| For exploring the specific microbes in endosphere and rhizosphere of wheat and those recruited by disease and insect pest infestation,we used the wheat cultivar Mingxian 169(resistance strip rust genes have not been found now)and Puccinia striiformis f.sp.tritici CY32 as our study objects.Based on above research model,the 16S rRNA metagenomes of microbiota in the endosphere and rhizosphere of wheat(normal and Puccinia striiformis f.sp.tritici Infected)will be sequenced to define the composition and abundance of different genera,therefore,leading to their identification of these specific and recruited microbes.In addition,four novel actinomycetes were identificated.The results are shown as below:For microbe in the rhizosphere of wheat,high throughput sequencing was performed.for analyzing the diversity of them in the control group and experimental group(control:normal;experimental group:Puccinia striiformis f.sp.tritici infected).The results showed that microbes at the level of phylum in the two groups mainly included Proteobacteria?Acidobacteria?Bacteroidetes?Firmicutes?Gemmatimonadetes?Actinobacteria and Verrucomicrobia,which had different proportions in different groups.The species were more abundant in the control group at the second,seventh and twelfth day of the two groups;Diversity and richness were also greater than those in the experimental group except the seventh day.The results of ?-diversity comparison and statistical analysis indicated that the microbial community structure of the two groups was significantly different at the second and twelfth day,but not the seventh day;At the levels of phylum,family and genus of microbes in the rhizosphere at the second day,there were three phyla(Acidobacteria,Verrucomicrobia and Chloroflexi),fourteen families(Sphingomonadaceae,Cytophagaceae,Chitinophaceae,Sinobacteraceae,Rhodospirillaceae,Erythrobacteraceae,Syntrophobacteraceae,Rubrobacteraceae,mb2424,[Chthoniobacteraceae],[Entotheonellaceae],NB1-I,Mitochondria and Streptomycetaceae)and seven genera(Flavisolibacter,Sphingobium,Skermanella,Adhaeribacter,Sphingobacterium,Rhodoplanes and Parasegitibacter)which had significant difference between the two groups.At the levels of phylum,family and genus of microbes in the rhizosphere at the seventh day,there are four families(Paenibacillaceae,Polyangiaceae,Geodermatophilaceae and Rhodobacteraceae)and six genera(Paenibacillus,Cohnella,Pseudomonas,Sphingopyxis,Arenimonas and Novosphingobium)which had significant difference between the two groups.At the levels of phylum,family and genus of microbes in the rhizosphere at the twelfth day,there were two phyla(Verrucomicrobia and Armatimonadetes),five families(Alteromonadaceae,Sphingobacteriaceae,Verrucomicrobiaceae,Cryomorphaceae and Bradyrhizobiaceae)and nine genera(Cellvibrio,Pedobacter,Luteolibacter,Pseudoxanthomonas,Niabella,Fluviicola,Sphingobacterium,Novosphingobium and Rhodoplanes)which had significant difference between the two groups.The result of analysis of these 4 phyla,23 families and 19 genera with significant difference at second,seventh and twelfth day showed that Verrucomicrobia,Alteromonadaceae,Sphingobacteriaceae,Verrucomicrobiaceae,Cryomorphaceae,Cellvibrio,Fluviicola,Luteolibacter,Niabella,Pedobacter and Sphingobacterium did not obviously change in earlier stage,but increasing rapidly at twelfth day.This result indicated these microbes were enriched by wheat when it was attacked by Puccinia striiformis f.sp.Tritici.For microbe in the endosphere of wheat,high throughput sequencing was also used for analyzing the diversity of them in the control group and experimental group.The results showed that microbes at the level of phylum in the two groups mainly included Proteobacteria,Bacteroidetes and Actinobacteria,which had different proportions in different group.The species were more abundant in the control group at the second and seventh day;Diversity and richness were also greater than those in the experimental group except the seventh day.The results of?-diversity comparison and statistical analysis indicated that the microbial community structure ofthe two groups was significantly different at the seventh day,but not the second and twelfth day;At the levels of phylum,family and genus of microbes in the endosphere at the second day,there was no species which had significant difference between the two groups.At the levels of phylum,family and genus of microbes in the endosphere at the seventh day,there were two phyla(Bacteroidetes and Actinobacteria),three families(Oxalobacteraceae,Flavobacteriaceae and Streptomycetaceae)and two genera(Janthinobacterium and Flavobacterium)which had significant difference between the two groups.At the levels of phylum,family and genus of microbes in the endosphere at the twelfth day,there was only one family,Paenibacillaceae which had significant difference between the two groups.The result of analysis of these 2 phyla,4 families and 3 genera with significant difference at the second,seventh and twelfth day showed that Actinobacteria,Streptomycetaceae,Micromonosporaceae,Promicromonosporaceae,Nocardioidaceae and Actinosynnemataceae were increasing more rapidly than those in the control group.This result indicated these microbes were also enriched by wheat when it was attacked by Puccinia striiformis f.sp.Tritici.The result of screening and isolation actinomycetes in the endosphere and rhizosphere of wheat roots(normal and Puccinia striiformis f.sp.tritici Infected)by culturable method showed thirty and twenty three strains were isolated from the rhizosphere and endosphere of wheat in the control group,respectively;Twenty and one hundred and nineteen strains were isolated from the rhizosphere and endosphere of wheat in the experimental group,respectively;This result indicated that actinomycetes were enriched in the endosphere of wheat roots.Preliminary identification and classification of these strains by morphological and cultural characteristics showed that there were three and nine different species of actinomycetes in the rhizosphere and endosphere of wheat respectively in the control group and five and thirty eight different species of actinomycetes in the experimental group,respectively.These results indicated that actinomycetes in the endosphere of wheat attacked by Puccinia striiformis f.sp.tritici was more abundant than that in the control group,but the richness of actinomycetes in the rhizosphere of the wheat exhibited little difference in the two groups.These strains were used to determine antifungal activities and found that actinomyecetes in the rhizosphere had no antifungal activity in the two groups;but there were 10(26.3%)strains in the experimental group had antifungal activity against at least two kinds of plant pathogenic fungi and 1(11.1%)strain in the control group had antifungal activity against four kinds of plant pathogenic fungi.These results indicated that actinomycetes in the rhizosphere of wheat had no difference in antifungal activity in the control group and experimental group,but actinomycetes in the endosphere of wheat roots were much more than that in the control group,so when wheat was attacked by Puccinia striiformis f.sp.tritici,the wheat could recruit beneficial microbes to enhance disease resistance ability.It is found that 4 strains maybe represented novel species after analysis of the 16S rRNA gene sequences.We characterized these four strains by using polyphasic approach.The results showed-that strain NEAU-Jh2-17 represented a novel species of the genus-Streptomonospora and for which the name Streptomonospora halotolerans is proposed;strain NEAU-Jh3-20 represents a novel species of the genus Streptomyces,for which the name Streptomyces tyrosinilyticus is proposed;strains NEAU-Jhl-3 and NEAU-Jh2-5 represent two novel species of the genus Actinomadura,for which the names Actinomadura jiaoheensis sp.nov.and Actinomadura sporangiiformans sp.nov.are proposed. |
PDF Full Text Request