Study Of Seed-borne Microbial Diversity And Plasma Disinfection Method In Tomato Seeds

In order to clarify the seed-borne microorganism community of tomato(Solanum lycopersicum L.)and explore its source,high-throughput pyrosequencing technology was used to clarify bacterial and fungal communities in tomato seeds,taking the cultivar Zhongza No.302 as experimental material.Community diversities of the bacterial and fangal assemblages from root zone and rhizosphere soil,as well as the epiphyte and endophyte in various tissues were compared and analyzed.Based on this,tomato seeds were treated by atmospheric pressure room-temperature plasma method and the sterilization effects of APRTP on the seed surface and internal bacteria were determined.The main results were as follows:1.Seed-borne bacteria were classified into 13 phyla 60 genera.At the genus level,Enterobacter(37.58%)and Acinetobacter(4.51%)from Proteobacteria,Weissella(36.27%)from Firmicutes,and Bacteroides(3.66%)from Bacteroidetes were predominant.Sequences from seed-borne fungi were identified as 2 phyla and 15 orders.Ascomycota was the most predominant fungi(81.85%),followed by Basidiomycota,which accounted for 18.15%.Four dominant fungal orders were detected in seed samples,including Capnodiales(37.13%),Malasseziales(13.64%),Saccharomycetales(10.64%)and Sordariales(10.06%).2.The traditional plate culture method,16 S rRNA copies analysis,denaturing gradient gel electrophoresis(DGGE)assay and high-throughput pyrosequencing technology were used to clarify the quantity distribution and community diversities of the bacteria isolated from the soil,as well as epiphytic and endophytic bacteria in various tissues.The number of soil samples bacteria were highest,followed by the epiphytic bacteria,and the endophytic bacteria were least.The highest similarity was detected among different endophytic samples,especially for the adjacent tissues,the similarities were above 0.6.High-throughput sequencing results showed that the structure of the bacterial communities differed in terms of both the predominant phylum and the relative abundance of each phylum among soil,tissues and seed samples.In soil samples,the main phylum included Proteobacteria(57.62%),Actinobacteria(14.63%)and Chloroflexi(9.83%).Proteobacteria(> 99%)was dominated in the tissue of stem and leaf.In Fruit,Proteobacteria was the most predominant bacteria(57.62%),followed by Firmicutes(22.76%).In seeds,Proteobacteria,Firmicutes and Bacteroidetes were predominant,contributing 64.73%,22.76% and 10.71%,respectively.According to the results of Venn analysis,soil samples,tissues epiphytic and endophytic samples shared 45,44 and 42 OTUs with seeds,accounted for 76.27%,74.58% and 71.19% of the total OTUs of seeds,only 10.17% of seeds OTUs were unique.These results indicated that seed-borne bacteria mainly originate from soil and tissue bacteria.3.High-throughput pyrosequencing technology were used to clarify the quantity distribution and community diversities of the fungi isolated from soils,as well as epiphytic and endophytic fungi in various tissues.The fungi diversity and richness decreased in the following order: soil samples >episphere > endosphere.Differences in the fungi compositions among samples were significant.Although Ascomycota was the most predominant fungi phylum in all samples,the fungi distributionpatterns were quite different at the order level.In soil samples,Sordariales(39.90%)and Microascales(9.55%)were predominant.In the epiphytic samples,the main phylum included Capnodiales(31.89%),Sordariales(21.84%),Saccharomycetales(18.44%)and Pleosporales(8.98%).For the endophytes of tissues,Capnodiales,Sordariales,Eurotiales,Pleosporales and Malasseziales accounted for 31.83%,10.13%,10.61,9.91% and 10.94%,respectively.Capnodiales(62.21%)and Eurotiales(11.75%)were predominant in seeds.According to the results of Venn analysis,all of the 17 OTUs localized in seeds were from soil samples,tissues epiphytic and endophytic samples.The study indicated that seed-associated fungi originate from soil and tissue fungi.4.To evaluate the application effects of atmospheric pressure room-temperature plasma(APRTP)during tomato seed treatment,the sterilization effects of APRTP on the seed surface and internal bacteria were determined.During 5 ~ 30 minof APRTP treatment,the sterilization rate was increased with the extention of processing time.After the treatment for 25 min,the sterilization rate could reach to more than 99%,for both the seed surface bacteria and internal bacteria.For the pathogens of tomato bacterial canker—Clavibacter michiganensis subsp.michiganensis(Cmm),APRTP treatment was also efficient.The sterilization rate to Cmm could reach to 99.84% at 25 min of treatment.Furthermore,detection of seed vigor showed that APRTP treatment could increase the seed vigor,and thereby improve the germination potentialand accelerateseed germination.