Identification Of Pathogenic Fusarium Species Causing Maize Ear Rot And Study On Host Resistance

Maize（Zea mays L.）is the highest yielding food crop in the word.As one of the major crops in China,it palys an important role in the food and energy supply,social economy and life science researches.Maize ear rot is one of the most important diseases in the late growth of maize,which poses a serious threat to food security and maize production.There are many kinds of pathogens causing maize ear rot,which are complex and changeable.Most of the maize varieties in production are susceptible to ear rot,and lack of effective resistance sources and available resistance genes in breeding,which are the important factors for the frequent occurrence of ear rot.Based on this,this paper studied the composition and distribution of Fusarium species of maize ear rot in Heilongjiang Province,China.Then the genetic diversity,population structure,and pathogenicity of the predominant Fusarium species identified in 16 provinces and cities were further analyzed.At the same time,a recombinant inbred lines（RIL）population stemed from an across between the resistant maize inbred lines Qi319 and susceptible Ye478 was used to mine and map gene/QTL with resistance to ear rot.The precise identification for resistance to maize ear rot caused by F.verticillioides was performed in multiple environments and quantitative trait locus（QTL）analysis was conducted,in order to explore the resistance gene insensitive to environmental condictions,and to provide resistance gene resources for breeding and production.The main results are as follows:1.The pathogenic Fusarium spp.causing maize ear rot in Heilongjiang Province are very complex,Fusarium graminearum species complex and Gibberella fujikuroi species complex are the main pathogens.Using seed health testing method,6 genuses of fungi,dominated by Fusarium spp.,were isolated and identified from 143 diseased maize samples,which were collected from 21 major maize producing areas and counties in Heilongjiang Province.A total of 200 Fusarium isolates were obtained and 12 Fusarium species were identified and determined according to the morphological and molecular biology methods,including F.graminearum,F.verticillioides,F.subglutinans,F.proliferatum,F.boothii,F.temperatum,F.andiyazi,F.incarnatum,F.sporotrichioides,F.poae,F.commune,and F.asiaticum,with the isolation frequency of 33.00%,16.00%,13.00%,13.00%,12.50%,7.50%,2.50%,2.00%,1.00%,1.00%,1.00%,and 0.50%,respectively.The study demonstrated the diversity of Fusarium species causing maize ear rot in Heilongjiang was relatively rich,F.graminearum and F.verticillioides were the predominant pathogens,and F.subglutinans,F.proliferatum and F.boothii distributed relative widely,while F.temperatum and F.andiyazi distributed in a relatively small range.F.sporotrichioides,F.incarnatum,F.poae and F.commune were isolated from diseased maize ear for the first time in Heilongjiang Province.It has important reference values for understanding the Fusarium species of corn ear rot in Heilongjiang Province.2.The toxin-producing types of Fusarium spp.in Heilongjiang Province are rich,has a strong potential to produce toxins.Toxigenic chemotype analysis in Heilongjiang showed that F.asiaticum belong to the NIV-producing type,whereas F.graminearum and F.boothii contained three kinds of genotypes,among which 15-AcDON was the most abundant,followed by 3-AcDON,and NIV type was the least.Among F.graminearum isolates,36 strains were 15-AcDON toxin-producing type,20 were3-AcDON toxin-producing type,and 4 were NIV toxin-producing chemotype.There were also three toxin-producing types among F.boothii,including 14 15-AcDON chemotypes,10 3-AcDON types,and1 NIV toxin-producing genotype.The key fumonisin-producing gene fum1 was detected in F.verticillioides,F.proliferatum,F.subglutinans,F.andiyazi,F.incarnatum,F.sporotrichioides,and F.poae,while F.temperatum and F.commune did not contain fum1 gene.3.Strong pathogenicity and rich genetic diversity of Fusarium graminearum species complex（FGSC）as well as F.verticillioides causing maize ear rot in China.From 2014 to 2018,representative strains of Fusarium species were isolated and identified from 15 provinces and cities（Hebei,Henan,Anhui,Shandong,Shaanxi,Chongqing,Jilin,Sichuan,Yunnan,Beijing,Ningxia,Liaoning,Hainan,Guangxi,Heilongjiang）.We investigated the genetic structure of F.verticillioides（n=53）and FGSC（n=45）flora based on molecular data generated by the simple sequence repeat（SSR）and variable number of tandem repeats（VNTR）markers,combined with the sequences of RPB2,TEF-1αandβ-tubulin genes.Phylogenetic analyses revealed that high genetic variability and diversity existed among the 7different geographical regions for both Fusarium species.Among FGSC,a total of 48 alleles were detected by SSR and VNTR primers,39 polymorphic sites were found,with a polymorphic band ratio of 91.67%among 45 strains,and the polymorphic bands ranged from 2 to 4.The average Shannon’s information index and Nei’s genetic diversity index of the 7 FGSC geographic populations were 0.41and 0.29,respectively,the genetic similarity in seven regions ranged between 0.6677 and 0.8797,with the genetic distances of 0.1282-0.4039,indicating there existed rich genetic diversity among the floras.The number of polymorphic loci in seven geographic populations of F.verticillioides ranged from 13.00to 43.00,with Shannon’s information index and Nei’s genetic diversity index of 0.46 and 0.32,respectively.The diseased area of maize ear caused by F.verticillioides was 2.8%-27.0%,with an average of 13.05%.Among FGSC species,F.graminearum exhibited the strongest pathogenicity,with an average diseased ear area of 20.79%.The average diseased areas of F.asiaticum,F.boothii,and F.meridionale were 15.79%,11.77%,and 8.12%,respectively.The sequence information of three genes showed FGSC population contained more SNP loci than F.verticillioides,with 141 and 42,respectively.Statistical analyses showed that there was no significant correlation between toxigenicity and origin for the two pathogens.In a word,there is a high level of genetic diversity among the seven regions and within each region,and most of the pathogens have toxigenic potential and infection ability.4.A stable QTL was detected for resistance to Fusarium verticillioides ear rot in maize.A RIL population containing 300 F₁₁ lines,stemed from an across between the susceptible maize inbred lines Ye478 and resistant Qi319,was used to mine resistant gene/QTL against F.verticillioides ear rot（FER）.In 2018 and 2019 years,the RIL population was precisely phenotyped in 5 different environments in Henan and Beijing.Combined with the high-density genetic linkage map of this population,the compound interval mapping method and the complete interval mapping method,QTL mapping for resistance were performed.A total of 18 QTLs were detected,two of them could be detected in single and multi environment at the same time.In 2019,a resistant QTL located between markers mk202 and mk203 on chromosome 1 was detected stably in three environments,with the physical location between43.50 and 44.35 Mb.The LOD value ranged from 4.36 to 9.88,the maximum phenotypic varience of14.80%,indicating that this QTL is insensive to environmental conditions.