Determination Of Pectobacterium Sensitivity To Benziothiazolinone And Streptomycin,as Well As The Resistant Mechanism Against Streptomycin

The strains from the genera of Pectobacterium are important pathogenic bactera distributing widely and having diverse hosts.During plant growth and post-harvest storage and transportation,it will cause soft rot and blackleg in the presence of suitable conditions.For the rapid multiplication and adaptive variation,the resistance is generally occurred very fast in ganthogenic bacteria against agents of bactericides.Thus,enhancing risk assessment of bacterial resistance to bactericides is very important.Benziothiazolinone is a high-efficiency,low toxicity and broad spectrum fungicide developed in China independently.On January 14,2021,12%water dispersible granule（WG）of benziothiazolinone was approved for the control of potato blackleg.This is the first agent of benziothiazolinone registered for Pectobacterium control.However,action mechanism of benziothiazolinone against Pectobacterium and whether benziothiazolinone has a cross-resistance with existing bactericides are still unclear.Although agrostreptomycin has been forbidden to be used as a pesticide in China since 2020,for it had been utilized for long time as a conventional agent for prevention and control of bacterial diseases before.Therefore,agrostreptomycin is still used as a reference in this study to assess the risk of developing resistance in Pectobacterium to benziothiazolinone.In this study,the strains of PccS1 and Pcb NJAU180 with complete genome are slected as materials,and flat stand-off method and turbidimetric method were used to determine the sensitivity of the two strains to benziothiazolinone.The results showed that the EC₅₀values obtained from flat stand-off method are handreds of times higher than that from turbidimetric method,indicating that turbidimetric method is more suitable for the determination of the susceptibility of bacteria to bactericides,although flat stand-off method is simple to operate and needs no special equipment.Subsequently,six strains of lab collection,Pcc NJAU110,PccS1,Pa NJAU2,Pcb NJAU130,Pcb NJAU180,Pcb BC1were selected to determine the sensitivity to streptomycin and benziothiazolinone.Among the 6 strains,Pcc NJAU110 with the lowest inhibitory concentration value was used as the reference strain,the results showed that all the strains are sensitive to streptomycin and benziothiazolinone,except Pa NJAU2 isolated from soft rot tissue in potted colored calla plants from Yunnan,having a ratio of resistance to streptomycin at 54.12 times than the reference strain.Which preliminarily proves that there is no cross-resistance between benziothiazolinone and streptomycin in Pectobacterium.To evaluate the risk of Pectobacterium resistance to the two pesticides,the media containing a low dose（EC₅）of the pesticides were used to culture the strains for a long time to induce resistance.The results showed that the resistance of PccS1 and Pcb NJAU180 to streptomycin was increased quickly,but the sensitivity to benziothiazolinone had not changed significantly.The strains,derivated from PccS1,with increased streptomycin resistance ratio of 124.41,4514.12 and 8908.53 were obtained,and named as PccS1-S1,PccS1-S2 and PccS1-S3,respectively.It is revealed that Pectobacterium is very easy to develop resistance to streptomycin and the resistance to benziothiazolinone is slow.To undertand formation mechanism of Pectobacterium resistant to streptomycin,the strains of streptomycin-induced resistance at three different levels were re-sequenced,and different mutation types were found in 34 genes.Based on the results of relevant studies on resistance of streptomycin,three genes 0772,2330and 0091 were selected as representatives to explore the relationships between these genes and the formation of streptomycin resistance.Compared with the sequence in wild type PccS1,0772 gene in the three strains with streptomycin resistance at different levels showed a mutation of C→T at position 247,encoding the amino acid residue changed from arginine to cysteine;0091gene all altered from C→T at position 1251,and no change in amino acid residues;2330gene only had a C→T mutation at position 2602 in the super-resistant strain PccS1-S3,and no amino acid residue change.It demonstrates that the base mutation of C→T is the key to streptomycin resistance in Pectobacterium.To further verify formation mechanism of streptomycin resistance in Pectobacterium,some strains with an insertion mutation in the genes slected as above were constructed for the genes are lethal ones.The results of streptomycin sensitivity test showed that the value of EC₅₀tested inΔPccS1＿0772,ΔPccS1＿2330 andΔPccS1＿0091 against streptomycin were2.65,1.94,1.82 times higher than that in the wild type,respectively,and the strains of the three mutants grew well on the plate with streptomycin at 10 mg/m L,while the wild type could not grow up.It revealed that mutations of 0772,2330 and 0091 genes are related to streptomycin resistance in Pectobacterium.In addition,phenotype observation of virulence showed that the virulence of PccS1-S1 did not change significantly and that of PccS1-S2was remarkable reduced when compared with that of the wild type,,ane the sex strains of PccS1-S3 lost the virulence completely;as well as a significant attenuant virulence was showed inΔPccS1＿0772,ΔPccS1＿2330,not inΔPccS1＿0091.It was confirmed by the exo-enzyme activities assayed with these strains.So it could be dedued that 0772,2330,and 0091 are related to the resistance of streptomycin in PccS1;and 0772,2330 are also involved in the virulence of PccS1.Additionally,among the 34 genes with different mutation types,there are some other genes related to the virulence.In conclusion,streptomycin-resistance is easily induced in Pectobacterium,and which is closely related to the genes 0772,2330,and 0091,especially 0772,a gene encoding ribosomal protein S12.Benziothiazolinone,a novel effective bactericide,induces resistance slower in Pectobacterium,can be selected to prevent crops to agaist soft rot and blackleg diseases.