Mechanism Of Bacillus Amylolyquefaciens T-5 Competition For Tomato Root Exudates With Ralstonia Solanacearum

Tomato wilt is a destructive soil-borne disease caused by R. solanacearum. As tomato is one of the main crops in the world, and bacterial wilt of tomato is seriously restricting the tomato production and economical benefits, the control of the disease is an urgent need. One of the biggest ecological challenges facing microbiologists and plant pathologists in the near future is the development of environmentally friendly alternatives to the extensive use of chemical pesticides for combating crop diseases. The use of beneficial microorganisms is considered one of the most promising methods for more rational and safe crop-management practices. Bacillus spieces have been successfully used to suppress many kinds of soilborne pathogens, their spore-forming ability also makes these bacteria some of the best candidates for developing efficient biopesticide products from a technological point of view. Bacillus spores have a high level of resistance to the dryness necessary for formulation into stable products.As the basal of all, the biocontrol agents should be have the ability to competition for the limited nutrients and niches from the plant roots, colonized on root systems and establishment the bacterial community, especially when there are pathogens in the soil. Members of the Bacillus genus are often considered microbial factories for the production of a vast array of biologically active molecules potentially inhibitory for phytopathogen growth. In our laboratory, a tomato root colonizing strain, Bacillus amylolyquefaciens T-5, isolated from healthy tomato roots showed strong inhibitory against R. solanacearum, and a new bio-organic fertilizer to control tomato wilt was developed. But the bio-control mechanism was still unclear. In this study, two exactly compounds in biological control of bacterial wilt pathogen R. solanacearum produced by B. amyloliquefaciens T-5 were purified, and a mimetic condition was established to study the competition between the bio-control agent T-5 and the pathogen QL-RFP.The results obtained were as follows:1. Carbon sources are one of the basic nutrition for bactierial growth, which is the key factor of bacterial competition in rhizosphere. We conducted 48 kinds of carbon sources to modulate tomato root exudates, investigating utilization of tomato root exudates by B. amylolyquefaciens T-5 and the pathogen QL-RFP. The results showed that both T-5 and QL-RFP were able to use some of carbon sources in tomato root exudates. T-5 and QL-RFP could use 19 and 23 carbon sources seperately. In addition,13 kinds of them could be utilized by both strains. The niche overlap index (NOI) between T-5 and QL-RFP is 0.56, demonstrating that nutritional competition exhibits between the bio-control agent T-5 and the pathogen QL-RFP. Additionally, growth of QL-RFP growed faster than T-5 in sole carbon sources condition, which means the rate of QL-RFP using carbon sources is rapidly than T-5, indicating that inhibition of T-5 against the pathogen is independent with nutritional competition.2. Bio-control agent T-5 could suppress growth of QL-RFP obviously under different carbon sources cinditions. With the increase of carbon source diversity, the antagonist ability against QL-RFP became stronger. Inhibition rate of T-5 against QL-RFP was 36.2% when co-cultured under sole carbon source at 23 h. Specifically, inhibition rates were 61.6% and 64.0% when cultured with L-proline and L-alanine respectively. Inhibition rate reached to 76.1% and 89.2% under 9 and 18 kinds of carbon sources conditions.3. Two kinds of compunds which suppress the pathogen were obtained from the fermentaion of T-5, using column chromatography and High Performance Liquid Chromatography (HPLC) with plate confrontation. After identified by HPLC-ES-MS these two compunds were determined as 7-O-malonyl macrolactin A (488 Da) and bacillaene (582 Da), which were belonged to polyketides and obviously influenced by carbon sources. The production of macrolactin A and bacillaene increased when the culture medium containing some of the amino acids, such as L-alanine, L-arginine, citrulline and L-proline, among which L-proline has the most positive impact on production of two antagonisitc compunds. Production of macrolactin A and bacillaene increased by 35.7 nad 11.2 fold than control, respectively. By adding L-Asparagine, production of macrolactin A significantly improved (4.7 fold increased compared with control), while no impact on Bacillaene.4. QL-RFP could be suppressed by T-5 significantly, while T-5 had no previliant on nutritional competition when co-cultured, indicating that there was interference competition between T-5 and QL-RFP, that was to say, T-5 produced antagonisitc compounds to inhibit the growth of Ralstonia by utilizing carbon sources in root exudates. With the increase of carbon sources, antagonistic ability of T-5 improved and became stable at last. After cultured 48 h, mean antagonistic ability of T-5 got to 36.1% under sole carbon source,57.5% under 9 carbon sources, and 57.1% under 18 carbon sources. Additionally, we found that production of T-5 antagonistic compunds and antagonistic gene expression increased with carbon source becoming more. Under 9 and 18 carbon sources condition, production of macrolactin A reached to 41.6% and 24.4%, Bacillaene reached to 92.9% and 145.7%. Expression of macrolactin A gene got to 21.1% and 24.1%, and of Bacillaene got to 18.2% and 18.6%, compared with sole carbon source condition.