Biol Fertil Soils (2003) 38:119–123DOI 10.1007/s00374-003-0599-0

S H O R T C O M M U N I C A T I O N

Veena Jain · Kaushalya Gupta

The flavonoid naringenin enhances intercellular colonizationof rice roots by Azorhizobium caulinodans

Received: 5 September 2002 / Accepted: 20 February 2003 / Published online: 19 June 2003� Springer-Verlag 2003

Abstract The intercellular colonization of rice roots byAzorhizobium caulinodans and other diazotrophic bacte-ria has been studied using strains marked with the lacZreporter gene. A. caulinodans were able to enter the rootsof rice at emerging lateral roots (lateral root cracks) bycrack entry and this was observed by light microscopy.After colonization of lateral roots, bacteria moved intointercellular space within the cortical cell layer of roots.Naringenin at 1�10-5 and 5�10-5 M concentration signif-icantly enhanced root colonization. The role of nodABCand regulatory nodD genes was also studied; lateral rootcrack (LRC) colonization of rice was shown to be Nodfactor and NodD independent. Lateral root crack coloni-zation of rice was also observed with similar frequencyfollowing inoculation with Azospirillum brasilense andthe colonization by A. brasilense was stimulated bynaringenin and other flavonoid molecules.

Keywords Azorhizobium caulinodans · Azospirillumbrasilense · Naringenin · Intercellular colonization · Ricelateral root cracks

Introduction

The inoculation of non-legume crops with diazotrophicbacteria can fix N2 gas and provide combined N to theplant for enhancement of the crop and this would reducethe dependence on chemical N fertilizers, with a contri-bution to the development of more sustainable agricul-ture.

An important requirement for efficient biologicalnitrogen fixation (BNF) is to have diazotropic bacteriagrowing endophytically within plants, as in legume-rhizobia (van Rhijn and Vanderlyden 1995) Parasponia-

Bradyrhizobium (Webster et al. 1995) and actinorhiza-Frankia (Benson and Silvester 1993) symbioses. In theseassociations, specialized organs called nodules are formedin which bacteria reduce N2 into NH4 and this fixed N istransferred to plants. One of the approaches to attempt toachieve BNF which we are investigating is to determinewhether naturally existing rhizobial strains which havethe ability to enter the legume plant by crack entry cancolonize non-legumes internally (Cocking et al. 1994). Incrack entry, rhizobia enter the host plant root systemsintercellularly between the adjacent cells and not byforming infection threads at the tip of root hairs. Amongthe rhizobial species used, Azorhizobium caulinodans,which induces root and stem nodules on the tropicallegume Sesbenia rostrata, is especially interesting sincein addition to forming nodules after crack entry invasion,it is able to fix N2 in its free-living state withoutdifferentiation into bacteroids (Kitts and Ludwig 1994).Here the ability of Azorhizobium caulinodans to colonizerice roots internally using bacteria marked with a lacZreporter gene has been investigated and quantified. Theeffect of flavonoids on the frequency of root colonizationhas been studied. The role of nodulation genes in crackentry has also been studied. Finally the nodulationabilities of Rhizobium meliloti and Azospirillum brasi-lense have also been evaluated and compared with that ofAzorhizobium caulinodans.

Materials and methods

Maintenance of bacterial strains and inoculation of plant culture

Strains of Azorhizobium caulinodans (ORS 571 and IRBG 314) andR. meliloti (2011) carrying lacZ reporter genes were constructed bytriparental mating using Escherichia coli (HB101 having pXLGD4;Leong et al. 1985; and helper plasmid pRK2013 in E. coli strainK12; Leong et al. 1982). Azorhizobium caulinodans and R. melilotiwere grown on TY media (Somasegaran and Hoben 1994),Azospirillum brasilense on YEP medium (Vanstockem et al.1987). Azospirillum brasilense carrying the lacZ fusion was kindlyprovided by Prof. Cocking, University of Nottingham, UnitedKingdom.

V. Jain ()) · K. GuptaDepartment of Biochemistry,College of Basic Sciences and Humanities,CCS Haryana Agricultural University,125 004 Hisar, Indiae-mail: rvjain@hau.nic.in

Rice (Oryza sativa cv. HRK120) seeds were surface sterilizedand grown aseptically in tubes containing 20 ml N-free F�hraeusmedium (F�hraeus 1957) semi-solidified with 0.8% (w/v) agar.Filter sterilized flavonoids dissolved in a minimum amount ofsodium hydroxide and the volume made up with sterile water wereincorporated into autoclaved plant growth medium giving a finalpH of 6.8. The 2-day-old rice seedlings were inoculated with 0.1 mlper tube of a 2-day-old bacterial culture (approx. 1�10–12 bacteriaL–1) and incubated at 22�C for 14 days. The uninoculated plantsserved as a control.

Detection of b-galactosidase associated with plant roots

Rice roots were harvested 14 days after inoculation and roots werewashed free of agar. The roots were infiltrated for 30 min undervacuum in a solution of prepared 2% (v/v) glutaraldehyde in 0.12 Msodium cacodylate buffer (pH 7.2) and fixed for a further 60 min inthe same solution at atmospheric pressure to inactivate the plants’endogenous b-galactosidase activity. After fixing, roots werewashed 3 times (15 min each wash) in 0.12 M sodium cacodylatebuffer (pH 7.2). Histochemical staining of bacterial b-galactosidasewas performed using X-Gal (5-bromo-4-chloro-3 indoyl-b-d-galactopyranoside) as substrate according to Boivin et al. (1990).Bacterial colonization was visualised by the dark blue precipitatesresulting from the degradation of X-Gal. The number of colonizedlateral root cracks (LRCs) was recorded and percentage of LRCscolonized per plant was calculated. When appropriate, sections cutto approximately 2 mm in thickness for light microscopy werestained with 0.5% (w/v) toluidine blue in 0.1% (w/v) sodiumtetraborate at 60�C for 3 min (Davey et al. 1993). Statisticalanalysis of the data was undertaken using Fisher’s test.

Re-isolation of bacterial endophytes from plant roots

Bacteria were re-isolated from colonized roots by surface steril-ization of excised roots in 95% (v/v) ethanol for 10 s, followed by4% (v/v) sodium hypochloride for 4 min. Roots were rinsedthoroughly with sterile distilled water. Sterile root pieces werecrushed in liquid TY medium and the resultant mixture streakedonto 20 ml 1.5% (w/v) agar-solidified TY medium. Cultures wereincubated at 25�C for 5 days and the bacterial colonies formed werecounted.

Results and discussion

Colonization of rice roots at lateral root cracksby Azorhizobium caulinodans

Colonization by Azorhizobium caulinodans with strainsORS 571 and IRBG 314 (pLXGD4) occurred on thesurface, at the tips and at the cracks found at the bases oflateral roots. The LRCs resulted from the emergence oflateral roots from the main roots. Colonization of LRCswas observed more frequently and found to be morereproducible than colonization of root surface or root tips.Sections of blue staining LRCs showed intercellularpockets of bacteria in the cortex of the main root in whichazorhizobia had multiplied extensively (Fig. 1a, b) as inthe first stage of nodulation of Sesbania rostrata (Ndoyeet al. 1994; Stone et al. 2001). b-Galactosidase activitywas detected at colonization sites of rice roots up to25 days after inoculation suggesting the persistent pres-ence of viable bacteria. The bacteria re-isolated fromsurface-sterilized roots were shown to be Azorhizobiumcaulinodans as they re-colonized rice and induced rootand stem nodules on its host legume Sesbania rostrata.

A high proportion of plants (61.1%) had colonizedLRCs with an average of four to six colonized sites perplant with strain ORS 571. A colonized site is heredefined as a LRC at any stage of colonization, and anyvisible signal was counted as positive. Similar resultswere obtained with IRBG 314 (Table 1). The proportionof LRCs colonized by both strains were expressed aspercentage of total number of LRCs per plant givingrespectively 5.2% and 4.9% for ORS 571 and IRGB 314.The use of lacZ reporter gene in the present studyfacilitated the detection of bacteria at the point of lateralroot emergence. Reporter genes have already been used astools to localize bacteria in the roots of several types ofplants (Webster et al. 1998). Azorhizobium colonizationof rice LRCs seems to share similarities with that of rootand stems of Sesbania rostrata (Stone et al. 2001).

Fig. 1a, b Light microscopy ofintercellular colonization of riceby Azorhizobium caulinodansstrain ORS 571 (pXLGD4). a Alongitudinal section showingpockets of bacteria (arrowed)contained within an intercellu-lar space at the site of lateralroot cracks (LRC). b Cross-section through a colonizedLRC showing bacteria withinan intercellular space (arrowed)near the vascular bundle (vb) ofthe main root

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Flavonoid stimulates colonization of rice LRCby Azorhizobium caulinodans

Flavonoids, a class of phenolics released by the plantroots, initiate transcription of their nodulation (nod) genes(Rolfe 1988) and consequently initiate the process ofrhizobial infection-thread formation and nodulation inlegumes. Naringenin is one of the most efficient inducersof nod genes in ORS 571 (Goethals et al. 1989). In orderto determine whether these molecules are also involved inLRC colonization of rice roots, different concentrations ofnaringenin were added to the growth medium. It wasfound that LRC colonization of rice by Azorhizobiumcaulinodans could be stimulated in the presence of5�10-5 M naringenin (Fig. 2A). A concentration below1�10–6 M had no effect on LRC colonization byAzorhizobium caulinodans. All subsequent experimentswere performed using 5�10–5 M concentration of narin-genin as we obtained higher reproducibility at thisconcentration.

Four families of flavonoids (flavone, flavonols, flava-none and isoflavones) have been shown to have rhizobialnod gene-inducing activity (Denarie et al. 1992). Com-pounds from each group (5�10-5 M) were tested for theirability to stimulate LRC colonization. The flavanonenaringenin significantly stimulated LRC colonization ofrice by ORS 571 but daidzein, apigenin and myricetin hadno effect (Fig. 2B). Syringaldehyde, a plant phenolicwhich induces the vir gene of Agrobacterium tumefaciens,had no effect. Malate and succinate, the best C sources forAzorhizobium caulinodans (Dreyfus et al. 1988) were alsotested and these had no significant effect on LRCcolonization of rice roots at 1�10–4 M. However, narin-genin at this concentration showed stimulation (Fig. 2B).As naringenin contains approximately four times as muchC as succinate or malate, this suggests that naringenin isnot stimulating LRC colonization by acting as a C source.Similar results have been reported in wheat (Webster etal. 1998).

LRC colonization does not require nod genes

Naringenin is one of the most efficient inducers of nodgene expression in Azorhizobium caulinodans regulatingtheir expression via nodD gene product, and nod genesare required for nodule formation in S. rostrata. It waspossible that naringenin was stimulating colonization ofrice via nod genes. Rice seedlings were inoculated with anodC mutant of Azorhizobium caulinodans which doesnot produce Nod factors. No difference was found in thelevel of colonization between nodC mutant and wild-typestrain. Colonization of rice roots by the mutant strain wasstimulated by naringenin (Fig. 2C). As it cannot be ruledout that activated NodD proteins could induce theexpression of rhizobial genes other than the nod genes,a nodD mutant was used to investigate the possibility thatLRC colonization of rice was mediated by NodD protein.The nodD mutant colonized rice and naringenin stimu-lated the LRC colonization at the same frequency as thewild-type strain, thus indicating no role of nodC andnodD genes in rice root colonization. The LRC coloni-zation has also been reported to be nod gene independentin wheat (Webster et al. 1998) and Arabidopsis thaliana(Gough et al. 1997).

In order to compare the ability of Azorhizobiumcaulinodans to colonize rice roots with that of otherplant-associated diazotrophic bacteria, rice plants wereinoculated with R. meliloti strain 2011 and Azospirillumbrasilense strain SPS 245 containing pXLGD4. R.meliloti could not colonize LRCs of rice even in thepresence of naringenin (Table 1). These results suggestsome specificity to the ability of Azorhizobium caulin-odans to colonize rice roots. However, Table 1 demon-strates that rice LRCs could also be colonized byAzopirillum brasilense and this colonization was stimu-lated by addition of 5�10–5 M naringenin to the growthmedia. Similar results have been reported in wheat(Webster et al. 1998).

In the present study, the colonization of rice was foundto be nod gene independent as reported in wheat (Websteret al. 1998) and Arabidopsis thaliana (Stone et al. 2001)but this does not exclude the possibility that flavonoids

Table 1 Colonization of lateral root cracks (LRCs) of rice roots by Azorhizobium caulinodans. Values are means of 36 replicates € SD, allstrains contained the plasmid pXLGD4 and three independent experiments were conducted

Strain % Plant colonized(colonized/tested)

Number of LRCsper plant

Number of colonizedLRCs per plant

% LRCs colonizedper plant

Azorhizobium caulinodans

ORS 571 61.1 (22/36) 53.8€17.2 2.8€1.9 5.2€3.9IRGB314 55.6 (20/36) 55.1€19.4 2.7€2.0 4.9€3.6ORS 571+nar 86.1 (31/36) 73.7€15.3 17.2€5.8 23.4€6.1

Azospirillum brasilense

SPS245 52.8 (19/36) 46.4€17.7 4.9€1.6 10.7€4.9SPS 245+nar 77.8 (28/36) 64.7€20.1 16.4€4.9 25.3€10.2

Rhizobium meliloti

2011 0.06 (2/36) 44.6€11.7 0.01€0.01 0.022011+nar 0.06 (2/36) 48.9€13.7 0.03€0.02 0.06

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may be inducing other bacterial genes responsible for rootcolonization. Recently flavonoids have also been demon-strated to induce the expression of other bacterial geneswith unknown functions which show no homology to nod

genes (Krishnan and Pueppke 1993; Perret et al. 1994).Naringenin has also been reported to enhance theefficiency of R. meliloti- alfalfa symbiosis (Jain et al.1990) although naringenin does not induce nod genes ofthis bacterium. Flavonoids have also been shown toinduce resistance in Bradyrhizobium japonicum andRhizobium fredii to the soybean phytoalexin, glyceollin(Parniske et al. 1991). This may suggest that the additionof naringenin might stimulate LRC colonization byincreasing the tolerance of inoculated bacteria to thesecretion of toxic plant compounds.

The ultimate aim of establishing endophytic interac-tion between diazotrophic bacteria and non-legumes is tofix N2 and transfer the fixed N to the plants. SinceAzorhizobium caulinodans is capable of N2-fixing in afree-living condition and in up to 3% (v/v) oxygen (Kittsand Ludwig 1994), it was anticipated that intercellularcolonization of rice might provide a niche for N2 fixation.However, under the experimental conditions employed inthis study, nitrogenase activity, as measured by acetylenereduction assay (Hardy et al. 1968), could not be detected.The rice plants inoculated with ORS 571 and grown inpots had higher dry weight when compared with theuninoculated control plants. It has been suggested thatxylem vessels are the sites of N2 fixation by diazotrophsin non-legumes as they provide the low pO2 and a site forexchange of metabolites necessary for N2 fixation (Jameset al. 1994). In rice in the present study, xylemcolonization of Azorhizobium caulinodans could not bedetected. It will now be interesting to study intercellularcolonization of azorhizobia, and to determine using thelacZ reporter gene whether these bacteria are able tospread deeper inside the root, particularly into the xylem,when plants are grown for longer under natural physio-logical conditions. The invasion of LRCs and subsequentcolonization of roots of rice provides important informa-tion to researchers attempting to extend rhizobial coloni-zation and endophytic nitrogen fixation to non-legumespecies.

Acknowledgements Authors are thankful to Dr. R.K. Jain,Veterinary Anatomy and Histology, CCS HAU Hisar, for his helpin light microscopy and to Professor E.C. Cocking, University ofNottingham, UK, for providing the A. brasilense strain.

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