Plant Growth Regulation 22: 145–149, 1997. 145c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Effects of european alder (Alnus glutinosa (L.) Gaertn) rhizobacteria onnodular metabolism and root development

A. Probanza, N. Acero, B. Ramos & F.J. Gutierrez ManeroDept. Biologıa Secc. Biologia Vegetal, Facultad CC, Experimentales y Tecnicas, Univ. San Pablo CEU, P.O. Box67, Boadilla del Monte, 28660 Madrid, Spain

Received 14 November 1996; accepted in revised form 24 February 1997

Key words: acetylene reduction activity (ARA), Alnus glutinosa, Frankia, nodular metabolism, rhizobacteria, rootdevelopment

Abstract

The effects of 3 Bacillus and 7 Pseudomonas strains on development of the root system and nodular metabolism,evaluating CO2 production and acetylene reduction activity (ARA) of Alnus glutinosa,were studied. All experimentswere done on nodulated plants (N) with the symbiont Frankia and on non-nodulated plants (NN).

An increase in root length (RL) and root surface (RS) was detected when growth culture media from threedifferent Bacillus free of bacteria were assayed, both in N and NN plants. However, Pseudomonas growth culturemedia reduced RS in N plants, and a decrease in RL parallel to an increase in RS in NN plants. Bacillus growthculture media caused an increase and CO2 production while Pseudomonas culture media caused lower ARA and anoticeable increase in nodular respiration. Results are discussed considering nutritional and/or hormonal (Bacillus)or phytotoxic factors (Pseudomonas).

Abbreviations: N plants = nodulated plants; NN plants = non nodulated plants; RS = root surface; RL = root length;TN = total nitrogen; ARA = acetylene reduction activity

Introduction

The influence of certain rhizobacteria on plant phys-iology has been widely reported in many studies [7,13, 19]. Certain bacterial genera such as Bacillus andPseudomonas have a wide associative spectrum, play-ing an important role in the rhizosphere of many plants[9, 12].

The effect of these rhizobacteria, either stimulat-ing or inhibiting plant growth can be accomplishedthrough several mechanisms such as nutrient mobiliza-tion [26], phytohormone production [20], productionof siderophores [14], production of antibiotcs [23] andproduction of HCN [19].

Additionally, Pseudomonas and Bacillus are bac-terial genera with a considerable influence on nitrogenfixation once nodulation has taken place [2, 8]. Inthe rhizospheric environment these genera may affect

those factors which strongly influence the physiologyof nitrogen fixation, both in legumes and non-legumes,such as O2 diffusion through the nodule cortex andtherefore nodular respiration [3], the effect of CO2

concentration on root environment [4], the concentra-tion of mineral nitrogen in soil and its characteristics[16] and hormone concentration on the root environ-ment [2].

A previous study [18] clearly demonstrated therhizobacteria isolated from alder either promoted(Bacillus strains) or inhibited (Pseudomonas strains)the growth of alder, a plant that is important in the firstsuccesional stages in humid soils with low nitrogencontents. The aim of the present work was to studythe effect of such rhizobacteria isolated from a naturalalder (Alnus glutinosa) population on root develop-ment and nodular metabolism (ARA and CO2 produc-tion).

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Material and methods

Bacterial strains

The ten bacterial strains assayed were isolated from therhizosphere of a riparian alder copse near “Las Tor-tolas” Creek (Avila, Spain, coordinate 40�2103000N,4�340000W). The selection was made from a group of600 colony forming units (cfu) sampled at random,sea-sonally during one year, from the rhizosphere of thecopse. The strains have been characterized at genericand specific levels in previous studies [1] and 20% ofthe strains were randomly selected; 3 Bacillus strains(1 Bacillus pumilus, isolate B.3 and 2 Bacillus licheni-formis, isolates B.12 and B.21) were obvious growthpromotors and 7 Pseudomonas fluorescens bv.II strains(isolates P.9, P.11, P.17, P.18, P.19 and P.20) wereinhibitors [18].

Growth medium for bacteria

The 10 bacteria were grown in the following liquidculture medium: 1 g of nutrient broth (Difco),10 mL of soil extract, 1 mL of oligoelements solu-tion (1L:Na2(MoO4), 2H2O: 0.05 g, K2B4O7.10H2O:0.05 g, FeCl3.6H2O: 0.05 g, Cd(NO3)2.4H2O:0.05 g, CoSO4.7H2O: 0.05 g, CuSo4.5H2O: 0.05 g,ZnSO4.7H2O: 0.05 g) and distilled water to 1 L [17].After determining the stationary growth stage by tur-bidimetry, each cfu was incubated at 27 �C in a shakingbath (85 rpm) until this stage was reached.

Once incubation was over, the culture medium wascentrifuged in sterile plastic tubes at 5000 rpm for5 minutes. Supernatant was kept at –20 �C in steriletubes. Before use, the medium samples were defrost-ed and filtered through 0.2 �m Millex-GS (Millipore)filters to remove bacteria.

Plant growth conditions and bacterial medium assay

Seed surfaces were sterilized for 2 minutes with aNaClO solution (10 g L�1) and rinsed 5 times for10 minutes each with distilled sterile water beforesowing on sterile vermiculite, previously brought tomaximum water holding capacity (WHC) with steriledistilled water. Until germination, seeds were wateredwith sterile distilled water; from thereafter, wateringwas done with Crone solution (Nitrogen Crone solu-tion (2.5 g L�1): KNO3: 1 g, CaSO4: 0.5 g, MgSO4:0.5 g, Ca3(PO4)2: 0.5 g, Fe(PO4)2: 0.25 g. Nitrogen-free Crone solution (2.5 g L�1): CIK: 0.75 g, CaSO4:

0.5 g, MgSO4: 0.5 G, Ca2(PO4)2: 0.25 g, Fe3(PO4)2:0.25 g, keeping vases at maximum WHC throughoutthe experiment. Once they had germinated, seedlingswere separated into two equal groups. 50% of plantswere inoculated with Frankia (strain A.g.89-1 from Dr.Moiroud’s collection) suspended in a 0.9% NaCl solu-tion and watered with nitrogen-free Crone solution,and the other 50% were watered with Crone solutionuntil plants developed four mature leaves.At this stage,both groups of plants were ready for the experiment inwhich they were distributed (3 per flask) in 800 mLflasks containing 75 g of sterile vermiculite.

Each bacterial-free culture medium was assayedat dilutions of 20% (46.5 mL of culture medium and184 mL of Crone solution) and 10% (23.5 mL of cul-ture medium and 207 mL of Crone solution). Flasks,with three plants each, were brought to maximum waterholding capacity (WHC) with the corresponding solu-tion in each case, and kept at 100% WHC with Cronemedium; nitrogen-free Crone was used for N plants andnitrogen-supplemented Crone for NN plants. Flaskswere covered with vented plastic paper and incubatedin an ASL culture chamber (Aparatos Cientıfıcos SL,Spain), with a 14 h light (25 �C, 5000 lux) and 10 hdark (15 �C) photoperiod. Controls were grown underthe same conditions (10 and 20%) with sterile culturemedium.

Growth measurements

After 25 days of incubation, plants were taken from theflasks, vermiculite was carefully removed from rootsand plants were pressed moist. The following parame-ters were assessed on each plant: total nitrogen, rootlength, root surface in NN plants and in N plants thesame parameters plus ARA and CO2 production. Forbiometrical analyses a system of image analysis (Delta-T, Devices Inc., England) with “Dias” software wasused. After Kjeldhal determination, using a microwavedigestor Maxidigest-MX 350 (Prolabo, Spain), totalnitrogen was assessed colorimetrically [22].

In order to evaluate ARA and CO2 production, nod-ules were separated from 25-day-old plant roots, andCO2 production was determined following Llinareset al. [15] by a gas chromatograph (KNK-HRGC-3000) provided with a thermic conductivity detector(TCD) and a 101 chomosorb column (80/100 mesh),200 cm long with a diameter of 0.2 cm under the fol-lowing conditions: column temperature (45 �C), injec-tor temperature (75�C), detector temperature (100�C),helium as carrier gas with a 1.8 mL m�1 flux and 1 mL

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Table 1. Effects of Bacillus pumilus (isolate B3), Bacillus licheniformis (isolates B12 and B21), and Pseudomonasfluorescens bv.II (isolate P9, P11, P17, P18, P19 and P20) culture medium free of bacteria on N plants. Values are themean of three replics and standard error appears by each mean value

Acetylene reduction assay CO2 reduction Root length Root surface Total nitrogen

Strain (nmol C2H2g�1h�1) (nmol CO2g�1h�1) (cm) (cm2) mg g�1

B3 953.9 � 15.2ax 1240.7 � 180.1a 7.89 � 0.22a 2.93 � 0.22a 3.07� 0.16a

B12 967.3 � 17.4a 1224.5 � 169.5b 8.00 � 0.45a 4.05 � 0.31b 2.56� 0.41a

B21 990.5 � 31.5c 1227.9 � 95.3b 7.90 � 0.31a 2.78 � 0.17b 3.42 � 0.32b

P9 332.8 � 21.3b 1544.4 � 147.1c 4.23 � 0.08bc 0.83 � 0.09c 0.67� 0.04c

P10 325.0 � 33.8c 1552.3 � 201.1c 4.22 � 0.05bc 0.81 � 0.11c 0.68� 0.11c

P11 332.6 � 9.3b 1548.6 � 86.4c 4.38 � 0.12b 0.85 � 0.07c 0.63� 0.02c

P17 333.7 � 41.0b 1541.7 � 123.3cd 4.31 � 0.24bc 0.89 � 0.08cd 0.62� 0.14c

P18 345.4 � 12.6b 1543.2 � 145.8cd 4.16 � 0.33bc 0.83 � 0.08c 0.53� 0.16c

P19 347.4 � 23.3b 1529.9 � 97.6cd 4.30 � 0.12bc 0.86 � 0.14c 0.63� 0.04c

P20 342.0 � 18.9b 1562.4 � 103.0cd 4.32 � 0.41bc 0.78 � 0.12c 0.64� 0.05c

Cont 512.2 � 29.1d 994.5 � 68.3d 4.20 � 0.13b 1.20 � 0.21d 2.21 � 0.17d

x Treatments sharing same letter(s) are statistically nonsignificant at p< 0.05 according to LSD.

samples. Ethylene production were determined in theconditions proposed by Grant and Binkley [10]. After1 hour of incubation at 10% acetylene atmosphere,1 mL samples were analyzed with the gas chromato-graph described above, provided with a flame ioniza-tion detector (FID) and a Porapack-R chomosorb col-umn (80/100 mesh), 200 cm long with a diameter of0.2 cm under the following conditions: column temper-ature (50 �C), injector temperature (100 �C), detectortemperature (200 �C), nitrogen as carrier gas with a20 mL m�1 flux.

Statistics

Results were analysed by unidirectional ANOVAs foreach of the evaluated parametres. Where significantdifferences were detected, as LSD test [22] was per-formed.

Results and discussion

A common topic in a number of studies on the plant-microorganism interaction in how this interaction isestablished under laboratory conditions, as a step priorto greenhouse and field experiments. The lack of thistype of study is due to intrinsic difficulties, given themany parameters that must be considered, such as thetype of inoculum, the colonization capacity and com-petition with other microorganisms. Our interest indetermining this interaction led us to search for bacter-ial growth metabolites. Therefore bacteria were grown

in a complete culture medium until the stationary stagewas reached, when metabolites released during bacter-ial growth were detected. A complete culture mediumwas used in order to avoid metabolic restrictions to theassayed strains.

Table 1 and 2 show data from these experiments.Each of the three culture media of Bacillus free ofbacteria, caused a sugnificant increase in root lengthcompared to sterile culture medium (control) andPseudomonas culture media. This effect was particu-larly obvious on N plants. Root surface also increasedunder the influence of three Bacillus strains althoughthis increase was not as striking as in root length, nei-ther in N nor in NN plants. The increase caused bystrain B3 and B21 was significantly different from allthe Pseudomonas treatments and control. Root lengthand surface did not increase in proportion to each otherin either group (N or NN plants). Root lengthening onN plants was more marked and some authors explainthis fact as an increase in plant capacity to explore newareas of soil [25], reaching a wider area for nutrientsand increasing the surface to contact the endophyte,therefore favouring the nodulation process. Since theassayed strains of Bacillus are able to synthesize andrelease auxins into culture media [11] the modifica-tions detected in roots could be attributed to these planthormones. However, the existence of either a poten-tial indirect effector or of substances related to plantnutrition [6] cannot be ruled out.

Root surface (RS) and root length (RL) was affecteddifferently under the influence of Pseudomonas growthculture media, free of bacteria. Root length was similar

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Table 2. Effects of Bacillus pumilus (isolate B3), Bacillus licheni-formis (isolates B12 and B21), and Pseudomonas fluorescens bv.II(isolates P9, P11, P17, P18, P19 and P20) culture medium freeof bacteria on NN plants. Values are the mean of three replics arestandard error appears by each mean value

Root length Root surface Total nitrogen

Strain (cm) (cm2) (mg g�1)

B3 6.21 � 1.12ax 3.11 � 1.14a 1.72 � 0.19a

B12 6.73 � 1.06b 2.78 � 0.89ab 1.73 � 0.23a

B21 7.64 � 2.00c 3.30 � 0.65a 1.04 � 0.03b

P9 2.45 � 0.21d 2.42 � 0.45b 0.51 � 0.05c

P10 1.48� 0.31f 2.43 � 0.32b 0.40 � 0.12c

P11 1.51� 0.25f 2.51 � 0.12b 0.48 � 0.16c

P17 1.43� 0.21f 2.50 � 0.37b 0.42 � 0.24c

P18 1.50� 0.12f 2.56 � 0.27b 0.44 � 0.01c

P19 1.48� 0.29f 2.45 � 0.42b 0.47 � 0.08c

P20 1.52� 0.34f 2.54 � 0.13b 0.41 � 0.02c

Cont 4.31 � 0.67c 0.97 � 0.12d 1.05 � 0.36bd

x Treatments sharing same letter(s) are statistically nonsignificant atp< 0.01 according to LSD.

to controls on N plants, althought there was a signif-icant reduction in root area; the root system appearedlees branched and had thicker ramifications. However,root length decreased while root surface increased sig-nificantly in NN plants; their root systems appearedshorter, more branched and with thinner ramifications.This last modification can be explained as a conse-quence of a nutritional deficit for which the plant triesto compensate by increasing its ability to profit fromavailable nutrients [25]. This phenomenon may be dueto several reasons among which the ability of thisPseudomonas strains to produce HCN [11], a metabo-lite known for its strong growth inhibition capacity canbe considered. The presence of HCN in the mediumby itself is unlikely to explain the differences detectedbetween N and NN plants in the root system. Howev-er, such differences could be explained if it is assumedthat HCN affects both groups of plants differently atthe point of nutrient absorption, especially in relationto nitrogen, due to the symbiosis in N plants. How-ever, our results do not rule out additional alternativesto explain growth inhibition, such as iron absorptionby siderophores [5] or a difference in response to acertain hormone concentrations.

As far as ARA and CO2 production are concerned,asignificant increase in nodular CO2 production parallelto a significant decrease in ARA and total nitrogen wasdetected plants treated with Pseudomonas fluorescensbv.II growth culture media free of bacteria. On the

contrary, plants treated with the Bacillus culture media,free of bacteria, showed a parallel increase in both para-meters and in total nitrogen content. Increased CO2

production and ARA could be expected as a responseto a great energy demand by the endophyte, althoughthe results obtained when assaying Pseudomonasfluorescens filtered culture media seem to invalidatethis hyplthesis. However, form studies on five acti-norrhizal genera, Tjepkema and Winship [24] foundthat 19% of the CO2 produced by nodular respirationis used to carboxylate phosphoenolpyruvate, yieldingdicarboxylic acids which could be used by the endo-phyte as an energy source. According to these data wewould expect and increase in CO2 release in responseto a lower energetic requirement since ARA is inhib-ited; this increase in CO2 would not be due to greaterproduction but to lower CO2 consumption by the nod-ules in the production of dicarboxylic acids.

Acknowledgements

We are grateful to Dr Moiroud (Lab Ecologie Microbi-enne, Univ Lyon II, Villeurbanne Cedex F-69622) forFrankia Strains and Carol F Warren for her linguisticassistance.

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