| Tobacco mosaic virus(TMV)has a wide host range,and it occurs widely in the world.It is one of the devastating pathogens that causes the yield and quality of solanaceous crops and other vegetables to decline.Plant growth-promoting rhizobacteria(PGPR)and plant natural products are potential sources of sustainable control of plant diseases.The contents of this study is:screening and identification of PGPR with the prospect to control TMV when rhizosphere application,isolation and identification of plant natural products with potential for TMV control when foliar spraying,exploring the characteristics and mechanism of PGPR and plant natural products when separated and combined application.The experiments in this study are expected to provide the basis and the reference for the sustainable control of TMV and other plant virus diseases.The results obtained are as follows:1. In order to enhance the rhizobacteria application value of PGPR for TMV control,the plate confrontation method was used to screen out the strain Ba168 which had a good antagonistic effect on the phytophthora nicotianae,the main pathogen of tobacco rhizosphere which causes tobacco black shank(TBS)disease.Ba168 and 5 strains of PGPR(Bacillus licheniformis DL,Bacillus subtilis BS,Brevibacillus laterosporus Y-1,Bacillus methylotrophicus LW-6,Bacillus pumilus BP-3)obtained from the laboratory pre-screening,were used to conduct the field test of TBS control.It was found that Ba168 had better control efficacy on TBS in two consecutive years,79.45%and 77.42%.To further study the effect of rhizosphere application of different PGPR on the resistance level of tobacco in vivo,P.nicotianae’s cake was used to inoculate the folia of Nicotiana benthamiana treated by different PGPR 3 days later.It was found that the local lesion of Ba168 treatment is relatively reduced by 83.10%.The control effect of different PGPR on TMV was detected by Nicotiana glutinosa half leaf method.It was found that the inhibition rate of Y-1 and Ba168(27.59%and 46.15%respectively)against TMV was higher than 1mg/m L 8%Ningnanmycin,and Y-1(1.12 mm~2)and Ba168(1.25 mm~2)had a significantly decrease in area of local lesions compared to the Ningnanmycin-treated group(1.83 mm~2).Further investigation revealed that the 48h fermentation supernatant of Y-1 and Ba168 could cause a small part of TMV particles to degrade.Staining experiments of nitrotetrazolium chloride(NBT),diaminobenzidine(DAB)and trypan blue have shown that Ba168 sterile fermentation supernatant can induce reactive oxygen burst and HR reaction.Finally,Ba168was identified as Bacillus velezensis by physiological,biochemical and molecular biological methods.2. The objectives in this part was to obtain a plant natural product with the potential to control TMV by foliar spraying.We isolated and identified honokiol,magnolol and osthole from the alcohol extract of the bark of Magnolia officinalis by using alkaline extraction and acid precipitation,NMR and mass spectrometry.The half-leaf method experiment found that honokiol has the highest TMV inhibition effect,which is 5.56%higher than the 1mg/m L8%Ningnanmycin.Quantitative real-time PCR(Q-RT-PCR)was used to detect the effect of three M.officinalis extracts on the relative expression level of responsive gene of salicylic acid(SA)pathway(pathogenesis-related protein 1a,PR1a and phenylalanine ammonia-lyase,PAL)and jasmonic acid(JA)pathway(plant defensin gene 1.2,PDF1.2),it was found that honokiol treatment significantly up-regulated the expression of PR1a,PAL and PDF1.2 gene,the level of up-regulation was higher than magnolol treatment and osthole treatment.The direct effect of different concentrations of honokiol on TMV particles was observed through transmission electron microscopy.It was found that honokiol can cause the aggregation and degradation of TMV particles.With the concentration of honokiol increased from 0.5mg/m L to 1mg/m L,3mg/m L,the degree of aggregation and degradation of TMV particles increased,indicating that the inhibitory effect of honokiol on TMV is concentration-dependent.3. In order to reveal the control efficacy,action features and possible mechanism when in separate and joint treatment of Ba168 and honokiol against TMV.The effects of the 3treatments on disease control efficiency,agronomic traits,susceptible symptoms,Coat protein(CP)and RNA accumulation of TMV,and the expression of disease resistance genes before and after TMV inoculation were analyzed.The three treatments had no significant effect on the plant height of N.benthamiana,and the stem diameter of N.benthamiana was significantly increased in the Ba168 treatment.The treatment with honokiol significantly reduced the leaf area of N.benthamiana,while the treatment with Ba168 significantly increased the leaf area.The leaf area of the combined treatment group was higher than that of the control group,but the difference was not significant.All the 3 treatments could significantly reduce the disease index of N.benthamiana,and the combined treatment group could further reduce the disease index of N.benthamiana compared with the single treatment group.Based on the statistical analysis of the symptoms,it was found that the symptoms in the Ba168 treatment were wilting of middle and lower leaves and sporadic mosaic leaves,and that in the honokiol treatment,the symptoms were mosaic leaves and sporadic wilting on the lower leaves of the whole plant,and the combined treatment further alleviated the symptoms of N.benthamiana.The results of ELISA showed that the accumulation of TMV CP in N.benthamiana and Nicotiana tabacum K326 after Ba168 treatment was significantly higher than that of honokiol treatment,and the accumulation level of TMV CP after joint treatment was lower than that of honokiol treatment,and the accumulation of TMV CP in K326 was about 1.5 times that of N.benthamiana after 5d of TMV inoculation.Q-RT-PCR was used to detect the accumulation of TMV RNA in infected tobacco plants,it is found that the down-regulation fold of TMV RNA accumulation in the Ba168 treatment,the honokiol treatment and the combined treatment was more than 40 times,and the TMV RNA accumulation in the combined treatment was lower than that in the separate treatment.Within 7 days before TMV inoculation(1d,3d,5d,7d)and 1d,7d and 21d after TMV inoculation,the Q-RT-PCR was used to detect the effects of 3 treatmets on the relative expression level of the responsive genes of SA pathways(PR-1a,PR1b,PR5 and PAL),JA pathway(PDF1.2 and Coronatine insensitive 1,COI1)and disease-related non-expressor genes(Non-expressor of pathogenesis related 1,NPR1).The results showed that the expression of responsive genes of SA pathway in the honokiol treatment and the combined treatment were significantly up-regulated and reached the peak on the 3rd day;the Ba168treatment was not significantly up-regulated,reached the peak on the 5th or 7th day.The responsive genes of JA pathway in the Ba168 treatment group were significantly up-regulated and higher than those in the honokiol and combined treatment groups,which reached their peaks on the 3rd and 5th days,respectively.The NPR1 gene of each treatment group was significantly up-regulated on the 5th day and reached a peak.On the 1st,7d,and21d after TMV inoculation,the expression trends of SA pathway responsive genes on the Ba168 treatment group and the combined treatment group on the 1st and 7th days were similar to those on the 3rd day before TMV inoculation,but the folds generally increased by more than 1.5 times.The expression of responsive genes in the JA pathway is contrary to the gene expression trend at the peak of expression before TMV inoculation.The expression level of NPR1 gene 1d after inoculation with TMV was more than 10 times higher than the peak before inoculation.After 21 days of TMV inoculation,except for Ba168 treatment,the expression level of PDF1.2 gene was up-regulated,and the expression levels of responsive genes of disease resistance pathways in the other treatment groups basically returned to normal levels. |
PDF Full Text Request