| Watermelon is one of the most important economic crops in China.The outbreaks of gummy stem blight significant affect watermelon's production,and cause serious economic losses.In order to prevention and forecast of gummy stem blight effectively,the polymorphisms of the fungi that cause this disease,and understands their genetic diversities and geographical population genetic structures were studied.Previous studied showed that gummy stem blight was caused by three morphologically indistinguishable species of Stagonosporopsis cucurbitacearum,S.citrulli,and S.caricae.Based on previous studies,this study used triple PCR to identify the species of Stagonosporopsis fungi in 7 regions of Southwest China,and further understand the distribution of Stagonosporopsis in these regions.Hereafter,genetic diversity analysis of 234 dominant S.citrulli strains was conducted based on the three difference factors of geographical distribution,collection years,and watermelon growth periods by using simple sequence repeat interval(Intel-simple sequence repeats,ISSR)molecular marker.Our findings provide a theoretical basis for further understanding the population biology,distribution,as well as disease forecast of the pathogens.The main results are as follows:1.Species identification and distribution of watermelon gummy stem blight pathogens in Southwest China.Gummy stem blight is mainly caused by three species of S.cucurbitacearum,S.citrulli,and S.caricae.In order to clarify the distribution of the three species in Southwest China,274 strains of Stagonosporopsis fungi isolated from watermelon fields were identified using triple PCR.The results showed that two species,S.caricae and S.citrulli,existed in these regions,including 234 dominant strains of S.citrulli(accounting for 84.17%),and remaining 40 strains of S.caricae(accounting for15.83%).However,S.cucurbitacearum was not found in these regions.Biological characteristics analysis results showed that no significant difference between S.caricae and S.citrulli in colony morphology,conidium morphology,growth rate,as well as the amount and rate of conidial sporulation.2.Genetic Structure of geographic population of Stagonosporopsis citrulli in Southwest ChinaGenetic diversity analysis of 234 S.citrulli strains using 12 pairs of high polymorphism of ISSR markers showed that a total of 142 loci were amplified,including 119 polymorphic loci.The average number of polymorphic sites per primer was 9.9,and the average polymorphic percentage was up to 83.80%..Each DNA sample can be amplified to 8-16 bands using per primer,and the fragment sizes of amplicons are mostly between 300-4000 bp,indicating that there is a certain genetic difference between the individuals of pathogen strain in Southwest China.Cluster analysis was performed to characterize the genetic difference using NTYSYSpc 2.10e software,and system clustering results indicated the genetic similarity coefficient was between 0.67and 0.92.When the genetic similarity coefficient was selected as 0.724,all of S.citrulli strains can be divided into 8 groups:?to?.A total of 216 strains were belonged to Group?class,accounting for 94.01%.Due to Group?contained all strains of geographical origin,the genetic coefficient was re-selected as 0.745,and Group?strains can be further divided into three subgroups.The third subgroup only contained strains from Yongchuan.In other small subgroups,strains from the same geographical origins were also basically tied together,indicating that the genetic structure of strains isolated from the same geographical regions showed similarities.In order to further understand the genetic structure differences of the geographic population of these pathogens,234 strains of S.citrulli were classified as Yongchuan(YC),Hechuan(HC),Luzhou(LZ)and Rongchang(RC),Wulong(WL),Beibei(BB),and Xifeng(XF)to study their genetic diversity correlation coefficients based on their sampling locations.The genetic diversity in the population was measured by the genetic diversity index(h)and the Shannon's index(I).BB population had the highest genetic diversity;the h-value and I-value were 0.2542 and 0.3891,respectively.LZ population had the lowest genetic diversity;h-value and I-value were 0.1531 and 0.2305,respectively.The genetic abundance of geographical populations was BB>WL>YC>XF>HC>RC>LC.Genetic distance and genetic similarity coefficient are the parameters that measure the distance between each population.The largest genetic distance among the seven geographical populations was 0.1164 between BB and LZ populations,indicating that the genetic relationship between BB population and LZ population was the longest.The smallest genetic distance was 0.0382 between RC and LZ populations,indicating that the genetic relationship between RC population and LZ population was closest.The genetic differentiation coefficient Gst and gene flow Nm among the 7 geographic populations in the Southwest China were 0.1848 and 2.2061,respectively,indicating that the genetic differentiation between these geographic populations was relative low,and the genetic exchanges of them were frequent.3.Temporal variation of genetic diversity of Stagonosporopsis citrulli populationsThe above results illustrated the genetic diversity of the geographical populations of S.citrulli in Southwest China,indicating that spatial isolation has a certain influence on the genetic structure of the populations.To further understand the temporal variation of genetic diversity of S.citrulli populations,the differences in genetic diversity among YC populations of 4 growth stages,WL populations of 4 years,BB populations of 3yearss were calculated.The results showed that the genetic index values among YC populations of 4 growth stages were close to each other,the genetic diversity index h-values were all small(between 0.1554 and 0.1737),without significant difference,indicating that the genetic diversity of these populations did not change with the growth period.In WL populations,both h-values and Shonnon's index I in 2016 were the highest,up to 0.2055 and 0.3083,respectively.The genetic abundance of four-year populations was 2016>2014>2017>2015,suggesting that population genetics diversity has no change with time.The genetic diversity of BB populations in 2015 was the most abundant,h-value was 0.2061,and the genetic diversity of BB population in2017 is the lowest,h-value was 0.1607.The maximum genetic distance was 0.1511between 2015 and 2017,and the minimum genetic distance was 0.0663 between 2015and 2016,indicating that the genetic diversity of BB populations had changed with time.With the time past,a certain amount of genetic differentiation had occurred.The genetic relationship between the strains had become farther,and the genetic richness had decreased.4.Biological characteristics of Stagonosporopsis citrulli with different ISSR groups in Southwest ChinaThe above studies clustered all S.citrulli strains into 8 ISSR groups.Analysis results of the biological characteristics of each ISSR group showed that there was no significant difference in growth rate,adaptability to temperature and pH values,as well as spore germination rate between different ISSR groups.However,there are different levels of differences in the pathogenicity and sporulation ability between different ISSR groups.The sporulation ability of Group?and Group?had significant differences with other groups;and the number of spores of Group?was the lowest,only 3.57×10~6±0.25 spores per mL.The number of spores in Group?was the highest,up to 6.47×10~6±1.74 spores per mL,the spore production of other groups were between 4.43×10~6±0.19-5.77×10~6±0.92 spores per mL.Group?had the lowest pathogenicity,the average lesion areas were only 4.60±0.66 cm~2,with a significant difference from Group?,?,?,and?;except for the first group and the seventh group.There was no significant difference among the other groups,the average lesion areas were between5.61±1.25 cm~2 and 7.10±0.42 cm~2.In summary,this study first identified the species of watermelon gummy stem blight pathogens in Southwest China,and clarified the distribution of them in these regions.Subsequently,the spatial and temporal genetic diversity of the major species were analyzed by using ISSR molecular markers,and the genetic structure of the population in Southwest China was finally revealed. |
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