| Pepper(Capsicum annuum L.)is native to South America,which is an herbaceous plant from the Capsicum genus in Solanaceae family.In China,the current field area of pepper industry is as high as 32 million mu.As an important vegetable and processed food in China,the annual output value of pepper also ranks first among the major vegetables.Although many pepper varieties have been bred through traditional breeding methods,the basic research on pepper is weak due to the huge genome and the difficulty of genetic transformation in pepper.There are many disease problems during pepper planting,which severely restrict the development of the pepper industry.Among them,pepper anthracnose is a serious disease with spreading fast a wide range and lasting long.The pepper anthracnose can cause a large area of production reduction,which is one of the main diseases that limit the high yield of peppers.However,the traditional anthracnose control methods have general effects and are not sustainable.As a result,screening and cultivating disease-resistant pepper resources is an effective way to prevent pepper anthracnose in agricultural industry.In this study,we first obtained the ‘HP’ pepper fruit that occurs pepper anthracnose naturally in the field,and found the pathogens causing pepper anthracnose by is Colletotrichum scovillei through isolation and identification.By using the ‘HP’ pepper mutant population obtained by EMS mutagenesis,we further screened 219 mutant lines against anthracnose in M1 generation.What’s more,in order to shorten the screening period,we established a simple and efficient resistant screening system against anthracnose at the seedling stage of pepper.By using this system,we screened the individual plants of pepper mutant lines in M2 generation at the seedling stage.Finally,we continued to screen the resistance of fruits in M2 generation,to obtain the stable resistant mutants.These pepper sources will lay a good foundation for our research group to map resistance genes and develop molecular markers in future.The detailed research results are as follows:1.A total of 60 pathogenic bacteria were isolated and cultured by pepper anthracnose fruits collected in the field.Through morphological observation and comparison of ITS reference gene sequences,we initially suspected 44 of them belonged to Colletotrichum genus.Then 13 strains were selected randomly for fruit inoculation,and we found all of them can cause pepper anthracnose,but the disease index is different among them.Afterward,we randomly selected 6 strains again to observe the morphological characteristics together with virulent C.scovillei strain CD,including the growth rate and color of colonies,as well as the size and shape of conidia andappressoria,and found these 6 strains are the same as CD.Finally,we confirmed that these 6 anthracnose pathogen strains all belonged to Colletotrichum Scovillei through phylogenetic analysis,which are consistent with CD.2.After establishing the standard for screening ‘HP’ pepper fruit resistance to anthracnose,green and yellow ripen pepper fruits of 219 mutant lines in M1 generation were screened respectively,and we found 10 mutant lines with the consistent resistance to anthracnose between green and yellow fruits,including 5 mutants resistant to C.Scovillei,and 5 mutants susceptible to C.Scovillei.3.In order to shorten the mutant screening cycle and overcome the restriction of fruit inoculation,we tried 2 different inoculation methods: injection and needle-point,5 spore suspension concentrations: 1×103,1×104,1×105,1×106 and 3×106 cells/ml,and 4 different pepper seedlings stages: cotyledon stage,1-2 true leaf stage,3-4 true leaf stage and 5-10 true leaf stage for disease observation.Finally,the seedling inoculation system of ‘HP’ pepper was established: by using needle-point method and 1×106 cells/ml concentration,we inoculated the pepper seedlings at the 3-4 true leaf stage.The result showed the plant incidence rates were 100%,and the disease lesions are nearly round,which can be used for quantitative statistics.4.Using the above inoculation system,we established the screening standard for for screening ‘HP’ pepper seedling resistance to anthracnose.Based on the 10 selected mutant lines from M1 generation,then we continued to screening the resistance against pepper anthracnose of single plants in M2 generation.And we found 8 lines were stay with the same resistance as M1 generation,with 4 of which keeping sensitive and another 4 mutant lines keeping resistant.Then 15 extremely resistant/sensitive plants were selected from each of the 8 mutant lines.After the diseased leaves were removed,the plants were transplanted in the field,and the M2 generation fruit resistance screening was carried out.Finally,three mutant lines with consistent and stable resistance were obtained,including H630,H662 were resistant and H204 was susceptible respectively. |
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