Biological Properties And Genetic Difference Analysis Among Four Isolates Of Cucumber Green Mottle Mosaic Virus

CGMMV has caused serious losses in agricultural production worldwide since it was reported in 1935.In 2007,it was listed as one quarantine pest in China.In this study,biological characteristics,molecular differentiation and phylogenetic relationship of four CGMMV isolates from distinct regions were analyzed.The main results are as follows:1.To clarify whether there were differences in the pathogenicity of four CGMMV isolates,two of which were respectively isolated from watermelon in Liaoning and Zhejiang provinces and the other two from cucumber in Shandong and Hunan provinces the biological characteristics of four CGMMV isolates were studied.In the host range assay,among the supplied plants from 12 species belonging to 3 families,the four isolates could cause different symptoms on the white gourd and loofah.In terms of white gourd leaves,it showed macular when infected by CGMMV-LN and CGMMV-SD,systemic mosaic when infected by CGMMV-ZJ,and no symptoms when infected by CGMMV-HN,but CGMMV-HN could be detected.In terms of loofah leaves,it showed systemic mosaic with macular when infected by CGMMV-LN and CGMMV-HN,systemic mosaic when infected by CGMMV-SD,and macular when infected by CGMMV-ZJ.It showed systemic mosaic when infected by the four isolates on the leaves of other Cucurbitaceous plants,while the four isolates caused no symptoms and no CGMMV could be detected on non-cucurbitaceous plants,showing that the four isolates could not infect non-cucurbitaceous plants.The above results showed that among all supplied plants,there was no difference in the scope of host plants,but different CGMMV isolates could cause different symptoms on some individual host plants.The analysis of stability in vitro showed that there was no significant difference among the four isolates:the lethal temperature was ranging from 95 to 100? dilution limit was between 10-7 and 10-8,and survival time in vitro was about 126 days.The results of pathogenicity differentiation showed that the four isolates led to different disease situation on different cucumber varieties:CGMMV-ZJ resulted in the highest disease index,followed by CGMMV-SD,and CGMMV-HN caused the lowest disease index.In the field test,the watermelon leaves infected by CGMMV-ZJ showed obvious chlorotic spots,but showed systemic mosaic symptoms when infected by the other three isolates.The necrosis time of watermelon flesh for the four isolates was orderly different.In the 14 days after pollination,the flesh of watermelon infected by CGMMV-ZJ firstly showed water-like symptoms with a slight red,followed by CGMMV-SD,and the slowest was CGMMV-HN.The interval time of disease occurrence for each isolate was about one week.The results of blood flesh rate of watermelon exhibited that the rate caused by CGMMV-ZJ ranked the highest with 96%,follow by CGMMV-SD,and CGMMV-HN with the lowest rate.The results showed that CGMMV-ZJ displayed the stronger pathogenicity than the other three isolates.2.The physiological and biochemical differentiation for the necrotic watermelon flesh caused by the four isolates were analyzed.The results of pectinase activity assay showed that there were no significant differences between the control group and the inoculation group within 7th-14th days.The activity of pectinase increased continuously during 14th-35th days in the healthy watermelon.The activity of pectinase was decreased in the inoculated watermelon during 14th-28th,while it was increased during 28th-35th days.The results of polygalacturonase activity assay showed that there was no significant difference between the control and the inoculation group during 7th-14th days.For the control group,the polygalacturonase activity was increased originally and then decreased.However,the polygalacturonase activity in the inoculation group was linearly increased during 7th-28th while decreased to some extent during 28th-35th days.The results of cellulase activity assay showed that the activity of Cx was increased in the watermelon infected by CGMMV during 7th-35th days and it was increased in the healthy watermelon during 7th-28th,while decreased in the 35th day.The comprehensive results showed that PE activity in the inoculation group was evidently lower than that in the healthy watermelon,but the activity of PG and Cx was higher.These factors were probably the reason for excessive hydrolysis,decay and fibrosis in watermelon flesh.The changes of these three enzymes activity were not significantly different in the watermelon flesh inoculated by the four isolates.But the extent of enzymes changes caused by CGMMV-ZJ infection was slightly higher,followed by CGMMV-SD,and CGMMV-HN with relatively lower level.The results were consistent with pathogenicity analysis in the previous studies,further verifying that there were differences among the four isolates.3.The homology and phylogenetic relationship of the amino acid and nucleotide sequence for two replication-related proteins,movement protein and coat protein genes from four isolates were analyzed.The nucleotide sequence similarity of P129,P186,MP and CP among four isolates were 98.75%,98.91%,99.94%and 99.59%,respectively.The amino acid sequence similarity were 99.44%,99.33%,98.14%and 100%,respectively.The results of NJ phylogenetic tree showed that all CGMMV isolates and the outgroup TMV were divided into two groups and all CGMMV isolates from Japan and Korea displayed genetically close relationship,while the isolate from Russia was far.In the phylogenetic tree based on P129 and P186 genes,the isolates from Asia were clustered into two groups:CGMMV-ZJ and CGMMV-LN were clustered into one group and closed to the isolates from Korean(AF417242)and Zhejiang(KM873788);CGMMV-HN and CGMMV-SD were clustered into another group in which CGMMV-HN and isolates from Shandong(KJ754197)were clustered into one clade.In the phylogenetic tree based on MP,CGMMV-ZJ was far from the other three isolates,followed by CGMMV-HN,and CGMMV-LN and isolates from Taiwan(FJ654658)were clustered into one clade.In the phylogenetic tree based on CP gene,the isolates from Asia were clustered into two groups:CGMMV-SD was in a group by itself,and was clustered into a clade with isolate from Anhui(KT236095);the other three isolates were inside another group in which CGMMV-HN was clustered together with CGMMV-ZJ,and CGMMV-LN and isolates from Korea was clustered into one clade.In the phylogenetic tree based on P186,P129 and CP,CGMMV-ZJ and CGMMV-LN were clustered into one group and were most genetically-close to the isolates from Korean(AF417242)and Zhejiang(KM873788)which were from watermelon,suggesting that they come from the same source and the evolution trends were similar in the same host.Taken together,there were high similarity in amino acid and nucleotide for the four CGMMV isolates;the results of phylogenetic relationship showed that there was a relatively far genetic distance between CGMMV isolates and TMV,showing the variation was evident in genera but stable and conservative in species;the isolates from Asia were more genetically-close with each other,showing their infection sources may be the same,however,the phylogenetic relationship for the isolates was not significantly correlated to hosts and geographical origins.