| Rice false smut,caused by the fungal pathogen Ustilaginoidea virens(teleomorph:Villosiclava virens),was a destructive disease throughout major rice-growing countries.In recent years,rice false smut was growing increasingly serious and had been one of the most important diseases with the changing of the rice cropping system and the replacing of varieties.Rice false smut not only reduced the rice production and lead to huge economic losses,but also reduced the rice quantity by toxins produced by the pathogen and directly threatened the health of human and animals.Currently,chemical control was the common treatment method for rice false smut.Therefore,it had great theoretical and realistic significance for strengthening the research about the pathogen monitoring and genetic diversity in scientific using pesticides.In this study,firstly,we had established a quantitative detection method of spore of U.virens in the field,which was based on the Real-time PCR technology.Secondly,we revealed the releasing rule of the spore of U.virens in the field.Finally,the genetic diversity of the strains of U.virens from Anhui province was studied by using ISSR marker.The main results were as follows:1.Developing a rapidly quantitative detection method for the U.virens sporeWe had established a standard curve of the spore of U.virens in the field with using the Real-time PCR technology: y =-0.2857 x + 10.403,(R2 = 0.9936,P<0.0001)(X:number of spore;y: relevant Ct value).The results of rationality test and reliability test,showed that there are no significant differences in standard curve which was established by different spores of U.virens.It showed that the quantitative detection method could be able to detect the chlamydospore and conidium of U.virens.The variation coefficient of technology repeated trials was between 0.61% and 1.48%,and the variation coefficient of biology repeated trials was 1.8%,which shows that the quantitative detection method has a good reproducibility.A significan linear relationship(R2=0.8966,P<0.0001)between spore number counted with a compound microscope and those determined with the real-time PCR assay was obtained,using the same samples of spore traps.2.Research for spores-releasing pattern of U.virens in FieldsAccording to the preliminary test results of the dynamic spores from July to September in 2014 through the real-time PCR assay,we found that the releasing of U.virens spores have two peaks in dynamic trends.The one peak was in mid-august,whichwas from booting stage to blooming stage of middle-season rice in the test field;the other is in the early September,which is during the milk stage,meanwhile,we could find the new rice false smut balls.And the 24 h monitoring results showed that there are two peaks of the spores’ number in cloudy weather,the one peak is from 22:00 to 24:00,the other is from 6:00 to 8:00;And there was significantly positive correlation between the number of spore released and rainfall in Rainy day.3.Analysis for genetic diversity of U.virensThe genetic diversity of the 49 strains of U.virens,which collected from 9 different rice production regions from Anhui Province in 2014,had been investigated by ISSR(Inter Simple Sequence Repeat)marker.In the study,we had obtain 11 primers which is good in ISSR-PCR from 20 primers,and the results showed that 153 fragments were amplified;polymorphic loci are 116 which accounted for 75.8% in the total amplified fragments.The UPGMA cluster analysis of all strains was analyzed by NTSYSpc(Version 2.10 e)software,the results showed that when the genetic similarity coefficient was 0.735,the tested strains can be divided into four ISSR groups,group A was dominant,at the same time there was also had secondary groups and specificity groups;The strains from the same area had high genetic similarity and closed genetic relationship;We analyzed the clustering results combined with the strain region source.And we found that,the strains from jianghuai region were classified as dominant A,the strains from northern Anhui were classified as a separate group,and diversity of the strains from region in the south of Anhui was relatively abundant,which was more secondary groups and specific group.The genetic variation of three groups which divided based on the geographical area was analyzed by POPGENE(version1.3.1)software,the results showed that the strains from different regions have different degrees of variation,the average coefficient of genetic differentiation(Gst)of three groups of U.virens was 0.3328,and the genetic diversity of the region in the south of Anhui is the highest(H = 0.272),and the genetic diversity of the region in the north of Anhui is the lowest,and the strain came from northern anhui to the strains came from the jianghuai region had the longest genetic distance;The all results showed that,there was a certain genetic differentiation of the U.virens from different geographical regions in Anhui province,and there was some correlation between ISSR groups and geographic regions. |
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