A rapid approach for isolating a single fungal spore from rice blast diseased leaves
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摘要
Single spore isolation is a fundamental approach in plant pathology and mycology to isolate and identify plant fungal pathogens from diseased samples. However, routine single spore isolation procedure is time-consuming and has a high risk of contamination by other microorganisms. In this study, we developed a rapid approach for isolating a single spore of the fungal pathogen, Pyricularia oryzae, from rice blast diseased leaves in the paddy field with low potential of contamination. First, rice blast leaves with single lesions were selected in the paddy field, and a single lesion was cut out and pressed and dragged gently across the surface of water agar. Next, a germinated single spore with a barely visible piece of agar was cut out of water agar with a dissecting needle under a stereomicroscope at approximately 120-fold magnification. Last, the germinated single spore with water agar was transferred onto oatmeal tomato agar for growth and preservation. Based on our experience, a skilled technician or student can successfully isolate single spore from over 150 independent diseased samples with nearly no contaminations in a single working day. This approach is also suitable for isolating single spore from other fungal disease samples that produce abundant aerial conidia.
Single spore isolation is a fundamental approach in plant pathology and mycology to isolate and identify plant fungal pathogens from diseased samples. However, routine single spore isolation procedure is time-consuming and has a high risk of contamination by other microorganisms. In this study, we developed a rapid approach for isolating a single spore of the fungal pathogen, Pyricularia oryzae, from rice blast diseased leaves in the paddy field with low potential of contamination. First, rice blast leaves with single lesions were selected in the paddy field, and a single lesion was cut out and pressed and dragged gently across the surface of water agar. Next, a germinated single spore with a barely visible piece of agar was cut out of water agar with a dissecting needle under a stereomicroscope at approximately 120-fold magnification. Last, the germinated single spore with water agar was transferred onto oatmeal tomato agar for growth and preservation. Based on our experience, a skilled technician or student can successfully isolate single spore from over 150 independent diseased samples with nearly no contaminations in a single working day. This approach is also suitable for isolating single spore from other fungal disease samples that produce abundant aerial conidia.
Single spore isolation is a fundamental approach in plant pathology and mycology to isolate and identify plant fungal pathogens from diseased samples. However, routine single spore isolation procedure is time-consuming and has a high risk of contamination by other microorganisms. In this study, we developed a rapid approach for isolating a single spore of the fungal pathogen, Pyricularia oryzae, from rice blast diseased leaves in the paddy field with low potential of contamination. First, rice blast leaves with single lesions were selected in the paddy field, and a single lesion was cut out and pressed and dragged gently across the surface of water agar. Next, a germinated single spore with a barely visible piece of agar was cut out of water agar with a dissecting needle under a stereomicroscope at approximately 120-fold magnification. Last, the germinated single spore with water agar was transferred onto oatmeal tomato agar for growth and preservation. Based on our experience, a skilled technician or student can successfully isolate single spore from over 150 independent diseased samples with nearly no contaminations in a single working day. This approach is also suitable for isolating single spore from other fungal disease samples that produce abundant aerial conidia.
引文
Callaway E.2016.Devastating wheat fungus appears in Asia for first time.Nature,532,421-422.
Chen X L,Yang J,Peng Y L.2011.Large-scale insertional mutagenesis in Magnaporthe oryzae by Agrobacterium tumefaciens-mediated transformation.Methods Molecular Biolology,722,213-224.
Choi Y W,Hyde K D,Ho W H.1999.Single spore isolation of fungi.Fungal Diversity,3,29-38.
Dean R A,Talbot N J,Ebbole D J,Farman M L,Mitchell T K,Orbach M J,Thon M,Kulkarni R,Xu J R,Pan H,Read N D,Lee Y H,Carbone I,Brown D,Oh Y Y,Donofrio N,Jeong JS,Soanes D M,Djonovic S,Kolomiets E,et al.2005.The genome sequence of the rice blast fungus Magnaporthe grisea.Nature,434,980-986.
Fang W W,Liu C C,Zhang H W,Xu H,Zhou S,Fang K X,Peng Y L,Zhao W S.2018.Selection of differential isolates of Magnaporthe oryzae for postulation of blast resistance genes.Phytopathology,108,878-884.
Gladieux P,Condon B,Ravel S,Soanes D,Maciel J L N,Nhani Jr A,Chen L,Terauchi R,Lebrun M H,Tharreau D,Mitchell T,Pedley K F,Valent B,Talbot N J,Farman M,Fournier E.2018.Gene flow between divergent cereal-and grassspecific lineages of the rice blast fungus Magnaporthe oryzae.MBio,9,pii:e01219-17.
Jia Y L.2009.A user-friendly method to isolate and single spore the fungi Magnaporthe oryzae and Magnaporthe grisea obtained from diseased field samples.Plant Health Progress,doi:10.1094/PHP-2009-1215-01-BR
Ou S H.1980.A look at worldwide rice blast disease control.Plant Disease,64,439-445.
Qiu X Y,Tang Z P,Zhang M,Jing H Y.2011.Research on the isolation method of single spore of most plant pathogenic fungi.Journal of Anhui Agricultural Science,39,5263-5264.(in Chinese)
Sesma A,Osbourn A E.2004.The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi.Nature,431,582-586.
Talbot N J.2003.On the trail of a cereal killer:Exploring the biology of Magnaporthe grisea.Annual Reviews in Microbiology,57,177-202.
Valent B.1990.Rice blast as a model system for plant pathology.Phytopathology,80,33-36.
Valent B,Chumley F G.1991.Molecular genetic analysis of the rice blast fungus,Magnaporthe grisea.Annual Review of Phytopathology,29,443-467.
Yang J,Xue F,Wang H F,Guo T,Jin G X,Wang J,Chen F,Zhang S Y.2017.Single spore isolation technology and common problem analysis of rice blast pathogen.Magnaporthe girsea.Shandong Agricultural Sciences,49,132-135.(in Chinese)
Yang J,Zhao X,Sun J,Kang Z,Ding S,Xu J R,Peng Y L.2010.A novel protein Com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae.Molecular Plant-Microbe Interactions,23,112-123.
Zhang S J,He Y Q.2003.A simple method to isolate single fungal spore.Journal of Yunnan Agricultural University,18,315-316.(in Chinese)
Chen X L,Yang J,Peng Y L.2011.Large-scale insertional mutagenesis in Magnaporthe oryzae by Agrobacterium tumefaciens-mediated transformation.Methods Molecular Biolology,722,213-224.
Choi Y W,Hyde K D,Ho W H.1999.Single spore isolation of fungi.Fungal Diversity,3,29-38.
Dean R A,Talbot N J,Ebbole D J,Farman M L,Mitchell T K,Orbach M J,Thon M,Kulkarni R,Xu J R,Pan H,Read N D,Lee Y H,Carbone I,Brown D,Oh Y Y,Donofrio N,Jeong JS,Soanes D M,Djonovic S,Kolomiets E,et al.2005.The genome sequence of the rice blast fungus Magnaporthe grisea.Nature,434,980-986.
Fang W W,Liu C C,Zhang H W,Xu H,Zhou S,Fang K X,Peng Y L,Zhao W S.2018.Selection of differential isolates of Magnaporthe oryzae for postulation of blast resistance genes.Phytopathology,108,878-884.
Gladieux P,Condon B,Ravel S,Soanes D,Maciel J L N,Nhani Jr A,Chen L,Terauchi R,Lebrun M H,Tharreau D,Mitchell T,Pedley K F,Valent B,Talbot N J,Farman M,Fournier E.2018.Gene flow between divergent cereal-and grassspecific lineages of the rice blast fungus Magnaporthe oryzae.MBio,9,pii:e01219-17.
Jia Y L.2009.A user-friendly method to isolate and single spore the fungi Magnaporthe oryzae and Magnaporthe grisea obtained from diseased field samples.Plant Health Progress,doi:10.1094/PHP-2009-1215-01-BR
Ou S H.1980.A look at worldwide rice blast disease control.Plant Disease,64,439-445.
Qiu X Y,Tang Z P,Zhang M,Jing H Y.2011.Research on the isolation method of single spore of most plant pathogenic fungi.Journal of Anhui Agricultural Science,39,5263-5264.(in Chinese)
Sesma A,Osbourn A E.2004.The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi.Nature,431,582-586.
Talbot N J.2003.On the trail of a cereal killer:Exploring the biology of Magnaporthe grisea.Annual Reviews in Microbiology,57,177-202.
Valent B.1990.Rice blast as a model system for plant pathology.Phytopathology,80,33-36.
Valent B,Chumley F G.1991.Molecular genetic analysis of the rice blast fungus,Magnaporthe grisea.Annual Review of Phytopathology,29,443-467.
Yang J,Xue F,Wang H F,Guo T,Jin G X,Wang J,Chen F,Zhang S Y.2017.Single spore isolation technology and common problem analysis of rice blast pathogen.Magnaporthe girsea.Shandong Agricultural Sciences,49,132-135.(in Chinese)
Yang J,Zhao X,Sun J,Kang Z,Ding S,Xu J R,Peng Y L.2010.A novel protein Com1 is required for normal conidium morphology and full virulence in Magnaporthe oryzae.Molecular Plant-Microbe Interactions,23,112-123.
Zhang S J,He Y Q.2003.A simple method to isolate single fungal spore.Journal of Yunnan Agricultural University,18,315-316.(in Chinese)