美国花生种质资源果腐病抗性分子标记的筛选及分析
|
摘要
花生(Arachis hypogaea)果腐病(pod rot)是一种由真菌引起的严重病害,直接影响花生的产量及品质。抗病育种是该病害防治最经济、有效的途径,而抗性相关分子标记的筛选和利用又是提高选择效率和加快育种进程的重要手段。本研究以引进的77份美国花生种质资源为材料,利用12对SSR标记和48对插入缺失(insertion-deletion, InDel)标记,通过Pearson相关分析和多元线性逐步回归筛选与花生果腐病抗性相关的标记,并利用构建的分离群体对相关标记进行了验证。结果表明,美国种质资源遗传多样性丰富,具有不同抗性级别的材料。利用12对SSR引物对77份美国种质资源进行扩增,共获得103条多态性条带,多态性比率在20%～100%,平均79.23%。从受害指数进行相关和多元线性回归分析,获得了4个与花生果腐病抗性显著相关的SSR标记,分别为3A8、GM1760、PM163和14H6。分离群体验证结果表明获得的4对SSR标记可用于花生果腐病抗性分子标记的深入研究。15对InDel标记在材料中具有多态性,通过果腐病抗性-标记相关性分析获得了2对InDel标记,分别是InDel-004和InDel-020。花生果腐病抗性相关分子标记的获得为分子辅助选择育种及抗病相关基因的挖掘提供了重要参考。
Peanut(Arachis hypogaea) pod rot, a worldwide fungal disease, has become one of the important diseases that directly affect the production and quality of peanut. Pod rot, as a kind of soil-borne disease, is difficult to be completely controlled with chemicals, cultivation methods or even biological techniques.Disease resistance breeding is an economical and effective way to control the pod rot. The screening and utilization of resistance related molecular markers will be an important means to improve the selection efficiency and speed up the breeding process. According to the results of the previous study, 12 pairs of SSR markers related to peanut disease resistance in references and 48 pairs of insertion-deletion(InDel) markers developed based on the result of stress resistance in this lab were selected to detect 77 American peanut germplasm resources, and the correlation between pod rot resistance and markers were analyzed. Pearson correlation analysis and multiple linear stepwise regression analysis were conducted based on the evaluation criteria of peanut pod rot resistance established in this laboratory. And the associated SSR markers of pod rot were validated with a following F2 segregation-population. The results revealed that there were abundant of genetic diversity and disease resistance in American germplasm resources, and inter-annual environmental factors had a significant impact on disease resistance of peanut germplasm. The results of SSR polymorphism showed that there were 103 polymorphic bands and the polymorphic rate of 20% to 100% with an average of79.23% among 77 peanut germplasm. The results of correlation and multiple linear stepwise regression analysis between injury index and SSR markers revealed that four markers including 3 A8, GM1760, PM163 and 14 H6 were significantly associated with peanut pod rot resistance. These 4 markers were considered could be well used in further study on the development of molecular markers related to peanut pod rot resistance.The validation results in F2 population showed that SSR marker 14 H6 had a close linkage with peanut pod rot which considered being the practical application value. 48 pairs of InDel markers were also used to screen American germplasm resources and 15 of 48 InDel markers showed polymorphism among the germplasms with the average polymorphic rate of 90.67%. The results of Pearson correlation analysis and stepwise multiple linear regression analysis showed that only 2 InDel(InDel-004, InDel-020) markers were found to be significantly correlated with peanut pod rot resistance. And the proportions of phenotypic variance explained by InDel-004 and InDel-020 were 5.1% and 5.9%, respectively, for what these two markers were not validated by F2 population due to low practical application value expectation. This study not only confirmed the resistance of peanut germplasm from different sources but also provided excellent materials forgenetic improvement of peanut disease resistance by rational utilization of germplasm. And furthermore, the acquisition of molecular markers related to peanut pod rot resistance could provide an important reference for molecular assisted selection breeding and the excavation of disease-related genes.
Peanut(Arachis hypogaea) pod rot, a worldwide fungal disease, has become one of the important diseases that directly affect the production and quality of peanut. Pod rot, as a kind of soil-borne disease, is difficult to be completely controlled with chemicals, cultivation methods or even biological techniques.Disease resistance breeding is an economical and effective way to control the pod rot. The screening and utilization of resistance related molecular markers will be an important means to improve the selection efficiency and speed up the breeding process. According to the results of the previous study, 12 pairs of SSR markers related to peanut disease resistance in references and 48 pairs of insertion-deletion(InDel) markers developed based on the result of stress resistance in this lab were selected to detect 77 American peanut germplasm resources, and the correlation between pod rot resistance and markers were analyzed. Pearson correlation analysis and multiple linear stepwise regression analysis were conducted based on the evaluation criteria of peanut pod rot resistance established in this laboratory. And the associated SSR markers of pod rot were validated with a following F2 segregation-population. The results revealed that there were abundant of genetic diversity and disease resistance in American germplasm resources, and inter-annual environmental factors had a significant impact on disease resistance of peanut germplasm. The results of SSR polymorphism showed that there were 103 polymorphic bands and the polymorphic rate of 20% to 100% with an average of79.23% among 77 peanut germplasm. The results of correlation and multiple linear stepwise regression analysis between injury index and SSR markers revealed that four markers including 3 A8, GM1760, PM163 and 14 H6 were significantly associated with peanut pod rot resistance. These 4 markers were considered could be well used in further study on the development of molecular markers related to peanut pod rot resistance.The validation results in F2 population showed that SSR marker 14 H6 had a close linkage with peanut pod rot which considered being the practical application value. 48 pairs of InDel markers were also used to screen American germplasm resources and 15 of 48 InDel markers showed polymorphism among the germplasms with the average polymorphic rate of 90.67%. The results of Pearson correlation analysis and stepwise multiple linear regression analysis showed that only 2 InDel(InDel-004, InDel-020) markers were found to be significantly correlated with peanut pod rot resistance. And the proportions of phenotypic variance explained by InDel-004 and InDel-020 were 5.1% and 5.9%, respectively, for what these two markers were not validated by F2 population due to low practical application value expectation. This study not only confirmed the resistance of peanut germplasm from different sources but also provided excellent materials forgenetic improvement of peanut disease resistance by rational utilization of germplasm. And furthermore, the acquisition of molecular markers related to peanut pod rot resistance could provide an important reference for molecular assisted selection breeding and the excavation of disease-related genes.
引文
崔顺立.2010.河北省花生(Arachis hypogaea L.)地方品种遗传多样性研究[D].硕士学位论文,河北农业大学,导师:刘立峰,pp.11.(Cui S L.2010.Studies on the genetic diversity of peanut(Arachis hypogaea L.)landraces in Hebei province[D].Thesis for M.S.,Hebei Agricultural University,Supervisor:Liu L F,pp.11.)
何美敬,刘阳杰,崔顺立,等.2018.花生种质资源果腐病的抗性评价[J].植物遗传资源学报,19(4):648-657.(He M J,Liu Y J,Cui S L,et al.Evaluation of peanut germplasm resources resistance to pod rot[J].Journal of Plant Genetic Resources,19(4):648-657.)
洪彦彬,李少雄,刘海燕,等.2009.SSR标记与花生抗黄曲霉性状的关联分析[J].分子植物育种,7(2):360-364.(Hong Y B,Li S X,Liu H Y,et al.2009.Correlation analysis of SSR markers and host resistance to Aspergil?lus flavus infection in peanut(Arachis hypogaea L.)[J].Molecular Plant Breeding,7(2):360-364.)
洪彦彬,温世杰,钟旎,等.2011.SSR标记与花生青枯病、锈病抗性的相关性研究[J].广东农业科学,(S1):61-63.(Hong Y B,Wen S J,Zhong N,et al.2011.Correlation analysis of SSR markers with bacterial wilt and rust resistance of peanut(Arachis hypogaea.L)[J].Journal of Guangdong Agricultural Sciences,(S1):61-63.)
胡宏霞.2013.河北省花生地方品种遗传多样性及性状-标记相关分析[D].硕士学位论文,河北农业大学,导师:刘立峰,pp.12-14.(Hu H X.2013.Genetic diversity and marker-trait analysis for the peanut landraces in Hebei[D].Thesis for M.S.,Hebei Agricultural University,Supervisor:Liu L F,pp.12-14.)
黄莉,任小平,张晓杰,等.2012.ICRISAT花生微核心种质农艺性状和黄曲霉抗性关联分析[J].作物学报,38(6):935-946.(Huang L,Ren X P,Zhang X J,et al.2012.Association analysis of agronomic traits and resistance to Aspergillus flavus in the ICRISAT peanut mini-core collection[J].Acta Agronomica Sinica,38(6):935-946.)
姜慧芳.2006.花生抗青枯病种质的遗传多样性及抗性分子标记[D].博士学位论文,华中农业大学,导师:傅廷栋.pp.66-73.(Jiang H F.2006.Genetic diversity among genotypes with bacterial wilt resistance and molecular markers linked to resistance in peanut(Arachis hypogaea L.)[D].Thesis for Ph.D.,Huazhong Agricultural University,Supervisor:Fu T D,pp.66-73.)
姜慧芳,廖伯寿,任小平,等.2007.用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较(英文)[J].遗传学报,34(6):544-554.(Jiang H F,Liao B S,Ren X P,et al.2007.Comparative assessment of genetic diversity of peanut(Arachis hypogaea L.)genotypes with various levels of resistance to bacterial wilt through SSR and AFLP analyses[J].Journal of Genetics and Genomics,34(6):544-554.)
郭峰,阮建,王莹莹,等.2017.利用变异系数分析花生品质性状应对环境变化的遗传稳定性研究[J].山东农业科学,49(9):25-31.(Guo F,Ruan J,Wang Y Y,et al.2017.Study on genetic stability of peanut quality characters in response environmental changes using coefficient of variation[J].Shandong Agricultural Sciences,2017,49(9):25-31.)
雷永.2004.花生对黄曲霉菌侵染抗性的分子标记[D].硕士学位论文,中国农业科学院,导师:廖伯寿,pp.1-42.(Lei Y.2004.Molecular marker for resistance to seed infection by Aspergillus flavus in peanut(Arachis hypogaea.L)[D].Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Liao BS.pp.1-42.)
雷永,廖伯寿,王圣玉,等.2006.花生黄曲霉侵染抗SCAR标记[J].遗传,28(9):1107-1111.(Lei Y,Liao B S,Wang S Y,et al.2006.A SCAR marker for resistance to Aspergillus flavus in peanut(Arachis hypogaea L.)[J].Hereditas,28(9):1107-1111.)
李术臣,陈丹,贾海民,赵聚莹,等.2010.花生果腐病研究进展[J].河北农业科学,14(9):74-75.(Li S C,Chen D,Jia H M,et al.2010.Research progress of peanut pod rot[J].Journal of Hebei Agricultural Sciences,14(9):74-75.)
李术臣,贾海民,赵聚莹,等.2011.河北省花生果腐病病原鉴定及致病性研究[J].河北农业科学,15(5):37-39.(Li S C,Jia H M,Zhao J T,et al.2011.Pathogens identification and pathogenicity of peanut pod rot in Hebei province[J].Journal of Hebei Agricultural Sciences,15(5):37-39.)
万书波.2003.中国花生栽培学[M].上海:上海科学技术出版社,pp.1-9.(Wan S B.2003,Peanut Cultivated Science in China[M].Shanghai Scientific and Technical Publishers,Shanghai,China,pp.1-9.)
王传堂,禹山林,于洪涛,等.2010.花生果腐病病原分子诊断[J].花生学报,2010,39(1):1-4.(Wang C T,Yu S L,Yu H T,et al.2010.Molecular diagnosis of peanut pod rot pathogens[J].Journal of Peanut Science,39(1):1-4.)
魏和清,罗良清.2011.实用统计学[M].北京:中国财政经济出版社,pp.49.(Wei H Q,Luo L Q.2011.Applied Statistics[M].China Financial and Economic Publishing House,Beijing,China,pp.49.)
徐志军.2016.花生青枯病抗性相关的QTL分析[D].硕士学位论文,中国农业科学院,导师:姜慧芳.pp.13-27.(Xu Z J.2016.Quantitative trait locus analysis of bacteria wilt resistance[D].Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Jiang H F,pp.13-27.)
徐志军,任小平,黄莉,等.2015.花生青枯病抗性相关SSR标记的筛选鉴定[J].中国油料作物学报,37(6):803.(Xu Z J,Ren X P,Huang L,et al.2015.Identification of SSR makers associated with bacterial wilt resistance of peanut through correlation analysis[J].Chinese Journal of Oil Crop Sciences,37(6):803.)
夏友霖,廖伯寿,李加纳,等.2007.花生晚斑病抗性AFLP标记[J].中国油料作物学报,29(3):318-321.(Xia Y L,Liao B S,Li J N,et al.2007.Identification of AFLPmarkers linked to resistance to late leaf spot in peanut(Arachis hypogaea.L)[J].Chinese Journal of Oil Crop Sciences,29(3):318-321.)
杨鑫雷,周晓栋,刘恒蔚,等.2013.AFLP标记与棉花重要农艺性状的关联研究[J].棉花学报,25(3):211-216.(Yang X L,Zhou X D,Liu H W,et al.2013.AFLPmarkers associated with important agronomic trait in cotton[J].Cotton Science,25(3):211-216.)
张成玲,张田田,吴翠霞,等.2016.花生果腐病病菌鉴定及生物学特性[J].花生学报,45(3):27-31.(Zhang C L,Zhang T T,Wu C X,et al.2016.Identification and biological characteristics of fungal isolates causing peanut pod rot pathogen[J].Journal of Peanut Science,45(3):27-31.)
周金超,杨鑫雷,崔顺立,等.2014.花生SSR标记与农艺性状的相关性[J].作物学报,40(7):1197-1204.(Zhou J C,Yang X L,Cui S L,et al.2014.Correlation between SSRmarkers and agronomic traits in peanut(Arachis hypo?gaea.L)[J].Acta Agronomica Sinica,40(7):1197-1204.)
Frank Z R.1972.Pythium myriotylum and Fusarium solanias cofactors in a pod-rot complex of peanut[J].Phytopathology,62:1331-1334.
Garcia R,Mitchel D J.1975.Interactions of Pythium myrioty?lum with several fungi in peanut pod rot[J].Phytopathology,65:1375-1381.
Guimar?es P M,Brasileiro A C,Morgante C V,et al.2012.Global transcriptome analysis of two wild relatives of peanut under drought and fungi infection[J].BMC Genomics,13(1):387.
Hayashi K,Yoshida H,Ashikawa I.2006.Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes[J].Theoretical and Applied Genetics,113(2):251-260.
Hollowell J E,Shew B B,Beute M K.1998.Occurrences of pod rot pathogens in peanut grown in North Carolina[J].Plant Disease,82(12):1345-1349.
Liu L F,Dang P M,Chen C Y.2015.Development and utilization of In Del markers to identify peanut(Arachis hypogaea.L)disease resistance[J].Frontiers in Plant Science,6.
Meng S,Yang X L,Dang P M,et al.2016.Evaluation of insertion-deletion markers suitable for genetic diversity studies and marker-trait correlation analyses in cultivated peanut(Arachis hypogaea L.)[J].Genetics&Molecular Research,15(3):1-12.
Sun W M,Feng L N,Guo W,et al.2012.First report of Neocosmospora striata causing peanut pod pot in China[J].Plant Disease,96(1):146.
Wheeler T A,Howell C R,Cotton J,et al.2005.Pythium species associated with pod rot on west texas peanuts and in vitro sensitivity of isolates to mefenoxam and azoxystrobin[J].Peanut Science,32:9-13.
何美敬,刘阳杰,崔顺立,等.2018.花生种质资源果腐病的抗性评价[J].植物遗传资源学报,19(4):648-657.(He M J,Liu Y J,Cui S L,et al.Evaluation of peanut germplasm resources resistance to pod rot[J].Journal of Plant Genetic Resources,19(4):648-657.)
洪彦彬,李少雄,刘海燕,等.2009.SSR标记与花生抗黄曲霉性状的关联分析[J].分子植物育种,7(2):360-364.(Hong Y B,Li S X,Liu H Y,et al.2009.Correlation analysis of SSR markers and host resistance to Aspergil?lus flavus infection in peanut(Arachis hypogaea L.)[J].Molecular Plant Breeding,7(2):360-364.)
洪彦彬,温世杰,钟旎,等.2011.SSR标记与花生青枯病、锈病抗性的相关性研究[J].广东农业科学,(S1):61-63.(Hong Y B,Wen S J,Zhong N,et al.2011.Correlation analysis of SSR markers with bacterial wilt and rust resistance of peanut(Arachis hypogaea.L)[J].Journal of Guangdong Agricultural Sciences,(S1):61-63.)
胡宏霞.2013.河北省花生地方品种遗传多样性及性状-标记相关分析[D].硕士学位论文,河北农业大学,导师:刘立峰,pp.12-14.(Hu H X.2013.Genetic diversity and marker-trait analysis for the peanut landraces in Hebei[D].Thesis for M.S.,Hebei Agricultural University,Supervisor:Liu L F,pp.12-14.)
黄莉,任小平,张晓杰,等.2012.ICRISAT花生微核心种质农艺性状和黄曲霉抗性关联分析[J].作物学报,38(6):935-946.(Huang L,Ren X P,Zhang X J,et al.2012.Association analysis of agronomic traits and resistance to Aspergillus flavus in the ICRISAT peanut mini-core collection[J].Acta Agronomica Sinica,38(6):935-946.)
姜慧芳.2006.花生抗青枯病种质的遗传多样性及抗性分子标记[D].博士学位论文,华中农业大学,导师:傅廷栋.pp.66-73.(Jiang H F.2006.Genetic diversity among genotypes with bacterial wilt resistance and molecular markers linked to resistance in peanut(Arachis hypogaea L.)[D].Thesis for Ph.D.,Huazhong Agricultural University,Supervisor:Fu T D,pp.66-73.)
姜慧芳,廖伯寿,任小平,等.2007.用SSR和AFLP技术分析花生抗青枯病种质遗传多样性的比较(英文)[J].遗传学报,34(6):544-554.(Jiang H F,Liao B S,Ren X P,et al.2007.Comparative assessment of genetic diversity of peanut(Arachis hypogaea L.)genotypes with various levels of resistance to bacterial wilt through SSR and AFLP analyses[J].Journal of Genetics and Genomics,34(6):544-554.)
郭峰,阮建,王莹莹,等.2017.利用变异系数分析花生品质性状应对环境变化的遗传稳定性研究[J].山东农业科学,49(9):25-31.(Guo F,Ruan J,Wang Y Y,et al.2017.Study on genetic stability of peanut quality characters in response environmental changes using coefficient of variation[J].Shandong Agricultural Sciences,2017,49(9):25-31.)
雷永.2004.花生对黄曲霉菌侵染抗性的分子标记[D].硕士学位论文,中国农业科学院,导师:廖伯寿,pp.1-42.(Lei Y.2004.Molecular marker for resistance to seed infection by Aspergillus flavus in peanut(Arachis hypogaea.L)[D].Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Liao BS.pp.1-42.)
雷永,廖伯寿,王圣玉,等.2006.花生黄曲霉侵染抗SCAR标记[J].遗传,28(9):1107-1111.(Lei Y,Liao B S,Wang S Y,et al.2006.A SCAR marker for resistance to Aspergillus flavus in peanut(Arachis hypogaea L.)[J].Hereditas,28(9):1107-1111.)
李术臣,陈丹,贾海民,赵聚莹,等.2010.花生果腐病研究进展[J].河北农业科学,14(9):74-75.(Li S C,Chen D,Jia H M,et al.2010.Research progress of peanut pod rot[J].Journal of Hebei Agricultural Sciences,14(9):74-75.)
李术臣,贾海民,赵聚莹,等.2011.河北省花生果腐病病原鉴定及致病性研究[J].河北农业科学,15(5):37-39.(Li S C,Jia H M,Zhao J T,et al.2011.Pathogens identification and pathogenicity of peanut pod rot in Hebei province[J].Journal of Hebei Agricultural Sciences,15(5):37-39.)
万书波.2003.中国花生栽培学[M].上海:上海科学技术出版社,pp.1-9.(Wan S B.2003,Peanut Cultivated Science in China[M].Shanghai Scientific and Technical Publishers,Shanghai,China,pp.1-9.)
王传堂,禹山林,于洪涛,等.2010.花生果腐病病原分子诊断[J].花生学报,2010,39(1):1-4.(Wang C T,Yu S L,Yu H T,et al.2010.Molecular diagnosis of peanut pod rot pathogens[J].Journal of Peanut Science,39(1):1-4.)
魏和清,罗良清.2011.实用统计学[M].北京:中国财政经济出版社,pp.49.(Wei H Q,Luo L Q.2011.Applied Statistics[M].China Financial and Economic Publishing House,Beijing,China,pp.49.)
徐志军.2016.花生青枯病抗性相关的QTL分析[D].硕士学位论文,中国农业科学院,导师:姜慧芳.pp.13-27.(Xu Z J.2016.Quantitative trait locus analysis of bacteria wilt resistance[D].Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Jiang H F,pp.13-27.)
徐志军,任小平,黄莉,等.2015.花生青枯病抗性相关SSR标记的筛选鉴定[J].中国油料作物学报,37(6):803.(Xu Z J,Ren X P,Huang L,et al.2015.Identification of SSR makers associated with bacterial wilt resistance of peanut through correlation analysis[J].Chinese Journal of Oil Crop Sciences,37(6):803.)
夏友霖,廖伯寿,李加纳,等.2007.花生晚斑病抗性AFLP标记[J].中国油料作物学报,29(3):318-321.(Xia Y L,Liao B S,Li J N,et al.2007.Identification of AFLPmarkers linked to resistance to late leaf spot in peanut(Arachis hypogaea.L)[J].Chinese Journal of Oil Crop Sciences,29(3):318-321.)
杨鑫雷,周晓栋,刘恒蔚,等.2013.AFLP标记与棉花重要农艺性状的关联研究[J].棉花学报,25(3):211-216.(Yang X L,Zhou X D,Liu H W,et al.2013.AFLPmarkers associated with important agronomic trait in cotton[J].Cotton Science,25(3):211-216.)
张成玲,张田田,吴翠霞,等.2016.花生果腐病病菌鉴定及生物学特性[J].花生学报,45(3):27-31.(Zhang C L,Zhang T T,Wu C X,et al.2016.Identification and biological characteristics of fungal isolates causing peanut pod rot pathogen[J].Journal of Peanut Science,45(3):27-31.)
周金超,杨鑫雷,崔顺立,等.2014.花生SSR标记与农艺性状的相关性[J].作物学报,40(7):1197-1204.(Zhou J C,Yang X L,Cui S L,et al.2014.Correlation between SSRmarkers and agronomic traits in peanut(Arachis hypo?gaea.L)[J].Acta Agronomica Sinica,40(7):1197-1204.)
Frank Z R.1972.Pythium myriotylum and Fusarium solanias cofactors in a pod-rot complex of peanut[J].Phytopathology,62:1331-1334.
Garcia R,Mitchel D J.1975.Interactions of Pythium myrioty?lum with several fungi in peanut pod rot[J].Phytopathology,65:1375-1381.
Guimar?es P M,Brasileiro A C,Morgante C V,et al.2012.Global transcriptome analysis of two wild relatives of peanut under drought and fungi infection[J].BMC Genomics,13(1):387.
Hayashi K,Yoshida H,Ashikawa I.2006.Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes[J].Theoretical and Applied Genetics,113(2):251-260.
Hollowell J E,Shew B B,Beute M K.1998.Occurrences of pod rot pathogens in peanut grown in North Carolina[J].Plant Disease,82(12):1345-1349.
Liu L F,Dang P M,Chen C Y.2015.Development and utilization of In Del markers to identify peanut(Arachis hypogaea.L)disease resistance[J].Frontiers in Plant Science,6.
Meng S,Yang X L,Dang P M,et al.2016.Evaluation of insertion-deletion markers suitable for genetic diversity studies and marker-trait correlation analyses in cultivated peanut(Arachis hypogaea L.)[J].Genetics&Molecular Research,15(3):1-12.
Sun W M,Feng L N,Guo W,et al.2012.First report of Neocosmospora striata causing peanut pod pot in China[J].Plant Disease,96(1):146.
Wheeler T A,Howell C R,Cotton J,et al.2005.Pythium species associated with pod rot on west texas peanuts and in vitro sensitivity of isolates to mefenoxam and azoxystrobin[J].Peanut Science,32:9-13.