Plant and Soil 182: 67-74, 1996. 1996 Kluwer Academic Publishers. Printed in the Netherlands.
The infuence of native rhizobacteria on European alder (Alnus glutinosa (L.) Gaertn.) growth H. Characterisation and biological assays of metabolites from growth promoting and growth inhibiting bacteria
EJ. Gutierrez Mafiero, N. Acero, J.A. Lucas and A. Probanza Departamento de Biologia Vegetal, Univ. San Pablo CEU, E-28660 Boadilla del Monte, Madrid, Spain*
Received 29 September 1995. Accepted in revised form 22 March 1996
Key words: Bacillus, DRB, hydrocyanic acid (HCN), indolacetic acid (IAA), PGPR, Pseudomonas
Metabolite production was investigated in four bacterial strains that promoted (plant growth promoting rhizobacteria -PGPR-, B. licheniformis, isolate B. 12 and B. pumilus isolate B.3) or inhibited (deleterious rhizobacteria -DRB-, P. fluorescens bv II, isolates P.9 and P.20) growth of nodulated and non-nodulatedAlnus glutinosa seedlings. These strains were isolated and characterized from the rhizosphere of a natural alder population, and their biological effects on plant growth determined on previous studies. Biological assays were performed to confirm the observed effects on aerial length (AL), aerial surface (AS), number of leaves (NL) and total nitrogen (TN). According to the high resolution gas chromatography (HRGC) results, PGPR strains produced auxin-like (IAA-1) compounds at levels of 1.736 and 1.790 mg IAA-1 L -1 culture growth medium; however, they did not produce HCN. These compounds are derived from IAA and not from the Trp originated by peptide degradation in culture media. The promoting effect is evidenced when comparing the effects of IAA and the filtered bacterial growth culture medium to control (increases of 64% in aerial surface, 277% in total N content and 32% in aerial length).
The deleterious strains produced HCN (1.6 and 2.4 mg kg -1 detected in growth culture medium) and they did not produce IAA-1 compounds. The bacterial culture's -free of bacteria-inhibiting effects were 7% in aerial surface, 240% in total nitrogen content and 15% in aerial length.
The results reported here suggest that the interactions that take place in the alder rhizosphere are in a delicate equilibrium. In view of this, the coexistence of PGPR and DRB strains in this environment is unquestionable, and does affect alder health in field conditions.
Abbreviations: N- nodulated, NN- non-nodulated, IAA-1- auxin-like, HRGC- high resolution gas chromatography, TLC- thin layer chromatography, DRB- deleterious rhizobacteria, PGPR- plant growth promoting rhizobacteria, AL- aerial length, AS- aerial surface, NL- number of leaves, TN- total nitrogen.
Among the number of interactions that define the complex rizospheric environment, many authors have reported on the coexistence of deleterious and promot- ing bacteria and their effects on the colonized plant
* FAX No. +3413510496
(Azad et al., 1985; Chanway et al., 1989; Sumner, 1990).
A number of rhizospheric bacterial strains with a positive effect on plant development Plant Growth Promoting Rhizobacteria (PGPR) have been report- ed. Many strains have been catalogued as PGPR due to their effect on plant pathogens (Mei et al., 1984; Schippers et al., 1991) or to their ability to induce
plant growth promotion. Most of these strains belong to Bacillus, Pseudomonas, Streptomyces, Azotobac- ter, Azospirillum and Clostridium genera (Reddy and Rahe, 1989). Phytohormones such as indol-3-acetic acid (IAA) or cytokinins (Brown, 1974; Hubbel et al., 1979; Kampert et al., 1975; MOiler et al., 1989) are among the plant growth promoting compounds often produced by bacteria. However, other compounds, known as auxin-like (IAA-1) and not IAA itself are often responsible for the promoting efects (0berhansli et al., 1990; Selvadurai et al., 1991).
As far as Deleterious Rhizobacteria (DRB) strains are concerned, many have been described among the genera Desulfovibrio, Pseudomonas, Erwinia, Agrobacterium, Enterobacter and Cro- mobacter (Lynch, 1990). Their deleterious effects are presumed to be due to a pool of metabolites among which the following can be found: aliphatic acids (Drew and Lynch, 1980), phenolic acids (Hartley and Withehead, 1985), sulfhydric acid (Gambrell and Patrick, 1978) and mainly, hydrocyanic acid (Alstrt~m and Bums, 1989; Dartnall and Bums, 1987).
In the first part of this study (Probanza et al., 1996), we isolated two PGPR strains (B. pumilus isolate B.3 and B. licheniformis isolate B.20) and two DRB strains (P. fluorescens bv II, isolates E9 and E20). The two Bacillus strains caused significant (p
lar effects to these hormones in biological assays, the IAA-like compound was evaluated by HRGC, and then characterized by its absorption spectra and TLC.
Extraction and evaluation by HRGC of lAA-like compounds on culture medium
The two strains were grown and incubated as described in the corresponding section. IAA-like compounds were extracted from 250 mL of growth culture medi- um, brought to pH 2.7 with HC1 1 N, as in Larque and Rodriguez (1993), in three consecutive extrac- tions (diethyl ether / culture medium, 1:1). The ether phase was eliminated "in vacuo" with a rotary evap- orator at 50 C in the dark. Dry residue was treated with 2 mL KOH 6 M in methanol for 10 minutes at 48 C. The alcoholic phase was eliminated "in vacuo" with a rotary evaporator at 50 C in the dark and the residue was suspended in 0.5 mL hexane. 2 #L sam- pies were analysed by HRGC following the modified McDougall and Hilman method (1978), with a chro- matograph KONIG HRGC-3000 provided with a FID detector and a chromosorb W 2.1 4 mm column with OV. 17 at 15% on 80-100 mesh, using nitrogen as the carrier gas at 40 mL min-I flux. Chromatograph temperature conditions were as follows: oven, 180 C, injector and detector, 270 C. This method differenti- ates between IAA-like substances and Trp although it does not discriminate IAA-like compounds.
In order to characterise IAA-like compounds, the absorption spectra of purified culture media were com- pared with the following patterns in diethyl ether: Trp, indol-3-acetic, indol-3-acetyl L-alanine, indol- 3-pyruvic, indol-3-acetamide, indol-3-acetyl glycine and indol-3-acetyl L-aspartic (Sigma-Aldrich). These spectra were obtained with a Beckman spectropho- tometer DU 600, from 190 nm to 370 nm.
Thin layer chromatography followed a modification of Leinhos and Birustiel (1988). The ether extracts of bacterial growth culture medium together with Trp and IAA patterns (Sigma) on diethyl ether were chro- matographed on a Merck silica gel layer. The carrier phase was isopropanol: ammonia 28%: water (8:1:1). The chromatography was carried out in the dark and indol compounds detected with UV light.
Plant growth conditions and bacteria medium assay
Prior to germination under aseptic conditions on sterile vermiculite (termite n3), the surface of Alnus seeds was sterilised with a NaC10 solution (10 g L -1) and washed 5 times with sterile distilled water. Accord- ing to reserve proteins analysis, there was no genet- ic variability (Probanza et al., 1996). Trays of seeds were watered with sterile distilled water, sufficient to bring vermiculite to maximum Water Holding Capaci- ty (WHC), and kept at 25 C until germination. At this point, half of the plants were supplied with a suspen- sion of Frankia (Strain A.g.89-1, from Dr Moiroud collection) in nitrogen-free Crone solution to maxi- mum WHC. The other half was supplied with a nitro- gen Crone solution (1 g of N as NO~- per L). Both sets of plants were kept at maximum WHC through- out the experiment, with nitrogen-free and nitrogen Crone solution, respectively. When nodulated plants had developed at least four mature leaves and one nodule and non nodulated plants had developed four mature leaves, three seedlings (both nodulated -N- plants and non-nodulated -NN- plants) were placed in 800 mL vials containing 75 g of sterile vermiculite in the conditions described above. In order to assay the growth culture medium at 20%, 46.5 mL of cul- ture medium free of bacteria suspended in 184 mL of nitrogen Crone solution was added to non-nodulated plants, and, in the other, 184 mL of nitrogen-free Crone to nodulated plants.
Simultaneously, two biological assays were made on N and NN alder plants with IAA (Sigma) and KCN. Non-inoculated bacterial culture medium was prepared and the following amounts of IAA added: 7.2, 3.6, 1.8, 0.9, 0.45 and 0 mg L -~ this latter being the con- trol; then these concentrations were assayed at 20% as described above (46.5 mL of non-inoculated culture medium with IAA in different concentrations, and 184 mL of appropriate Crone solution). For HCN, the same basic procedure was followed using 8, 4, 2, 1 and 0 (control) mg L-1 KCN.
Vials were covered with a slightly pierced plas- tic film and set in an ASL culture chamber (Aparatos Cientficos S.L., Spain) with a 14 hour light (25 C) and 10 hours dark 15 C) photoperiod. The lights used were Extra Light (Phillips HGM1/F) and provided 5000 lux. At the same time, controls were made with sterile bacterial culture medium, also at 20% on N and NN plants watered with nitrogen-free Crone and nitrogen Crone solution respectively, to maximum WHC.
After 25 days, plants were taken from the vials and vermiculite was carefully removed from their roots. Plants were pressed wet extending leaves and roots for image analysis.
The following parameters were studied in each plant: (i) total nitrogen -TN-, (ii) aerial length -AL- , (iii) aerial surface -AS- and (iv) number of leaves -NL. An image analysis system (