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Plant and Soil 182: 59-66, 1996.

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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

Abstract

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<0.05) increases in all biometric parameters were detected (163% on AS and 182% on AL). All 7 Pseudomonasfluorescens strains had a significant (p<0.05) negative influence on plants, evidenced by a decrease in the value of different parameters when compared to control values.

The obvious effect of the assayed bacterial strains on alder growth drawn from our results should be considered as the starting point for a deeper study of the plant-rhizobacteria interaction, the final aim being to improve production of any forestry species, particularly that ofAlnus glutinosa.

Abbreviations: N-nodulated, NN-non-nodulated, AL-Aerial length, AS-Aerial surface, NL-number of leaves, TN-total nitrogen.

Introduct ion

Many studies show the role played by numerous rhizospheric microorganisms in plant growth and physi- ology (Astrt~m and Gerhardson, 1988; Frankenberger and Arshad, 1995; Griffin, 1990; Kloepper et al., 1980; Schippers et al., 1991 ; Shishido et al., 1995). A vari- ety of studies have been carried out with rhizospheric bacteria involved in phytopathogen control (Burr and Caesar, 1984; Carruthers et al., 1995; Dowling and O'Gara, 1995; Liu et al., 1995) or in the optimiza- tion of the nodulation process (Derylo and Skorpuska, 1993; Freitas et al., 1993). The aim of all these studies

* FAX No: +3413510486

was the improvement of root environmental conditions so as to enhance plant growth.

The use of biological systems to improve plant growth has lately gained special importance for two reasons: first, the use of fertilizers and consequent pol- lution can be reduced, and second, these studies can also help to identify some deleterious non pathogenic bacterial strains which may have a harmful effect on harvest yields (Frederickson and Elliot, 1985).

In the case of actinorhizal plants, such as Alnus glutinosa, the use of indigenous rhizospheric strains to improve primary production is of relevant importance, basically because Alnus glutinosa can be applied to forestry management (Homann et al., 1992), for its importance as a pioneer plant (Bermudez de Cas-

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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

Bacterial strains

Strains were isolated from the rhizosphere of a ripar- ian alder population by "Las Tortolas" creek (Avi- la, Spain, coordinates 40°21'30"N,4°34'O"W). The selection was made from a group of 600 isolates sampled 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 medium, 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 - 2 0 °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

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 n°3), the seed surface was ster- ilized with a NaC10 solution (10 g L - t ) and washed 5 times with sterile distilled water. Before germination, 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 experiment. 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 capacity (WHC) with the corresponding solution in each case, and kept at 100% WHC with Crone medium; nitrogen-free Crone was used for N-plants and nitrogen-supplemented Crone for NN plants. Vases were covered with vented plastic paper and incubated 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.

Growth measurements

After 25 days of incubation, plants were taken from the vases and vermiculite was carefully removed from

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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 processing

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 measurements). 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 compared with each other and with controls by unidirec- tional ANOVAs for each parameter assessed; when significant differences existed, an LSD test was performed (Sokal and Rholf, 1979).

Results

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 medium from 7 Pseudomonas and the 3 Bacillus strains. As shown by the PCA, the culture medium concentration 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

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Figure I. SDS-PAGE electrophoresis of Ahus glutirwso seeds reserue proteins, M: Proteins marker, Ovoalbumine v fraction (65 Kd)

AA

% 4

-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

0 5.8

Oc x0.

NLOA6

h8 l 8 0. 0. l o

0 0 Oo 0

0 0

0

I.9

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

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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<0.05 according to LSD.

development and the inhibitory effect of the 7 Pseu- domonas strains (group A on PCA). The three Bacillus strains assayed (B.3, B. 12 and B.21) caused significant (p<0.05) increases in all the parameters measured, both in N and NN plants, although the effects were more marked in N plants. Nevertheless, the inhibition caused by the 7 Pseudomonas strains is more marked in NN plants, in which these Pseudomonas determine significant differences between treatments (10 and 20%) and control in all biometrical parameters. However, in N plants, non significant differences were detected for NL and only strains P9, PI 0 and PI 1 caused significant variations on AS.

The characterization tests mentioned before lead us to identify the three Bacillus strains as B. licheniformis (B.3 and B.21) and B. pumilus (B.12). All the Pseu- domonas strains were identified as P fluorescens bv.II. The last biochemical tests for bacteria characterization at a species level, used the DNA polymerase chain reaction and RAPD-markers in order to detect genetic variation among strains of the same species. All Kit-B primers were assayed: 1 out of 5 gave good banding patterns. The clearest banding patterns corresponded to primers 3 and 13 for Bacillus and 4 and 17 for Pseu- domonas (Fig. 3). Six of the seven P. fluorescens had genetic homogeneity, only the E20 strain showed a different banding pattern. The two B. lichenformis strains had identical genetic fingerprints. However, there were differences between the type strains and those from the same species isolated from the rhizosphere (Fig. 3).

It should be noted that the three Bacillus strains were able to grow in the nitrogen-free medium and were able to reduce acetylene to ethylene while this activity was not detected in any of the Pseudomonas tested.

Discussion

The analysis of reserve proteins shows no internal variation in the plant population studied. Therefore, growth alterations must be caused by culture medium and can only be due to bacterial strains. This prior test is of basic importance in this type of study since different genotypes can give a different answer to the same external stimulus, such as inoculation with bacterial strains (Astr0m and Gerhardson, 1988).

Deleterious effects caused by the Pseudomonas growth medium are more marked in NN plants. These differences reflect the greater physiological resistance of N plants and the dificulties NN plants have for nitrogen uptake, as shown by the symptoms of slight leaf chlorosis.

There are several studies that consider several Pseudomonas strains and species as Deleterious Rhi- zobacteria (DRB) (Suslow and Schroth, 1982; Schip- pers et al., 1986). In some cases, this deleterious effect is caused by the HCN these strains release (Schip- pers et al., 1991). These compounds affect the res- piratory metabolism and inhibit cytochrome oxidase

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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)promoted 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 nitrogen 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 aerobic 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 licheniformis 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.

Acknowledgements

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.

References

Acero N, Probanza A, Blanco B and Gutienez Mafiero J 1994 Sea- sonal changes in physiological groups of bacteria that participate in nitrogen cycle in the rhizosphere of alder. Geomicrobiol. J. 11, 133-140.

Astrrm B and Gerhardson B 1988 Differential reactions of wheat and pea genotypes to root inoculation with growth-affecting rhizosphere bacteria. Plant and Soil 109,263-269.

Atkinson W A and Hamilton W I 1978 The value of red alder as a source of nitrogen in Douglas-fir/alder mixed stands. In Utilization and Mangementofalder. Eds. D G Griggs, D S DeBell and W A Atkinson. pp 337-351, USDA for Serv.Gen.Tech.Rep. PNW-70.

Bermtidez de Castro F 1981 Diazatrofia como trcnica en agronomfa y silvicultura. In Productividad Vegetal. Ed. C Vicente. pp 191- 225. Univ. Complutense, Madrid, Spain.

Brown M E 1972 Plant growth substances produced by microorganisms of soil and rhizosphere. J. Appl. Bact. 35, 443451.

Burr T J and Caesar A 1984 Beneficial plant bacteria. Crit. Rev. Plant Sci. 2, 1-20.

Carruthers F L, Shum-Thomas T, Conner A J and Mahanty H K 1995 The significance of antibiotic production by Pseudomonas auret~fiwiens PA 147-2 for biological control of Phytophthoru megasperma root rot of asparagus. Plant and Soil 170, 339-344.

Chanway C P, Nelson L M and Holl F B 1988 Cultivar-specific growth promotion of spring wheat (Triticum aestivum L.) by coexistant Bacillus species. Can. J. Microbiol. 34, 925-929.

Claus D and Berkley R C W 1989 Genus Bacillus. In Bergey's Man- ual of Systematic Bacteriology, Vol. 1. Eds. R N Krieg and J G Holt. pp I 105-1139. Williams and Wilkins Company, Baltimore, USA.

Curl E A and Truelove B 1986 The Rhizosphere. Advances series in Agricultural Sciences, 15. Eds. D F R Bommer, B R Sabey, Y Vaadia, G W Thomas and L D Van Vleck. Springer-Verlag, Berlin, Germany. 80 p.

Derylo M and Skorpuska A 1993 Enhacement of symbiotic nitrogen fixation by vitamin-secreting fluorescentPseudomonas. Plant and Soil 154, 211-217.

Dowling D N and O'Gara F 1995 Metabolites of Pseudomonas involved in the biocontrol of plant disease. Trends Biotechnol. 12, 133-141.

65

Frankenberger W T and Arshad J R M 1995 Biological active substances in soil. In Phythormones in Soils. Marcel Dekker, Inc. New York, USA. 503 p.

Frederickson J K and Elliot L F 1985 Effects of winter wheat seedling growth by toxin-producingrhizobacteria. Plant and Soil 83,399- 4O5.

Freitas J R, Gupta V V S R, and Germida J J 1993 Influence of Pseudomonassyringae R25 and P putida R 105 on the growth and N2 fixation (acetylene reduction activity) of pea (Pisum sativum L.) and field bean (Phaseolus vulgaris L.). Biol. Fertil. Soils 16, 215-220.

Gilbert G S, Parke J L, Clayton M K, and Handelsman J 1993 Effects of an introduced bacterium on bacterial communities on roots. Ecology 74, 840-854.

Gordon J C 1978 Biological components of alder yield improvemenL In Utilization and Management of Alder. Eds. D G Griggs, DS DeBell and W A Atkinson. USDA Forest Service Gen Tech Rep PNW-70, 321-325.

Grant D and Binkley D 1987 Rates of free-living nitrogen fixation in some piedemont forest types. For. Sci. 32, 548-551.

Griffin G I 1990 Importance of Pythum ultimum in a disease syn- drome of cv. Essex soybean. Am. J. Plant Pathol. 12, 135-140.

Halverson L J and Handelsman J 1991 Enhancement of soybean nodulatin by Bacillus cereus W85 in field and growth chamber. Appl. Environ. Microbiol. 57, 713-718.

Harman J H 1967 Modem Factor Analysis Univ. Chicago Press. Chicago, 133 p.

Harrington C A 1984 Red alder: an American wood. USDA For Serv Publ. FS-215.

Homann P S, Van Miegroet H, Cole D W and Wolfe G V 1992 Cation distribution, cycling and removal from mineral soil in Douglas-fir and red alder forests. Biogeochemistry 16, 121-150.

Kampert M and Strzelczyk E 1984 Effect of pH on production of citokinin-like substances by bacteria isolated from soil, rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.). Acta Microbiol. Pol. 33, 77-85.

Kloepper J W, Schroth M N and Miller T D 1980 Effects of rhizosphere colonization by plant rowth-promoting rhizobacteria on potato plant development and yield. Phytopatol. 70, 1078-1082.

Liu L, Kloepper J W and Tuzun S 1995 Induction of systemic resistance in cucumber by plant growth promoting rhizobacteria: d ura- tion of protection and effect of host resistance on protection and root colonization. Phytopathology 85, 1064-1068.

Lynch J M 1990 The Rhizosphere. John Willey and Sons, Chichester, UK. 458 p.

Noller E C and Hartsell S E 1961 Bacteriolysis of Enterobactefi- aceae. J. Bacteriol. 81,492-499.

Palleroni N J 1989 Family Pseudomonadaceae. In Bergey's Manual of Systematic Bacteriology, Vol. 1. Eds. R N Krieg and J G Holt. pp I 105-1139. Williams and Wilkins Company, Baltimore, USA.

Payane J M, Cornfield K G, Holt L M and Blackman J A 1981 High molecular-weight subunits of glutenin and bread making quality in pathogeny aF six crosses of bread whear. J. Sci. Food Agric. 32, 51-60.

Pochon J and Tardieux P 1962 Techniques d'analyse en microbiolo- gie du sol. Editions de la Tourelle, Saint Mandr, Siene, France.

Rytter L 1995 Effects of thinning on the obtainable biomass, stand density and tree diameters of intensively grown grey alder plan- tations. For. Ecol. Manag. 73, 135-143.

Schippers B, Bakker A W, Bakker, P A H M, Weisbeck P J and Lugtenberg B J 1986 Plant growing-inhibiting and stimulating rhizosphere microorganisms. In Microbial Communities in Soil. Ed. V Jensen, A Kjoler and L H Sorensen. pp 447-468. Elsevier Appl. Publishers, London, UK.

66

Schippers B, Bakker P A H M, Bakker A W and Peer R 1991 Ben- eficial and deleterious effects of HCN-producing Pseudomonas on rhizosphere interactions. Plant and Soil 129, 76-83.

Selvadurai E L, Brown E A and Hamilton J T G 1991 Production of indole-3-acetic acid analoges by strains of Bacillus cereus in relation to their influence on seedling development. Soil Biol. Biochem. 23, 401-403.

Shishido M, Loeb B M and Chanway C P 1995 External and internal root colonization of lodgepole pine seedlings by two growth-promoting Bacillus strains originated from different root microsites. Can. J. Microbiol. 41,707-713.

Smith M R 1980 A phenol-hypochlorite manual determination of ammonium-nitrogen in Kjeldhal digests ofplanttissue. Soil Sci. Plant. Anal 11,709-722.

Sokal R R and RohlfF J 1979 Biometrie. Ed. H Blume, Barcelona, Spain. 832 p.

Solomonsson L P 1981 Cyanide as a metabolite inhibitor. In Cyanide in Biology. Ed. B Benesland. pp 11-28. Academic Press, London, UK.

Suslow T V and Schroth M N 1 982 Rhizobacteria of sugar beets: effects of seed application and root colonization on yield. Phy- topatology 73, 13-22.

Section editor: R 0 D Dixon