| Maize(Zea mays L)is not only an important grain-feeding crop,but also an important energy plant and industry material,and plays an important role in the national economy in China.With the change of climate,variety change and the change of farming system,the occurrence and damage of maize stalk rot are obviously aggravated.With mechanized harvesting and direct grain harvesting,stalk rot has become an urgent problem to be solved.Therefore,this study investigated the occurrence and damage of maize stalk rot in different ecological regions of Gansu,collected samples,and then studied on the pathogen species,genetic diversity of dominant pathogens,toxigenic types and resistance gene mining,mainly includes the following aspects:(1)In 2015 and 2017,a systematic survey was conducted on the distribution range and degree of damage of maize stalk rot in Gansu.The results showed that the disease was distributed in Huating County,Zhuanglang County,Lingtai County,Handan District,Luanchuan County and Huachi County.County,Zhenyuan County,Heshui County,Qingcheng County,Ning County,Qingshui County,Qinzhou District,Qin'an County,Gangu County,Maiji District,Zhangjiachuan County,Cheng County,Diebu County,Kang County,Zhouqu County,Linxia County,Guanghe County,Pingchuan District,Jingyuan County,Huining County,Tongyu County,Anding District,Linyi County,Ganzhou District,Gaotai County,Linze County,Suzhou District and Liangzhou District.The average disease field rate was 28.6% and 100%,and the disease rate was 3.6% and 31.5% for 2 years,respectively.(2)In order to identify the pathogenic species and pathogenic groups of maize fusarium stalk rot in Gansu province,42 samples of maize stalk rot were collected from four biome(Longnan region,Longdong region,Longzhong region and Hexi corridor)in 2015 and 2017,pathogens were isolated by organize separation method,and then separation purification and single spore isolation.Based on the morphological,pathogens were identified by reference Leisle classification system.The results showed that a total of 253 strains of Fusarium species were isolated.Among 253 strains,150 strains of Fusarium graminearum species complex(FGSC),29 strains of F.verticillioides,26 strains of F.equiseti,15 strains of F.subglutinans,12 strains of F.proliferatum,10 strains of F.incarnatum,5 strains of F.tricinctum,3 strains of F.temperatum,2 strains of F.acuminatum and 1 strain of F.oxysporum,which showed 59.3%,11.5%,10.3%,5.9%,4.7%,4.0%,1.9%,1.2%,0.8% and0.4% isolation frequencies,respectively.In accordance with Koch's rule,the pathogenicity was determined on maize variety Ganyu 2 by dish method,pot method and field experiment.Ten kinds of Fusarium species were confirmed as pathogen by dish method and pot method.Among them,the FGSC and F.verticillioides were the dominant species of maize stalk rot in Gansu province.F.equiseti,F.subglutinans,F.proliferatum,F.incarnatum,F.tricinctum,F.temperatum,F.acuminatum and F.oxysporum were first reported in Gansu as the pathogen of maize stalk rot.(3)Representative strains HCSZ4-9,HHXHZ15-7,ZY2-2,PC14-1,YJ8-1,ZY7-1,ZY11-1,KTQ3-1,ZQDC13-3,HA26,TW30,ZY13-2,KTQ16,KTQ19,ZJCZC14-5,ZJCZC14-2,TW26,TW40 and HCSZ4-19 were selected for sequence analysis of EF-1?(tef)gene.The PCR products were recovered and sequenced and compared in GenBank.The results showed that strain HHXHZ15-7 with F.boothii,strains HCSZ 4-9 with F.graminearum,strains ZY2-2 and PC-14-1 with F.verticillioides,strains YJ 8-1 and ZY7-1with F.equiseti,strains ZY11-1 and KTQ3-1 with F.subglutinans,strains ZQDC13-3 and HA-26 with F.proliferatum,strains TW30 and ZY13-2 with F.incarnatum,strains KTQ16 and KTQ19 with F.tricinctum,strains ZJCZC14-5 and ZJCZC14-2 with F.temperatum,strains TW26 and TW40 with F.acuminatum,strains HCSZ 4-19 with F.oxysporum were located in the same branch of phylogenetic tree,respectively,indicating that the results of molecular identification are consistent with the results of morphological identification.(4)In this study,species identification of 150 FGSC were performed by using specific primers of Fusarium.The results showed that a total of 110 strains of F.boothii and 40 strains of F.graminearum were detected.In this study,we have mastered the population structure of the FGSC of maize Fusarium stalk rot in Gansu province,which is composed of two groups of F.boothii and F.graminearum,accounting for 73.33% and 24.67%,respectively,with a ratio of about 3:1.(5)In order to determine the populations genetic diversity of maize Fusarium stalk rotin four ecological regions,Gansu province.In this study,the populations genetic diversity of FGSC was analyzed by 8 VNTR markers and 10 SSR markers in 4 ecological regions,Gansu province.The results showed that 26 allelic sites and 26 polymorphic loci were detected in 114 strains of FGSC by the 18 pairs of primers,and the percentage of polymorphic bands was 100%.The number of alleles in 4 geographical populations was1.9519,the effective number of alleles was 1.7140,the diversity index of Nei's gene was0.3939 and Shannon information index was 0.5691,the number of polymorphic sites was24.75 and the percentage of polymorphic loci was 95.19%.The Nei's genetic similarity and genetic distance of 4 populations were 0.8880-0.9674 and 0.0331-0.1188,respectively.The group of FGSC was divided into three groups,group one was composed of Longnan region,and Group two was composed of Hexi region,and Group three was composed of Longdong and Longzhong regions.The genetic variation of FGSC species mainly came from within the population,accounting for 90.71% percent of the total.(6)The toxin types of 114 strains of FGSC were tested.The results showed that 42 strains produced 15-AcDON,34 strains produced 3-AcDON,20 strains produced NIV,and18 strains did not produce toxin,accounting for 36.84%,29.82%,17.54% and 15.79%,respectively.The results also showed that both F.boothii and F.graminearum produced15-acdon,3-acdon and NIV,and the three toxins were distributed in 4 ecological regions.(7)In order to investigate the biological characteristics of Trichoderma brevicompactum and biocontrol of T.brevicompactum against plant disease,a Trichoderma strain GAS1-1 was isolated from rhizosphere soil of continuously cropped potato in Jingtai County,Gansu province.Taxonomic status of strain GAS1-1 was confirmed by morphologic observation,rDNA-ITS and EF-1? sequence analysis.Nutrition growth and sporulation conditions of strain GAS1-1 were ascertained with biological method.The inhibitory effect of the strain GAS1-1 against five plant pathogenic fungi was determined by dual culture.The results of morphological and sequence analysis showed that the strain GAS1-1 was identified as T.brevicompactum,which is a firstly recorded Trichoderma species in Gansu province.The strain showed strong antagonistic activity against Fusarium graminearum,F.verticillioides,F.oxysporum,Pythium inflatum and Botrytis cinerea.In particular,itproduced the highest antifungal activity against P.inflatum with inhibition ratio of 100%.The results of biological characteristics showed that the optimum carbon and nitrogen sources of the strain are glucose and yeast extract for mycelial growth and sporulation,respectively.The strain can grow at 15~35?,of which the optimal hyphae growth and sporulation temperature were 30? and 25?,respectively.The strain can grow at pH 5.0~12.0,of which the optimal hyphae growth and sporulation were pH 5.0.The condition of 24 h growth in dark was most favorable to its mycelial growth,while the condition of alternative12 h light and 12 h dark was most favorable to its sporulation.The spore lethal temperature was 69? for 10 min.It is proved that this strain of T.brevicompactum has biocontrol potential against plant diseases.(8)This project will construct a F2 population of maize by hybridization of high resistance Fusarium graminearum stalk rot inbred lineX178 and high susceptible inbred line B73,will test its pathogenecity of this population by artificial inoculation in the big bell period,and clear the characteristics of its phenotypes.The survey results showed that the resistance-susceptible ratio was about 2.38:1.According to the phenotypic results,50 resistance materials and 50 susceptible materials,and 2 parents were built database,respectively,and then perform WGS whole genome resequencing and localization analysis by the BSA trait.The candidate genes identified based on SNP-index and InDel-index were6 and 33,respectively.By integrating the information of SNP and InDel,it was found that a non-synonymous mutation occurred in Zm00001d035153 on chromosome 6,and a frameshift mutation occurred in Zm00001d040332 on chromosome 3.In other word,the sequence of proteins in the coding region of these 2 genes was changed.Therefore,Zm00001d035153 and Zm00001d040332 are two major disease resistance candidate genes.On the basis of the research,this study aims to dig out the target genes of maize resistant to Fusarium graminearum stalk rot,will provide effective gene resources and function marker for fast-assisted selection,hence will speed up breeding and construct breeding system of maize to Fusarium graminearum stalk rot. |
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