阿拉尔地区枣园不同时期枣花、枣果黑斑病菌带菌量检测
|
摘要
【目的】检测新疆南疆阿拉尔市枣园不同时期枣花、枣果上黑斑病菌的带菌量,确定枣果黑斑病的侵入时期和侵入部位,明确防治该病害的关键时期。【方法】在阿拉尔市周边3个枣园分别采集不同时期的枣花、枣果,采用常规组织分离法对其进行分离,并对分离物进行鉴定,用交链格孢菌特异性引物采用实时荧光定量PCR方法,检测不同时期枣花及枣果各部位带菌量。【结果】在不同时期采集的枣花及枣果各部位均分离到了链格孢菌,累计获得276个分离物,经鉴定均为A.alternata;实时荧光定量PCR检测表明,在枣花上就能检测到枣果黑斑病菌,其平均含量为2.11μg/100 mg,在栆果上果肉中链格孢菌含量最高,其中胴部果肉含量最高为20.12μg/100 mg,果柄处果肉为9.34μg/100 mg,胴部果皮11.48μg/100 mg,果柄处果皮为3.84μg/100 mg。【结论】枣果黑斑病菌在红枣开花期就可以感染枣花,病菌可在枣果果肉中大量潜伏侵染,待黑斑病显症时,果肉中已有大量病菌存在,对枣果黑斑病的药剂防治应提前至红枣开花前期。
【Objective】To clarify different infection periods and infection locations of jujube black spot disease in the Alar area. The hyphae amount of A. alternata in flowers and fruits of jujube was detected with RT-PCR in the hope of providing theoretical basis to predict and control the disease.【Method】Jujube flowers and fruits of different periods in the 3 surrounding orchards in Alar City area were collected and the conventional tissue separation method was used to isolate them,after that,these isolates were identified. And the fungi in flowers and fruits at different periods were detected with the specific primers of A. alternata by real-time fluorescence quantitative PCR method.【Result】A total of 276 isolates were obtained and these isolates were identified as A. alternata by morphological characteristic and β-tubulin,EF-1α gene sequencing analysis. The real-time fluorescence quantitative PCR detection results showed that A. alternata was checked and the amount of A. alternata was 2. 11 μg/100 mg in the flower. The highest amount of A. alternata was checked in sarcocarp of fruit body and amount of fungi was 20. 12 μg/100 mg. The amount of A. alternata was 9. 34μg/100 mg in the sarcocarp of fruit body carpopodium. The amount of A. alternata was 11. 48 μg/100 mg in the pericarp of fruit body and 3. 84 μg/100 mg in the carpopodium pericarp.【Conclusion】Jujube black spot disease pathogens in flowering period can invade flowers. When the disease has appeared,there has been a large number of A. alternata in the sarcocarp of fruit body,so chemical control of it should be advanced to the earlier flowering stage of red jujube.
【Objective】To clarify different infection periods and infection locations of jujube black spot disease in the Alar area. The hyphae amount of A. alternata in flowers and fruits of jujube was detected with RT-PCR in the hope of providing theoretical basis to predict and control the disease.【Method】Jujube flowers and fruits of different periods in the 3 surrounding orchards in Alar City area were collected and the conventional tissue separation method was used to isolate them,after that,these isolates were identified. And the fungi in flowers and fruits at different periods were detected with the specific primers of A. alternata by real-time fluorescence quantitative PCR method.【Result】A total of 276 isolates were obtained and these isolates were identified as A. alternata by morphological characteristic and β-tubulin,EF-1α gene sequencing analysis. The real-time fluorescence quantitative PCR detection results showed that A. alternata was checked and the amount of A. alternata was 2. 11 μg/100 mg in the flower. The highest amount of A. alternata was checked in sarcocarp of fruit body and amount of fungi was 20. 12 μg/100 mg. The amount of A. alternata was 9. 34μg/100 mg in the sarcocarp of fruit body carpopodium. The amount of A. alternata was 11. 48 μg/100 mg in the pericarp of fruit body and 3. 84 μg/100 mg in the carpopodium pericarp.【Conclusion】Jujube black spot disease pathogens in flowering period can invade flowers. When the disease has appeared,there has been a large number of A. alternata in the sarcocarp of fruit body,so chemical control of it should be advanced to the earlier flowering stage of red jujube.
引文
[1]王兰,冯宏祖,熊仁次,等.新疆红枣病虫害发生现状及对策[J].中国植保导刊,2014,34(6):73-75.WANG Lan,FENG Hong-zhou,XIONG Ren-ci,et al.(2014).Situation and countermeasures on pests and disease of jujube in Xinjiang[J].China Plant Protection,34(6):73-75.(in Chinese)
[2]向征,钟聪慧,黄家风,等.新疆枣树实生苗枯萎病病原鉴定[J].植物保护学报,2014,41(2):253-254.XIANG Zheng,ZHONG Cong-hui,HUANG Jia-fen,et al.(2014).Identification of thepathogen of Fusarium wilt of jujube seedlings in southern Xinjiang[J].Journal of Plant Protection,41(2):253-254.(in Chinese)
[3]Xinwen,Xuedong,Zheng,&Zhenjiang.(2015).Current development situations of ziziphus jujuba industry in south xinjiang and recommendations.Asian Agricultural Research,(4):37-41.
[4]董宁,冯宏祖,王兰,等.南疆骏枣黑斑病症状表现及病原菌鉴定[J].植物保护学报,2016,43(6):922-927.DONG Ning,FENG Hong-zu,WANG Lan,et al.(2016).The disease symptom and the pathogen identification of jujube black spot in southern Xinjiang[J].Journal of Plant Protection,43(6):922-927.(in Chinese)
[5]向征,钟聪慧,胡军,等.新疆枣果黑斑病病原鉴定[J].新疆农业科学,2013,50(5):845-850.XIANG Zheng,ZHONG Cong-hui,HU Jun,et al.(2013).Identification of jujube black spot pathogens in Xinjiang[J].Xinjiang Agricultural Sciences,50(5):845-850.(in Chinese)
[6]郭东起,蒋卉,牛宁宁,等.南疆骏枣黑斑病病原菌的分离、鉴定及控制研究[J].中国植保导刊,2015,35(12):5-8,85.GUO Dong-qi,JIANG Hui,NIU Ning-ning,et al.(2015).Separation and identification of jujube black spot pathogens and its controlling in southern Xinjiang[J].China Plant Protection,35(12):5-8,85.(in Chinese)
[7]包慧芳,王宁,侯敏.新疆红枣黑斑病病原菌鉴定及拮抗细菌筛选[J].新疆农业科学,2016,53(9):1 667-1 673.BAO Hui-fang,WANG Ning,HOU Min.(2016).Identification of the pathogen and screening of the antogonistic bacteria of jujube black spot disease in Xinjiang[J].Xinjiang Agricultural Sciences,53(9):1,667-1,673.(in Chinese)
[8]吴玉柱,季延平,刘慇,等.冬枣黑斑病发生规律的研究[J].山东林业科技,2004,(3):1-3.WU YU-zhu,JI Yan-ping,LIU Yin,et al.(2004).The study on outbreak regularity of black patch disease in cv.Dongzao jujube[J].Shandong Forestry Science and Technology,(3):1-3.(in Chinese)
[9]陈小飞,熊仁次,徐崇志,等.红枣黑斑病研究现状与展望[J].黑龙江农业科学,2013,(10):141-144.CHEN Xiao-fei,XIONG Ren-ci,XU Cong-zhi,et al.(2013).Current situation and prospect on jujube black spot disease[J].Heilongjiang Agricultural Sciences,(10):141-144.(in Chinese)
[10]马荣,刘晓琳,梁英梅,等.新疆枣果黑斑病发生规律及温湿度对其的影响[J].植物保护学报,2016,43(5):805-811.MA Rong,LIU Xiao-lin,LIAN Ying-mei,et al.(2016).The growth and decline regularity of jujube black spot pathogen and the influence of temperature and humidity in Xinjiang[J].Journal of Plant Protection,43(5):805-811.(in Chinese)
[11]孙洁,池振江,赵思峰,等.新疆南疆红枣花腐病病原鉴定[J].西北农业学报,2014,23(2):176-180.SUN Jie,CHI Zhen-jian,ZHAO Si-feng,et al.(2014).Identification of pathogen causing flower rot on jujube in southern Xinjiang[J].Acta Agriculturae Boreali-occidentalis Sinica,23(2):176-180.(in Chinese)
[12]符泽.新疆阿拉尔垦区矮化密植枣园红枣黑斑病发病规律的初步研究[D].石河子:石河子大学硕士学位论文,2015.FU Ze.(2015).Preliminary study on incidence regulation of black spot disease of jujube in reclamation area of Alar[D].Master Thesis.Shihezi University,Shihezi.(in Chinese)
[13]孙炳剑,陈清清,袁虹霞,等.SYBR Green I实时荧光定量PCR检测小麦纹枯病菌体系的建立和应用[J].中国农业科学,2015,48(1):55-62.SUN Bin-jian,CHEN Qing-qing,YUAN Hong-xia,et al.(2015).Establishment of SYBR green I real-time PCR for quantitatively detecting rhizoctonia cerealis in winter wheat[J].Scientia Agricultura Sinica,48(1):55-62.(in Chinese)
[14]肖蕊,余真真,Elsharawy A A,等.土壤中棉花黄萎病菌SYBR Green I荧光RT-PCR定量检测技术研究[J].菌物学报,2011,30(4):598-603.XIAO Rui,YU Zhen-zhen,Elsharawy A A,et al.(2011).SYBR green I real time RT-PCR assay for quantitatively detecting the occurrence of Verticillium dahliae of cotton in naturally infested soil[J].Mycosystema,30(4):598-603.(in Chinese)
[15]Guo,Y.,Li,W.,Sun,H.,Wang,N.,Yu,H.,&Chen,H.(2012).Detection and quantification of rhizoctonia cerealis,in soil using real-time PCR.Journal of General Plant Pathology,78(4):247-254.
[16]林惠娇,王卫芳,易建平,等.Taq Man探针实时荧光PCR快速检测苹果牛眼果腐病菌方法的建立[J].植物检疫,2016,30(2):55-62.LIN Hui-qiao,WANG Wei-fang,YI Jian-ping,et al.(2016).Development of Taq Man probes real-time fluorescence PCR method for the rapid detection of the causal fungal agents of bull's-eye rot on apple[J].Plant Quarantine,30(2):55-62.(in Chinese)
[17]王凤军,张祥林,谭志文,等.枣褐斑病菌实时荧光PCR快速检测方法研究[J].食品工业科技,2014,35(14):56-58,63.WANG Feng-jin,ZHANG Xiang-lin,TAN Zhi-wen,et al.(2014).Real-time fluorescence PCR method for detection of the pathogen causing brown spot on jujube[J].Science and Technology of Food Industry,35(14):56-58,63.(in Chinese)
[18]方中达.植病研究方法[M].第3版.北京:中国农业出版社,1998.FANG Zhong-da.(1998).Research methods of plant pathology[M].3rd edition.Beijing:China Agriculture Press.(in Chinese)
[19]张天宇.中国真菌志:链格孢属[M].北京:科学出版社,2003.ZHENG Tian-yu.(2003).Chinese fungi:Alternaria[M].Beijing:Science Press.(in Chinese)
[20]王勇.细基格孢属、葡柄霉属及其近似属的形态学与分子系统学的研究[D].泰安:山东农业大学博士学位论文,2010.WANG Yong.(2010).Morphological and molecular phylofenetic studies of Ulocladium,Stemphylium and allied genera[D].Ph D Thesis.Shandong Agricultural University,Tai'an.(in Chinese)
[21]Carbone,I.,&Kohn,L.M.(1999).A method for designing primer sets for speciation studies in filamentous ascomycetes.Mycologia,91(3):553-556.
[22]李菲菲,龙超安.柑橘炭疽病菌的分离、鉴定及在果实上的潜伏侵染特性[J].果树学报,2015,32(1):108-114.LI Fei-fei,LONG Chao-an.(2015).solation,identification and latent infection characteristics of citrus anthracnose pathogens[J].Journal of Fruit Science,32(1):108-114.(in Chinese)
[23]贾娜娜,翟立峰,白晴,等.腐烂病菌的GFP标记及其在梨叶片组织中的侵染和扩展观察[J].果树学报,2015,32(6):1 195-1 200.JIA Na-na,ZHAI Li-feng,et al.(2015).Marking of Valsa pyri with GFP and observing the infection and extension of marked strains in pear leaf tissues[J].Journal of Fruit Science,35(6):1,195-1,200.(in Chinese)
[2]向征,钟聪慧,黄家风,等.新疆枣树实生苗枯萎病病原鉴定[J].植物保护学报,2014,41(2):253-254.XIANG Zheng,ZHONG Cong-hui,HUANG Jia-fen,et al.(2014).Identification of thepathogen of Fusarium wilt of jujube seedlings in southern Xinjiang[J].Journal of Plant Protection,41(2):253-254.(in Chinese)
[3]Xinwen,Xuedong,Zheng,&Zhenjiang.(2015).Current development situations of ziziphus jujuba industry in south xinjiang and recommendations.Asian Agricultural Research,(4):37-41.
[4]董宁,冯宏祖,王兰,等.南疆骏枣黑斑病症状表现及病原菌鉴定[J].植物保护学报,2016,43(6):922-927.DONG Ning,FENG Hong-zu,WANG Lan,et al.(2016).The disease symptom and the pathogen identification of jujube black spot in southern Xinjiang[J].Journal of Plant Protection,43(6):922-927.(in Chinese)
[5]向征,钟聪慧,胡军,等.新疆枣果黑斑病病原鉴定[J].新疆农业科学,2013,50(5):845-850.XIANG Zheng,ZHONG Cong-hui,HU Jun,et al.(2013).Identification of jujube black spot pathogens in Xinjiang[J].Xinjiang Agricultural Sciences,50(5):845-850.(in Chinese)
[6]郭东起,蒋卉,牛宁宁,等.南疆骏枣黑斑病病原菌的分离、鉴定及控制研究[J].中国植保导刊,2015,35(12):5-8,85.GUO Dong-qi,JIANG Hui,NIU Ning-ning,et al.(2015).Separation and identification of jujube black spot pathogens and its controlling in southern Xinjiang[J].China Plant Protection,35(12):5-8,85.(in Chinese)
[7]包慧芳,王宁,侯敏.新疆红枣黑斑病病原菌鉴定及拮抗细菌筛选[J].新疆农业科学,2016,53(9):1 667-1 673.BAO Hui-fang,WANG Ning,HOU Min.(2016).Identification of the pathogen and screening of the antogonistic bacteria of jujube black spot disease in Xinjiang[J].Xinjiang Agricultural Sciences,53(9):1,667-1,673.(in Chinese)
[8]吴玉柱,季延平,刘慇,等.冬枣黑斑病发生规律的研究[J].山东林业科技,2004,(3):1-3.WU YU-zhu,JI Yan-ping,LIU Yin,et al.(2004).The study on outbreak regularity of black patch disease in cv.Dongzao jujube[J].Shandong Forestry Science and Technology,(3):1-3.(in Chinese)
[9]陈小飞,熊仁次,徐崇志,等.红枣黑斑病研究现状与展望[J].黑龙江农业科学,2013,(10):141-144.CHEN Xiao-fei,XIONG Ren-ci,XU Cong-zhi,et al.(2013).Current situation and prospect on jujube black spot disease[J].Heilongjiang Agricultural Sciences,(10):141-144.(in Chinese)
[10]马荣,刘晓琳,梁英梅,等.新疆枣果黑斑病发生规律及温湿度对其的影响[J].植物保护学报,2016,43(5):805-811.MA Rong,LIU Xiao-lin,LIAN Ying-mei,et al.(2016).The growth and decline regularity of jujube black spot pathogen and the influence of temperature and humidity in Xinjiang[J].Journal of Plant Protection,43(5):805-811.(in Chinese)
[11]孙洁,池振江,赵思峰,等.新疆南疆红枣花腐病病原鉴定[J].西北农业学报,2014,23(2):176-180.SUN Jie,CHI Zhen-jian,ZHAO Si-feng,et al.(2014).Identification of pathogen causing flower rot on jujube in southern Xinjiang[J].Acta Agriculturae Boreali-occidentalis Sinica,23(2):176-180.(in Chinese)
[12]符泽.新疆阿拉尔垦区矮化密植枣园红枣黑斑病发病规律的初步研究[D].石河子:石河子大学硕士学位论文,2015.FU Ze.(2015).Preliminary study on incidence regulation of black spot disease of jujube in reclamation area of Alar[D].Master Thesis.Shihezi University,Shihezi.(in Chinese)
[13]孙炳剑,陈清清,袁虹霞,等.SYBR Green I实时荧光定量PCR检测小麦纹枯病菌体系的建立和应用[J].中国农业科学,2015,48(1):55-62.SUN Bin-jian,CHEN Qing-qing,YUAN Hong-xia,et al.(2015).Establishment of SYBR green I real-time PCR for quantitatively detecting rhizoctonia cerealis in winter wheat[J].Scientia Agricultura Sinica,48(1):55-62.(in Chinese)
[14]肖蕊,余真真,Elsharawy A A,等.土壤中棉花黄萎病菌SYBR Green I荧光RT-PCR定量检测技术研究[J].菌物学报,2011,30(4):598-603.XIAO Rui,YU Zhen-zhen,Elsharawy A A,et al.(2011).SYBR green I real time RT-PCR assay for quantitatively detecting the occurrence of Verticillium dahliae of cotton in naturally infested soil[J].Mycosystema,30(4):598-603.(in Chinese)
[15]Guo,Y.,Li,W.,Sun,H.,Wang,N.,Yu,H.,&Chen,H.(2012).Detection and quantification of rhizoctonia cerealis,in soil using real-time PCR.Journal of General Plant Pathology,78(4):247-254.
[16]林惠娇,王卫芳,易建平,等.Taq Man探针实时荧光PCR快速检测苹果牛眼果腐病菌方法的建立[J].植物检疫,2016,30(2):55-62.LIN Hui-qiao,WANG Wei-fang,YI Jian-ping,et al.(2016).Development of Taq Man probes real-time fluorescence PCR method for the rapid detection of the causal fungal agents of bull's-eye rot on apple[J].Plant Quarantine,30(2):55-62.(in Chinese)
[17]王凤军,张祥林,谭志文,等.枣褐斑病菌实时荧光PCR快速检测方法研究[J].食品工业科技,2014,35(14):56-58,63.WANG Feng-jin,ZHANG Xiang-lin,TAN Zhi-wen,et al.(2014).Real-time fluorescence PCR method for detection of the pathogen causing brown spot on jujube[J].Science and Technology of Food Industry,35(14):56-58,63.(in Chinese)
[18]方中达.植病研究方法[M].第3版.北京:中国农业出版社,1998.FANG Zhong-da.(1998).Research methods of plant pathology[M].3rd edition.Beijing:China Agriculture Press.(in Chinese)
[19]张天宇.中国真菌志:链格孢属[M].北京:科学出版社,2003.ZHENG Tian-yu.(2003).Chinese fungi:Alternaria[M].Beijing:Science Press.(in Chinese)
[20]王勇.细基格孢属、葡柄霉属及其近似属的形态学与分子系统学的研究[D].泰安:山东农业大学博士学位论文,2010.WANG Yong.(2010).Morphological and molecular phylofenetic studies of Ulocladium,Stemphylium and allied genera[D].Ph D Thesis.Shandong Agricultural University,Tai'an.(in Chinese)
[21]Carbone,I.,&Kohn,L.M.(1999).A method for designing primer sets for speciation studies in filamentous ascomycetes.Mycologia,91(3):553-556.
[22]李菲菲,龙超安.柑橘炭疽病菌的分离、鉴定及在果实上的潜伏侵染特性[J].果树学报,2015,32(1):108-114.LI Fei-fei,LONG Chao-an.(2015).solation,identification and latent infection characteristics of citrus anthracnose pathogens[J].Journal of Fruit Science,32(1):108-114.(in Chinese)
[23]贾娜娜,翟立峰,白晴,等.腐烂病菌的GFP标记及其在梨叶片组织中的侵染和扩展观察[J].果树学报,2015,32(6):1 195-1 200.JIA Na-na,ZHAI Li-feng,et al.(2015).Marking of Valsa pyri with GFP and observing the infection and extension of marked strains in pear leaf tissues[J].Journal of Fruit Science,35(6):1,195-1,200.(in Chinese)