Plant and Soil 182: 59-66, 1996. 1996 KluwerAcademic Publishers. Printed in the Netherlands.
The influence of native rhizobacteria on european alder (Alnus glutinosa (L.) Gaertn.) growth 1. Characterization of growth promoting and growth inhibiting bacterial strains
A. Probanza, J.A. Lucas, N. Acero and EJ. Gutierrez Mafiero Departamento de Biologfa 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: Alnus glutinosa, Bacillus, DRB, PGPR, Pseudomonas
The effects on plant growth of 27 bacterial strains (7 Pseudomonas and 20 Bacillus) isolated from the rhizosphere of a natural alder stand, were studied. The chosen bacteria were selected from the dominant genera found in that habitat in each season. These bacteria were grown in a complete culture medium, and removed (centrifuged and filtered) prior to testing on nodulated (N) and non-nodulated (NN) aider plants. Tests were made in order to determine their effects on the following biometric parameters: aerial surface (AS), aerial length (AL), number of leaves (NL) and total nitrogen (TN). Among the 20 Bacillus strains tested, three promoted growth; two were B. pumilus strains and one was B. licheniformis. Significant (p
tro, 1981), high production of biomass (Rytter, 1995), excellent growth (Gordon, 1978), application in indus- try (Harrington, 1984) and for being independent of nitrogen supply (Atkinson and Hamilton, 1978).
The characterization of rhizobacteria in an alder copse, located in the center of the Iberian Peninsula, and determination of the dominant genera throughout the year has been carried out by Acero et al. (1994) on a previous study. The aims of the present study were: (i) to determine the effect on biometric parameters (AL, AS, TN and NL) of the rhizobacteria growth medium free of bacteria, on nodulated and non-nodulated plants and (ii) characterize strains affecting alder growth.
Material and methods
Strains were isolated from the rhizosphere of a ripar- ian alder population by "Las Tortolas" creek (Avi- la, Spain, coordinates 4021'30"N,434'O"W). The selection was made from a group of 600 isolates sam- pled at random, during one year, from the rhizosphere of the alder copse. Strains were characterized at a generic level in a previous study (Acero et al., 1994); 20% of the strains were randomly selected among the most abundant genera in each season (Pseudomonas or Bacillus), resulting in a total of 27 strains, 20 of which belonged to Bacillus and 7 to Pseudomonas.
Specific Kits were used to identify the strains of Bacillus (API-50 CHB, version D, API-20E, Api Sys- tems SA, France) and Pseudomonas (Kit API 10), as well as additional assays for Bacillus (growth in NaCI 5%, growth at 50 C, growth in a nitrogen free medi- um, spores round and growth at pH 5.7 in nutrient broth) and for Pseudomonas (synthesis of pigments: King's A and B, medium to detect indigoine; and the arginine dihydrolase reaction) from Bergey's Manual (Claus and Berkley, 1989; Palleroni, 1989).
Additionally, all strains were inoculated in Pochon and Tradieux's medium (1962) for aerobic nitrogen fixing microorganisms. After incubation (15 days), 1 mL was transferred to 15 mL sterile flasks and 10% of the atmosphere was replaced by acetylene, according to Grant and Binkley (1987). After 24 h at 28 C, I mL of the atmosphere was sampled to determine acetylene reduction by gas chromatography with a 3000 KONIK chromatograph HGRC, provided with a flame ioniza- tion detector and a Porapack R column.
DNA isolation and PCR amplifcation
DNA was isolated from bacteria grown in a nutritive culture broth, and separated by centrifugation. Bacteria were lysed following the Noller and Hartsell method (1961); then 2 mL of extraction buffer (10 mM Tris, HCI pH=7, 100 mM NaC1, 10 mM EDTA and 1% w/v SDS) were added and incubated for lh at 60 C. The sample was centrifuged at 3000 rpm, and 3 mL of Phenol/Chloroform 1:1 was added to the supernatant, which was shaken gently to form an emulsion and then centrifuged at 3000 rpm. One mL of sodium acetate and of ethanol (at -20 C) were then added to the supernatant. Precipitated DNA was resuspended in Tris HCI pH=7.6.
Amplification reactions contained 10 mM Tris HCI pH=8.3, 50 mM KC1, 4 mM MgC12, 0.001% gelatin, 200/~M of dATP, dCTP and dTI'P, 1.25 units TAQ DNA polymerase (Perkin Elmer) and 2 mM primer (random 10-mers, Kit B, Operon Technologies, Alameda, Calif.)
DNA amplification was performed in a Perkin Elmer Cetus DNA Thermal Cycler programmed for an initial step cycle of 5 minutes at 95 C (to separate all DNA) and 45 cycles of 1 minute at 94 C (denat- uralization), 2.5 minutes at 35 C (annealing) and 2 minutes at 72 C (elongation). Amplification products were analyzed by electrophoresis in 1% agarose gels and visualized by ethidium bromide staining.
This assay was done with the 10 strains that showed activity on plant growth and with typed strains of Bacil- lus licheniformes, Bacillus pumilus and Pseudomonas fluorescens obtained from the Spanish Collection of type cultures (CECT) n o C20, C29 and C378 respec- tively, from 10341, 10337 and 10038 of the NCTC.
Growth medium for bacteria
Each of the 27 isolates were grown in the following liq- uid culture medium: 1 g of nutrient broth (Difco), 999 mL of soil extract and 1 mL of oligoelements solution (Pochon and Tradieux, 1962). After determining the stationary growth stage by turbidimetry, each isolate was incubated at 27 C in a shaking bath (85 rpm) until the stationary stage was reached.
Once incubation was over, each culture medium was centrifuged in sterile plastic tubes at 5000 rpm for 5 minutes. Supernatant was kept at -20 C in sterile tubes. Before use, the medium samples were defrosted and filtered through a 0.2 #m Millex-GS (Millipore) to remove the last traces of bacteria. This was done for
each of the 27 bacterial growth culture media that will be assayed as described below.
Seeds for this experiment were collected from the same alder copse where bacteria were isolated. In order to determine genetic variability, electrophoresis on SDS- polyacrylamide gels of reserve proteins from 240 seeds were made following the Payane et al. (1981) method.
Plant growth conditions and bacteria medium assay
Prior to germination in aseptic conditions on sterile vermiculite (termite n3), the seed surface was ster- ilized with a NaC10 solution (10 g L - t ) and washed 5 times with sterile distilled water. Before germina- tion, seeds were watered with sterile distilled water; once they germinated two groups were made. 50% of the plants were inoculated with Frankia (strain A.g.89-1 from Dr Moiroud's collection suspended in a 0.9% NaCI solution) and watered with nitrogen- free Crone medium until plants developed four mature leaves and at least one nodule. The other 50% were watered with nitrogen-supplemented Crone medium until plants developed four mature leaves. At these stages, both groups of plants were ready for the exper- iment. Plants were distributed (3 per flask) in 800 mL flasks containing 75 g of sterile vermiculite and then the experiment was carried on.
Each culture medium free of bacteria was assayed at 20% (46.5 mL of culture medium and 184 mL of Crone) and 10% (23.5 mL of culture medium and 207 mL of Crone). Vases, with three plants each, were brought to maximum water holding capac- ity (WHC) with the corresponding solution in each case, and kept at 100% WHC with Crone medi- um; nitrogen-free Crone was used for N-plants and nitrogen-supplemented Crone for NN plants. Vases were covered with vented plastic paper and incubat- ed in an ASL culture chamber (Aparatos Cientfficos SL, Spain), with a 14 h light (25 C, 5000 lux) and 10 h dark (15 C) photoperiod. Controls were grown under the same conditions (10 and 20%) with sterile culture medium with no bacterial growth.
After 25 days of incubation, plants were taken from the vases and vermiculite was carefully removed from
roots. They were pressed moist, extending roots and leaves.
The following parameters were assessed on each plant: total nitrogen (TN), length of aerial part (ALL aerial surface (AS) and number of leaves (NL). For these analyses a system of image analysis (Delta T, Devices Inc., England) with "Dias" software was used. After Kjeldhal digestion using a microwave digestor Maxidigest-MX 350 (Prolabo, Spain), total nitrogen was assessed colorimetrically, as in Smith (1980).
Data was organized in a 112x4 matrix (28 bacteria growth cultures - 27 bacteria growth cultures and one control-, at 2 concentrations (10 and 20%), 2 types of plants (N and NN plants) and 4 growth measure- ments). A Principal Component Analysis (PCA) was made with this matrix (Hartman, 1967), using Stat- graphics v 2.1 computer program (Statistical Group Corp).
Those strains segregated by the PCA were com- pared with each other and with controls by unidirec- tional ANOVAs for each parameter assessed; when significant differences existed, an LSD test was per- formed (Sokal and Rholf, 1979).
The analysis of the seed reserve proteins did not show genetic variability. Band patterns were identical in the 240 seeds selected at random from the bank used to obtain plants for this study. Figure 1 shows one of the gels with the band patterns for 20 seeds.
The PCA distributed the treatments into three groups (Fig. 2) that reflected both the concentration of the medium for bacterial cultivation and its effect on N and NN plants. Group A includes the medium for seven Pseudomonas strains; Group B includes the medium for three Bacillus strains; and Group C the medium for controls and the other strains.
ANOVAs and LSD's reflect effects of the medi- um from 7 Pseudomonas and the 3 Bacillus strains. As shown by the PCA, the culture medium concentra- tion did not seem to have an influence on the effects observed. Therefore, the ANOVAs were carried out using the average of the two concentrations of each strain; N and NN plants were considered separately.
Table 1 shows the clear activating effect of the three Bacillus strains (group B on PCA) on plant
Figure I. SDS-PAGE electrophoresis of Ahus glutirwso seeds reserue proteins, M: Proteins marker, Ovoalbumine v fraction (65 Kd)
-4.2 0 0
I q.. 0 OO 0
0 O O0
l O * 8.. do0
AL 0.47 0 A8050 0 0 TN 0.30
h8 l 8 0. 0. l o
0 0 Oo 0
Fi~ure2. Representation of axis I and II on PCA of plant parameters(dntn from medium assayed at lOand 20%). Weight valueon axis I appears by the different parameters considered. Location corresponds to position on axis II. AL: Aerial Length; AS: Aerial Surface; TN: Total Nitrogen; NL: Number of Leaves; * Pseudomotm medium assayed on N and NN plants; l Bacillus assayed on N plants; 0 Brrcill~s assayed on NN plants; f~ Controls. A: inhibitory strains, B: promoting strams. C: no effect strains. For details see text.
Table 1. Effects of Bacillus, PGPR (B.3, B.12 and B.2I) and Pseudomonas, DBR (R9, P.10, P.I 1, RI7, P.18, P.19 and P20) culture medium free of bacteria on AInus glutinosa growth
N Plants z NNPlants
Strains AL y AS NL TN AL AS NL TN (cm) (cm 2 ) (/~g g- J ) (cm) (cm 2 ) (,ugg -1 )
B.3 7.834-.23a x 9.30=LI.02a 8.604-.16a 3.07-t-.1 la 8.134-.33a 9.32+.58a 9.304-.33a 1.724-.25a B. 12 7.77-t-.34a 9.66+.45b 9.30+.33a 2.56-t-.09a 8.89-1-.21 b 8.90+.56b 9.45-t- 1.04a 1.734- .31 a
B.21 7.384-.45c 9.634-.86b 9.15+.30a 3.4+.43b 9.01+.21b 9.384-.24a 9.45+.16a 1.944-.12a E9 4.624-.32b 5.21+.14c 7.304-.16b 0.674-.31c 3.864-.18c 3.95=L16c 4.834-.33b 0.51 :Iz.02c E l 0 4.69-k.45b 5.20-k.05c 7.45+.33b 0.684-.09e 3.914-.26c 3.944-1-.95c 5.16=[:.33b 0.404-.02c E l 1 4.9d::L 19b 5.224-.27c 7.604-.66b 0.634-.08c 3.92+.33c 3.994-t-.24c 5.16-k.32b 0.484-t-.01 c
R 17 4.934-.46b 5.37=t=.0cd 7.80+.33b 0.62+.05c 3.97+.45c 3.96+.85c 4.994-.16b 0.42=1:.06c
P 18 4.834-.23b 5.45+.54cd 7.30-1-. 16b 0.534-. 11 c 3.96+.64c 3.96-k-.32c 4.98::k.99b 0.444-.01 c El9 4.92+.23b 5.55zt:.78cd 7.60+.33b 0.63+.1 lc 3.92+.16c 3,92::L02c 4.83zL16b 0.474-.01c
E20 4.89:k.08b 5.48+.05cd 7.45+.16b 0.64+.09c 4.06=L12c 4.06+.06c 5.164-.33b 0.41+.12c
Control 5.404-. 19d 5.684-. 16d 7.154-.33b 2.214-.12d 4.934-.26d 5.34.36d 7.66=/=.33c 1.05-t-.20b
z N plants-nodulated plants, NN plants-non nodulated plants. ~AL Aerial Length, AS-Aerial Surface, NL-Number of leaves, TN-Total Nitrogen. :~Treatments sharing same letter(s) are statistically nonsignificant at p
Figure 3. Banding pattern by PCR and RAPDs markers of bacteria assayed. Bacillus lichen@-mis (strains B.3 and B.21), Bacillus pumilu.7 (B. 12), Pseudomoncrs~uorescensbv.11 (P.9.. P. IO., P. I I ., I? 17.. P. 18.. P. 19. and P.20.). and typified strains from Spanish Collection Type Cultures of each bacteria: C29 (Bacillus pumilus), C20 (Bacillus lichen~formis) and C378 (Pseudomonaspuorescens). M: DNA marker, Lambda DNA Hind III digest. In left margin appears the number of base pair (bp).
(Solomonson, 1981) which causes a general and inspe- cific inhibition. Immediate effects are a reduction of plant metabolic activities and nitrogen uptake (either root absorption or symbiotic fixation) and therefore, the significant decrease in growth revealed by our results.
The threeBacillusstrains(B.3, B.12 andB.2l)pro- moted growth as the increase in the different biometric parameters point out. The effect was more marked in N plants, which showed a higher increase in total nitro- gen content, probably due to the physiological benefits and amplification effects of the nodules.
Bacillus strains B.3, B.12 and B.21 increased aerial surface 163% and aerial length 182%. Simi- lar to these results, there are many other studies on growth promoting Bacillus strains used to improve agricultural production (Chanway et al., 1988; Halver- son and Handlesman, 1991; Kloepper et al., 1980). Many other studies also report that Bacillus produces growth promoting metabolites such as vegetal hor- mones, e.g. citokinins (Kampert and Strzelezyk, 1984) and quantitatively more important, auxins (Brown, 1972; Sevadurai et al., 1991) or vitamins (Curl andTru- clove, 1986). Besides the already mentioned beneficial characteristics, the three Bacillus strains are nitrogen fixers. Previous studies (Acero et al., 1994), showed that up to 10% of alder rhizospheric Bacillus were aero- bic nitrogen fixers. This capacity increased our interest
in identifying the agents causing the effects described in this study.
The DNA polymerase chain reaction and RAPD- markers technique were used to determine if the 7 strains of Pseudomonas jluorescens had the same genetic fingerprint and, therefore, all would have the same metabolic capacities. If this were so, we could assume that growth inhibiting or activating mecha- nisms would be identical. In this respect, the exist- ing homogenicity among all the Pseudomonas strains except for P20, as well as that of the Bacillus licheni- formis strains should be noticed. It is also worth men- tioning that they show a different band pattern when compared to the respective type strains. This is a very typical aspect in rhizospheric studies since strains that inhabit this environment have a considerable adaptive capacity, which leads to strains of the same species having different metabolic characteristics (Gilbert et al., 1993). These adaptations may give advantages to rhizospheric colonization.
This study reports, strong alder responses to metabolites in rhizospheric bacterial growth medium. The observed effects and the coexistence of these strains in the rhizosphere, demonstrate tridirectional control (Lynch, 1990). This relation occurs from plant to rhizobacteria through exudates, from rhizobacteria to plant through metabolites and among rhizobacteria themselves by competition.
The results are a step to the possible use of bacterial strains in optimizing production and colonization in Alnus glutinosa or other forest species.
We are grateful to Dr Moiroud (Lab. Ecologie Micro- bienne, Univ Lyon) for Frankia strains, to Dr Uruburu (Spanish Collection of Standard Cultures) for Pseu- domonas and Bacillus Strains and Beatriz Ramos (Dpto Biologia Vegetal, Univ San Pablo CEU) and Carol F Warren for their linguistic assistance.
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