Antimicrobial Activities of Cover Crops against Rhizobium vitis, the Grapevine Crown Gall Pathogen

Abstract

The aim of this research was to evaluate the antimicrobial activity of chinese milk vetch (Astragalus sinicus), forage rape (Brassica napus), orchard grass (Dactylis glomerata), ryegrass (Lolium perenne), italian ryegrass (Lolium multiflorum) and hairy vetch (Vicia villosa), and then to isolate and characterize the bioactive compounds from the most active cover crop species. And also to enhance existing and induced defense responses in “Campbell Early’ and ‘Kyoho’ grapevine using tested cover crops extracts. Crude extracts from the shoot and root tissue of chinese milk vetch (Astragalus sinicus), forage rape (Brassica napus), orchard grass (Dactylis glomerata), ryegrass (Lolium perenne), italian ryegrass (Lolium multiflorum) and hairy vetch (Vicia villosa) were investigated for their antimicrobial activity against Rhizobium vitis, Bacillus subtilis, Aspergillus niger, Botrytis cinerea, Colletotrichum gloeosporioides, C. acutatum, and Elsinoe ampelina. Extracts were prepared from dried and powdered shoots and roots using common solvents (methanol, ethyl acetate, and water) with different degrees of polarity. The diameter of inhibition zones of the shoot (2,000 pg/disc) and root (2,500 ug/disc) extracts in water and ethyl acetate solvents against R. vitis and B. subtilis were found to be in the range of 9.8 1.3 to 18.0 + 2.0 mm and 9.0 + 0.7 to 16.4 + 0.5 mm, respectively. However, methanolic extract did not show any inhibitory activity. All the extracts against A. niger showed a minute inhibition zone which was not measurable, whereas the other tested fungi were not inhibited by the extracts. The minimum inhibitory concentration values of shoot and root extracts against the tested bacteria were found to be in the range of 3.12 to 12.5 and 3.12 to 25 mgmL”, respectively. A preliminary phytochemical screening of all the tested extracts indicated the presence of alkaloids, flavonoids, saponins, cardiac glycosides, steroids and terpenoids. Total polyphenol contents of extracts positively correlated with their inhibitory activities, suggesting their potential antibacterial efficacy. Three antibacterial compounds isolated from hairy vetch (Vicia villosa Roth) shoots against grapevine crown gall pathogen. The hairy vetch shoot powder was sequentially extracted and screened for antibacterial activity by the disc diffusion method. Ethyl acetate extract from the hairy vetch shoots was subjected to column chromatography on silica gel. The structures of the compounds were then elucidated by spectroscopic analysis and chromatographic assay. Three compounds were isolated and identified as bis (2-ethylhexyl) phthalate (compound 1), diethyl phthalate (compound 2), and p-hydroxybenzoic acid (compound 3). The compounds showed potential antibacterial effect as diameters of inhibition zones (7.3 + 0.3 to 11.2 + 0.4 mm) against Rhizobium vitis and Bacillus subtilis. The minimum inhibitory and minimum bactericidal concentrations of these compounds against the target bacteria were found to range from 62.5 to 125 and 125 to 250 pg-mL”", respectively. The compounds also showed bactericidal activity based on the viable counts of the tested strain. These findings indicate that isolated compounds could be used as possible candidates for control of crown gall disease in grapevines caused by R. vitis. Water based crude extracts from hairy vetch (Vicia villosa) and ryegrass (Lolium perenne) were investigated for their antimicrobial activity against Rhizobium vitis, the causal agent of crown gall of grapevine (Vitis spp.) grown in the greenhouse. One hundred ml of each extract (500, 1,000 and 2,000 g/4 L) from hairy vetch and ryegrass prepared from fresh and pounded shoots and roots were applied by water irrigation. The shoot and root extracts displayed remarkable in vivo antibacterial effects on reduction of gall formation by R. vitis by up to 24.97 to 75.02% in ‘Kyoho’ (susceptible) and 100% in ‘Campbell Early’(moderately resistant to crown gall) grapevines compared with the untreated control. The expression of several defense-related genes was investigated by quantitative real-time and semi-quantitative RT-PCR in two grapevine cultivars. The treatment of an extract from hairy vetch and ryegrass showed the induction of defense response-related genes in both cultivars of grapevines. Treatment of tested plant extracts exhibited differential induction of the accumulation of defense related genes including B-1,3-glucanase (PR-2), phenylalanine ammonia-lyase (PAL), thaumatin-like protein (ZZP), leucine reach-repeat (LRR), polygalacturonase inhibiting protein (PG/P), stilbene synthase (StSy) and catalase (CAT). Expression analysis of defense related genes revealed noticeable differences between ‘Kyoho’ and ‘Campbell Early’ grapevines. Based on the reduction of crown gall and induction of defense-related genes by hairy vetch and ryegrass extracts, extracts from tested cover crops can act as efficient biological inducers in an alternative strategy of grapevine crown gall protection. Stilbenes are natural compounds occurring in a number of plant families, including Vitis vinifera L., which is the most important species grown worldwide for grape and wine x1 production. Extract of hairy vetch and ryegrass showed induction of stilbene synthase gene in the grapevine leaves in previous study. The present study investigated the change of stilbene compounds (trans-and cis- resveratrol and piceides, and piceatannol) and induction of gene expression related with their synthesis by treatment of extracts from hairy vetch and ryegrass in the berries of ‘Campbell Early’ and ‘Kyoho’ grapes. Trans-resveratrol was present in substantial amounts (4.1 to 5.3 g/g FW) in ‘Campbell Early’ and (7.6 to 10.4 g/g FW) in ‘Kyoho’, whereas trace amounts of cis- resveratrol were detected in the range of 0.8 to 1.0 ug/g FW in treated grape berries. Besides, the concentration of piceid and piceatannol in berries of both cultivars was markedly enhanced in response to treatment of hairy vetch and ryegrass extracts. Accumulation of stilbene compounds in grape berries increased in response to tested extract, 24 h after treatment. Berries treated with extracts showed the increase in transcript levels of genes coding enzymes involved in stilbene compound biosynthesis (phenylalanine ammonia lyase, chalcone synthase and stilbene synthase). Changes in stilbene compounds content were correlated with levels of StSy mRNA, indicating a transcriptional control of stilbene synthase activity. The results suggest that stilbene compounds were accumulated by hairy vetch/ryegrass extracts through the induction of StSy transcription in grape berries The results of our study demonstrate that the secondary metabolites (crude extract and purified bioactive molecules) derived from tested cover crop species possessed a broad spectrum of biological activities, and act as an efficient biological induces in an alternative strategy of grapevine crown gall protection.