Phenotypes Analysis for T-DNA Insertional Mutants of Paecilomyces lilacinus

MA Jian-wei; WANG Xi-zhuo; WANG Lai-fa; WANG Yuan; GOU Da-ping; WANG Yan-ju

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Phenotypes Analysis for T-DNA Insertional Mutants of Paecilomyces lilacinus

1.
Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry

Received Date: 2013-12-27

Abstract

The T-DNA insertional mutants of Paecilomyces lilacinus were studied and the phenotypes were identified. It may contribute to understanding the pathogenic mechanism of P. lilacinus and the clone genes related to pathogenicity. All of 14 mutants generated by T-DNA insertion were tested with PCR for their insertional sequence. The morphology, colony growing rate, spore production, conidia germination rate, dry weight of mycelium and pathogenicity against Meloidogyne incongnita of phenotypes of mutants were analyzed by comparing with wild-type strain 20-7. The results demonstrated that the genomes of these mutants contained the sequence of beta-tubulin gene and T-DNA had been integrated into wild-type genome of P. lilacinus. Compared with wild-type strain 20-7, the morphologies of these colonies changed at different degrees. The colony growing rate of 85.71% strains speeded up obviously, only the strain BN-11 slowed down its colony growing rate distinctly. The strain BN-11 and BN-12, accounting for 14.29% of the total, increased the spore production. 78.57% mutants changed their conidia germination rate significantly. Both the rates of mutants that dry weight of mycelium reduced and increased were 21.43%. One mutant with increased pathogenicity was obtained, and the pathogenicity of 11 mutants decreased enormously.
- Paecilomyces lilacinus,
- insertional mutagenesis,
- biological characteristics,
- pathogenicity

References

[1]	Williamsona V M, Hussey R S. Nematode Pathogenesis and Resistance in Plants[J]. The Plant Cell, 1996, 8: 1735-1745
[2]	汪来发, 杨宝君, 关文刚, 等. 淡紫拟青霉和厚壁轮枝霉防治南方根结线虫[J]. 四川农业大学学报, 1998, 16(2): 231-233
[3]	Khan A, Williams K L, Nevalainen H K M. Control of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum in pot trials[J]. Biocontrol, 2006, 51: 643-658
[4]	Mendoza A R, Sikora R A. Biological control of Radopholus similis in banana by combined application of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus[J]. Biocontrol, 2009, 54: 263-272
[5]	Kiewnick S, Neumann S, Sikora R A, et al. Effect of Meloidogyne incognita Inoculum Density and Application Rate of Paecilomyces lilacinus Strain 251 on Biocontrol Efficacy and Colonization of Egg Masses Analyzed by Real-Time Quantitative PCR[J]. Phytopathology, 2011, 101(1): 105-112
[6]	孙漫红, 刘杏忠, 晋治波. 淡紫拟青霉对大豆胞囊线虫卵及2龄幼虫的影响[J]. 植物保护学报, 2002, 29(1): 57-61
[7]	Ramos B, Gonzalez-Melendi P, Sanchez-Vallet A, et al. Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana[J]. Molecular Plant Pathology, 2013, 14(1): 44-57
[8]	Rogers C W, Challen M P, Green J R, et al. Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus Coniothyrium minitans[J]. FEMS Microbiology Letters, 2004, 241: 207-214
[9]	Li M X, Gong X Y, Zheng J, et al. Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens[J]. FEMS Microbiology Letters, 2005, 243: 323-329
[10]	Duarte R T D, Staats C C, Fungaro M H P, et al. Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum[J]. Letters in Applied Microbiology, 2007, 44: 248-254
[11]	孙文良,胡晓璐,吴萌章,等. 根癌农杆菌介导的深绿木霉菌T23遗传转化研究[J]. 上海交通大学学报: 农业科学版,2009, 27(5): 489-493
[12]	赵培宝,任爱芝,胡明江,等. 农杆菌介导的拟康氏木霉遗传转化及T-DNA插入突变体的筛选[J]. 植物保护,2010, 36(6):74-76
[13]	Liu Y G, Whittler R F. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking[J]. Genomics, 1995, 25: 674-681
[14]	王曦茁. 根癌农杆菌介导的淡紫拟青霉遗传转化体系的建立. 北京: 中国林业科学研究院,2010
[15]	汪来发,杨宝君,李传道. 寄生真菌对根结线虫的致病力评价[J]. 林业科学,1999, 35(3): 41-47
[16]	de-Groot M J A, Bundock P, Hooykaas P J J, et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. [J] Nature Biotechnology, 1998, 16: 839-842
[17]	Michielse C B, Hooykaas P J J, van-den-Hondel C A M J J, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J]. Current Genetics, 2005, 48: 1-17
[18]	Furlaneto M C, Paiāo F G, Pinto F G S, et al. Transformation of the entomopathogenic fungus Metarhizium flavoviride to high resistance to benomyl[J]. Canadian Journal of Microbiology, 1999, 45: 875-878
[19]	黄亚丽,蒋细良,田云龙,等. 哈茨木霉厚垣孢子产生突变体的筛选及T-DNA标签序列的克隆[J]. 华北农学报,2009, 24(5): 35-39
[20]	王璇,邢继红,赵斌,等. 灰葡萄孢分生孢子产生相关基因的克隆及功能分析[J]. 微生物学通报,2013, 40(3): 533-543
[21]	Mullins E D, Chen X, Romaine P, et al. Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer[J]. Phytopathology, 2001, 91: 173-180
[22]	Sugui J A, Chang Y C, Kwon-Chung K J. Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption[J]. Applied and Environmental Microbiology, 2005, 71(4): 1798-1802
[23]	Liu Y G, Chen Y L. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. Biotechniques, 2007, 43: 649-656

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Phenotypes Analysis for T-DNA Insertional Mutants of Paecilomyces lilacinus

1. Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry

Received Date: 2013-12-27

Abstract: The T-DNA insertional mutants of Paecilomyces lilacinus were studied and the phenotypes were identified. It may contribute to understanding the pathogenic mechanism of P. lilacinus and the clone genes related to pathogenicity. All of 14 mutants generated by T-DNA insertion were tested with PCR for their insertional sequence. The morphology, colony growing rate, spore production, conidia germination rate, dry weight of mycelium and pathogenicity against Meloidogyne incongnita of phenotypes of mutants were analyzed by comparing with wild-type strain 20-7. The results demonstrated that the genomes of these mutants contained the sequence of beta-tubulin gene and T-DNA had been integrated into wild-type genome of P. lilacinus. Compared with wild-type strain 20-7, the morphologies of these colonies changed at different degrees. The colony growing rate of 85.71% strains speeded up obviously, only the strain BN-11 slowed down its colony growing rate distinctly. The strain BN-11 and BN-12, accounting for 14.29% of the total, increased the spore production. 78.57% mutants changed their conidia germination rate significantly. Both the rates of mutants that dry weight of mycelium reduced and increased were 21.43%. One mutant with increased pathogenicity was obtained, and the pathogenicity of 11 mutants decreased enormously.

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Reference (23)

[1]	Williamsona V M, Hussey R S. Nematode Pathogenesis and Resistance in Plants[J]. The Plant Cell, 1996, 8: 1735-1745
[2]	汪来发, 杨宝君, 关文刚, 等. 淡紫拟青霉和厚壁轮枝霉防治南方根结线虫[J]. 四川农业大学学报, 1998, 16(2): 231-233
[3]	Khan A, Williams K L, Nevalainen H K M. Control of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum in pot trials[J]. Biocontrol, 2006, 51: 643-658
[4]	Mendoza A R, Sikora R A. Biological control of Radopholus similis in banana by combined application of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus[J]. Biocontrol, 2009, 54: 263-272
[5]	Kiewnick S, Neumann S, Sikora R A, et al. Effect of Meloidogyne incognita Inoculum Density and Application Rate of Paecilomyces lilacinus Strain 251 on Biocontrol Efficacy and Colonization of Egg Masses Analyzed by Real-Time Quantitative PCR[J]. Phytopathology, 2011, 101(1): 105-112
[6]	孙漫红, 刘杏忠, 晋治波. 淡紫拟青霉对大豆胞囊线虫卵及2龄幼虫的影响[J]. 植物保护学报, 2002, 29(1): 57-61
[7]	Ramos B, Gonzalez-Melendi P, Sanchez-Vallet A, et al. Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana[J]. Molecular Plant Pathology, 2013, 14(1): 44-57
[8]	Rogers C W, Challen M P, Green J R, et al. Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus Coniothyrium minitans[J]. FEMS Microbiology Letters, 2004, 241: 207-214
[9]	Li M X, Gong X Y, Zheng J, et al. Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens[J]. FEMS Microbiology Letters, 2005, 243: 323-329
[10]	Duarte R T D, Staats C C, Fungaro M H P, et al. Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum[J]. Letters in Applied Microbiology, 2007, 44: 248-254
[11]	孙文良,胡晓璐,吴萌章,等. 根癌农杆菌介导的深绿木霉菌T23遗传转化研究[J]. 上海交通大学学报: 农业科学版,2009, 27(5): 489-493
[12]	赵培宝,任爱芝,胡明江,等. 农杆菌介导的拟康氏木霉遗传转化及T-DNA插入突变体的筛选[J]. 植物保护,2010, 36(6):74-76
[13]	Liu Y G, Whittler R F. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking[J]. Genomics, 1995, 25: 674-681
[14]	王曦茁. 根癌农杆菌介导的淡紫拟青霉遗传转化体系的建立. 北京: 中国林业科学研究院,2010
[15]	汪来发,杨宝君,李传道. 寄生真菌对根结线虫的致病力评价[J]. 林业科学,1999, 35(3): 41-47
[16]	de-Groot M J A, Bundock P, Hooykaas P J J, et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. [J] Nature Biotechnology, 1998, 16: 839-842
[17]	Michielse C B, Hooykaas P J J, van-den-Hondel C A M J J, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J]. Current Genetics, 2005, 48: 1-17
[18]	Furlaneto M C, Paiāo F G, Pinto F G S, et al. Transformation of the entomopathogenic fungus Metarhizium flavoviride to high resistance to benomyl[J]. Canadian Journal of Microbiology, 1999, 45: 875-878
[19]	黄亚丽,蒋细良,田云龙,等. 哈茨木霉厚垣孢子产生突变体的筛选及T-DNA标签序列的克隆[J]. 华北农学报,2009, 24(5): 35-39
[20]	王璇,邢继红,赵斌,等. 灰葡萄孢分生孢子产生相关基因的克隆及功能分析[J]. 微生物学通报,2013, 40(3): 533-543
[21]	Mullins E D, Chen X, Romaine P, et al. Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer[J]. Phytopathology, 2001, 91: 173-180
[22]	Sugui J A, Chang Y C, Kwon-Chung K J. Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption[J]. Applied and Environmental Microbiology, 2005, 71(4): 1798-1802
[23]	Liu Y G, Chen Y L. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. Biotechniques, 2007, 43: 649-656

Phenotypes Analysis for T-DNA Insertional Mutants of Paecilomyces lilacinus

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通讯作者: 陈斌, bchen63@163.com

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Phenotypes Analysis for T-DNA Insertional Mutants of Paecilomyces lilacinus

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