2002, 440(2), 160~165.
Kay G J,Cooke L R.A PCR-based method to characterize and identify benzimidazole resistance in Helminthosporium solani.FEMS Microbiology letters,1997,152: 371-378.
Kavallaris M, Bukhart C A, Horwitz S B. Antiisense oligonucleotides to class β-tubulin sensitize drug-resistant cells to Taxol.Bri J Cancet,1999,80(7):1020~1025.
Khodjakov A, Riedet C L.The sudden recruitment of γ-tubulin to the centrosome at the onset of mitosis and its dynamic exchang throughout the cell cycle does not require microtubues. J Cell Biol,1999146(3):585~596.
Locke T.Current incidence in the UK of fungicide resistance in pathogens of cereals.proceedings 1986 British Crop protection ,1986.781-786.
Leroux P,Gredt M.Effects du barbane, chloryelAme, du chlorpropham et du prophams sur diverses souches de Botrytis cinerea Pers.Et de Penicillium expansum Link sensibles ou resistantes au carbendazime et au thiabendazole.C R A cad.Sci.Paris,Ser.1979, 289.691~693.
Luduena R F,Woodward D O.α-and β-tubulin;separation and partial sequece analysis.Annal of the New York Academy of Science,1975,253:272～283.
Luck J E,Gillings M R.Rapid identification of benomyl resistant strains of Botrytis cinerea using the polymerase chain reaction.Mycological Research,1995,99(12):1483-1488.
Linhartova I, Novotna B, Sulimenko V et al.,Gamma-tubulin in chicken crythrocytes:changes in localizatiln during cell differerntion and characterization of cytoplasmic complexes.Dev Dyn,2002,223(2)229~240.
Leguy R, Mellki R, Pantaloni D, et al. Monomeric γ-tubulin nucleates microtubules.J Biol Chem, 2000,275(29):21975~21980.
Molnar A.Hornok L,Miklo’s P. The high level of benomyl tolerance in Fusarium oxysporum is determined by the synergistic interaction of two genes .Experimental Mycology, 1985.9(3):326~333.
May G S,Tsang M L-S,Smith H.Aspergillus nidulans β-tubulin genes are unusually divergent.Gene,1987,55:231～243.
Martin L A ,Fox R T V,Baldwin B C.Rapid method for detection of MBC resistance in fungi:Immunological approach.Proceedings of 10th Inernational Reinardsbrunn Symposium,1992.
Martin L A,Fox R T V,Baldwin B C .Use of polymerase chain reaction for the diagnosis of MBC resistance in Botrytis cinerea.In:1992 Brighton Crop Protection Conference-Pests and Diseases.Surry:BCPC publications, 1992, 207-214.
McKay G J,Cooke L R.A PCR-based to characterize and identify benzimidazole reisistance in Helminthoporium solani.FEMS Microbiology Letters, 1997, 152:371-378.
Menendez M, Rivas G, Diaz J F, et al. Control of the structural stability of the tubulin dimmer by ong high affinity bound magnesium ion at nucleotide N-site.J Biol. Chem, 1998,273(1):167~176.
Moritz M,Agard D A. γ-tubulin complexes and microtubule nucleation.Curr Opin Stru Biol,2001,11(2):174~181.
Murfphy S M,PREble A M, Patel U K, et al.GCP6:Two new members of the human γ-tubulin complex.Mol Biol Cell,2001,12(11):3340~3352.
Nakata A,Snao S, Hashimoto S,Hayakawa K, Nishikawa H, Yasuda Y. Negatively correlated cross-resistant to N-Phylformamidoximes in benzimidazole-resistant phytopathogenic fungi. Annals of the Phytopathological Society of Japan, 1987, 53: 659~662.
Neff N F,Thomas J J,Grisafi P et al.Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo.Cell,1983,33:211～219.
Nogales E,Wolf S G,Downing K H.Structure of the α β tubulin dimmer by electron crystallography.Nature,1998,391(6663):199～203.
Nogales E,Whittaker M, Milligan R A et al.,Hingh-resolution model of microtubule.Cell, 1999,96(1):79~88.
Osmani,S A and OaKley,B R .Cell cycle and tubulin mutations in filamentous fungi.In: Bennet,J.W. and Lasure,L.L.,(Eds),More Gene Manipulations in Fungi.Academic Press,NY,1991,107～125.
Orbach M J ,Porro E B ,Yanofsky C.Cloning and characterizationof the gene for β-tubulin froma benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.Mol.Cell.Biol,1986,6:2452-2461.
Orita M,Suzuki Y,Sekiya T.Rapid and sensitive detection of point mutations and DNA polymorphisms using polymerase chain reaction.Genomics,1989,5:874-879.
Oakley B R. An abundance of tubulins.Tren Cell Biol,2000,10(12):537~542.
Oakley C E, Oakley B R. ldentification of r-tubulin , a new member of the tubulin superfamily encoded by mipaA gene of Aspergillus nidulans. Nature, 1989,338(6217):662~664.
Oakley B R. Г-tubulin, Curr Top Dev Biol,2000,49(1):27~54.
Oakley B R. An abundance of tubulons. Tren Cell Biol,2000,10(12):537~542.
Panaccione,D G .McKiernan,M and Hanau,R M .Colletotrichum graminicola transformed with homologous and heterologous benomyl-resistant genes retains expected pathogenicity to corn.Mol.Plant-Microbe Interact. 1988,I,113～120.
Rosenberger D A, Meyer F W. Negatively correlated cross-resistance to diphenylamine in benomyl-resistant Penicillium expansum. Phytopathol, 1985. 75: 74~79.
Pereira G,Grueneberg U, Knop M et al., lnteraction of the yeast γ-tubulin complex binging protein Spc72p with Karl p is essential for microtubule function during karyogamy. EMBO J,1999,18(15):4180`4196.
Rose V L.Herodima formation and tubulin function in Saccharomyces cereviside.PhD Thesis,Messachusetts institute of Technology,1999.
Ruiz F, Krzywicha A, Klotz C, et al. The SM19 gene,required for duplication of basal bodies in Paramecium, encodes a novel tubulin, -tubulin. Curr boil,2000,10(22): 1451~1454.
Schroeder W T ,Provvidenti R.Resistance to benomyl in powdery mildew of cucurbits. Plant disease Reporter,1969,53:271-275.
Shabi E, Katan T, Marton K. lnheritance of resistance to benomyl in isolates of Venturia inaegualis from lsrael. Plant Pathology, 1983,32:207~211.
Stover,R H .Extranuclear inherited tolerance to benomyl in Mycosphaerella fijiensis var.difformis, Trans.Br. Mycol.Soc, 1977,68(1):122.
Sheir-Neiss G,Lai M H,Morris N R.Identification of a gene for β-tubulin in Aspergillus nidulans.Cell,1978,15:639～647.
Spalding D H .Resistance of mango pathogens to fungicides used to control postharvest diseases.Plant Disease,1982,66:1185～1186.
Sekiya T,Orita M, Lwahana H, et al. An efficient method of detecting nucleotide sequence polymorphisms of human genome by gel electrophoresis as single-strand conformation polymorphisms.J Cell. Biochem, 1989,Sppl. 304.
Susan K D. The tubulin fraternity:alpha to eta. Curr Opin Cell Biol,2001,13(1):49~54. Schiebel E. γ-tubulin complexes:binding to the centrosome, regulatiln and microtubule nucleation.Curr boil,2000,12(1):113~118.
Takeuchi T. Studies of the epidemiology of fungicide resistant gray mold strains.Special Bulletion of the Chiba-Ken Agricultural Experiment Station, 1987.14:1~75.
Thomas J H,Neff N F,Botstein D.Isolation and characterization of mutation in the β-tubulin gene of Saccharomyces cerevisiae.Genetics. 1985,112:715～734.
Van Tuyl J M.Genetics of fungicide resistance.Mededelingen Landbouwhogeschool, Wageningen:1977,77(2):236.
Vonk J W,Sijpesteijin A K. Methyl benzimidazole-2-ylcarbamate,the fungitoxic principle of thiophanate-methyl. Pesticide Science,1971,2:160-164.
Wicks T. Tolerance of the apple scab fungus to benzimidzole fungocides. Plant Disease Relorer, 1974,58; 886~889.
Wilson L,Bryan J.Biochemical and pharmacological properties of microtubules. Advances in Cell and Molecular Biology,1974,4:21～72.
Wheeler I E,Kendall S I,Butters J.Using allete-specific digonucleotide probes tocharacterize benzimidazole resistance in Rhynchosporium secalis.Pestic Sci,1995,43: 201-209.
Wiese C, Zheng Y. γ-tubulin complexes and their interaction With microtubule-organizing centers.Curr Opim Stru Biol,1999,9(2):250~259.
Wiese C .Zheng Y. A new function for the gamma-tubulin ring complex as a microtubule minus-end cap. Nat Cell Biol,2000,10(12):537~542.
Yanden O,Katan T.Mutations leading to substiutions at a mino acids 198 and 200 of beta0tubulin that correlate with benomyl-resistance pheno-types of field strains of Botrytis cinerea.Phytopathology, 1993,83(12):1478～1483.
Yank, Dickman M B. Isolation of a β-tubulin gene from Fusarium moniliforme that confers cold sensitive benomyl resistance. Applied and Environmental Microbiology, 1996, 62: 3053～3056.
Zhao Z, Huang X, Li N et al., Direct cloning of cell differential expression genes with full-lrngth by a new strategy based on the multiple rounds of”long distance” polymerase chain and magnetic based mediated subtraction.J Biotechnol,1999,73(1):35~41.
Zheng Y,Wong M L, Alberts B M et al., Nucleation of microtubule assembly by aγ-tubulin-containing ring complex.Nature,1995,378(6557):78583.
Zhou J Shu H B, Joshi H C.Regulatiln of tubulin synthesis and cell cycle progression in mammalian cell by gamma-tubulin-mediated microtubule nucleation. J Cell Biochem, 2002,84(3):472~483.
韩贻仁主编。分子细胞生物学(第二版)。北京:科学出版社,2002
黄朝豪 主编.热带作物病理学. 中国农业出版社, 1997, 95～96.
金冬雁等译.分子克隆实验指南[第二版]. 北京:科学出版社,1995
陆悦健,周明国,叶钟音,王建新.抗苯并咪唑的小麦赤霉病菌β-tubulin 基因序列分析与特性研究.植物病理学报,2000,30(1):30-34.
刘波等.南京农业大学学报,1993,16(3):50～54.
刘勇等.中国甜菜,1992,(1):27～33.
刘波,苗荣,Hollmon D W.大麦云纹斑病菌中抗多菌灵乙霉威株系及其抗药性对致病性作用分子分析.植物病理学报,1996,26(3):211～215.
陆家云主编.植物病害诊断.北京:中国农业出版社,1997.
戚佩坤.广东果树真菌病害志. 北京:中国农业出版社,2000.
魏景超.真菌鉴定手册.上海:上海科学技术出版社,1979.
王关林,方宏筠. 植物基因工程原理与技术. 北京:科学出版社,1998.
许志刚主编.普通植物病理学.北京:中国农业出版社,1997.
肖倩纯,余卓桐,陈绵香.芒果炭疽病化学防治研究.热带作物学报, 1999,(3):25-30.
杨谦.植物病原菌抗药性分子生物学. 北京:科学出版社, 2003.9.
易克贤,黄俊生,刘国道等.中国拄花草炭疽病原菌遗传多态性的 RAPD 分析.微生物学报,2003,43(3):379-387.
周明国,叶钟音.植物病原菌对苯并咪唑类及相关杀菌剂的抗药性. 植物保护,1987,(2):31～33.
周明国,叶钟音,刘经芬.杀菌剂抗药性研究进展.南京农业大学学报, 1994, 17(3): 33~41.
周明国,王建新。禾谷镰孢霉对多菌灵的敏感性基线及抗药性菌株生物学性质的研究。植物
赵善欢.植物化学保护.北京,中国农业出版社,2002.
Amsellem Z. Long term dry preservation of active mycelia of two mycoherbicidal organisms.Crop protection. 1999,18:643-649.
ANG B T, YEN D F. Studies on the entomogenous fungus, Metarhizium anisoplise Sorokin[J]. Memorirs of the College of Agriculture National Taiwan University.1972,13(2):31～46.
Armstrong J L ,Harris D L. Biased DNA integration in Colletotrichum gloesporioides f. sp. aeschynomene transformants with benomyl resistance. Phytopathology. [St. Paul, Minn. : American Phytopathological Society] ,v. 1993,83 (3) p. 328-332.
Bagga S, Hu G,Screen S E,St Leger R J.Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene, 2004,Vol.324, p. 159-169.
Bidochka M J,McDonald M A,StLeger R J et al.,Differentiation of species and strains of entomopathogenic fungi by random amplication of polymorphic DNA (RAPD).CurrGenet,1994,25:107~113.
Bridge P D,William M A J, Prior C et al.,Morphological, biochemical and molecular characteristics of Metarhizium anisopliae and M.flaroviride.Gen Microbiol, 1993, 239: 1163~1169.
Bogo M. Henning Vainstein M. Lima Aragao F.J. Rech E. Schrank A. 1996.High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae. [Journal Article; Summary] FEMS Microbiology Letters. 142(1):123-127.
Burland T G,Schedl T,Gull K. 1984.Genetic analysis of resistance to benzimidazoles in Physarum:differential expression of β-tubulin genes. Genetics, 108: 123-141.
Butters J A, Hollomon D W. 1999. Resistance to benzimidazole can be caused by changes in beta-tubulin isoforms. Pesticide Science. 55: 4, 501-503. 6 ref.
Bundock P,Hooykaas P J J.1996.Integration of Agrobacterium tumefaciens T-DNA in the saccharamyes cerevisiae genome by illegitimate recombination. Proc. Natl. Acad.sci.USA,93:15272-15275.
Bundock P,Mroczek K,Winkler A A,Steensma H Y.Hooykaas P J J.1999.T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromydes lactis.Mol.Gen.Genet,261:115-121.
Bridge PD, Prior C, Sagbohan J, Lomer CJ, Carey M, Buddie A. Molecular characterization of isolates of Metarhizium from locusts and grasshoppers. Biodiversity and Conservation.,1997,6: 177–189.
Cobb B D,Clarkon J M. Detection of molecular variation in the insect pathogenic fungus Metarhizium using RAPD-PCR.FEMS Microbiol Lett,1993, 112: 319~324.
Cantone F A, Vandenberg J D. Use of the green fluorescent protein for investigations of Paecilomyces fumosoroseus in insect hosts. Journal of Invertebrate Pathology, 1999,74:193-197.
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.
Charnley A K. Entomopathogenic fungi and their role in pest control. In:The Mycota IV Environmental and microbial Relationships, Wicklow/Soderstrom (Eds.), 1997,pp 185-201.
Curran J, Driver F, Ballard J W O, Milner R J.Phylogeny of Metarhizium: sequence analysis of the internally transcribed and 5.8s region of the ribosomal DNA repeat. Mycological Research .1994,9: 547-552.
Driver F, Milner R J, Trueman W H. A taxonomic revision Metarhizium of based on a phylogenetic analysis of rDNA sequence data.Mycol Res,2000,104:134~150
Dickman, M. B. 1988. Whole cell transformation of the alfalfa fungal pathogen Colletotrichum trifolii. Curr. Genet. 14:243-246.
De-Groot MJA,Bundock P,Hooykaas PJJ,Beijersbergen AGM,1998.Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nature Biotechnol,16:839-842.
Douthwaite B, Langewald J, Harris J, Development and Commercialization of the Green Muscle Biopesticide. IITA,Cotonou, Benin,2000.
Daoust,R.A.Effect on formulation on the Viability of Metarhizium anisopliae conidia. J. Invertebr.Pathol. 1983,41:151-160.
Fegan M, Manner J M,MacLean D J. Random amplication of polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var.anisopliae.J Gen Microbiol,1993,139:2075~2081.
Fungaro M H P, Vieira M L C, Pizziranikleiner A A et al.Diversity among soiland insect isolates of Metarhizium anisopliae var. anisopliae detected by RAPD.Lett Appl Microbiol,1996,22:389~392.
Fernandez-Larrea J., and U. Stahl. Transformation of Podospora anserina with a dominant resistance gene. Curr.Genet. 1989,16:57-60.
Fincham J R S. Transformation in fungi. Microbiology Review, 1989,Mar,148-170.
Goettel M S, Leger R J S, Bhairi S,Jung M K, Oakley B R,Roberts D W, Staples R C. Pathogenicity and growth of Metarhizium anisopliae stably transformed to benomyl resistance. Current Genetics. [Berlin, W. Ger. : Springer International], 1990,17(2):129-132.
Goldman G H, Temmermann W, Jacobs D, Contreras R, Montagu M, van Herrera Estrella A. A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate. Molecular and General Genetics,1993,240(1): 73-80.
Gouka R J, Gerk C, Hooykaas P J J, Wouter V C T, de Groot M J A. Transformation of Aspergillus awamori by Agrobacterium tuemfaciens-mediated homologous recombination.Nature Biotechnology, 1999,17:598-601.
Goettel M S, Hajek A E, Siegel J P, Evans H C. Safety of Fungal biocontrol agents.In:Fungi as biocontrol agents,Butt T M,Jackson C, Magan N(eds),CAB International, 2001,PP347-375.
Glare T R, Milner R J, Beaton C D, Variation in Metarhizium, a genus of fungal pathogens attacking Orthoptera: is phialide morphology a useful criterion? Journal of Orthoptera Research . 1996,5: 19–27.
GUO H L, YE B L,YUE Y Y, Three new species of metarhizium. Acta Mycologica Sinicanisopliae, 1986,5(3):177-184.
Haenel H. Preliminary field tests on the use of Metarhizium anisopliae for the control Nasutitermes exitiosus (Isoptera: Termitidae). Bull. Entomol.Res. 1983, 73: 305-314.
Henson J M, Blake N K, Pilgeram A L. Transformation of Gaeumannomyces graminis to benomyl resistance.Curr. Genet,1988,14:113-117.
Ho H H, Ann P J, Chang H S.The Genus Phytophthora in Taiwan.Taipei.Published by Institute of Botany,Academia Sinica,1～86, 1995.
Hawksworth DL. Mycologist’s handbook. England: CAB Press, 1971.
Hernández-Crespo P, Santiago-álvarez 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.
Inglis P W, Aragao F J L, Frazao A H, Magalhaes B P,Valadares-Inglis M C. Biolistic co-Transformation of Metarhizium anisopliae var. acridum strain CG423 with green fluorescent protein and resistance to glufosinate ammonium. FEMS Microbiology Letters. Elsevier Science B.V., Amsterdam, Netherlands: 2000.191(2):249-254.
John E.Hamer;William E,Timberlake. Functional Organization of the Aspergillus nidulans trpC Promoter.MOLLECULAR AND CELLULAR BIOLOGY, July 1987,7(7): 2352-2359.
Koenradt H,Jones A L. Resistance to binomyl conferred by mutation in codon 198 or 200 of the beta-tubulin gene of Neurospora crassa and sensitivity to diethofencarb conferred by codon 198.Phytopathlogy, 1993,83(8):850～853.
Kachroo P ,Potnis A,Chattoo B B. Transformation of the rice blast fungus Magnaporthe grisea to benomy resistance. World Journal of Microbiology & Biotechnology. 1997,13(2):185-187.
Koenraadt H,Sonerville S C,Jones A L. 1992.Characterization of mutations in the beta-tubulin gene of benomy resistant fields strains of Venturia inaepualisans other plant pathogenic fungi.Phytopathology, 82(11):1348～1354.
Leal S C M, Bertioli DJ , Butt T M, et al. Amplification and restriction endonuclease digestion of the Pr1 gene for the detection and charactization of Metarhizium strains.Mycol Res,1997,101:257~265.
Leal S C M, Bertioli D J, Butt T M et al., Characterization of isolates of the entomopathogenic fungus Metarhizium anisopliae by RAPD-PCR.Mycol Res, 1994, 98:1077~1081.
Lomer C J, Bateman R P, Johnson D L, Langewald J, Thomas M, Biological control of locusts and grasshoppers. Annual Review of Entomology . 2001,46: 667–702.
LIU A Y,.A new record of the genus Metarhizium. Acta Mycologica Sinicanisopliae 1988.7(3): 191-192.
Mavridou A, Types M A. Intraspecific polymorphism in Metarhizium anisopliae var.anisopliae revealed by analysis of rDNA gene complex and mtDNA RFLPs.Mycol Res,1998,102(10):1233~1241.
Mullins E D,Chen X,Romaine P,Raina R,Geiser D M,Kang S. Agrobecterium -mediated transformation of Fusarium osysporum:and efficient tool for insertional mutagenesis and gene transfer.Phytopathology, 2001,91:173-180.
Marcel J A. et al..Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology Volume 16 September 1998.
Marshall K J,Vargo A M ,Fatuesi S .Application of a New Strain of Metarhizium anisopliae (Fungi Imperfecti) 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.
Milner R J. Mycoinsecticides:making the most of microbes. Australian Microbiologist . 1998,19: 11-12.
Milner R J, Hunter D M, Recent developments in the use of fungi as biopesticides against locusts and grasshoppers in Australi anisopliae.Journal of Orthoptera Research 2001,10: 271–276.
Milner R J, Samson P R, Bullard G K.FI 1045: A Profile of a Commercially Useful Isolate of Metarhizium anisopliae var. anisopliae Biocontrol Science and Technology, 2002,12(1):43-58.
Milner R J, Lozano L B, Driver F, Hunter D. A comparative study of two Mexican isolates with an Australian isolate of Metarhizium anisopliae var. acrider – strain characterisation, temperature profile and virulence for wingless grasshopper, Phaulacridium vittatum. BioControl 2003,48: 335–348.
Magalhaes B P, De Faria M R, Lecoq M, Schmidt F G V, Silva J B T, Frazao H S, Balanc A G, Foucart A.The use of Metarhizium anisopliae var. acridum against the grasshopper Rhammatocerus schistocercoides in Brazil. Journal of Orthoptera Research , 2001,10: 199–202.
Neff N F,Thomas J J,Grisafi P.Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo.Cell, 1983,33:211-219.
NINA OSSANNAt ,SUE MISCHKE. Genetic Transformation of the Biocontrol Fungus Gliocladium virens to Benomyl Resistance. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1990. p. 3052-3056.
Nigel Dunn-Coleman , Huaming Wang. Agrobacterium T-DNA: A silver bullet for filamentous fungi? Nature Biotechnology, 1998,16:817-818.
Orbach M J , Porro E B, Yanofsky C. Cloning and characterization of the gene for β-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.Mol.Cell.Biol. 1986,6:2452-2461.
O'Donnell K. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris).Curr Genet. , 1992,22(3):213-220.
Peter W. Inglis, Myrian S. Tigano and M. Cléria Valadares-Inglis.Transformation of the entomopathogenic fungi, Paecilomyces fumosoroseus and Paecilomyces lilacinus (deuteromycotina: hyphomycetes) to benomyl resistence. Genet. Mol. Biol. 1999,vol.22n.1S?o Paulo Mar.
Panaccione D G, McKiernana M, Hanau R M. Colletotrichum graminicola transformed with homologous and heterologous benomyl-resistance genes retains expected pathogenicity to corn. Mol. Plant-Microbe Interact. 1988,1:113-120.
Picknett T M ,Saunders G. Transformation of Penicillium chrysogenum with selection for increased resistance to benomyl. FEMS Microbiol. Lett. 1989,10:165-168.
Punt P J, Oliver R P, Dingemanse M A,Pouwel P H,van den Hondel C A M J J. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli.Gene, 1987,56:117-124.
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.
Rombach M C, Humber R A ,Roberts D W. Metarhizium flavoviride var.minusvar. nov.,a pathogenic of plant and leaf hoppers on rice Philippine sand Solomon Islands.Mycotaxon,1986,27:87~92.
Rath A C,Carr C J,Graham B R. Characterization of Metarhizium anisopliae strains by carbohydrate utilization(AP150CH).J Invert Pathol,1995,65:152~161.
Rkotonirainy M S,Dutertre M,Cariou M L et al., Phylogenetic relationships with in the genera Metarhizium based on 28S rRNA sequences and isozyme comparison.MycolRes,1994,98:225~230.
Rupesh Thakur,Hu S S. Development of Transformation system for entomopathogenic fungus Metarhizium anisopliae (ENT 12) based on cnx-gene. Indian Journal of Microbiology, 2003,43(3):187-192.
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.
Singh S P ,Rethinam P . Coconut hispid beetle Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae). Cord, 2004,Vol.20,p.1-20.
Sandhu S S,Kinghorn J R,Rajak R C, Unkles S E. Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.Indian Journal of Experimental Biology, 2001,39(7): 650-653.
Steven E,Screen,Gang Hu,Raymond J, St.Leger. Transformants of Metarhizium anisopliae sf.anisopliae Overexpressing Chitinase from Metarhizium anisopliae sf.acridum Show Early Induction of Native Chitinase but Are Not Altered in Pathogenicity to Manduca Sexta. Journal of Invertebrate Pathology,2001, 78,260-266.
St Leger R J, May B, Allee L L, et al,. Genetic differencesinal lozymesand information of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae.J Inverterb Pathol,1992,60:89~101.
St.Leger R J,Shimizu S,Joshi L,Bidochka M J ,Roberts D W. Co-Transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants. FEMS Microbiology Letters. [Amsterdam, The Netherlands : Elsevier Science B.V], 1995, 131(3):289-294.
St.Leger R J,Screen S E. Prospects for strain improvement of fungal pathogens of insects and weeds.In:fungi as biocontrol agent. CABI international, 2001,pp219-237.
Souza Dias E , Vieira de Queiroz M , Gomes Cardoso P , Goncalves de Barros E , Fernandes Araujo E. Transformation of Penicillium expansum with a heterologous gene which confers resisance to benomyl. World Journal of Microbiology & Biotechnology. 1999,15, (4): 513-514.
SEIP E R,WOLOSHUK C P,PAYNE G A ,CURTIS S E .Isolation and sequence Analysis of a β-Tubulin Gene from Aspergillus flavus and Its Use as a Selectable Marker.APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec.1990, p.3686-3692.
Susumu Shimizu,Motoko Yamaji,2002.Pathogenicity of Entomopathogenic Fungi to the Termite, Reticulitermes speratus .Japanese Journal of Applied Entomology and Zoology.46(2):89-91.
Tulloch M.Thegenus Metarhizium.Trans Brit Mycol Soc,1976,66:407~411.
Tzean S S,Hsieh L S,Chen J L et al., Nomuraea cylindrosporacomb nov.Mycologia, 1993, 85:514~519.
Thomas J H,Neff N F,Botstein D.Isolation and characterization of mutation in the β-tubulin gene of Saccharomyces cerevisiae.Genetics. 1985,112:715-734.
Thompson C J,Movva N R,Tizard R,Crameri R,Davies J E,Lauwereys M,Botterman J. Characterization of the herbicide-resisitance gene bar from streptomydes hygroscopicus. EMBO J, 1987,6:2519-2523.
Tarasco E, Bievre C de, Papierok B, Poliseno M, Triggiani O. Occurrence of entomopathogenic fungi in soils in Southern Italy. Entomologica, Bari 1997; 31: 157–166.
Tigano-Milani M S, Faria M R de, Martins I, et al. Ocorrência 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.
Ulhoa C J , Vainstein M H, Peberdy J F.Transformation of Trichoderma species with dominant selectable markers. Curr. Genet.1992,21: 23-26.
Valadares-Inglis M C, Inglis P W. Transformation of the entomopathogenic fungus, Metarhizium flavoviride strain CG423 to benomyl resistance. FEMS Microbiology Letters. 1997. 155: 2, 199-202.
Weiguo Fang,Yan Pei,Michael J Bidochka. Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens.Canadian Journal of Microbiology; Jul 2006; 52(623-626).
Williams J G K,Kubelik A R,Livak K J et al., DNA polymorphism samplified by arbitary primers are useful as genetic markers.Nucl Acids Res,1990,18: 6531~6535.
Wikardi E A. Protects for controlling coconut hispids with Metarhizium sp. [Plesispareichei, and Brontispa longissima]. Pemberitaan Lembaga Tanaman Ind. Bogor: Lembaga,1982, 8(44):35～38.
Yanden O,Katan T.Mutations leading to substiutions at a mino acids 198 and 200 of beta-tubulin that correlate with benomyl-resistance pheno-types of field strains of Botrytis cinerea. Phytopathology, 1993,83(12):1478～1483.
ZHAN Ru-lin, HUANG Jun-sheng. Cloning and Detecting of a High Carbendazim- Resistant Gene From Colletotrichum gloeosporioides of Mango in South China. African Journal of Biotechnology Vol. 6 (2), pp. 143-147, 18 January, 2007. Zimmerman G. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biological agent. Pesticide Science 1993; 37:375–379.
迟玉杰,杨谦,杜金哲.tub2 基因鉴定及其在毛壳菌中的转化.北京林业大学学报, 2003, 25 (4): 30-34.
陈国相.微生物及其在农业上的应用.河北人民出版社,石家庄,1980,130.
陈祝安,黄基荣.不同来源绿僵菌对云斑金龟蛴螬致病力评价.微生物学通报,1997,24(2):81～83.
丁中,刘峰,慕立义.紫外光诱导哈茨木霉产生腐霉利抗性菌株的研究.中国生物防治, 2002,18(2):75～78.
丁立孝,李文泽,宋子红,徐丽娟,梅汝鸿.双抗性标记花生内生菌的诱变与筛选.莱阳农学院学报,1997,14(4):235～239.
董辉,苏红田,高松.绿僵菌对蝗虫及其捕食性天敌的影响.中国生物防治,2005,21(1):60～62.
方卫国,张永军,肖月华,杨星勇,范艳华,侯磊,罗小英,裴炎. 2002a.根癌土壤杆菌介导球孢白僵菌的遗传转化.首届中国青年学者微生物遗传学学术研讨会论文摘要集.
方卫国,张永军,杨星勇,裴炎.2002b.根癌土壤杆菌介导真菌遗传转化的研究进展.中国生物工程杂志,22(5):41-45.
郭好礼,叶柏龄,岳莹玉等.绿僵菌属的三个新种.真菌学报,1986,5(3):177~184.
郭超,樊美趁.中国虫生真菌的研究与应用.1993,3:14-20.
贾春生.昆虫病原真菌对荔枝蝽的致病性.吉林林业科技,34(3): 32-36.
黄原.分子系统学原理、方法及应用.北京:中国农业出版社,1998
黄大昉主编,农业微生物基因工程.科学出版社,2001.
胡景江,樊美珍.绿僵菌免疫特性的研究.安徽农业大学学报,1996,23(3):433~437
李梅,杨谦,宋金柱.多菌灵抗性基因在球毛壳菌中的转化.高技术通讯,2002(2):43-45.
李维,张义正.1999.以潮毒素 B 磷酸转移酶基因为遗传标记的启动子探针载体的构建.四川大学学报(自然科学版),36(4):736-740.
李宝笃,沈崇尧.植物病原真菌对杀菌剂的抗性对策.植物病理学报,1994,24(3):193-196.
李红霞,陆悦健,王建新,周明国.四种不同植物病原真菌与多菌灵抗药性相关基因突变的比较.南京农业大学学报,2002,24(3):41-44.
李梅,杨谦,宋金柱.多菌灵抗性基因在球毛壳菌中的转化.高技术通讯,2002(2):43-45.
李维,张义正.1999.以潮毒素 B 磷酸转移酶基因为遗传标记的启动子探针载体的构建.四川大学学报(自然科学版),36(4):736-740.
刘波,苗荣,Hollmon D W.大麦云纹斑病菌中抗多菌灵乙霉威株系及其抗药性对致病性作用分子分析.植物病理学报,1996,26(3):211～215.
刘爱英,梁宗琦,曹蕾.双型孢绿僵菌的分离鉴定研究.贵州农学院学报,1989(2):27~31.
刘兆伟,郭好礼,KurtzmanCP.由部分核糖体 RNA序列确定的绿僵菌属种系统发育关系.真菌学报,1994,13(2):139~151.
刘少华,陆金萍,朱瑞良,戴富明,2005. 一种快速简便的植物病原真菌基因组 DNA 提取方法. 植物病理学报 35 (4): 362-365
刘爱英,1988.绿僵菌属的新记录种.真菌学报.7(3):191-192.
刘萍萍,闫艳春.微生物农药研究进展.山东农业科学,2005(2):78～80.
梁宗琦,刘爱英,刘杰麟.虫草一新种及其绿僵菌无性型.真菌学报, 1991, 10(4): 257~262.
雷仲仁,问锦曾.绿僵菌治蝗研究进展.植物保护,2004,30(4):14-17.
卢辉,张礼生,张泽华,沈有孝,吴坤宏,龙瑞军.绿僵菌侵染椰心叶甲的部位及侵染过程的电镜观察. 植物检疫,2005,19(4):201-204.
林华峰,李世广,张磊,王萍莉,周义文.绿僵菌大孢变种的生物学特征及其对蛴螬的毒力研究应用生态学报. 2006,17(2): 351~335.
樊美珍,郭超.金龟子绿僵菌对几种林木害虫的致病性及防治应用试验. 生物防治通报, 1987,4(1):29-32.
李贵芳,高贤华.绿僵菌 Metarhizium SP.防治天麻双叉犀金龟及其应用研究.贵州农学院学报,1994, 13(1):103-104.
农向群,吴正铠,包建中.金龟子绿僵菌原生质体形成和再生的研究.微生物学通报,1990,17(2):76-80.
蒲蛰龙.应用昆虫的病原真菌防治害虫 . 生物防治通报, 1985,1(1):27-31.
蒲蛰龙主编.昆虫病理学.广东科技出版社,1992.
蒲蛰龙,李增智.昆虫真菌学. 安徽科技出版社,1996,P93、378.
轻工部甘蔗糖业科学研究所.绿僵菌(Metarhizium anisopliae)防治黑色蔗龟(Alissonotum spp.)试验报告. 1978.
邱式邦.蝗虫生物防治的新进展.植保参考,1992,6:13-14.
任志红,徐平,王富强,苏彩云,丰玫玫,张佳,戴梦,韦春玲,穆岷,贺建功.产黄青霉工业生
产菌种基因报告系统的构建及启动子效率的评价.菌物学报,2005,24(3):376～384.
孙乃恩,孙东旭,朱德煦编著.分子遗传学.南京大学出版社,2002 年,P161～165.
孙晓君,朱婷婷,秦智,蒋忠平,魏金芝,张艳,薛滨泰.紫外诱变获得耐高浓度丙烯腈菌株的研究. 哈尔滨工业大学学报,2005,37(10):1376～1393.
王音,雷仲仁,张青文.绿僵菌侵染小菜蛾体表过程的显微观察.昆虫学报,2005, 48(2):188-193.
王四宝,黄勇平,张心团,刘云鹏,樊美珍,李增智.松褐天牛成虫高毒力病原真菌筛选及林间感染试验. 中国森林病虫,2004 年,23(6):13-16.
王万立,王勇,刘耕春,郝永娟,刘春燕,陈静.天津市茄子灰霉病菌的抗药性检测.华北农学报,1998,13(4):107～111.
王芳,徐秉良,曹奎荣.深绿木霉紫外光诱导耐低温突变菌株的研究.甘肃农业大学学报, 2005,40(4):507～511.
武觐文.侵染金龟子的几种真菌. 北京林学院学报 1982,(3):88-93.
熊道雅.绿僵菌防治甘薯华叶虫(Colasposoma metallicum).植物保护,1981,7:36.
杨洪俊,宗兆锋,郭小芳.紫外线诱导提高重寄生放线菌PR和F46活性的研究.西北农业学报,2005,14(3):22～25A.
杨谦,赵小岩.多菌灵抗性基因在木霉菌中的转化方法.科学通报,1998,43(22): 2423-2426.
赵善欢.植物化学保护.北京:中国农业出版社,2002,118-154.
赵俊生,郭素萍,武慧贞.利用绿僵菌防治鳞翅目害虫研究.山西农业科学2000,28(2):3～6.
张永军,方卫国,裴颖,裴炎.两株球孢白僵菌 T-DNA 插入突变体的生物学特性.首届中
国青年学者微生物遗传学学术研讨会论文摘要集, 2002 年.
张承来,欧晓明.植物病原物对杀菌剂的抗药性机制概述.湖南化工,2000,30(5):7-10.
张道海,王未名,陆文华,等.生防真菌的分子生物学研究.中国虫生真菌研究与应用.第 4卷.北京:中国农业科技出版社,1997.32~36.
周明国,叶钟音.杀菌剂抗性研究进展.南京农业大学学报.1994,17(3):33-41.
周明国,叶钟音.植物病原菌对苯并咪唑类及相关杀菌剂的抗药性.植物保护,1987,(2):31～33.
周燚,王中康,喻子牛主编.微生物农药研发与应用.化学工业出版社,北京,2006,56-70.
詹儒林,李伟,郑服丛.芒果炭疽病菌对多菌灵的抗药性.植物保护学报,2005(1): 71-75.
詹儒林,许天委,张世清,黄俊生.紫外光的照射与绿缰菌对多菌灵敏感性变化的关系.热带作物学报, 2006,26(4):91-94.