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 (Delta-T Devices Inc., England) with a "Dias" type software was used to mea- sure the last three. Total nitrogen was determined by Kjedhal digestion with a Maxidigest MX 350 (Pro- labo, Spain), and N was determined colorimetrically as in Smith (1980).
Stat is t ics
Unidirectional ANOVAs were used to compare the effects of the different concentrations of IAA to those of growth culture medium from strains B3 and B21 on the biometric parameters studied (AL, AS, NL and TN) in N and NN plants (Sokal and Rholf, 1979). The same was done to compare the effects of CN to those of strains P9 and P20. If there were any significant differences, an LSD test was done (Sokal and Rholf, 1979).
Resu l ts
Products of bacterial metabolism
DRB: On the presumptive assay for HCN production the two Pseudomonas isolates assayed showed their strong cyanogenic potential by turning the indicator paper to reddish brown. Isolate P.9 produced 2.4 mg L- t and isolate 1:'.20, 1.6 mg L- l, as evaluated per the conditions proposed by Lambert et al. (1975), modified by Dartnall and Burns (1987).
PGPR: Both Bacillus strains were IAA-like pro- ducers. The amounts of IAA-like compounds detected by HRGC were 1.79 mg L-1 for B. licheniformis and 1.73 mg L- l for B. pumilus. On the other hand, the amounts of Trp detected were 0.23 mg L- l and 0.25 mg L- l respectively.
The TLC chromatogram for the bacterial growth culture medium showed IAA-like compounds with 0.55 Rf, other than Trp (0.48) or IAA (0.55). These
IAA-like compounds from B. licheniformis and B. pumilus showed the same Rf.
The absorption spectrophotometer analysis showed that the IAA-like produced by both Bacillus strains were identical, and peaked at 246, 296 and 321 nm. However, the peaks observed in the IAA spectrum dif- fered from each of all the 7 patterns studied, although those from indol-3-acetyl L-alanine (247, 297, 337 nm) and indol-3-pyruvic (247, 290, 366 and 354 nm) were quite close to the peak for IAA-like (Figure 1).
DRB: NN plants showed lower values for growth mea- sures and in total nitrogen content than N plants (Table 1). In N plants there was a significant decrease in AS and TN, parallel to the increase in the concentration of KCN assayed; NL and AL responses were far from this trend. NN plants showed the same trends as N plants except for AL, which in this group of plants followed the general trend. The influence of bacteri- al growth culture medium is more pronounced in NN plants except for the marked decrease in total nitro- gen in N plants. Although the banding patterns for the two P. fluorescens bv II obtained by RAPDs-PCR (Probanza et al., 1996) were different, no differences were found when the filtered bacteria growth culture medium themselves were assayed.
KCN was lethal when assayed at 8 mg L- l for N and NN plants. However, N plants were more sensitive than NN plants to KCN at 4 mg L - l , which was the concentration below lethal. The control containing 2 mg L- l KCN, which was approximately the amount produced by the two strains assayed, only showed sig- nificant differences for total nitrogen contents in N plants when the effects of the two Pseudomonas strains were compared.
PGPR: growth measurements were quite similar in the N and NN plants, except for total nitrogen content, which was noticeably higher in N plants in all cases (Table 2).
All biometric measurements increased significant- ly with the increment in IAA from 0.45 to 1.8 mg L- t ; above this concentration, values decreased. The only parameter that showed significant differences when comparing bacterial growth medium with 1.8 mg L- 1 IAA control was total nitrogen content in the NN plants, which was lower in the control. While total N increased in the 1.8 mg L-1 IAA control on N plants, it decreased in corresponding NN control plants. The
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190 WAVELENGTH(nm) 370
Figure I. Absorption spectra analysis of IAA-1 standards and diethyl ether extracts of Bacillus pumilis (isolate B.3) and Bacillus lichen(/hrmis (isolate B. 12) growth culture medium. The n umbers represent: Indol-3-acetamide ( 1 ), Indol-3-pyruvic (2), [ndol-3-acetyl L-alanine (3), lndol-3- acetyl L-aspartic (4), IAA-I of diethyl ether extracts of Bacillus (5), IAA (6), lndol-3-acetyl L-glycine (7) and Yryptophan (8).
Table 1. Effects of KCN and HCN-producing bacteria (Pseudomonasfluorescens bv II, isolates P9 and P.20) on Alnus glutinosa growth
Treatment N plants' NNplants
AL y AS NL TN AL AS NL TN (cm) (cm ~ ) (ag g- k ) (cm) (cm 2 ) (~zg g- l )
8 mg L -~
4mgL -1 2.32 n 2.01 n 6.00 n 0.41 n 2.84+.21a 3.06+,03a 4.00-]--. 16a 01.12=[z.01a 2 ,ngL -L 6.514- .21a x 5.204-.17a 7.454-.33b 0.304-.12b 3.95+.19b 3.984-.21b 5.014-.33b 0.45~.05b lmgL -I 4.79+.23b 5.39+.13b 7.80+.16b 0.814-.05b 4.234-.23b 4.164-.07b 5.15+.16b 0.504-.03b 0.5 mgL -] 5.02+.18b 5.50+.23b 7.804-.16b 1,00+.09b 4.934-.51c 5.34+.18c 7.604-.33c 0.724-.09c 0mgL - I 5.344-.16b 5.564-.21b 7.004-.33a 2,28-t-,17a 5.28+.39c 5.414-.29c 6.33:[:.33c 1.04-F.12c R9 4,62+.32b 5.214-.14a 7.304-.16ab 0.67-t-.31 b 3.864-.18b 3.95:J:.16b 4.83~.33b 0.51.02b E20 4.894-.08b 5.484-.05b 7,454-.16b 0.64:[:.09b 4.064-.12b...