Letters in Applied Microbiology 1998, 26, 311–316

Plant mediated interactions between Pseudomonasfluorescens, Rhizobium leguminosarum and arbuscularmycorrhizae on pea

G. Andrade, F.A.A.M. De Leij and J.M. LynchSchool of Biological Sciences, University of Surrey, Guildford, Surrey, UK

1655/97: received 30 September 1997 and accepted 3 December 1997

G. ANDRADE, F.A.A.M. DE LEIJ AND J.M. LYNCH. 1998. Using undisturbed sandy loam soil coresheavily infested with mycorrhizae, the effects of the antibiotic-producing Pseudomonasfluorescens strain F113 and its non-antibiotic derivative Ps. fluorescens F113G22 on nodulationby introduced and indigenous Rhizobium were studied. Furthermore, the effects of thedifferent microbial inocula on the colonization of the pea roots by mycorrhizae were studied.It was found that Ps. fluorescens F113 enhanced nodulation by Rhizobium fourfold, while thenodules produced were much larger and strongly pigmented (pink) compared with those inother treatments. The proportion of roots colonized by arbuscular mycorrhizae was notsignificantly affected by the different treatments.

INTRODUCTION

The beneficial effects of Rhizobia, mycorrhizae and flu-orescent pseudomonads have been studied extensively inrelation to plant nutrition and plant protection. However, theinteractions between these organisms in the rhizosphere havereceived less attention. Nevertheless, the interactions withinmicrobial communities associated with plant roots are ofconsiderable interest, because such interactions might eitherenhance or inhibit the beneficial effects of the individualspecies (Germida and Walley 1996). Meyer and Linderman(1986) showed that Pseudomonas putida strain R-20, a sidero-phore producing plant growth promoting rhizobacterium,enhanced nodulation and nitrogen fixation by Rhizobium onsubterranean clover. The mechanism postulated was thateffective scavenging for iron by this strain reduced popu-lations of micro-organisms that were deleterious toRhizobium, while simultaneously providing the plant withextra iron. Similarly, Staley et al. (1992) showed that thisstrain increased nodulation on alfalfa.

The ability of specific microbial agents to stimulate thegermination and growth of arbuscular mycorrhizae has beenwell documented (Azcon-Aquilar et al. 1986 ; Mayo et al.1986 ; Garbaye 1991) and has recently led to the conceptof ‘Mycorrhizal Helper Bacteria’ (Garbaye 1994). Severalmechanisms by which bacteria enhance mycorrhizae have

Correspondence to : F.A.A.M. De Leij, School of Biological Sciences, Universityof Surrey, Guildford, Surrey GU2 5XH, UK (e-mail : F.De-Leij@surrey.ac.uk).

© 1998 The Society for Applied Microbiology

been proposed, including removal of self-inhibitors frommycorrhizal spores (Mosse 1959 ; Daniels and Trappe 1980)or by the production of volatile chemicals that trigger sporegermination (Azcon 1987 ; Mugnier and Mosse 1987). Ano-ther mechanism by which rhizobacteria might stimulate rootcolonization by mycorrhizae is by producing compounds thatincrease root cell permeability (Bowen 1980). This wouldlead to enhanced exudation rates, which would enhance themycorrhizal growth and ability of the fungus to penetrate itshost (Mosse 1962 ; Azcon-Aquilar and Barea 1985).

Interactions between arbuscular mycorrhizae and Rhizo-bium in the rhizosphere of legume plants often lead to anincrease in nitrogen uptake by the plant (Smith et al. 1979 ;Pacovsky et al. 1986). The general consensus is that nodu-lation is enhanced in nutrient deficient soils because ofincreasing P absorption by plants colonized by mycorrhizae(Barea and Azcon-Aquilar 1983 ; Cluett and Boucher 1983).This view is supported by the fact that increasing availablefertilizer phosphorus frequently increases N2 fixation by Rhizo-bium in the absence of mycorrhizae. It is also thought thatthe plant-Rhizobium system benefits from the presence ofarbuscular mycorrhizae because the mycorrhizae correct notonly P deficiency but also any nutrient deficiency in the plantthat might be limiting to the Rhizobium (Pacovsky 1986 ;O’Hara et al. 1988). Increased mineral nutrient levels in theplant would not only benefit Rhizobium directly, but wouldalso lead to increased photosynthesis, making a greater pro-portion of photosynthates available to the Rhizobium nodules(Pang and Paul 1980 ; Harris et al. 1985). This paper is

312 G. ANDRADE ET AL.

concerned with the interactions between Rhizobium leg-uminosarum and an antibiotic (DAPG) and non-antibioticproducing Tn5 mutant of Pseudomonas fluorescens inoculatedonto pea plants planted in undisturbed grassland soil con-taining extensive mycelial networks of arbuscular mycor-rhizae associated with the old grass roots.

MATERIALS AND METHODS

Experimental systems

Undisturbed soil cores were taken from a permanent pasturesite (×15 year grassland) at Merrist Wood Agricultural Col-lege (Surrey, UK). The soil was a sandy loam (Holiday Hillsseries ; 81% sand, 9% silt and 10% clay) with an organicmatter content of 1·6% (w/w) and a pH (H2O) of 5·36.

PVC tubes (15 cm diam ; 20 cm long) were pushed intothe soil to a depth of 25 cm. The soil core, together with theassociated grassland vegetation, was extracted andsubsequently, the vegetation was removed by taking off thetop 5 cm of the soil to leave an undisturbed soil profile takenfrom 5 to 25 cm depth with mainly grass roots in each soilcore. All grass roots were extensively (80–100%) colonized byarbuscular mycorrhizae, providing an extensive mycorrhizalnetwork of hyphae in each of the soil cores. The bottom ofeach core was covered with 10 mm nylon mesh. The soil coreswere stored in open plastic bags at 20 °C for 2 weeks beforeeach core was planted with five pre-germinated pea seedlings.

Organisms used

Pseudomonas fluorescens (strain F113) was isolated from theroot hairs of sugar beet and was characterized as a fluorescentnon-fastidious motile rod that was oxidase- and catalase-positive (Fenton et al. 1992). This strain produces the anti-biotic 2,4-diacetylphloroglucinol (DAPG) which inhibits thegrowth of a range of bacteria and fungi (Shanahan et al.1992). Using Tn5 mutagenesis, a DAPG negative derivative(F113G22) of this strain was created (Shanahan et al. 1992).Both strains were chromosomally marked with the lacZYgene cassette to facilitate detection by simple plating methods(Drahos et al. 1986). Expression of the lacZY genes for lactoseutilization by this strain allowed detection on agar mediacontaining the chromogenic substrate 5-chloro-4-bromo-3-indolyl-b-D-galactopyranoside (X-gal), as this compound iscleaved by the action of b-galactosidase to yield a blue prod-uct.

Rhizobium leguminosarum biovar. Viciae (strain 1112) wasobtained from the University of Padua, Italy. This strainwas chromosomally marked with the lacMer gene cassetteallowing easy differentiation from indigenous Rhizobiumstrains on media containing X-gal and/or mercury.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 311–316

Culturing and inoculation

Bacterial cells were stored at −70 °C on beads impregnatedwith cryo-preservatives (Technical Service Consultants,Lancs, UK). The two Ps. fluorescens strains were grown onTryptic Soy agar (Oxoid, Basingstoke, UK) while Rh. leg-uminosarum was grown on Yeast Manitol agar (Vincent 1970).Inoculated plates were incubated at 25 °C for 2 and 4 d,respectively. Bacteria were subsequently harvested by flood-ing the plates with 10 ml sterile 0·25 strength Ringer’s solu-tion and the bacterial mat was brought into suspension witha sterile glass spreader. Bacteria were collected in 100 mlbottles and the concentration of the different strains wasadjusted to approximately 109 cfu ml−1. By mixing Pseudo-monas strains F113 and F113G22 with Rhizobium strain 1112,a total of six treatments was obtained, including a controltreatment without bacteria (1. control ; 2. Ps. fluorescens F113 ;3. Ps. fluorescens F113G22 ; 4. Rh. leguminosarum 1112 ; 5.F113¦1112 ; and 6. F113G22¦1112). By mixing each treat-ment with 1·5% (w/v) guar (Fluid Drilling Ltd, Stratfordupon Avon, UK), viscous suspensions were obtained whichwere used to inoculate pre-germinated pea seeds (Pisum sati-vum, cv Montana). Pea seeds were washed five times in steriledistilled water, and pre-germinated for 3 d at 25 °C on wetfilter paper. The seedlings were subsequently inoculated byimmersing each seedling in the appropriate bacterial sus-pensions.

Experimental set up and sampling

Each soil core was planted with five pea seedlings. For eachtreatment, four replicate cores were used. The soil cores thusplanted were placed in a completely randomized design in acontrolled environment chamber (Vindon, Oldham, UK) setat a photoperiod of 16 h (500 mmol m−2 s−2) with day andnight temperatures of 22 and 12 °C and a relative humidityof 80%. For a period of 8 weeks, every 2 weeks, one pea plantat a time was removed from each soil core with a 5 cmdiameter soil corer (Coremaster, Southampton, UK). Aftereach sampling, the holes were filled with fresh soil. Soiladhering to the roots was removed by washing in tap water.Washed roots were blotted dry and weighed. Subsequently,the shoot of each plant was removed, and each root systemwas cut into approximately 2 cm pieces and mixed thoroughlyby hand. Effects of the different treatments on plant growthwere estimated using shoot dry weights.

Microbial populations

Rhizobium populations. The number of Rhizobium nodulespresent on a well mixed 1 g sample taken from each rootsystem was determined. Subsequently, from each rootsystem, 10 randomly chosen nodules were removed with a

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scalpel. These nodules were surface sterilized for 1 min in a5% solution of sodium hypochlorite followed by three rinsesin sterile distilled water to remove the hypochlorite. Thenodules thus surface-sterilized were crushed in 0·1 ml 1/4strength Ringer’s solution and plated onto Yeast ManitolAgar (Vincent 1970) amended with 50 mg X-gal ml−1. After5 days of incubation at 25 °C, the Rh. leguminosarum coloniesthat originated from each nodule were assessed for b-gal-actosidase activity (conversion of X-gal into a blue product)in order to assess the proportion of nodules that originatedfrom the genetically modified Rhizobium.

Arbuscular mycorrhizal populations. To estimate colon-ization of the roots with mycorrhizae, washed roots werecleared and stained using the methodology of Philips andHayman (1970). Colonization by arbuscular mycorrhizae wassubsequently estimated using the gridline intersect methodof Giovanetti and Mosse (1980) by examining the roots at×40 magnification.

Fluorescent pseudomonads populations. Indigenous andgenetically modified Pseudomonas populations were estimatedfor each sample by preparing from a well mixed, macerated1 g root sample a 10-fold dilution range in 1/4 strengthRinger’s solution. Aliquots of 0·1 ml of appropriate dilutionswere plated onto fluorescent Pseudomonas selective agar(Katoh and Itoh 1983) amended with 50 mg ml−1 X-gal. Theplates were incubated for 7 d at 25 °C and blue (recombinants)and white fluorescent colonies (indigenous Pseudomonas) wereenumerated on each plate. The populations of indigenousand lacZY positive colonies were expressed as colony formingunits (cfu) per gram root fresh weight.

Statistical analyses

All data were analysed using analyses of variance (ANOVA).Non-normal distributed data sets were log transformed toobtain a normally distributed set of data that could be sub-sequently analysed using ANOVA. Significant differencesbetween means were analysed using LSD (Least SignificantDifference) tests.

RESULTS

Effects of microbial inocula on shoot weight

Inoculation with Ps. fluorescens F113 or Ps. fluorescens G22reduced plant shoot weight significantly compared to thenon-inoculated control treatment. Co-inoculation of the twoPs. fluorescens strains with Rhizobium negated this effect to a

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large extent in the case of Ps. fluorescens F113G22, but notwith strain F113 (Table 1).

Effects of microbial inocula on nodulation

Compared with the control treatment, nodulation by Rhi-zobium was significantly enhanced in the presence of theDAPG producing Ps. fluorescens F113 (Table 1). This effectwas especially large (P³ 0·001) when Rh. leguminosarum1112 was added together with Ps. fluorescens F113, resultingin four times the number of nodules per gram root comparedwith the control treatment. Not only were there many morenodules per gram root compared with the other treatments,but also the nodules themselves were large and pink, whilethose in the other treatments were small and white. Inocu-lation of seedlings with either Rhizobium alone, Ps. fluorescensG22 alone or a combination of Ps. fluorescens G22 and Rhi-zobium 1112 did not result in a significant increase in thenumber of nodules per gram root. However, inoculation witheither of the Ps. fluorescens strains enhanced the establishmentof the introduced Rhizobium strain (Table 1).

Effect of microbial inocula on rhizosphere colonizationwith Pseudomonas

Whereas especially Ps. fluorescens F113 led to increased estab-lishment of the introduced Rh. leguminosarum 1112, presenceof Rh. leguminosarum 1112 reduced the density of the twoPseudomonas strains in general (Table 1). This effect wasmore pronounced (P³ 0·05) in the combined Rhizobium/Ps.fluorescens G22 treatment. No significant difference in rhi-zosphere competence of the two Pseudomonas strains couldbe detected. Densities of indigenous fluorescent Pseudomonaspopulations were on average 2 log units lower (P³ 0·001) inthe rhizosphere where plants were inoculated with the non-DAPG producing Ps. fluorescens G22, whereas the DAPGproducing Ps. fluorescens F113 had no significant effect onindigenous fluorescent Pseudomonas populations (Table 1).

Effect of microbial inocula on arbuscular mycorrhizae

Pea roots were rapidly colonized by mycorrhizae present inthe soil environment. After 2 weeks, between 20 and 30% ofthe total sampled root length in all treatments containedarbuscular mycorrhizae, rising to ×70% 56 days after sowing(Table 2). Overall, the proportion of roots colonized by AMwas not significantly affected by the different treatments.However, inoculation with the two Pseudomonas strains, aloneor in combination with Rhizobium, led to a transient butsignificant increase in mycorrhizal colonization 4 weeks aftersowing (Table 2).

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Table 1 Establishment of fluorescent pseudomonads (introduced and indigenous), Rhizobium (introduced and indigenous) on pea rootsgrown in undisturbed sandy loam soil—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

LacZY positive LacZY negative Number of lacZY positiveShoot dry weight Pseudomonas Pseudomonas Rhizobium Rhizobium nodules

Treatment (g) (log10 cfu−1 root) (log10 cfu−1 root) nodules g−1 root (%)—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Control 1·41d — 5·34b 5·1a —Rhiz. 1112 1·24c,d — 5·24b 7·9a,b 43·3Ps. F113 1·02a,b,c 6·31b 4·86b 9·9b —R1112¦F113 0·89a,b 6·10b 5·21b 20·3c 84·2Ps. G22 0·81a 6·13b 3·13a 7·9a,b —R1112¦G22 1·22b,c,d 5·59a 3·15a 6a,b 84·6

P ³0·001 ³0·001 ³0·001 ³0·001 ³0·001LSD 0·11 0·11 0·18 0·9 5·0—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Pea seedlings were inoculated with either 1·25% (w/v) guar gel inoculated in water (control), Rhizobium leguminosarum 1112 (R112),Pseudomonas fluorescens F113 (F113), a combination of Rhizobium and Ps. fluorescens (R112/F113), Ps. fluorescens F113G22 (G22) or acombination of F113 and Rhizobium (G22/R1112). Data represent average establishment over four samplings (2, 4, 6 and 8 weeks aftersowing ; n � 16). Different letters represent a significant difference (P ³ 0·05) between data sets.

Table 2 Colonization (%) of pea roots by naturally occurringmycorrhizae that originated from colonized grass rootspresent in the soil—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Time (days) after plantingTreatment T� 14 T � 28 T � 42 T � 56—–––––––––––––––––––––––––––––––––––––––––––––––––––––Control 20·3 44·7a 63·0 70·9R1112 25·5 53·3a,b 54·8 70·6F113 25·0 58·7b 61·2 66·9R1112/F113 32·8 58·0b 63·2 73·0G22 24·0 57·0b 51·0 71·3R1112/G22 26·2 57·0b 53·0 70·5

P NS* ³0·05 NS NS—–––––––––––––––––––––––––––––––––––––––––––––––––––––

* No significant difference between treatments.Pea seedlings were treated either with no bacteria (control),Rhizobium (R1112), Pseudomonas fluorescens F113 (F113), Ps.fluorescens F113G22 (G22) or combination of either F113 or G22with Rhizobium (R1112/F113 and R1112/G22). Pea roots weresampled at 2-weekly intervals over a period of 56 days (n � 4).Different letters represent a significant difference (P ³ 0·05)between data sets.

DISCUSSION

As DAPG is a relatively non-specific antibiotic involved inthe inhibition of a range of fungi and bacteria, it was expectedthat inoculation of pea seedlings with this strain would inhibitmycorrhizal development, nodulation and/or indigenous

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Pseudomonas populations. In fact, inoculation with Ps. fluor-escens F113 increased nodulation significantly and enhancedearly mycorrhizal infection of pea roots (but not colonization),while indigenous Pseudomonas populations were not affected.In contrast, the DAPG negative Tn5 mutant had no sig-nificant effect on nodulation while indigenous Pseudomonaspopulations were much lower. This suggests that DAPG wasresponsible for the observed positive effects on the differentmicrobial populations that were monitored. A possible expla-nation could be that the effect of this metabolite on the plantwas greater than its effect on microbial populations. Previousinvestigations (Naseby and Lynch 1998) showed that Ps.fluorescens F113 reduced shoot/root ratios of pea plants. Likemany metabolites produced by micro-organisms, DAPGmight increase root cell permeability, resulting in enhancedroot exudation (Barber and Martin 1976). Higher levels ofeasily degradable carbon sources in the rhizosphere leads toenhanced establishment of zygomogenic organisms, such asPseudomonas spp. (Bowen 1980 ; De Leij et al. 1993). This issupported by the fact that the total population of indigenousfluorescent pseudomonads was significantly higher on plantsinoculated with Ps. fluorescens F113 compared with thoseinoculated with Ps. fluorescens F113G22. Increased root exu-dation would also mean that compounds, such as flavonoids,which are involved in triggering nodulation activity in Rhi-zobium (Phillips et al. 1995), would be more readily available,leading to higher densities of Rhizobium nodules per gram ofroot. Also, increased leaching of root exudates implies thatplants divert a greater proportion of the available photo-synthates to the root system. This would mean that more

PLANT MEDIATED INTERACTIONS ON PEA 315

photosynthates would be available for the Rhizobium popu-lation infecting those roots. The availability of extra photo-synthates would explain why the nodules on roots colonizedby Ps. fluorescens F113 were larger and pink in colour com-pared with those on the other treatments which were smalland white.

ACKNOWLEDGEMENTS

The authors are grateful for the support of this work fromthe OECD Co-operative Research Programme and BiologicalResource Management for Sustainable Agricultural Systems,the Ministry of Agriculture Fisheries and Food (contract :CSA 2739) and the European Community (IMPACT II pro-gramme). The authors wish to thank Merrist Wood Agri-cultural College for the soil used in this experiment, ProfessorFergal O’Gara (University College Cork, Ireland) for pro-viding the genetically modified Pseudomonas strains, and Pro-fessor Marco Nuti (University of Padova, italy) for providingthe genetically modified Rhizobium strain. Dr Richard Jack-son and Professor David Read are also thanked for theiradvice.

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