INTRODUCTION

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Rhizobia of the strain Azorhizobium caulinodans ORS571 are nitrogen-fixing (diazotrophic) bacteria that initiate and invade stem and root nodules on their legume host Sesbania rostrata (22, 33). Azorhizobia initially invade Sesbania roots by entering lateral root cracks (LRCs), the natural epidermal fissures which form around emergent lateral roots (22), whereas most legumes are invaded by rhizobia through a highly specialized root hair infection process. Entry into the root system by ORS571 is apparently not dependent on rhizobial Nod (nodulation) factors (signal molecules inducing nodule formation), since an ORS571 Nod factor-deficient mutant retained the ability to enter LRCs of Sesbania (23). In addition, Nod⁺ and Nod forms of ORS571 (and other rhizobia) colonize LRCs in several nonlegumes (13, 25, 34), indicating a degree of independence from the legume host. Reporter gene assays developed for the quantitative assessment of LRC colonization in nonlegumes have been employed to demonstrate that exogenous flavonoids significantly enhance colonization by ORS571 (12, 34). Accordingly, A. caulinodans has become an established tool in studies of colonization of nonlegumes.

Oilseed rape, a member of the family Brassicaceae (Cruciferae), is of considerable agronomic importance (30). In the United Kingdom, winter-sown oilseed rape is the third most extensively cultivated crop after wheat and barley (21). Some crucifers contain glucosinolates and erucic acid in their seed (28), which may pose health risks in human food or animal feed (30). Consequently, oilseed rape varieties have been developed to produce seed low in these two compounds in order to conform to European Union regulations which reduced the permissible concentrations in seed intended for nutritional use (21). Products of the hydrolysis of glucosinolates are bioactive, being strongly bactericidal (5). However, azorhizobia are able to colonize LRCs in the crucifer Arabidopsis thaliana (13), despite the presence of glucosinolates in this plant (6). Apparently, some nonrhizobial bacteria dwell as endophytes in certain varieties of Brassica napus growing under field conditions, possibly entering host plants after colonization of the rhizosphere (11).

Certain sugarcane varieties benefit from their association with Acetobacter diazotrophicus, which dwells as an obligate endophyte within those plants (3), implying that endophytic colonization is an important property in such nitrogen-fixing, beneficial interactions. Endophytic Acetobacter organisms are probably carried over into sugarcane plants during vegetative propagation and do not readily colonize plants following root inoculation (3). However, root colonization is likely to be an important factor in potentially beneficial interactions between nonlegume crops and soil-dwelling bacteria. Consequently, studies on colonization of the roots of plants by diazotrophs make important contributions to the aim of extending the benefits of biological nitrogen fixation to nonlegumes. This is especially true with respect to crucifers, since glucosinolates in those plants may inhibit or prevent bacterial colonization. The present study examined whether A. caulinodans is able to colonize oilseed rape, assessing the effect of in planta glucosinolates and erucic acid by utilizing plants grown from seeds differing in their concentrations of those two compounds; this study also assessed whether exogenous flavonoids affect the colonization of B. napus. This is apparently the first published study utilizing a quantitative in situ colonization bioassay together with chemical analyses to examine the effects of glucosinolates in roots on bacterial colonization of oilseed rape.

	MATERIALS AND METHODS

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Plant germination and culture. Seeds of varieties of B. napus differed in their erucic acid and glucosinolate contents. Each variety of seed contained either low concentrations of erucic acid and glucosinolates, high concentrations of both compounds, or high concentrations of one compound and low concentrations of the other (Table 1). Seeds were surface sterilized in 10% (vol/vol) Domestos bleach (Lever Industrial Ltd., Runcorn, United Kingdom) for 10 min, rinsed in sterile water, and then germinated aseptically on 0.8% (wt/vol) water agar (Sigma Chemical Co., Poole, United Kingdom) for 1 day in the dark at 28°C. Germinated seeds were transferred aseptically to sterile tubes (25 by 150 mm, 60-ml capacity), each containing 20 ml of nitrogen-free medium (8) semisolidified with 0.8% (wt/vol) agar. Some tubes contained a 50 µM concentration of one of four flavonoids. Seedlings were grown under Daylight fluorescent tubes (37 microeinsteins of illuminance m² s¹) in a growth room (25°C day, 22°C night) with a 16-h photoperiod. After 1 day, seedlings were inoculated with 0.2 ml of a suspension of azorhizobia (carrying a lacZ reporter gene) in sterile water at a density of approximately 10⁸ bacteria ml¹. Plants were removed from tubes after 2 weeks, and the number of LRCs colonized by azorhizobia was quantified using a lacZ -galactosidase assay.

Flavonoids. Chrysin (flavone), naringenin (flavanone), quercetin (flavonol) (all from Sigma) and the isoflavone daidzein (Apin, Abingdon, United Kingdom) (Fig. 1) were dissolved individually at the highest possible concentration in water (adjusted with sodium hydroxide to approximate pH 9.5) and filter sterilized immediately prior to use. Flavonoids from these stock solutions were added aseptically to tubes (at 50 µM) containing cooled (40°C) Fåhraeus agar medium (final pH = 6.6).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Structural formulae of flavonoids used in this study.

Culture of azorhizobia. A. caulinodans ORS571(pXLGD4), supplied by J. Dénarié (Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique, Castanet-Tolosan, France), contained a constitutively expressed lacZ reporter gene and was cultured on TGYE medium (16) semisolidified with 0.8% (wt/vol) agar, with 10 µg of tetracycline ml¹. ORS571(pXLGD4) bacteria nodulated their host plant, Sesbania rostrata, and fixed nitrogen as assessed by the acetylene reduction assay (data not shown).

lacZ -Galactosidase assay. Insertion of the lacZ gene confers -galactosidase activity on recipient bacteria. Plants inoculated with azorhizobia containing lacZ were removed from tubes and excess agar removed from their roots before intact root systems were excised. Root systems were treated with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) as described previously (4), except that roots were fixed in 3% (vol/vol) glutaraldehyde for 3 h at atmospheric pressure, with sodium cacodylate being used at 0.12 M in all procedures. Colonies of azorhizobia in secondary LRCs became visible as a result of the dark blue precipitate formed by degradation of X-Gal after expression of azorhizobial -galactosidase. The extent of LRC colonization was quantified by calculating the mean percentage (per plant) of secondary lateral roots with blue LRC colonies (12).

Reisolation of bacteria from seeds and plants. Seeds and plants were surface sterilized by immersion for 10 min in 10% (vol/vol) Domestos adjusted to pH 8.0, rinsed, and macerated in 10 ml of sterile water. After serial dilution of the supernatant, bacteria were plated onto TGYE medium (16) semisolidified with 0.8% (wt/vol) agar, either with or without the addition of Congo red dye (nonrhizobial bacteria take up the dye [31]).

Sectioning and microscopy. Roots with extensive areas of blue LACZ precipitate, which denotes high bacterial density, were excised, fixed, and processed for light microscopy (7). A phenolic-magenta stain (K. J. O'Callaghan, unpublished work) was produced by dissolving 4 g of basic fuchsin chloride in 12 ml of phenol at 80°C with stirring, adding 25 ml of 95% (vol/vol) ethanol at 45°C, and increasing the volume to 300 ml with distilled water. After 1 month the solution was passed through a GF/C filter (Whatman, Maidstone, United Kingdom) and applied to sections on glass slides for 10 s at room temperature before rinsing. This stain did not bind blue LACZ precipitate, allowing visualization of plant cell walls (red) and blue-staining bacteria.

Glucosinolate determinations. Approximately 40 14-day-old seedlings from each treatment were removed from tubes. Their roots were removed, frozen immediately in liquid nitrogen, stored at 70°C, and freeze-dried for 4 days. The freeze-dried material was ground immediately, returned to the drying unit for 24 h, and removed for extraction. Intact glucosinolates were extracted from freeze-dried roots using boiling 80% (vol/vol) aqueous methanol and converted enzymatically to desulfo-glucosinolates by aryl-sulfatase (15). The concentrations of the individual glucosinolates were determined by high-pressure liquid chromatography (HPLC), utilizing reported experimental parameters and methods (17, 32). The four major glucosinolates (2-hydroxy-3-butenyl, 3-indolymethyl, 4-methoxy-3-indolylmethyl, and 1-methoxy-3-indolylmethyl) were identified by coelution with authenticated standards supplied by R. K. Heaney (Food Research Institute, Norwich, United Kingdom) and N. P. Botting (Department of Chemistry, University of St. Andrews, St. Andrews, United Kingdom). The minor constituent (4-methylthiobutyl glucosinolate), which accounted for less than 4% of the total glucosinolates, was identified tentatively on the basis of its relative retention time. Response factors for the glucosinolates were determined by the glucose release method (18). The results are expressed as micromolar units gram of freeze-dried matter¹.

Culture of azorhizobia with ITC and naringenin. Since isothiocyanates (ITCs) are probably the most bioactive glucosinolate products in the roots of B. napus and affect the growth and survival of bacteria (5), the effect of 2-phenylethyl-ITC on the growth of ORS571 was examined. Azorhizobia cultured on semisolidified TGYE medium (as described above) were suspended in water at approximately 10⁸ bacteria ml¹. A 100-µl aliquot from this suspension was added to each of 24 flasks separated equally into eight treatments, consisting of TY medium (31), TY with 50 µM naringenin, TY with 2-phenylethyl-ITC (1, 10, and 50 µM), and TY with both 2-phenylethyl-ITC (1, 10, and 50 µM) and 50 µM naringenin. Naringenin was dissolved as described previously for flavonoids; ITC was dissolved in methanol (an appropriate volume of methanol was added to flasks not receiving ITC). Cultures were incubated at 27°C for 48 h, and optical density at 600 nm (OD₆₀₀) was recorded. In other experiments, two equal volumes (one supplemented with 50 µM naringenin) from an aqueous suspension of azorhizobia (10⁸ cells ml¹) were incubated at 27°C for 30 min, serially diluted, and spread onto TGYE medium semisolidified with 0.1% (wt/vol) agar and also the same medium with 50 µM 2-phenylethyl-ITC.

	RESULTS

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Inoculation of oilseed rape plants with Azorhizobium. Tube-grown plants of oilseed rape inoculated with A. caulinodans ORS571(pXLGD4) and treated subsequently with X-Gal had distinct regions of blue precipitate in some LRCs, which were observed readily by light microscopy (Fig. 2A). Uninoculated plants did not have blue precipitate in LRCs. Sections of blue LRCs observed by light microscopy showed numerous azorhizobia (Fig. 2B). Surface-sterilized seed (n = approximately 30) of Askari ground in sterile water formed a slurry, which was spread on semisolidified TGYE medium. However, bacterial growth was not observed on the medium after 7 days' incubation at 28°C. Surface-sterilized and milled Askari plants inoculated previously with ORS571(pXLGD4) were also spread on TGYE medium. Bacteria identical to azorhizobia used for plant inoculation grew without uptake of Congo red dye.

View larger version (106K): [in this window] [in a new window]   FIG. 2.   Light micrographs showing A. caulinodans ORS571(pXLGD4) in LRCs of oilseed rape. During treatment with X-Gal, colonies of azorhizobia carrying the lacZ gene formed a dark blue, highly stable precipitate (A), the presence of bacteria being confirmed in sections (B) of plant tissues excised from those blue-staining regions. Bars = 500 µm (A) and 8.7 µm (B).

Colonization of oilseed rape varieties differing in seed glucosinolate content. Tube-grown, 14-day-old oilseed rape plants inoculated with A. caulinodans ORS571(pXLGD4) were treated with X-Gal, and the mean percentage of colonized LRCs was calculated. Colonization was poor in high-glucosinolate-seed (HGS) varieties but extensive in most low-glucosinolate-seed (LGS) varieties (Fig. 3). The LGS varieties Apex and Express exhibited a level of colonization greater than in HGS but less than in other LGS varieties. These same differences in colonization among varieties were observed in replicate experiments. Therefore, data appeared to be clustered into three sets, with the greatest colonization always observed in LGS plants (Fig. 3). Data showed normal distributions, and analysis of variance (ANOVA) between varieties (with percentage of colonization as the response variable) confirmed that means were not from the same population (Fig. 3). The number of secondary lateral roots and the number of blue LRCs were not correlated in any experiment (r [Pearson's correlation coefficient] of 0.1 to 0.3), showing that the size of the root system had little or no influence on colonization. Apparently, the concentration of erucic acid in the seed did not affect colonization, since varieties differing in erucic acid content were not colonized in any systematic manner (Fig. 3).

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Colonization by A. caulinodans ORS571(pXLGD4) of LRCs in 14-day-old tube-grown plants of 10 varieties of B. napus grown from seed designated as low (L) or high (H) in glucosinolates and erucic acid. In one to three replicate experiments for each variety, plants from HGS were always colonized less than those from LGS. The data revealed an overall pattern of colonization, with plants clustering into L and H glucosinolate groups and with erucic acid concentration apparently not affecting colonization. The LGS varieties Apex and Express were both colonized more or less than HGS or other LGS varieties, respectively. ANOVA revealed differences between variety means derived from the pooled data (P < 0.001).

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Colonization by A. caulinodans ORS571(pXLGD4) of LRCs in varieties of B. napus (as in Fig. 3), either without (white bars) or with (black bars) 50 µM naringenin. Naringenin promoted colonization in HGS varieties that usually showed minimal colonization.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Colonization by A. caulinodans ORS571(pXLGD4) of Apex and Zongyou. Flavonoids other than naringenin did not significantly stimulate colonization of LRCs. Apex (white bars), an LGS variety, was colonized more in the presence of naringenin (P = 0.008; n = 24). Among the LGS varieties, such significant increases occurred only in plants of Apex and Express (Fig. 3). The LRCs of Zongyou plants (striped bars) were colonized more (as were all other HGS varieties) in the presence of naringenin (P < 0.001; n = 24): bars show 1 standard error of the mean.

Effects of ITC and naringenin on growth of azorhizobia. 2-Phenylethyl-ITC at 50 µM had a marked effect on the absorbance (OD₆₀₀) of azorhizobial cultures (Table 2), suggesting that azorhizobia are susceptible to the antibacterial effects of ITCs. Although naringenin promoted colonization by azorhizobia in HGS varieties of oilseed rape, this flavonoid did not eliminate or retard the antibacterial effects of ITCs on azorhizobia in culture (Table 2). Also, azorhizobia incubated with naringenin for 30 min before serial dilutions of bacteria were spread on TGYE plates containing ITC showed 36 to 48 CFU plate¹ (n = 2 plates), a number similar to that formed on TGYE-ITC plates by azorhizobia which had not been exposed to the flavonoid (42 to 46 CFU; n = 3 plates). Colonies of flavonoid-treated and untreated azorhizobia were morphologically identical.

HPLC analysis of root glucosinolates. In previous tests using the -galactosidase bioassay, untreated plants of Askari, Express, and Alaska showed relatively low, medium, and high numbers of azorhizobial colonies, respectively (Fig. 3). Two independent HPLC studies were performed in which plants of these three varieties were assessed quantitatively for the presence of glucosinolates to obtain data on the possible link between colonization and glucosinolate concentration. One cannot assume that concentrations of glucosinolates in plant tissues will be correlated with those in the seed (R. Mithen [John Innes Centre, Norwich, United Kingdom], personal communication). In both HPLC studies, plant material was derived from four treatment groups, namely, untreated plants and those treated with naringenin, azorhizobia, or naringenin and azorhizobia. Five glucosinolates were detected in these studies (Fig. 6), the major constituent being 2-hydroxy-3-butenyl glucosinolate (progoitrin). A comparison of results from the two studies revealed that plants from corresponding treatments showed similar proportions of glucosinolates (data not shown), although overall concentrations of glucosinolates were lower in the second HPLC study. A trend was apparent in which the total concentration of glucosinolates was progressively higher among untreated plants of Alaska, Express, and Askari (Fig. 7). However, in each HPLC study, quantitatively similar relative increases in glucosinolates between the three varieties were also observed in plants treated only with naringenin (data not shown), suggesting that addition of this flavonoid did not eliminate the pattern of glucosinolate concentrations observed in untreated plants. Moreover, inoculated plants of Askari, which had shown highly significant increases in root colonization by ORS571(pXLGD4) after treatment with naringenin, contained similar levels of glucosinolates in the presence and absence of naringenin. This result, obtained in the first HPLC study, was confirmed in each of several replicate treatments (Fig. 8). A two-tailed t test of these data showed that the mean (n = 3 replicates) total glucosinolate levels in inoculated Askari plants (with or without naringenin) were not significantly different (P = 0.17). As in the data from all other treatments, there were no obvious major changes in the relative proportions of individual glucosinolates after naringenin treatment (Fig. 8). Therefore, in untreated plants, a pattern of glucosinolate concentrations between three oilseed rape varieties explained a correlated pattern of colonization by A. caulinodans (Fig. 3), although naringenin-stimulated increases in colonization (Fig. 4) were apparently not mediated by an induced reduction in glucosinolate concentrations.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Formulae and chemical names (trivial names in parentheses) of glucosinolates. These compounds form a class of organic anions exhibiting a sulfated thioglucose moiety (A) and are distinguished on the basis of side attachments (R); five different glucosinolates (B) were present in plant material from this study. ITCs are considered to be the most biologically important products of the degradation of intact glucosinolates. After hydrolytic cleavage of the glucose moiety (A) and production of HSO4, any of several products retaining the side chain (R-) may be formed, ITCs (R-NCS) being favored under certain conditions (14).

View larger version (21K): [in this window] [in a new window]   FIG. 7.   Concentrations of total glucosinolates in three varieties of oilseed rape, determined in the first of two HPLC studies performed several months apart. Although total concentrations were lower in the second study (data not shown), there was a consistent trend of increasingly higher concentrations among Alaska, Express, and Askari. FDM, freeze-dried matter.

View larger version (30K): [in this window] [in a new window]   FIG. 8.   HPLC-determined mean (n = 3 experiments) concentrations of individual and total glucosinolates in inoculated plants of the HGS variety Askari. Plants treated with naringenin did not contain lower concentrations of glucosinolates in root tissues. Symbols: horizontally striped bars, 2-hydroxy-3-butenyl; white bars, 4-methylthiobutyl; black bars, 3-indolylmethyl; diagonally striped bars, 4-methoxy-3-indolylmethyl; horizontally cross-hatched bars, 1-methoxy-3-indolylmethyl; diagonally cross-hatched bars, total glucosinolates. FDM, freeze-dried matter.

	DISCUSSION

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The majority of analytical phytochemical studies investigating glucosinolates focus on tissues from leaves and stems, which possibly are easier than roots to screen for microbial infection. For example, necrotic leaf lesions may be observed following inoculation (20). The present study has demonstrated that microbial invasion into the roots of B. napus may be studied quantitatively using a readily assayable reporter gene alongside chemical analysis of root glucosinolates. One of the dominant glucosinolates in the leaves and stem of B. napus is progoitrin (5), which may be especially prevalent in seedling tissues (10). The detection of relatively high concentrations of progoitrin in all samples assessed in this study indicates that this compound is also a major glucosinolate constituent of the roots of young plants of B. napus.

Results from this study suggest that the high or low glucosinolate content of the seed of some varieties of B. napus correlates positively with glucosinolate levels in the roots, at least during the early stages of in vitro plant development. A. caulinodans, genetically marked with a reporter gene and shown in this study to be susceptible to ITCs in vitro, colonized plants grown from LGS more effectively than those from HGS. Moreover, HGS roots consistently yielded higher levels of glucosinolates than LGS roots. Such an experimental system may work effectively only with young plants, since glucosinolate concentrations per unit of tissue in mature plants are sometimes markedly different from those in the seed (19, 20). Although glucosinolate concentrations for three oilseed varieties substantially differed in two HPLC studies performed at different times, both of these studies showed a consistent pattern in the relative proportions of glucosinolates, both between and within varieties. Therefore, if the differences observed between those HPLC studies resulted partly from the methods or conditions employed, such effects introduced no apparent bias into the data. The ages of plants assessed in these HPLC investigations were uniform, and the growth conditions for plants were rigorously controlled. However, it is important to note that variation in glucosinolate concentrations in tissues of plants of the same variety is frequently observed and is influenced by growth conditions (19, 29). The stage of plant maturation also influences tissue concentrations of glucosinolates (27), 10-fold differences sometimes being observed during the first few weeks of growth (17). Frequently, considerable variation is also encountered between tissues in the same plant (9).

Although naringenin reproducibly promotes LRC colonization in B. napus, primarily in HGS plants, evidence could not be found for a naringenin-induced reduction of glucosinolate concentrations in such plants. 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). In addition, naringenin was reproducibly the most effective of several flavonoids utilized in this study, although the amount of carbon in each of those compounds is similar. Furthermore, naringenin does not promote the growth of azorhizobia in culture (13).

Superoxide (O₂) and hydrogen peroxide are involved in protection of plants against avirulent pathogens (1). Although many flavonoids act as antioxidants, the results of this study suggest that naringenin does not promote colonization by an antioxidative mechanism (but see reference 2). Glucosinolates, present in relatively high concentrations in HGS plants, also act as antioxidants (36). Furthermore, quercetin, a flavonoid with greater antioxidant activity than naringenin (26), did not promote root colonization in the present studies. Nevertheless, there may be scope for specific assessments of the oxidative status of plant tissues or exudates treated with naringenin. An alternative subject for study is whether naringenin renders azorhizobia resistant to the effects of glucosinolate products. A similar situation exists in isoflavonoid-induced resistance of soybean rhizobia to the plant defense compound glyceollin (24). However, azorhizobia pretreated with naringenin did not survive ITC treatment better than controls pretreated only with water. It may be that naringenin does not influence colonization directly; plants might have modified the exogenous flavonoid, with one of the products stimulating bacterial colonization of roots.

Since oilseed rape is important economically but contains antibacterial glucosinolates, future strategies designed to improve growth of the crop by using plant growth-promoting bacteria are likely to benefit from advances in our understanding of bacterial colonization of its roots. Results from this study suggest that the early establishment of bacteria in roots of young plants is influenced negatively by glucosinolates, which are found in higher concentrations in some HGS plants. Nevertheless, azorhizobia were able to effectively colonize young plants of several LGS varieties. The quantitative methods used in this study provided a novel means of studying the effects of glucosinolates on bacterial colonization of roots; the approach resembled conceptually that employed in biological monitoring. The extent of bacterial colonization of LRCs, quantified using a simple reporter gene assay in this study, can apparently be used to monitor (albeit coarsely) the glucosinolate content of the roots of B. napus. This approach could be modified easily to enable studies of other phytochemicals affecting bacteria in the rhizosphere and confirms the usefulness of molecular marker genes for microbial ecology (37). 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.