The flavonoid naringenin stimulates the intercellular colonization of wheat roots by Azorhizobium caulinodans

Abstract

We have studied intercellular colonization of wheat roots by Azorhizobium caulinodans and other diazotrophic bacteria, using strains marked with the lacZ reporter gene to facilitate their detection and identification. A. caulinodans was observed by light and electron microscopy to enter the roots of wheat at high frequency at the points of emergence of lateral roots (lateral root cracks). After lateral root crack colonization, bacteria moved into intercellular spaces within the cortical cell layer of roots. The flavonoid naringenin at 10 and 100 mmol m -3 significantly stimulated root colonization. The roles of the structural nodABC genes and the regulatory nodD gene were also studied; lateral root crack colonization of wheat was shown to be Nod factor-and NodD-independent. Similar frequencies of lateral root crack colonization were observed following inoculation of wheat with Azospirillum brasilense. Colonization by A. brasilense was stimulated by naringenin and also by other flavonoid molecules.Key-words: Azorhizobium caulinodans ORS571; Azospirillum brasilense; crack entry; endophytic nitrogen fixation; intercellular colonization; lateral root cracks; naringenin; nonlegume; reporter genes; rhizobia. INTRODUCTIONBiological nitrogen fixation (BNF) is the microbiological process which converts atmospheric dinitrogen gas into a plant-usable form. An important requirement for efficient BNF is to have diazotrophic bacteria growing endophytically within plants, as in the legume-rhizobia, Parasponia-Bradyrhizobium and actinorhizal-Frankia symbioses (Van Rhijn & Vanderleyden 1995;Webster, Davey & Cocking 1995a;Benson & Silvester 1993). In these associations, specialized organs, called nodules, are formed in which bacteria reduce nitrogen into ammonia and this fixed nitrogen is transferred to the plant. One of the approaches taken to attempt to achieve BNF in nonlegume crops is to determine whether certain naturally occurring rhizobial strains, which have the ability to enter legume plants by crack entry, are able to internally colonize the roots of non-legumes (Cocking et al. 1992(Cocking et al. , 1994a(Cocking et al. ,b, 1995Spencer et al. 1994). Crack entry infection involves rhizobia entering host plants intercellularly between adjacent cells and not by the formation of infection threads at the tips of root hairs. Among the rhizobia used, Azorhizobium caulinodans, which forms nodules on the stems and roots of the tropical legume Sesbania rostrata, is especially interesting. A. caulinodans forms nodules after crack entry invasion (Tsien, Dreyfus & Schmidt 1983;Ndoye et al. 1994) and is able to fix nitrogen in the freeliving state without differentiation into bacteroids, in up to 30 mmol O 2 mol -1 (Kitts & Ludwig 1994). Here, we have investigated the ability of A. caulinodans strain ORS571 to colonize internally wheat roots, using bacteria marked with a lacZ reporter gene. We describe an experimental system in which colonization has been quantified and the effect studied of a range of flavonoids on the frequency o...

Effects of Glucosinolates and Flavonoids on Colonization of the Roots of Brassica napus by Azorhizobium caulinodans ORS571

“…Apparently, some other mechanism mediates the naringenin colonization effect. This corroborates the observation that naringenin promotes colonization in wheat (35), which lacks glucosinolates. Previous studies on interactions between azorhizobia and the crucifer A. thaliana, and with wheat and rice, have shown that naringenin does not simply act as a carbon source for stimulation of colonization, since succinate (a carbon source favored by A. caulinodans) does not promote colonization (12,34).…”

“…It may also have potential as a screening assay for antibacterial phytocompounds. Notably, the present experiments have shown that naringenin-induced enhancement of colonization by A. caulinodans, previously demonstrated in cereals (34,35), still occurs in HGS oilseed rape, despite the susceptibility of azorhizobia to glucosinolate products. This shows the considerable potential of flavonoids to enhance potentially beneficial interactions between bacteria and crop plants, even when the latter exhibit some resistance to colonization.…”

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island

“…Naringenin is widespread in legumes and nonlegumes (28), which probably explains why transfer is enhanced in the rhizosphere of several nonhost plants, and largely involved in communication with plant-associated bacteria. In addition to its crucial role in the nodulation process, it was shown to stimulate wheat and Arabidopsis thaliana colonization by A. caulinodans (29,30). It was previously reported that opines produced by crown gall tumors initiated on plants by the pathogen Agrobacterium tumefaciens are required for Ti plasmid conjugal transfer among agrobacteria (31) and that root exudates enhance bacterial conjugal gene transfer in the rhizosphere (32,33).…”

Phenotypic Variations of Endophytic Bacteria Associated with Carob Tree (Ceratonia Siliqua L.)

“…The faculty to infect and colonize roots has been considered the major factor that determine inoculum efficacy both for crop yield enhancement and for disease control [15]. Root colonization by endophytic bacteria occurs preferentially through the natural fissures resulting from the emergence of lateral root like lateral root cracks (LRCs) or on the root surface and root tips [16]. The flavonoids (naringenin and daizein) were found to stimulate significantly the LRC colonization of Wheat and Arabidopsis thaliana by Azorhizobium caulinodans ORS571, by Azospirillum and by Herbaspirillum [16,17].…”

“…Root colonization by endophytic bacteria occurs preferentially through the natural fissures resulting from the emergence of lateral root like lateral root cracks (LRCs) or on the root surface and root tips [16]. The flavonoids (naringenin and daizein) were found to stimulate significantly the LRC colonization of Wheat and Arabidopsis thaliana by Azorhizobium caulinodans ORS571, by Azospirillum and by Herbaspirillum [16,17]. Inside the plant tissues, bacteria form a large intercellular infection pockets in which the endophyte divide, develop infection threads and interchange with host [18].…”