Isolation, Purification Of The Active Component Against TMV From An Antagonistic Bacteria And Study On Its Mechanism

The virus disease is one main disease on crops. The control effects and security of antiviral agent are not as good as people's demands till now. New antiviral agents are in dire need of researching. Developing antivirus products with high efficiency and high activity is the key mission of plant disease biocontrol for sustainable development, though chemical pesticide is still the main body of pesticide. The microbial pesticide is the main body of developing biopesticide.In this study, we isolated 127 bacterial strains from healthy and sick tobacco leaves and seeds in spring and summer. After zymolysis and antagonist action test with half-leaf method in lab, from the fermented broth we got a bacteria strain which has strong antagonist activity to tobacco mosaic virus (TMV). This bacteria strain was identified as Pseudomonas fluorescens based on physiological and biochemical characteristics and in 16S rDNA methods and named CZ. The anti-virus rate of CZ is up to 96.73%. Initial study on fermented broth showed that the antivirus substance is protein named CZp. Fermented condition of CZ is as follows, NB culture medium, original pH 8.0, ventilation volume 30ml/100ml, 25℃, 60h.At the same time, we isolated and purified activated protein CZp. Ammonium sulfate fractionation was used for separation and purification to obtain crude protein which has ultraviolet absorption band in 280 nm by ultraviolet scanning (220～340nm) and it was confirmed as protein substance. After crude deposition with ammonium sulfate and pretreatment of fermented broth, active material was purified with cation exchange chromatography and molecular sieve chromatography and the relative molecular mass of subunit of which is 13.5kD tested in SDS-PAGE. Preliminary analysis of active material's partial physical and chemical characteristics showed that active protein CZp has good stability and is sensitive to acid and alkali but not to temperature completely. So it can't suffer from durative high temperature for long time. It is not sensitive to light, but its activity will decay along with prolonging the photoperiod. It is not sensitive to proteinase K or trypsin but it can keep activity.In this study Nicotiana tabacum var.samsun NN, Nicotiana tabacum var.NC89 and TMV are the host plant and pathogen respectively. Inhibiting mechanism on active protein CZp isolated from antagonistic bacteria CZ to TMV is studied preliminarily. The main results are as follows: CZp has the ability to inhibit multiplication of TMV in tobacco. Experiment result in round leaf method showed that the quantity of rot spots on round leaf suspending in CZp crude extracts reduced 89.19% compated with that in distilled water after abrading and inoculating. We can conclude that CZp has the ability to inhibit multiplication of virus in tobacco.CZp has the ability to induce host to cause systemic resistance to TMV. The lower and upper leaves of Nicotiana tabacum var.samsun NN were treated with CZp and TMV respectively and lower leaves treated with water as CK. The results showed that CZp has the ability to induce Nicotiana tabacum var.samsun NN to cause systemic resistance to TMV, of which the resistance rate to rot spots in the 4th day is up to 81.76% which is the highest and the emergence time of rot spots prolong more than 10 hours.Nicotiana tabacum var.NC89 was inoculated with TMV in rubbing after being treated with CZp for 12 hours. The activities of POD and PAL, chlorophyll content and MDA content of treated leaves were tested in 1st, 5th, 8th, 12th, 16th day respectively. The result showed that CZp can enhance the activity of cytophylaxis enzyme such as POD and PAL in common tobacco and MDA content in NC89 tobacco leaves treated with cZp reduced but chlorophyll content increased.The result of scanning electron microscopy indicated that CZp can break regular arrangement of TMV virion by its combining irreversibly with TMV virion and weaken infection ability of TMV.