In vitro Bioassay Of The Antagonistic Activity Of Some Bacteria Isolated From Compost Extracts

Received: 14 March, 2014 Accepted: 2 June, 2014 Published: 20 June, 2014 In the present study, nine kinds of compost extracts were tested primarily for their activity against the causal agent of crow gall Agrobacterium tumefasiens (strain C58). The second step consisted on isolating bacteria, from the most efficient extracts. For this, twenty-seven bacteria were isolated and investigated in vitro with the objective of selecting efficient antagonists against this disease. The bacterial activity is compared to the reference antagonist Agrobacterium rhizogenes K84 by the double layer method. In vitro analyzing the antagonistic activity revealed that, after incubation at 27°C with the pathogen, antagonists tested exhibited considerable inhibitory activity in vitro and reduced the development of the strain C58 of Agrobacterium tumefasiens with variable degrees. Statistical analyses revealed four groups of antagonistic isolates. The first group contains the isolates that didn’t induce inhibition zone; the second group is composed by isolates showing no significant activity compared to the control, whereas the third group is composed by the less efficient isolates and the final group is composed by the most efficient isolates. The highest level of inhibition zone diameter was observed with C5B2 (30.25 mm) against 19.37 mm in the control. Reduction of pathogen growth reached 38% compared to control. Compost extracts isolates tested in this study may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for crown gall disease management. © 2014 PSCI Publisher All rights reserved. Key Word: compost extracts, antagonists, Agrobacterium rhizogenes K84, Agrobacterium tumefaciens, inhibition, control. Introduction Crown gall caused by Agrobacterium tumefaciens is considered as an economically important bacterial disease. Over the world, it affects dicotyledonous plants from almost 100 different families (Belaskri, 2006; Matthysse, 2006), including stone fruits, grapevines, roses, some ornamental species, forest trees and tomato (Pulawska, 2001). Infected plants, especially those with tumors on the main roots and collar, are unfit for marketing and must be disposed of (Pulawska, 2010). In Tunisia, the crow gall has frequently been observed on bitter almond (Rhouma et al., 2005). The disease has spread rapidly with the expansion of fruit tree cultivation and the establishment of new nurseries without adequate phytosanitary standards. Tunisian farmers are now facing problems in raising healthy stone fruit plants in nurseries, due to the lack of information about this disease and difficulties in identifying diseased stocks at an early stage. In spite of the preventive measures, that are being taken, crown gall continues to cause important damage in nurseries and in the field (Rhouma et al., 2008). Biological control has been successfully applied using the non pathogenic strain Agrobacterium rhizogenes K84 (Ryder et al., 1991; Gupta et al., 2010) for almost three decades. It was the first example of biocontrol against pathogenic strains of Agrobacterium in different hosts and countries all over the world (Moore and Canfield, 1996; Rhouma et al., 2004). Nevertheless, the use of K84 has certain problems (Moore and Canfield, 1996). The failure of this strain is mainly due to transfer of genes controlling agrocin 84 production and so to the development of resistance to K84. Therefore, search for others antagonistic microorganisms with high activity for managing the crown gall, is necessary. Composts and compost extracts have been reported to control plant diseases caused by pathogens such as fungi (Hoitink and Fahy, 1986; Kerkeni et al., 2007a; KhanfirBenJenana et al., 2009), nematodes (Kerkeni et al., 2007b), bacteria (Al-Dahmani et al., 2003) and virus (Wahyuni et al., 2010). Inhibition induced by composts and extracts resulted from a combination of chemical and biological mechanisms. Biological factors included especially microflora (fungi and bacterial species) contained in these products (Nelson and Boehm, 2002). In fact, the effectiveness of microorganisms isolated from Sci. Agri. 6 (3), 2014: 120-125 121 composts and compost extracts against different pathogens was confirmed in several studies (Naidu et al., 2010). Bacteria of the genus Bacillus, Pseudomonas and Serratia and filamentous fungi of the genus Trichoderma were the most isolated and were known as biocontrol agents (Hoitink et al., 1991; Hoitink et al., 1997; Zhang et al., 1998; Quarles, 2001; Ingham, 2002; Camozzi, 2003). Antifungal activity of microorganisms isolated from compost and compost extracts was widely investigated but studies about their antibacterial activity are fewer. The aim of this study was to evaluate, in vitro, the antibacterial activity of some composts extracts and then the individually effect of some bacteria isolated from these compost extracts against Agrobacterium tumefaciens strain C58, and to compare their activity to the reference antagonist Agrobacterium rhizogenes K84. Material And Methods Compost extracts Nine extracts prepared from different composts (C1, C2, C3, C4, C5, C6, C7, C8 and C9) and primarily composed of different animal manures (poultry, sheep, cattle and horse manures) were used (Table 1). Original composts were produced in the composting unit of the Technical Center of Organic Agriculture of Chott-Mariem (Tunisia), according to an aerobic process. Extract-production consists on suspending composts in tap water (1:5, v/v) in 20-liter plastic container and stirring the mixture daily for about 10 min during an extraction period of 5 days (Weltzien, 1992). After the incubation period, the mixtures were filtered through cheesecloth (250 μm) and the obtained extracts were stored at 4°C. They were taken out 30 min before use. Table 1. Composition of composts used for extracts preparation