Biocontrol Mechanisms Of Agrobacterium Vitis Strain E26 On Grapevine Crown Gall Disease

Crown gall of grapevine, caused by pathogenic strains of Agrobacterium vitis, has been a problem with economical losses for extensive damage to grape health and quality all over the word. Since chemical pesticides are seldom effective and not environmentally friendly, biological control is receiving increased attention as a useful sustainable alternative. Nonpathogenic A. vitis strain E26 was naturally suppressive to crown gall bacteria both in vitro and in vivo tests, and was suggested to be a potential biocontrol agent of grapevine crown gall disease. The overall goal of this study was to determine mechanisms involved in the biological control process by strain E26.The effect of strain E26 dosage and applying time on suppression of tumor formation by a virulent strain of K308 on sunflower (Helianihus annus) was investigated in greenhouse. The suppression of tumor formation was more effective when E26 was inoculated either prior to strain K308 or at a higher concentration. In field tests conducted at two independent regions, E26 similarly exhibited strong effect of reducing the tumor index on grape (Vitis vinifera cv Muscat humburg) when co- inoculated with K308 at the same concentration. The controlling index was over 70% as compared with the check treatment.The dynamics of strain E26 were studied in grape root surface and rhizosphere under field conditions, using green fluorescence protein gene (gfp)-marked strains. Root colonization and survival in the rhizosphere of strain E26 was observed. Average populations of 104 cfu/g (fresh weight) of root and 104 cfu/g (dry weight) of soil of E26 were found 5 months after planting. The attachment and interaction of strain K308 and E26 directly on grapevine wound surfaces were also examined in vitro. E26 and K308 attached at a similar level to both stem and root explants. The attachment of strain K308 to grape cells was blocked by E26 in both systems. Scanning electronic microscope showed that strain E26 could attach to grape root wounded cells with the same manner as that of strain K308. Strain E26 and K308 were shown to grow competitively in grape stem wound exudates. There was also similarity in utilizing carbon and nitrogen nutrients reported to present in grape tissues in vitro between strain E26 and K308. These results indicated that E26 was an efficient colonizer of wound sites and suggested to exclude A.vitis from site essential for infection by competition for space and nutrients.Strain E26 was found to produce an agrocin that inhibited the growth of strain K308 on culture plate. This agrocin was isolated and purified from culture broth. Preliminary chemical analyses indicated that agrocin E26 was a nucleotide analogue. Agrocin E26 showed a broad host range of activity on tumorogenic bacteria. Mode of action of agrocin E26 was determined using strain K308.lt was shown to inhibit DNA and RNA syntheses.To determine the role of agrocin E26 in the control of crown gall, two Tn5- induced mutants, ME005 and ME084, that had lost their ability to inhibit A. vitis on plates were compared with the wild-type strain for their ability to control crown gall in greenhouse and fields. Although the two mutants grew both in culture medium and in grape stem explants at a rate similar to that of the wild-type strain and were active in suppressing crown gall, neither of them controlled crown gall as effective as strain E26 did. The data implied that agrocin production is an important factor of the biological control of grape crown gall by strain E26, but that another mechanism(s) is involved.The results obtained suggested that the biological control of grape crown gall by strain E26 is a complex process with a number of factors playing roles involved in. Agrocin production, efficient colonization on roots and wound sites, and competition for sites and nutrients are all one of these factors. The relative contribution of individual factors in the biocontrol situation is unknown at this time.