Biological Control Of Eucalypt Bacterial Wilt By Endophytes From Non-host Plants Of Ralstonia Solanacearum

In this study, biological control of bacterial wilt in Eucalyptus urophylla was investigated through suppression of the disease with culturable endophytic from non-host plants of Ralstonia solanacearum, selection of effective endophyte population from non-host plants for controlling of bacterial wilt, and control of eucalypt bacterial wilt by endophytic bacteria of different varieties of Allium sativum Linn. And finally, the colonization of the endophytic bacteria from four varieties of A. sativum was determined in eucalyptus seedling by 16S rDNA PCR-DGGE techniques. The main results of this research are as follows:1 Biological control of eucalypt bacterial wilt by culturable endophytic bacteria from non-host plants of R. solanacearumThis part mainly investigated the inhibition of culturable endophytic bacteria isolated from non-host plants to R. solanacearum, their control effect against bacterial wilt in E. urophylla, the colonization dynamics in the seedlings of E. urophylla, and the inhibitive interactions among the endophytic bacterial strains. The endophytic bacteria were isolated from A. sativum, A. fistulosum, A. cepa and Vitis vinifera by dilution-plate method. In vitro antagonism between endophytic bacteria and R. solanacearum was determined on Kelman agar plates. Three different ways were applied to assess the ability of strains to control bacterial wilt. Firstly, seedlings were cultured in a suspension mixture with each of the biocontrol strains and the pathogen in 1/10 MS; secondly, seedlings were first dipped in a bacterial suspension of biocontrol agent for 24 h followed by transferring them into suspension with pathogen in 1/10 MS; and thirdly, roots of seedlings were dipped in bacterial suspension of biocontrol agents for 20 min followed by transferring them into potting soil infected with pathogen. Fourteen bacterial strains were isolated, in which only four strains, designated as SH, SB, PT, C1 and C2, respectively, had inhibition against R. solanacearum. The diameter of growth inhibition zone against R. solanacearum induced by strain PT was 28 mm in average, and inhibition effect was PT> C2> C1> SH> SB. In the interactive inhibition test, strain C2 had strong growth inhibition against SH, and strain SH had slight inhibition against C1. The disease suppression of endophytic bacterial strains SH, SB and PT was evaluated in three kinds of bioassays. Inoculum density of the pathogen at 5×107 CFU/mL was adopted for further experiments and the biocontrol bacterial inoculum density was set at 1×109 CFU/mL. SH had the best control effect in the test of hydroponic growing system, especially biocontrol bacteria inoculation firstly, it significantly reduced bacterial wilt by 48% compared with the control treatment (CK) (α= 0.05). Strain PT had better effect than others in a potting soil system, and the percentages of wilted seedlings reduced by 28% compared to CK. Strain SH also has a certain effect in soil system. The non-host endophytic bacterial strain SH had the colonization ability in eucalypt seedlings and bacterial cells were capable of moving to the upper part of the stem, when the seedlings were inoculated with 108 CFU/mL of SH suspensions.2 Suppression of eucalypt wilt by endophyte population and root exudates from non-host plants of R. solanacearumThe endophyte population with positive control of eucalypt bacterial wilt was extracted from A. sativum, A. fistulosum, A. cepa, Citrus reticulate, Citrus junos and C. grandis. Centrifugal method was used after grinding the plant tissue for the separation of endophytic population and plant extract. The inhibitory effect of endophyte and plant extract on R. solanacearum was determined by the Oxford plate method and dilution plate method. Endophytic population, plant extract and plant slurry were used in hydroponic system and soil system for the control of bacterial wilt in E. urophylla. The impact of root exudates from non-host plants, Phaseolus radiatu, Triticum aestivum and Zea mays,and host plants, Lycopersicon esculentum, Capsicum frutescens, E. urophylla, E. camaldulensis and E. dunnii, on the growth of R. solanacearum was determined by adding the pathogen to the solution with root exudate. The results showed that, separation of the endophytic population and plant extract is to spin down the supernatant liquor at 12,000 rpm for 10 min (4 times), after spinning down the plant slurry at 900 rpm for 10 min. Only endophytic population of A. sativum, A. fistulosum and A. cepa have certain inhibitive activity on R. solanacearum on Kelman plate. Plant extracts show obvious inhibitive activity on R. solanacearum on KB plate with the order of A.sativum > A. cepa > A. fistulosum > C. junos. Pathogen can not grown on Kelman agar plates after co-culture with plant extracts. In two kinds of bioassays, endophytes and extract from garlic have obvious effects on eucalypt wilt, and incidence rate reduced by 44% compared to CK at 10th days after inoculating endophytes from garlic. The statistical analysis at 10th day showed that, in the level ofα= 0.05, the percentage of wilted seedlings treated with garlic endophytes were significantly lower than that of control. Root exudates of non-host and host plants did not inhibit the growth of R. solanacearum. On the contrary, they promoted the growth of the pathogen, especially in the presence of root exudates of maize, which promoted the growth of pathogen most. The impact of root exudates from different species of Eucalyptus spp. on the growth of R. solanacearum is essentially the same. 3. Control effect of eucalypt bacterial wilt by entophytes in different varieties of garlic.The entophytes from different varieties of garlic were used to control the bacterial wilt of E. urophylla. Four varieties of garlic, Luyou No.4, Lusuanwang No.1, Shuangfeng No.1 and Siliuban were used in this experiment. The grinding and centrifugal methods were used to separate the endophyte and its extract, and then their inhibiting effects to the pathogen were determined through Oxford plate method and dilution plate method. Endophytes from four varieties of garlic were evaluated in hydroponic system and soil system. In the determination of antagonistic action, Luyou No.4, Suanwang No.1 and Siliuban have slight effect, and Shuangfeng No.1 had no antagonism. The number of R. solanacearum did not decrease significantly compared with the control, after co-culturing with entophytes of garlic. Bacterial cells of the pathogen reached 107 ~ 108 CFU/ml 96 h later. The extract showed strong bacteriostasis activity, with the order of Luyou No.4 > Siliuban > Lusuanwang No.1 > Shuangfeng No.1. The result also showed that entophytes from different varieties of garlic all have control effect, in which the role of Siliuban was the most powerful. The incidence of eucalypt seedlings in the hydroponic system which was treated with Siliuban, was significantly lower than that of the control from 6thday after inoculation, and at the tenth day the disease rate reduced by 36% compared with control. And in the soil system, the incidence on the tenth day was reduced by 32%, with a significantly differences compared with the control. At the same time the incidence of wilted eucalyptus seedlings dipped in entophytes of Shuangfeng No.1 in the hydroponic system was significantly lower than the control from the 8th day, and that dipped in entophytes of Lusuanwang No.1 was significantly lower than the control from the 10th day. In the soil system, the incidence of the eucalyptus seedlings treated with entophyes of Luyou No.4, Lusuanwang No.1 and Shaungfeng No.1 was reduced, but without significant differences.4. Detection of the effective endophytes from non-host plants in E. urophylla determined by 16S rDNA PCR-DGGE technique.The study identified the suitable tissue culture medium and rooting medium for the protocorm-like body of E. urophylla, and after 4 times of subculture a large number of the same background eucalypt seedlings were cultured. Modified CTAB method was used to extract the DNA of the endophytic bacteria in plant. Vertical slab gel electrophoresis methods and combination of different concentrations of denaturing were used to determine the 16S rDNA gene denaturing gradient gel electrophoresis detection system of garlic endogenous flora. The selected detection system was applied to detect the endogenous flora of E. urophylla inoculated with garlic endophyte. E. urophylla seedlings inoculated exogenous endogenous flora by the method of root dipping for co-culture of the endogenous bacteria of eucalyptus seedlings and exogenous endogenous bacteria. The PCR-DGGE detection system selecting 16S rDNA used 10% polyacrylamide gel concentration, 35% ~75% of the denaturant concentration, in 1×TAE buffer electrophoresis under 50V, 60°C running 15h. The fragment of 230 bp was were all amplified from E. urophylla and four garlic varieties. The patterns of endophytic bacteria were slightly different because of the different backgrounds of the E. urophylla seedlings. indicating the necessity to use the same background during the experiment. The amplified DNA bands of different garlic varieties were basically in the same position. This phenomenon showed that the endophytic bacteria from different garlic varieties had essentially the basic pattern, and rich diversity, similarity of 67%. The method of co-culture enabled the exogenous endophytic flora to enter the eucalypt's inner tissues, and after that process the DNA band number of the endophytic bacteria in eucalypt increased, which had the same rate of flow (RF) as the amplified bands of the endophytic bacteria in garlic, indicating that the endophytic bacteria of garlic could get into the body of eucalypt. The reason that those amplified bands were not coincide with that of the garlic may be that the stimulation of endophyte of garlic broke the balance system in which the original endogenous flora in the tissue of eucalypt kept. Especially in the eucalypt seedlings treated with Siliuban five bands could be amplified which had the same RF as those in garlic.Based on the above results, conclusion can be drawn that endophytes from non-host plants had the potential to be used as biocontrol agents to suppress eucalypt bacterial wilt.