Screening Of Agrobacterium Tumefaciens Antagonistic Fungi, Fermentation And Organic Tin New Compounds Resistance To Tmv, The Anticancer Mechanism

Plant diseases caused by virus are of common occurrence and cause serious threats and economic loss to the farmers dependent heavily on the production of agricultural crops. Pathogenic Agrobacterium tumfaciens is known for its ability to adversely infect the rhizome by forming crown galls and thereby weakening and retarding the growth of the diseased plant.This eventually affects the economy,shortens the life of the fruit crop and lowers the output and crop yield.In tobacco leaves infected by TMV,the metabolism is seriously affected,leaves become dry and yellow,chlorosis gets uneven leading to photosynthetic rate decline,plant dwarfing and decrease of accumulation of dry matter.Over the last few years,it has become a major agricultural issue to tackle with in China.The present investigation is mainly divided in two parts.Owing to the disadvantages associated with low drought tolerance and shot life of biocontrol bacterium,in the first part of the study,the biocontrol fungi strains of antagonism A.tumfaciens from root soil were screened and the fermentation conditions were established.Again it is well known that by using the commonly employed reagents it is difficult to inhibit the proliferation without hurting the host.Therefore,in order to develop reference compound with high activity and low toxicity, the second part involved studies on the mechanism,anti-tumor and anti-TMV activities of 04-W0319,an organotin compound provided by Academician Li Zhengming of Nankai University.For the first part of the work,we isolated 152 strains of fungi from the soils of 12 different provinces in China,and could obtain 2 strains of antagonism A.tumefaciens T-37 from our collection by employing agar column.At the same time,the antagonism were confirmed by filter paper method.According to the classification standard of Raper(1965),NO.1 strain is generally in good agreement with the standard of holotype Aspergillus niger V.Tieghen 1867,therefore,we identified and named the type culture as Aspergillus niger xj.According to the classification standard of Brown & Smith(1957),NO.2 strain is a new species,we identified and named the type culture as Paecilomyces verticillatus 49-01.We obtained 8 monoconidium strains from xj with single spore separation technique.They were marked as xj-1,xj-2,xj-3,xj-4,xj-5,xj-6,xj-7,xj-8,the xj-5 was considered as the dominant monoconidium strain after comprehensively comparing the situation of colony growth,sporulation, the germination of conidia and the inhibition activity.Subculture characters were found to be relatively more stable and the diameter of inhibition(20.13±0.09mm) was longer than the contrast (18.28±0.21mm).Subsequently,fermentation process condition was studied with xj-5 serving as the test strain.Six different kinds of carbon and nitrogen sources were subjected to the single factor screening study.They were 2%of maltose,glucose,starch,cane sugar,lactose,glycerol,and 3% of peptone,beef extract,yeast extract,ammonium nitrate,ammonium chloride and urea in culture medium.Maltose,a sugar as the carbon source,yeast extract and peptone as the nitrogen source were designed for orthogonal experiment L₉(3⁴) in order to optimize the C/N ratio.2%maltose, 2%cane sugar,3%yeast extract and 1%peptone were considered as the fermentation media.Different liquid inoculums were inoculated into fermentation medium,15%(v/v) was selected from an economic point of view.The effect of different liquid volume in the flask,10% (v/v) was found to be the optimum.Anti-bacterial activities at various temperatures were also studied.Results showed that the longest diameter was at 25℃.For the production and accumulation of antibacterial substances,natural pH was convenient for media preparation of xj-5.Based on our experimental results,the flask was inoculated with xj-5 and the dynamic fermentation curve was drawn after testing the biomass and diameter of inhibition per every 24h. Results showed that xj-5 followed a direct exponential growth phase without displaying any lag phase.Perhaps due to the strong activity of the liquid inoculum and lack of variation in the culture condition,the strain could quickly adapt the fermentation and go into the growth phase as fast as possible.The time was long(1-8d) and corresponding biomass could reach the peak(161.24 g/L) on the 8^th day.The antibacterial components were secreted and accumulated at the same time. Then the lag phase(9-12d) lasted for an appreciably longer time,the metabolites reached the peak and the diameter was 54.48 mm on the 10^th day.With the exhaustion of the nutrient,aging phase started to appear(13-15d),the biomass sharply decreased and toxicity enhanced with the accumulation of autolysis.The pH value varied due to the outflow of the cellular content and production of cellular fragments,which affected the antibacterial activity and caused dramatic decrease in the value of the diameter.Thus,in accordance with the dynamic fermentation data,the optimal condition to produce active antibacterial active substance was set as follows:2%maltose,2%sugar,3%yeast extract, 1%peptone,natural pH with the addition of 15%inoculation volume into 10%(v/v) flask at 25℃under 120rpm,cultured for 10 days.After collecting the concentrated fermentation liquid,the diameter was found to be 54.48 mm by filter paper method.The second part of the study concerned with Nicotiana glutinosa as the material.We determined the living therapy,protection and inactivation effect of 04-W0319 against TMV,the treatment effect was 60.53%,inactivation effect was 75.97%and protection effect was 54.85%.Electron microscope studies revealed that the virus particles were fractured when 500μg/mL 04-W0319 was treated with TMV for 30 minutes in vitro.TMV-RNA showed degradation in vitro which was detected by agarose gel electrophoresis.The results indicated that 04-W0319 could inhibit the polymerization of TMV-CP in vitro.The activity of PAL,POD and SOD of Nicotiana tabacum K326 was enhanced by at different degrees when treated with 04-W0319 at 500 mg/L.The results of PAL activity showed that PAL was significantly increased by 04,and achieved the peak on the 7^th day,but fell to the lowest value on the 9^th day.PAL activity was also found to increase when K326 was vaccinated with TMV before being treated with 500 mg/L 04-W0319.The results of POD activity showed that POD achieved the peak on the 7^th day when K326 was treated with 04-W0319,but the peak was attained on the 9^th day when K326 was vaccinated with TMV before treatment with 04-W0319,and the value was higher as compared to other treatments.The results of SOD activity on the other hand showed that SOD achieved its first peak on the 5^th day when K326 was treated with TMV(Control),then attained a second peak on the 9^th day.Treatment with 04-W0319 before being vaccinated with TMV and the control,revealed the same pattern in the SOD activity of K326.SOD activity also achieved the peak on the 5^th day when K326 was treated with 04-W0319, but the peak was higher than those observed with other treatments.The results of SDS-PAGE showed that the induction of PR protein of K326 was relatively low by 04-W0319,and intercellular protein was not affected by 04-W0319.The role of PR protein to induce plant resistance was perhaps remarkable with their coexistence.It should also be noted that the chlorophyll content of the tobacco leaf infected by TMV has been found to decrease sharply due to the destruction of chloroplast by the virus.However,the issue can be addressed partially by treatment with 04-W0319 which helps to develop disease resistance of the host thereby enhancing the chlorophyll content. Further studies using 04-W0319 on different cancer cells e.g.PC3,Bcap-37 and BGC823 showed that 04-W0319 had different inducing effects towards various cells.The IC₅₀ of 04-W0319 was 0.13μg/mL when the PC3 cells were incubated for 72h;0.18μg/mL when the Bcap-37 cells were incubated for 48h and 0.17μg/mL when the BGC823 cells were incubated for 48h.04-W0319 could reduce the mobility of three cancer cells and inhibit the growth of cancer cells.From the results derived from cellular morphology and LDH assay,we noticed that apoptosis in 72h was the main method of inducing the death of PC3 and Bcap-37 cells and apoptosis in 48h was primarily responsible for inducing the death of BGC823 cells.In addition, the results of FCM further indicated that 0.15μg/mL 04-W0319 could arrest PC3 or Bcap-37 cells at G₂/M stage in 48h.However,with the increase of time and concentration,04-W0319 mainly induced the apoptosis of cells.BGC823 cells could be arrested by 0.15μg/mL 04-W0319 at G₀/G₁ phase of cell cycle in 48h.This was followed by the inhibition of the growth of the cells by induction apoptosis.However,with the elapse of time,04-W0319 could arrest BGC823 cells at G₂/M phase,with the death of BGC823 cells.The results of western blot indicated that 04-W0319 could up-regulate the expression of p21 in PC3 and Bcap-37,in addition to hindering the cell to overpass G₂/M restriction point and inhibit its growth.However,the concentration of 04-W0319 required to up-regulate the expression of p21 in PC3 was 0.1μg/mL,and only 0.05μg/mL for Bcap-37.But for BGC823,04-W0319 at a concentration of 0.2μg/mL could mainly down-regulate the expression of CyclinD.The cells were then hindered through the G₀/G₁ restriction point,and growth of BGC823 cells was also inhibited. As a matter of fact,04-W0319 had practically no influence on CDK₂ and CDK₄ of PC3,Bcap-37 and BGC823 cells,suggesting that the target of 04-W0319 is relatively specific.