Biological Control of Ralstonia solanacearum using Ralstonia phage and the influence of diversity of soil bacteriums

Transcription

1 Biological Control of Ralstonia solanacearum using Ralstonia phage and the influence of diversity of soil bacteriums Fenglong Wang Tobacco Research Institute, Chinese Academy of Agricultural Sciences

2 Tobacco bacterial wilt, one of the main diseases of tobacco, caused by Ralstonia Solanacearum is a destructive soil-borne disease. Presently, the main method to control this disease is chemicals,use. The control s result is not only poor, but also leads to some new problems, such as environment pollution, posing a threat to the safety of the human and animals. Introduction

3 Phages can specially lyse bacteria and lead them to the death. The phages are safe to the environment and their private specificity obviously implies that they do not have remarkable influence to the other microorganisms, keeping the soil microorganisms in balance. So isolating a phage able to effectively lyse R. Solanacearum will bring us a new thought in terms of the control of Tobacco bacterial wilt.

4 Presently the biological control of Tobacco bacterial wilt is still in preliminary stage. No biological agent being high efficient, pollutionfree and easy to use is available on market. Bacteria, Fungi and Actinomycetes are the most biocontrol resources against this disease in China. But lately a few researches on R. solanacearum phages have been reported abroad, such as ΦRSA1 ΦRSB1 ΦRSL1 ΦRSM1 ΦRSS1 ΦRSX and M_DS1. Even though the researchers studied the Phages biological properties and their control of R. Solanacearum, the biological agent hasn t been developed yet. Hardian S. Addy et al., 2012

5 Our team has isolated a strain of Phage RS-1 which can lyse R.Solanacearum from the soil of tobacco bacterial wilt nurseries from experimental fields of Tobacco Research Institute of CAAS, Jimo District. Phage RS-1 showed good bactericidal efficacy with a short latent period and strong-lysis ablitity to R. Solanacearum. Fig.1 Plaques of phage RS-1 in this study Fig.2 Electronic microscope of phage RS-1 in this study

6 The control efficiency of root-irrigation with RS-1 culture on R. solanacearum Because RS-1 culture was made on NA liquid medium, our experiment designed NA liquid medium as Treatment Group 3 to eliminate the possible effect on the results. Table1 showed no significant differences of disease index and control efficiency between treatment group 3 and CK1 group during the whole experiment time. This indicated that the seedlings growth were almost the same in Treatment Group 3 and CK1 Group after inoculatation. Therefore NA liquid medium showed no effect on the infection of R. solanacearum.

7 Table1 showed 7 days after the inoculation, the tobacco seedling of CK Group developed first diagnostic symptoms, but those treated with RS-1 culture and agricultural streptomycin had no symptoms yet. This means both RS-1 culture and agricultural streptomycin played some role on R. solanacearum.

8 14 days after the inoculation, the tobacco seedling treated with agricultural streptomycin developed some symptoms, but those treated with RS-1 culture still had no symptoms yet. Obviously, At this time 100% control efficiency of RS-1 culture is better than 53.33% control efficiency of agricultural streptomycin. 28 days after the inoculation, the tobacco seedling treated with RS-1 culture developed symptoms of the desease too. The control efficiency of RS-1 culture and agricultural streptomycin is 94.87% and 43.59% respectively. 35 days after the inoculation, no more affected plants treated with RS-1 culture were observed. Its control efficiency is 94.87% compared with 37.55% of agricultural streptomycin only.

9 The control efficiency of root-dipping with RS-1 culture on R. solanacearum Table2 Showed that after the seedling were treated with root-dipping while transplanted, NA liquid medium (Group 3) and sterile water (CK1 Group) showed no significant difference on the infection of R. solanacearum.

10 Seven days after the inoculation, the tobacco seedling of CK Group developed first diagnostic symptoms, but those treated with RS-1 culture and agricultural streptomycin had no symptoms yet. This means both RS-1 culture and agricultural streptomycin played some role on R. solanacearum. 14 days after the inoculation, both the tobacco seedling treated with RS-1 culture and agricultural streptomycin developed some symptoms. MOCK NA Streptomycin RS-1

11 Their control efficiency were 49.17% and 48.33% respectively. That means they both played the same role on the control of R. solanacearum. 28 days after the inoculation, the control efficiency of RS-1 culture was 41.88%, the same as 35days. So was agricultural streptomycin of 7.69%. Even though the control efficiency of RS-1 culture was 41.88% only, it was still much higher than 7.69%, the control efficiency of agricultural streptomycin. MOCK NA Streptomycin RS-1

12 Analysis of bacteria community biodiversity in 6 soil samples Figure1 showed that the bacteria groups were mainly distributed in 10 different divisions, including Proteobacteria, Firmicates, Acidobacteria, Flanctomycetes, Bacterodetes, Chloroflexi and Gemmatimonadetes. The bacteria group distribution of the 6 samples were similar shown in the figure. The abundance of Proteobacteria, R.solanacearu belonging to, was more than 50%, the highest in each sample, without showing significant difference in the CK samples and in the STJ samples. But abundance of Acidobacteria and Actinobacteria in STJ samples were much higher than that in CK samples, showing a significant difference between the 2 groups.

13 Species relative abundance of the 6 soil samples at the level of division Others: the sum of relative abundance in other divisions except the 10 divisions shown in the figure

14 Analysis of biocoenosis structure of these samples Figure 2 Species relative abundance of the 6 soil samples at the level of genus Others: the sum of relative abundance in other genus except the 10 genus shown in the figure These results showed that the bacteria groups were mainly distributed in 10 different genus, incuding Pseudoxanthomonas, Rhodoplanes, Clostridium, Candidatus Koribacter, Rhodanobacter, Salinispora, Burkholderia, Kaistobacter, Ralstonia and Symbiobacterium. The bacteria group distribution of the 6 samples were similar shown in the figure

15 Combined with the following Table 3 and Table 4, the abundance of Symbiobacterium in Sample STJ2 showed the highest in all the 6 soil samples. The abundance of Symbiobacterium in STJ samples was higher that in CK samples. The abundance of Ralstonia in CK Samples obviously was higher than that in STJ Samples. The abundance of Kaistobacter didn t shown a significant difference in the 6 samples, with the highest in Sample STJ3. The abundance of Burkholderia, Salinispora, Rhodanobacter, Clostridium and Pseudoxanthomonas in STJ Samples were higher than those in CK Samples. The abundance of Candidatus, Koribacter and Rhodoplanes in CK Samples were a little bit higher that those in STJ Sample.

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18 Our experiments showed that the isolated Phage RS-1 had good control efficiency to R.solanacearum. Meanwhile It almost had no influence to bacterial community biodiversity of the soil, considered safe to the whole Soil Ecosystem. These advantages of Phage RS-1 will provide scientific basis for the further development of biological agent against R.solanacearum.

19 Thank you very much