Screening, Genome Sequencing And Optimization Of Medium Of PGPR From The Rhizosophere Of Crassulaceae Plants

The succulent plant is a new economic plant that is suitable for family planting and plays an increasingly important role in landscaping.Its cultivation market is also expanding and prospering.Plant rhizosphere growth-promoting bacteria can promote plant growth,improve plant quality,and suppress pests and diseases.In this study,we conducted work on the microorganisms of the roots of succulent plants of the Crassulaceae,and isolated pathogens of the black rot of the succulent plants of the Crassulaceae and screened antagonistic strains.Phosphorus-dissolving strains,protease-producing bacteria and IAA producing strains were used to verify the growth-promoting and antagonistic effects of the obtained strains through pot experiments,the better-promoted strains were combined with different bio-organic materials to select suitable carriers.The development of bio-organic fertilizers provided a reference,the optimization of the culture medium was carried out on antagonistic strains with relatively good effects,whole genome sequencing was performed on a relatively effective antagonistic bacteria.A fungus was screened from the diseased and healthy tissues of the Crassulaceae leaves in Shandong Province,which was infected with black rot,and it was identified as Fusarium inflexum.It was verified by a back-to-back test that the fungus can cause black rot in succulents.Thirty-three strains of antagonistic bacteria were screened from the rhizosphere soil samples of the succulent plants of sedum.The morphological observation and 16S rRNA gene sequence analysis of JTJK9,JTYP1,JTYP2,and JTYP3 were performed.JTJK9 was identified as Bacillus subtilis,and JTYP2 was identified as Bacillus velezensis,identifying JTYP1 and JTYP3 as Bacillus glycinifermentans.From the rhizosphere soil,7 strains with better progesterone production ability were screened out,11 strains with better proteolytic capacity were selected,and 9 inorganic phosphate strains were screened for better effect.JIA2,JIA3,JIA6,and JIA7 were subjected to morphological observation and 16S rRNA gene sequence analysis.JIA2 and JIA3 were initially identified as Bacillus flexus,JIA6 as Cellulosimicrobium cellulans,and JIA7 as Micrococcus aloeverae.Antagonistic leaf antagonism test was carried out,in which JTJK9,JTYP1,JTYP2,and JTYP3 had better antagonistic effects against black rot pathogens and could reduce the incidence and disease index of black rot.Pot experiment was conducted to verify the effect of promoting growth,among which JIA2,JIA3,JIA6,and JIA7 can increase the number of leaves and crown of succulent plants,increase the biomass of aboveground and underground parts,increase the absorption area of roots,and increase the chlorophyll content of leaves.content.It was proved that the screened and promoted bacteria can promote the growth of succulent plants.Extract the JTYP2 DNA sample and use the assembly software smrtlink（version 4.1）to assemble the PacBio down data to get the assembly result.Statistical results of the assembled sequences showed that the JTYP2 genome was 3,929,789 bp in length,3,656 predicted genes,46.5%of GC content,and 259 non-coding RNAs of JTYP2,including 90 rRNAs,86 tRNAs,and 83 other non-coding RNAs.Among the 12 secondary metabolite gene clusters of JTYP2,6 were highly similar to those reported previously.Co-linear analysis of JTYP2 and Bacillus velezensis 9912D（CP017775.1）,Bacillus velezensis M75（CP016395.1）,Bacillus velezensis G341（CP011686.1）,and common endemic gene analysis indicated that different species of Bacillus velezensis are different.Select test strains JIA2,JIA6,donkey dung,mushroom mushroom residue,sheep manure,mushroom mushroom residue,apricot mushroom mushroom residue,washed cow dung,conventional cow dung,peat moss,worm manure,fermented chicken manure,worm manure:mushroom=1:1,maggots:Flammulina velutipes=4:1,Alum oysters:Pleurotus ostreatus=1:1,Alum oysters:Pleurotus ostreatus=4:1 as a carrier,and the two strains were selected as the most suitable bioorganic fertilizer carrier.The optimal carrier for determining JIA2 was maggot:the mushroom slag=4:1.The optimal carrier for JIA6 is maggot:Pleurotus ostreatus=1:1.The five kinds of organic carriers were used for the toxicity test on the vermicompost:Flammulina velutipes=4:1,worm feces:Pleurotus ostreatus slag=4:1,vermicompost:oyster mushroom slag=1:1,cow manure,and fermented chicken manure.It is proved that in practical applications,the carrier will not be toxic to the seeds and will not affect the germination of the seeds.The medium was optimized for JTYP2 and its optimal medium ratio was determined.Through single-factor experiments,the optimal carbon source of JTYP2 was sucrose,the optimum organic nitrogen source was soybean meal,the optimal inorganic nitrogen source was sodium nitrate,and the optimal inorganic salt was MgSO₄.The optimal medium for JTYP2 was 3%sucrose,1%soybean meal,1.5%sodium nitrate,and 0.5%MgSO₄.The number of viable cells after optimization was 3.7×10⁹cfu/m L,which was significantly higher than the 1.35×10⁸cfu/mL viable cells in the medium of bean sprouts.