Characterization of new bacterial biocontrol agents Acinetobacter, Bacillus, Pantoea and Pseudomonas spp. mediating grapevine resistance against Botrytis cinerea
Introduction
Grapevine (Vitis vinifera L.) is highly vulnerable to several fungal diseases, among them gray mold caused by Botrytis cinerea Pers.; Fr., which leads to serious damage in French vineyards, particularly in the regions where the climate is cool and humid. This fungus infects flowers, setting fruits, mature fruits, and leaves. Currently, gray mold is controlled before harvest by preventive fungicides. However, because of the increasing worldwide concern about pesticide use due to environmental problems and pathogens developing resistance, alternative plant protection strategies are becoming increasingly attractive. This has promoted the consideration of biological disease control and induction of plant resistance strategies by using either non-pathogenic plant-associated microorganisms (Van Loon et al., 1998, Bargabus et al., 2003, Tjamos et al., 2005) or components derived from microorganisms and plants (Aziz et al., 2003, Nürnberger et al., 2004).
A great number of reports indicated that certain bacterial strains are beneficial for the growth of plants; these are called plant growth-promoting rhizobacteria (PGPR). Colonization of roots with PGPR can also induce resistance in parts of the plant that are spatially separated from the inducing microorganism (Maurhofer et al., 1994, Van Loon et al., 1998). An important trait of these bacteria is their ability to maintain a stable relationship with the associated plant species (Smith and Goodman, 1999, Miethling et al., 2000). Consequently, the plant material can have a significant influence on the composition of the microflora obtained, as well as on the probability of finding isolates with biocontrol activities. Microorganisms isolated from the rhizosphere or from tissues of a specific plant are non-exotic, thereby presenting no risk of proliferation of a new microorganism in the environment. Furthermore, they may be better adapted to that plant and therefore provide better control of diseases than organisms originally isolated from other plant species (Handelsman and Stabb, 1996).
Biocontrol bacteria may protect plants against pathogens by direct antagonistic interactions between the biocontrol agent and the pathogen, as well as by induction of the host resistance. The biocontrol depends on a wide variety of traits, such as the production by the biocontrol strain of various antibiotic compounds, iron chelators and exoenzymes such as proteases, lipases, chitinases, and glucanases (Leong, 1986, Maurhofer et al., 1994, Chin-A-Woeng et al., 1998, Dunlap et al., 1998, Raaijmakers and Weller, 1998, Trejo-Estrada et al., 1998); as well as competitive root colonization (Chin-A-Woeng et al., 2000, Lugtenberg et al., 2001), and induced resistance in the host plant (Baker et al., 1985).
In recent years, considerable attention has been focused on induced resistance as an important phenomenon that occurs when plants develop enhanced defensive capacity upon appropriate elicitation. Induced defense reactions can be restricted to the tissues close to the site of elicitation or can be expressed systemically throughout the tissue or the whole plant. Bacteria-induced systemic resistance (ISR) has been demonstrated in a variety of plant species against a broad spectrum of pathogens (Hammerschmidt and Kuc, 1995, Van Loon et al., 1998, Magnin-Robert et al., 2007). In some cases, ISR is associated with the expression of some defense genes such as those encoding for pathogenesis-related (PR) proteins (e.g. chitinase) and also phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX) pathways (Maurhofer et al., 1994, Van Loon and Van Strien, 1999). LOX is required for the synthesis of the precursors of jasmonates, compounds that may act as the signal factor in plant defense responses (Creelman and Mullet, 1997, Pieterse et al., 1998). PAL is a key enzyme concerned with the synthesis of salicylic acid and phenolic compounds which were proposed to reduce incidence of plant disease through antifungal activity and stimulation of plant defense responses (Lee et al., 1995, Reymond and Farmer, 1998, Shadle et al., 2003). The relative importance of all these mechanisms differs considerably among strains of biocontrol bacteria (Neiendam-Nielson et al., 1998, Van Loon et al., 1998).
In grapevine, much of research reported on the use of the fungi Trichoderma spp. and Gliocladium spp. to control gray mold (Elmer and Reglinski, 2006). Nevertheless, a possible control of this disease by a Burkholderia sp. originally isolated from onion has been reported and attributed to a systemic spread of the bacterium into the aerial parts of the plant (Compant et al., 2005). Recently, a commercial biofungicide Serenade, which contains a Bacillus subtilis strain (QST 713), was reported to be effective against various pathogenic fungi (http://www.agraquest.com).
Our goals were to: (1) screen, identify, and characterize non-pathogenic bacteria isolated from the rhizosphere and tissues of healthy grapevine plants for their effectiveness to control gray mold on grapevine leaves caused by a highly virulent B. cinerea isolate and (2) quantify elicitation of some defense-related responses in grapevine leaves by selected bacterial strains.
Section snippets
Isolation of bacterial strains
Bacteria were isolated during the growing season from the rhizosphere, roots, leaves and stems of healthy grapevine plants (V. vinifera L., cv Chardonnay) from a vineyard located in the Champagne area (Marne, France). Leaves, root and stem sections were surface disinfected (20 s with 70% ethanol, and then for 10 min in a 2% sodium hypochlorite solution for root and stem sections or for 20 s in a 2% sodium hypochlorite solution for leaves) and severely washed with sterile aqueous NaCl (0.85%). Each
Isolation and screening of bacterial strains
To select populations of potential biocontrol bacteria associated with grapevine plants, rhizospheric soil and tissues of healthy plants were collected and bacteria were counted. Significant differences in population sizes were observed between rhizospheric and endophytic communities. A majority of the microorganisms (1 × 105 to 2.2 × 107 CFU/g soil) was obtained from rhizospheric soil, while 8.5 × 104 CFU/g were recovered from roots, 2.0 × 104 CFU/g from stems, and 1 × 102 CFU/g from leaves. Based on
Discussion
In this study, 282 bacterial strains have been isolated from the rhizosphere and tissues of healthy grapevines located in an area naturally affected by B. cinerea. Twenty-six isolates with high biocontrol activity against B. cinerea were selected by using detached leaves from in vitro-grown plantlets. Sequencing of 16S rRNA provided consistent results in identifying a set of seven bacterial strains. They have emerged as distinct strains belonging to P. fluorescens (designed PTA-268 and
Acknowledgements
This work was supported by a grant from Europol’Agro (Reims, France) through the Vineal program.
We are grateful to Pr. P. Grimont and Dr. A. Leflèche (CIMB, Institut Pasteur, Paris) for their help with molecular identification of bacteria and valuable discussion. Thanks are also due to Pr. C. Clément for allowing PTA to use some of his laboratory facilities and Dr. S. Gogniès for her help with bacterial samplings.
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