| Screening for excellent rhizobium strains with dry-tolerance ability and establishing a rapid identification method are extremely important in the development and application of rhizobium industry,including new technology of rhizobium coating inoculant,intellectual property protection for functional strains,as well as large-scale application.In this study,100 strains of soybean rhizobia isolated from different soybean producing areas in China were analyzed by a glass bead-drying method and vermiculite potting experiments,compared with commercial strains as reference.An excellent strain 5873 was screeninged and shown a good ability in symbiotic nitrogen fixation and dry-tolerance.Moreover,the specific molecular markers of strain 5873 were used to establish a specific and rapid PCR identification technique,which could be used to evaluate the competitive nodulation ability of rhizobium strains.In addition,the mothed of rhizobium coating inoculant was analyzed,including the functional materials and the optimization of coating composite.The results were presented as follows:1.A superior strain 5873 was screeninged and shown a good ability in symbiotic nitrogen fixation and dry-tolerance.After drying for 24 h,the survival rate of strain 5873 was 0.44%,which was significantly higher than other soybean Rhizobium and the commercial strains,indicating a better dry-tolerance characteristic.Based on the 16S rDNA sequence phylogenetic analysis and ANI calculation,the strain5873 was identified as the B.japonicom.Then,the biological function of strain 5873 was evaluated in greenhouse and the results showed that the strain 5873 could nodulate with the 9 main soybean varieties,which proved that the strains 5873 have extensive-host advantage in symbiotic nodulation.Compared with the non-inoculation control,the dry weight,total nitrogen and chlorophyll of the plants that treated with 5873 were increased,expecially Diandou 5,Xudou 18 and Jidou 17.Hence,strain 5873 that have the characters of good resistance to desiccation and better nitrogen fixation bioactivity can be used for soybean inoculant industry through seed coating.2.A rapid identification mothed based on strain-specific PCR for strain 5873 was established.In this section,the whole genome sequence of strain 5873,as well as other sequences of Bradyrhizobium japonicum in NCBI was analyzed to search for a unique primer sequence for strain 5873.Meanwhile,we focused on the differential fragments of B.japonicum E109 and B.japonicum USDA 6~T that were highly homologous to strain 5873(the ANI value of the genome is greater than 99.95%).The specific sequences and differential fragments were input into the primer-BLAST in NCBI for specific primer design and a group of PCR primer sequence was designated which was developed based on specific PCR amplification from the whole genome of a dry-tolerant B.japonicum 5873 and bioinformatic analysis.A set of strain-specific primers 4-4 and Q1 were designed.After the optimization of PCR reaction conditions and the detection of sensitivity and specificity,a rapid detection method for dry-resistant soybean rhizobium 5873was established.The results demonstrated that,355 bp PCR fragment was only generated from strain5873 not from all other B.japonicum strains(except B.japonicum E109 and B.japonicum USDA 6~T)tested in the study,which indicated that the primer had a good intraspecific specificity(4-4-F 5’-GATAAGGCCACGGGTGAACA-3’/4-4-R 5’-CACTCGATAAGCTCCGCTGT-3’and Q1F 5’-CCGGTCGTGACTGGAATGAT-3’/Q1R:5’-TCGAGGCCTACAAGAACGTC-3’).Sensitivity of this PCR based detection is about 1500 cfu/μL.In addition,the size of the amplified fragment using primer Q1 could further clearly distinguish the strain 5873 from B.japonicum E109 and B.japonicum USDA 6~T.The specific PCR rapid identification technology could also use crushed nodulation solution and bacteria culture solution as templates.The method could avoid the traditional method of extracting bacterial DNA steps and greatly improve the detection efficiency.The ability of compete for nodulation of strain 5873was determined by PCR.The results showed that strain 5873 had stronger ability to compete for nodulation.3.The functional materials and the optimization of coating composite were studied.The results showed that when the survival rate was 1.0%and the shelf life was one month,the soybean seeds could be mixed with strain 5873 and 0.2 g sodium molybdate per kg.When Junxianke that was used as a green medicament to kill root nematodes and rhizobium were coated with soybean,we must ensure that the rhizobium was mixed with the protectant first,and then the Junxianke was coated with the mixture.The results of this study provide a basis for screening and evaluating excellent drought-resistant strains and improving the new technology of soybean seed coating for rhizobium. |
