| Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease in the areas of tropical, subtropical and temperate zone. This pathogen has a large range of hosts with more than 200 species including approximately 50 families. Bacterial wilt appears in many provinces in China. With the appearance of new hosts and global warming in recent years, bacterial wilt is getting more and more popular and severe worldwide. It is very important to study the mechanisms of bacterial pathogenesis and plant resistant, and to find efficient methods to control bacterial wilt.Biological control to bacterial wilt has been studied with an increase interest and has been recently reviewed. Biological control used to be thought as antagonism against the pathogen via production of antibiotics or hydrolytic enzymes, or through competition for nutrients to inhibit multiplication and growth of the pathogen. In the past years, many progresses have been made on bacterial wilt management with nonpathogenic mutant obtained by gene engineering from pathogenic strain. It has been found that nonpathogenic mutant of R. solanacearum generated by knocking out DNA fragment of its hrp gene has very good effect on controlling pathogenic isolates based on the observation carried out in greenhouse, however, the study in biocontrol mechanism is limited. The objective of this paper is to set up the biocontrol system, to find the effects and mechanisms of hrp- mutant as biological agents to tomato bacterial wilt.First of all, tomato varieties were inoculated with the pathogenic strain of GMI1000 by using two methods named traditional root inoculation and improved root inoculation, respectively. It was indicated that the response of tomato varieties to GMI10000 is similar in both inoculations. Since the improved root inoculation has the characteristics of saving manpower, material and operating room, and of shortening cycle time between the inoculations, the improved root inoculation method was adopted in this work. In addition, it also provided a rapid and efficient method in bacterial wilt-resistant identification and screening.Pre-inoculating tomato zhongshu No.6 with hrp- mutant before it was challenged with pathogenic strain GMI1000 different times after pre-inoculation, it was shown that inoculating the plants with pathogenic pathogen 6h-48h after pre-inoculation with hrp- mutant could significantly increase the resistance, whereas the protective effects of treatments of 72h and 7d after pre-inoculation fell down, even though the plants involved in these treatments still performed rather resistant to R. solanacearum.To better understand the effectiveness of hrp- mutant in preventing disease development, bacterial multiplication of GMI1000 in aerial parts of the plant were tested. After single inoculation with GMI1000, the bacterium were detected two days after inoculation and the pathogen multiplied extensively, reaching densities of 1015cfu/g eight days after inoculation. In contrast, the bacterium of GMI1000 multiplied slowly when the plants were pre-inoculated with hrp- mutant, which displayed not only on the time of appearance of the pathogen (8 days after inoculation) but also on its concentration (106 cfu/g in the 10th day after inoculation).No changes of cell structure including tyloses were detected after ultrastructural observation. Possibly reasons are indicated as follows: either the sections observed were not appropriate or resistant structures formed in the plant cell are not the major way to prevent the multiplication of pathogenic bacteria. This work need to be done in the future.The expression of total tomato RNA by using RT-PCR indicated that pathogenesis related protein PR-1 expressed both in plant inoculated with hrp- mutant, GMI1000 as well as in the double inoculation, but the time and quantity of the expression are different. Therefore, this result together with the expression of other PR genes and defense genes need to be confirmed by RT-PCR and Northern blotting.
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