Study On The Occurrence Regularity Of Black Spot And Disease Resistance Mechanism Of Marigold

Marigold(Tagetes erecta L.)is an important ornamental plant as well as a functional crop used for extraction of lutein.Marigold is cultivated over a wide geographic area in China.In recent years,marigold has played an important role in rural revitalization and rural amenity horticulture.However,black spot disease occurs in plots continuously cropped with marigold.As the disease progresses,brown lesions on the leaves coalesce to form irregular black lesions on the stems and flowers,which are often followed by plant death.In addition,black spot epidemics greatly reduce the yield of marigold biomass for lutein extraction,which has impeded the development of the marigold industry in China,especially in Beijing.Two strategies to address this problem are(1)to identify the pathogenic factor responsible for the disease,and to prevent and control the incidence of black spot disease through chemical control and cultivation management practices in the short term,and(2)to accelerate the development of resistant cultivars using molecular breeding to assist traditional hybridization breeding.In the present study,through isolation and identification of the pathogen responsible for marigold black spot in Beijing and neighboring production areas,systematic investigation of black spot incidence in the Beijing-Yanqing area,assessment of disease-resistant cultivation techniques,and the screening of control agents,we studied the etiology,incidence,and control of marigold black spot in the study area.We sequenced the transcriptome of disease-resistant and-susceptible marigold genotypes at different stages before and after inoculation with the pathogen.We screened candidate genes for resistance to black spot,and studied the defense mechanism to black spot infection by differential gene expression analysis,gene expression trend analysis,and weighted gene co-expression network analysis(WGCNA)of associated traits based on the RNA sequencing data.This study lays a foundation for future prediction and control of marigold black spot,and provides information invaluable for molecular breeding of marigold for disease resistance.The main findings are as follows:(1)We isolated Alternaria tagetica from symptomatic marigold plants collected from Beijing and Hebei province,and investigated the pathogen characteristics and meteorological factors that influence disease development.The optimum temperature for growth of the fungus was 20-25℃.(2)Field experiments conducted from 2012 to 2014 to evaluate diseased residual branches from the previous year indicated that marigold residues from the previous year are a likely source of primary inoculum.Regression analysis,structural equation modeling,and error-correction modeling showed that temperature and precipitation were explanatory factors for disease development from 2013 to 2015.The results indicated that crop rotation may play an important role in the management of marigold black spot;in addition,increased rainfall and reduced temperature provided ideal conditions for infection and spread of the disease.Assessment of irrigation and fertilization management showed that cultivation of plants in wide and narrow rows in combination with drip irrigation reduced disease incidence.Screening of fungicide application in the field revealed that 0.01%picoxystrobin and 0.025%boscalid were optimal for the prevention and control of marigold black spot.The results of this study provide a reference for future prediction and control of marigold black spot in the field.(3)In 2013～2015,we evaluated the resistance of 100 natural marigold germplasm resources to black spot in the field,and screened gene expression patterns in the highly resistant inbred line Ts and the highly susceptible inbred line Ma to investigate the mechanism of resistance to black spot in conjunction with artificial inoculation of the pathogen.After inoculation of Ts and Ma with A.tagetica,we calculated the leaf disease index and observed spore behavior on the leaf surface by means of scanning electron microscopy.The sampling times for transcriptome sequencing were defined as follows:TsO,Ma0(pre-inoculation CK);Ts1,Ma1(2,4,8,and 24 h after inoculation);Ts2,Ma2(48 h after inoculation);and Ts3,Ma3(75 h after inoculation).Analysis of differentially expressed genes and functional annotation of resistance genes(R genes)revealed that the transcription factor genes associated with marigold black spot disease resistance were members of the AP2,DOF,EIL,ERF,HSF,MYB,NAC,and WRKY families.Among the R genes detected,the N,NL,RLP,and TNL families were represented by a large number of differentially expressed genes at different stages before and after inoculation of the two genotypes with A.tagetica.Among the unique differentially expressed Unigenes,23 R genes were detected.(4)Using the transcriptome sequencing data,trend analysis and WGCNA were carried out to analyze gene expression patterns.The results showed that the genes for which expression levels continued to decrease from Ts0 to Ts3 were enriched in the phenylpropanoid biosynthesis,sesquiterpene and triterpenoid biosynthesis,starch and sucrose metabolism,and cyanoamino acid metabolism pathways.The genes that continued to increase in expression level from TsO to Ts3 were enriched in pathways such as ribosomes,eukaryotic ribosomal biosynthesis,and proteasome.At an early stage of A.tagetica infection,the PLDS gene was activated and expressed in the disease-resistant Ts genotype and the disease-susceptible Ma genotype,which activated the recognition of plant material for A.tagetica and further activated plant defense-associated genes.The resistant genotype Ts initiated the expression of the disease-resistance transcription factor WRKY25 at 48 h after infection,and expression of the ethylene response factor EFR1 and the Aux/IAA family gene IAA9 was detected at 75 h after infection.The expression level of the late defense,SA-responsive gene PR I increased significantly during this period.In the susceptible Ma genotype,transcripts of the jasmonate signal-repressing gene JAZ were expressed at 24 h,and peaked at 48 h,in response to pathogen infection.The results of WGCNA of the disease index revealed that 11 disease-associated Hub genes were screened in four target modules,including the jasmonic acid pathway inhibitor JAZ and the auxin receptor gene TIR1.The present results lay the foundation for molecular breeding of marigold for resistance to black spot disease.