Bacillus amyloliquefaciens BNM122, a potential microbial biocontrol agent applied on soybean seeds, causes a minor impact on rhizosphere and soil microbial communities

applied soil ecology 41 (2009) 185–194

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Bacillus amyloliquefaciens BNM122, a potential microbial biocontrol agent applied on soybean seeds, causes a minor impact on rhizosphere and soil microbial communities Olga S. Correa a,*, Marcela S. Montecchia a,b, Marı´a F. Berti a, Marı´a C. Ferna´ndez Ferrari a, Norma L. Pucheu b, Norma L. Kerber b, Augusto F. Garcı´a b a

Ca´tedra de Microbiologı´a Agrı´cola, Facultad de Agronomı´a, Universidad de Buenos Aires, Av. San Martı´n 4453 (C1417DSE), Ciudad Auto´noma de Buenos Aires, Argentina b Instituto de Investigaciones Bioquı´micas y Fisiolo´gicas (IBYF-CONICET), Av. San Martı´n 4453 (C1417DSE), Ciudad Auto´noma de Buenos Aires, Argentina

article info

abstract

Article history:

The increase in soybean productivity has contributed to a greater use of agrochemicals,

Received 31 March 2008

which cause major problems, such as soil and water pollution and reduction of biodiversity,

Received in revised form

and have a negative impact on non-target species. The development of microbial biocontrol

2 October 2008

agents for soybean diseases can help to reduce pesticide abuse. Bacillus amyloliquefaciens

Accepted 18 October 2008

BNM122 is a potential microbial biocontrol agent able to control the damping-off caused by Rhizoctonia solani when inoculated in soybean seeds, both in a plant growth chamber and in a greenhouse. In this study, we report the effect of soybean seed treatments with strain

Keywords:

BNM122 or with two fungicides (thiram and carbendazim) on the structure and function of

Soil microbial community

the bacterial community that colonizes the soybean rhizosphere. Also, soybean root

r- and K-strategists

nodulation by Bradyrhizobium japonicum, mycorrhization by arbuscular mycorrhizal fungi

Community-level physiological

and plant growth were evaluated. We used the r- and K-strategist concept to evaluate the

profiles

ecophysiological structure of the culturable bacterial community, community-level phy-

Denaturing gradient gel

siological profiles (CLPP) in BiologTM EcoPlates to study bacterial functionality, and the

electrophoresis

patterns of 16S RNA genes amplified by PCR and separated by denaturing gradient gel

Soybean nodulation

electrophoresis (PCR–DGGE) to assess the genetic structure of the bacterial community.

Arbuscular mycorrhizal fungi

Neither the ecophysiological structure nor the physiological profiles of the soybean rhizosphere bacterial community showed important changes after seed inoculation with strain BNM122. On the contrary, seed treatment with fungicides increased the proportions of rstrategists and altered the metabolic profiles of the rhizosphere culturable bacterial community. The genetic structure of the rhizosphere bacterial community did not show perceptible changes between treated and non-treated seeds. Regarding the bacterial and fungal symbioses, seed treatments did not affect soybean nodulation, whereas soybean mycorrhization significantly decreased (P < 0.05) in plants obtained from seeds treated with strain BNM122 or with the fungicides. However, a higher negative effect was observed in plants which seeds were treated with the fungicides. Plant growth was not affected by seed treatments.

* Corresponding author. Tel.: +54 11 4524 8061; fax: +54 11 4514 8741. E-mail address: [email protected] (O.S. Correa). 0929-1393/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2008.10.007

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It can be concluded that soybean seed treatment with B. amyloliquefaciens BNM122 had a lesser effect on soil microbial community than that with the fungicides, and that these differences may be attributed to the less environmental persistence and toxic effects of the strain, which deserve further studies in order to develop commercial formulations. # 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Diseases, pests and weeds are the most important biotic factors that limit soybean yields in Argentina (Satorre, 2003). Annually, around 10% of the production loss is attributed to diseases that represent between 2.8 and 3.5 millions of tons (Andrade and Sadras, 2002). As a result, the increase in soybean productivity also contributes to a greater use of agrochemicals. It is known that in Argentina 32% of the products marketed by the agrochemical companies are used in soybean crops (SAGPyA, 2001). Thus, in this scenario, the indiscriminate use of agrochemicals represents a risk to sustainability due to soil and water contamination, air quality deterioration through volatilization of active products, and the negative impact caused on the biodiversity because of their ability to also affect non-target species (Viglizzo et al., 2002). In this respect, it is desirable to consider other alternatives to reduce the negative impact of agrochemical abuse. The most important soil-borne pathogens that affect soybean seedling emergence and vegetative growth are fungi. Rhizoctonia solani is one of the main fungal pathogens that can cause pre- and post-emergence damping-off, root rot, hypocotyl lesions and a reduction in soybean yield as much as 48% in small plots (Bradley et al., 2001). Damage caused by Rhizoctonia is commonly observed in areas where there is a long history of soybean production or during weather conditions not favorable for fast seed germination and rapid growth of seedlings (Dorrance et al., 2003), as it is frequently the case in soybean fields in Argentina. Argentinean farmers are used to inoculating soybean seeds with commercial inoculants based on Bradyrhizobium japonicum in order to take advantage of biological nitrogen fixation. In addition to the bacterial inoculant, two fungicides (carbendazim and thiram) are also applied on soybean seeds to improve emergence of seeds and protect seedlings from soil-borne fungi (Perticari et al., 2003). Soybean roots also develop a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which results in a key association for the supply of phosphorus (P) in soils with low P availability. In fact, P deficiency is one of the main factors that limit soybean productivity in the agricultural systems of Argentina (Garcı´a, 2001). Seed treatment with fungicides may negatively affect these two symbioses and thus determine a decrease in soybean yield. Early reports have shown that there are differences in the effect of different fungicides on the soybean–rhizobium symbiosis. Captan and carboxin applied on soybean seeds severely reduce the survival of rhizobia and the number of nodules as compared to inoculated seeds without fungicide, whereas thiram and pentachloronitrobenzene (PCNB) did not have any effects (Chamber and Montes, 1982; Mallik and Tesfai, 1985; Tesfai and Mallik, 1986). These effects have been attributed to differences in fungicide tolerance between rhizobial strains

(Ramos and Ribeiro, 1993). Also, interactions of AM fungi and fungicides are known to depend on fungus-fungicide combinations and on environmental conditions. It has been previously demonstrated that benomyl and PCNB are more toxic to AM fungi than captan, reducing sporulation, hyphal growth and root colonization in Pisum sativum (Schreiner et al., 1997). Less information exists in the literature about the effects of seed fungicides on AM symbiosis in soybean crops. Several studies have reported the use of microbial biocontrol agents (MBCA) as an environmentally friendly alternative to reduce or replace the chemical control of plant diseases (Emmert and Handelsman, 1999; Compant et al., 2005). However, the addition of large numbers of microorganisms on seeds or directly into the soil, as it is needed for an effective disease biocontrol, can also have non-desirable effects on other organisms including the host plant that the MBCA is supposed to protect. In this sense, it is important to study the effects of an introduced MBCA on non-target organisms before considering its extensive use. In Argentina, there are few reports concerning microbial biocontrol of plant diseases (Cavaglieri et al., 2005; Prı´ncipe et al., 2007), although no commercial product has been registered yet. One possible reason for that is the lack of connection between the research in the laboratory and its application in the field. Also, it is necessary to develop policies concerning the safe release of large populations of microorganisms into the environment and a faster register of commercial products. In our laboratory, we isolated, identified and functionally and genetically characterized the BNM122 strain of Bacillus amyloliquefaciens and proposed it as a potential MBCA. This strain excretes metabolites with antifungal activity that have been partially identified as surfactin and iturin-like compounds (Souto et al., 2004). Strain BNM122 showed in vitro antifungal activity against R. solani, Fusarium oxysporum and Sclerotinia sclerotiorum and in vivo, the coating of soybean seeds with strain BNM122 had a protective effect against the damping-off produced by R. solani. In greenhouse, a compost-based formulation with BNM122 applied onto soybean seeds also increases significantly the mean plant stand per pot when compared with the pathogen check, in addition, biocontrol treatment is as effective in controlling R. solani damping-off in soybean as the application of the fungicide PCNB in the soil (Souto et al., 2004). In order to continue with our studies, the aim of this work was to determine whether the coating of soybean seeds with BNM122 strain alters the structure and function of the microbial communities associated with roots, as well as to compare and contrast BNM122 treatment and the common seed treatment with two fungicides. We characterized the structural changes in the bacterial community by applying the concept of r- and K-strategists (De Leij et al., 1993). The functional attributes of the microbial communities were

applied soil ecology 41 (2009) 185–194

characterized by the community-level physiological profiles (CLPP) using BiologTM EcoPlates (Insam, 1997). Changes in the genetic structure of bacterial community were evaluated by the patterns of 16S RNA genes amplified by PCR and separated in denaturing gradient gel electrophoresis (PCR–DGGE) (Heuer et al., 1997). Also, we evaluated the effects of both treatments on root nodulation by B. japonicum, mycorrhization by AM fungi and vegetative growth of soybean plants.

2.

Material and methods

2.1.

Bacterial strain and culture conditions

B. amyloliquefaciens BNM122 was isolated from a sclerotium of S. sclerotiorum withdrawn from a sunflower (Heliantus annus L.) capitulum as described in Souto et al. (2004). Strain BNM122 was grown at 28 8C for 72 h in nutrient broth (peptone 5 g l1, meat extract 3 g l1) on a rotary shaker (150 rpm). Cells were harvested by centrifugation (10,000  g for 10 min) to remove the culture medium and the bacterial pellet was resuspended in sterile saline solution (SSS: 0.9% NaCl) at 3  108 colonyforming units (cfu) ml1.

2.2.

Soybean seed inoculation

Twenty soybean seeds were submerged in 20 ml of the above cell suspension of BNM122 (control seeds were submerged in 20 ml of SSS) in 125-ml Erlenmeyer flasks. Three flasks with inoculated seeds and three flasks with control seeds were incubated for 2 h in a rotary shaker (80 rpm, 25 8C) for allowing bacterial cells to adhere to seeds. After incubation, excess of inoculum was removed onto sterile filter paper and seeds were immediately sowed in sterile vermiculite or soil (for colonization assays or for studying the impact of the BNM122 strain on microbial community, respectively).

2.3. Seed and root colonization assays in sterile vermiculite. The experiments were carried out according to the procedure of Simon et al. (2001). Both inoculated and non-inoculated seeds (one seed per tube, tubes 30 cm in height, 4 cm in diameter) grew in a growth chamber at 25 8C with a 16/8 h light/dark period and seedlings were watered twice a week with 1/4 Hoagland’s solution. In order to study the colonization of seeds by strain BNM122, three soybean varieties were used: (i) Nidera A4910RG, (ii) Don Mario 4800 and (iii) Asgrow 4500. Five seeds of each soybean variety, either noninoculated or inoculated with BNM122, were collected for each sampling time. Seeds were suspended in 5 ml of 10 mM sterile phosphate buffer (pH 7.0) and sonicated in an ultrasonic bath (Grant XB2) to release adhering bacteria, and then serial dilutions (1/10) were plated on nutrient agar (NA). Petri dishes were incubated for 5 days at 28 8C and the number of cfu per seed was determined at inoculation time (0 h) and after 24 h, 48 h and 5 days from inoculation time. Colonization of soybean rhizosphere by BNM122 was only evaluated in Nidera A4910RG variety. Fifteen and thirty days after sowing (DAS) five seedlings of each treatment were withdrawn, and roots

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were separated from shoots and vermiculite and submerged in 10 ml of SSS and bath-sonicated to remove the adhering bacteria. Serial dilutions (1/10) in SSS were plated on NA and incubation and cfu counting were as above. Data were logtransformed and analyzed by ANOVA. Two experiments were run under the same conditions.

2.4.

Greenhouse experiment

Soil (Cox 1.97%, total N 0.23%, P 60 ppm, Ca 13.25 cmolc kg1, Mg 2.4 cmolc kg1, Na 0.56 cmolc kg1, K 1.57 cmolc kg1, pH 5.6, electrical conductivity 0.78 mmhos cm1, cationic exchange capacity 22 cmolc kg1) was collected from the upper 20 cm of a grassland field, being wheat the last crop five years before this experiment. The sampled soil was mixed thoroughly and stored in plastic bags at 10 8C for 2 months. After that, the soil was sieved (2 mm) and placed in 5-l plastic pots, 20 cm height. AM fungi, multiplied on Trifolium repens, were applied to each pot (50 g of a mixture of 50% Glomus intraradices and 50% Gigaspora rosea) before sowing the seeds. All soybean seeds (Nidera A4910RG) were inoculated with a liquid commercial inoculant of B. japonicum USDA 138 strain (2  108 cfu ml1) following the manufacturer’s instructions. There were three treatments: (i) seeds non-inoculated with BNM122 (control), (ii) seeds inoculated with BNM122 and (iii) seeds treated with the fungicides thiram (35%) and carbendazim (15%) at doses recommended by the manufacturer. Six seeds were sowed per pot and, after germination, four seedlings were left in each pot. Pots were watered with tap water in order to maintain the soil at 60% of field capacity. After sixty DAS, soil samples were removed from the rhizosphere of plants growing in pots with a 20 mm  200 mm core borer. We established that due to the large radical system developed by plants, no distinction could be made between rhizosphere and bulk soil. Soil without roots was sieved (2 mm) and sub-samples were used for DNA extraction and the other studies. To estimate the different components of the cultivable bacterial community, 10 g of soil samples were used to prepare ten-fold serial dilutions in SSS. Appropriate dilutions were plated (100 ml) onto 0.1-strength tryptic soy broth (TSB, Merck) with 1.5% of agar (TSA) plus cycloheximide (100 mg ml1). Plates were incubated at 28 8C for 10 days. r-strategists or ‘‘fast growers’’ were defined as bacteria that produced visible colonies at 28 8C onto 0.1 TSA within 2 days and K-strategists or ‘‘slow growers’’ as those that grew after that. Soil cores plus roots sampled with the 20 cm coring device were kept in plastic bags at 10 8C for mycorrhizal determinations.

2.5.

Analyses

2.5.1.

Bacterial community structure

Bacterial colonies appearing on 0.1 TSA were enumerated on a daily basis for 6 consecutive days and then on days 8 and 10, in this way, eight counts (or classes) were obtained per plate. Plates with 30–150 colonies were selected for enumeration and colonies that were visible at low magnification (6) were marked. The data obtained from the counting were used to calculate two indices: the colony development index (CD) used for

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bacterial colonies by Kozdro´j et al. (2004) and the ecophysiological (EP) index (Shannon’s diversity index) as described by De Leij et al. (1993). The CD was calculated as follows: CD ¼

  N1 N2 N3 N4 N5 N6 N8 N10 þ þ þ þ þ þ þ  100 1 2 3 4 5 6 8 10

N represents the proportions of bacterial colonies appearing the different days (1–10). A high CD value indicates a greater proportion of r-strategists, with a CD varying from 50 (if all colonies appear on day 2) to 10 (if they appear on day 10). The EP index was calculated as follows: EP ¼ 

X ðPi  log10 Pi Þ

Pi is the proportion of bacterial colonies appearing on counting day i (i = 1, 2, up to10). The more even the distribution of the colony classes, the higher the EP index, with EPmax = 0.9 and EPmin = 0.

2.5.2.

Bacterial community functionality

Suspensions of 10 g of rhizosphere soil in sterilized distilled water (SDW) were left 18 h at room temperature according to Go´mez et al. (2004). After that, serial dilutions (1/10) were performed in SSS and 150 ml of the 104 dilution was loaded in BiologTM EcoPlates. Five independent replicates of each treatment were analyzed. Microplates were incubated at 28 8C for 6 days and the absorbance at 590 nm was recorded every 12 h with a Multiskcan1 EX (Thermo). Richness was calculated as the number of oxidized C substrates, and the Shannon–Weaver P index (H: Diversity) was calculated as follows: H =  pi(ln pi), where pi is the ratio of the activity on each substrate (Ai) to the P sum of activities of all substrates ( Ai). Evenness was: H/ln S, where S is substrate richness. Carbon substrates were grouped in six guilds as suggested by Dobranic and Zak (1999).

2.5.3. Soybean root nodulation, mycorrhization and plant growth Nine plants per treatment were sampled at sixty DAS (R2 phenological stage) and the nodules counted in main and secondary roots. Then, plants were placed at 70 8C for 5 days to determine the dry weight of shoots and roots. For mycorrhizal determinations roots were separated from the soil, washed with tap water, cleared with 10% KOH and stained with 0.05% trypan blue as described by Phillips and Hayman (1970). Mycorrhizal colonization was estimated as described by Trouvelot et al. (1986) for two parameters: M% intensity of the mycorrhizal colonization and A% arbuscule abundance, both in the root system. Eight plants per treatment and thirty root fragments (1.0 cm each) per plant were analyzed.

2.5.4. DNA extraction and DGGE analysis of amplified 16S rRNA gene fragments Total DNA was extracted from 0.25 g of rhizosphere soil samples (three independent replicates per treatment) using the Power Soil DNA isolation kit (Mo Bio Laboratories, Inc.). The yield of purified DNA was checked by agarose gel electrophoresis (1%, w/v, agarose in Tris-borate–EDTA buffer) as well as in a spectrophotometer at 260 nm (GeneQuant

DNA/RNA calculator, Pharmacia Biotech). Hot-start PCR was performed in a 50-ml reaction mixture containing 20 ng of soil DNA, 1.5 mM MgCl2, 5% DMSO, 0.25 mM of each primer (GCF984 and R1378) (Heuer et al., 1997), 0.2 mM of each dNTPs, 2.5 U Platinum Taq DNA Polymerase (Invitrogen) and the buffer (1) provided with the enzyme. Amplification was carried out in an MJ Research PTC-100 thermocycler with the following temperature program: 5 min at 95 8C, 35 cycles consisting of 1 min at 94 8C, 1 min at 55 8C, and 2 min at 72 8C, and finally 30 min at 72 8C. The amplification product was checked by agarose gel electrophoresis. DGGE analysis was performed as previously described (Heuer et al., 1997) but with a double gradient polyacrylamide gel containing 6–9% acrylamide with a denaturing gradient of 40–60%. The additional acrylamide gradient was used to enhance the band’s resolution and sharpness (Gomes et al., 2005). The gels were run 1600 V/h in Tris-acetate–EDTA buffer at 60 8C, stained with SYBR Green I nucleic acid stain (Molecular probes) and photographed under UV light with a EDAS120 (Kodak). Comparative analysis of DGGE profiles was performed with GelCompar II v. 3.0 (Applied Maths, Belgium) using Pearson’s product–moment correlation analysis to calculate pairwise similarity coefficients among pattern densitometric profiles and similarity matrices were clustered using the unweighted pair group method with averages (UPGMA) algorithm (Sneath and Sokal, 1973).

2.5.5.

Statistical analysis

Data of seed and rhizosphere colonization, EP and CD indices, nodulation and mycorrhization were analyzed by means of ANOVA procedures and means were compared by the Tukey’s test. (STATISTICA for Windows, v. 5.5). Data regarding colonization and nodulation were log10 transformed before being analyzed by ANOVA. CLPP were analyzed by principal components (PCA) using PC-ORD software, v. 4 and data from the principal components (PC1 and PC2) were analyzed by ANOVA. Richness, evenness and diversity indexes were analyzed by ANOVA. All ANOVA and Tukey tests were performed at a 0.05 significance level.

3.

Results

3.1. Soybean seed and rhizosphere colonization by B. amyloliquefaciens BNM122 strain Seeds of three soybean varieties were inoculated with a culture of strain BNM122 and the number of viable cells per seed was determined at different times after inoculation. Table 1 shows that strain BNM122 colonized soybean seeds and no differences (P > 0.05) were observed in the number of bacteria per seed between soybean varieties. We found that the number of inoculated bacteria increased in approximately three magnitude orders, from 0 to 48 h. Thus, the average value of cfu per seed for the three soybean varieties increased from 3.0  104 to 1.1  107 from 0 to 48 h, respectively, and after that, the cfu per seed remained unchanged up to 5 days post inoculation (Table 1). Having established that strain BNM122 colonized soybean seeds without showing differences between plant varieties,

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Table 1 – Seed and rhizosphere colonization of soybean (Glycine max [L.] Merr.) varieties by Bacillus amyloliquefaciens strain BNM122. Seed colonizationa

Soybean varieties 0h Asgrow 4500 Don Mario 4800 Nidera A4910RG

4

2.0  10 Aa 1.2  104 Aa 5.5  104 Aa

Rhizosphere colonizationb

24 h

48 h

120 h

5

7

6

4.0  10 Bb 9.0  104 Bb 1.3  105 Ba

1.1  10 Cc 1.4  107 Cc 8.9  106 Cc

8.5  10 Cc 1.1  107 Cc 2.0  107 Cc

15 DAS ND ND 1.3  106 a

30 DAS ND ND 4.3  106 b

Data are from one representative experiment. Values are the mean of five replicates. Different capital letters indicate significant differences (P < 0.05) between soybean varieties (columns). Different lower case letters indicate significant differences (P < 0.05) between sampling times (rows). ND: not determined. DAS: days after sowing. a Data are expressed as cfu per seed. b Data are expressed as cfu g1 of root dry weight.

we next examined the ability of this bacterial strain to colonize the rhizosphere of soybean Nidera A4910RG variety. Table 1 shows that strain BNM122 was able to colonize the rhizosphere of soybean and the data revealed that the number of bacterial cells increased (P < 0.05) from 15 to 30 DAS (1.3  106 and 4.3  106 cfu g1 of root dry weight, respectively).

3.2.

Impact on rhizosphere microorganisms

3.2.1.

Ecophysiological structure of the bacterial community

The culturable bacterial community obtained from the rhizosphere of soybean plants was characterized by means of the percentage of colonies appearing on 0.1 TSA plates over a period of 10 days. Fig. 1 shows that the bacterial community in both control and treated plants was dominated by slow growing bacteria (K-strategists), those that formed colonies after 2 days on 0.1 TSA medium (71% for control plants and those treated with BNM122 and 65% for plants treated with fungicides). However, the bacterial community of plants obtained from seeds treated with the two fungicides differed (P < 0.05) from the bacterial community of the other plants in the 8th counting day (15% for fungicide, 28% for BNM122 treated and 33% for control plants). Although the bacterial

community structure in the rhizosphere of treated plants changed toward faster growth bacteria (r-strategists), the changes were statistically significant only in the fungicidetreated plants (Fig. 1). The ecophysiological structure of the bacterial community associated with the soybean rhizosphere was studied by means of the strategist concept using the EP and CD indices. Table 2 shows that there was a significant decrease (P < 0.05) in the EP index of the rhizosphere bacterial community in plants treated with the fungicides (EP = 0.65), whereas no differences were observed between control plants and those inoculated with BNM122 strain (EP = 0.79 and 0.77 for control and BNM122 treatments, respectively). The EP index represents the evenness, which is the degree of similarity in abundance between the different colony classes, and is one of the components of the bacterial diversity index (diversity = richness + evenness). Thus, a decrease in the EP index indicates that seed treatment with the fungicides reduced the bacterial diversity on the soybean rhizosphere. Also, the CD index, which represents the predominance of r- or K-strategists, changed when the bacterial community of treated plants was compared with that of control plants (29.14, 30.83 and 35.90 for control, BNM122, and fungicide treatments, respectively). Both seed treatments determined an increase in the CD index, thus resulting that a higher proportion of r-strategists than of control plants colonized the rhizosphere of plants treated either with the BNM122 or with the two fungicides. Despite the increase observed between control and treated plants, the differences in the CD index were only significant (P < 0.05) between control and fungicide treated plants (Table 2).

Table 2 – Ecophysiological (EP) and colony development (CD) indices of culturable heterotrophic bacteria from the soybean rhizosphere as affected by seed treatments with B. amyloliquefaciens BNM122 or two fungicides (carbendazim + thiram). Fig. 1 – Percentage of culturable bacterial colony counts on 0.1 TSA medium appearing over a period of 10 days. Bacterial community was obtained from the soybean rhizosphere of control plants = non-treated with B. amyloliquefaciens BNM122 (&), B. amyloliquefaciens BNM122-treated (^) and fungicide-treated (~) plants. Data represent the means of six independent replicates and bars are the standard deviations (SD).

Treatments Control BNM122 Fungicides

EPa 0.79 a 0.77 a 0.65 b

CDa 29.14 a 30.83 ab 35.90 b

Different letters indicate significant differences (P < 0.05) between treatments. a Values are the mean of six independent replicates.

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Fig. 2 – Principal component analysis (PCA) of the catabolic profiles of the bacterial community obtained from the rhizosphere of control plants = non-treated with B. amyloliquefaciens BNM122 (&), B. amyloliquefaciens BNM122-treated (^) and two fungicide-treated (~) soybean plants. Data are the means of five independent replicates and bars represent the standard deviations (SD).

3.2.2.

Catabolic profiles of the bacterial community

Fig. 2 shows the PCA results of the functional characterization of the bacterial community associated with the rhizosphere of soybean plants, where PC1 accounted for 60% and PC2 accounted for 12% of the data variability. PCA revealed that the catabolic profiles of the bacterial community associated with the plants treated with the two fungicides were significantly different from those of control and BNM122inoculated plants only at PC2 level, while no differences were detected between control and BNM122-inoculated plants. Fig. 2 also shows that the catabolic profiles obtained from fungicide-treated plants showed the highest variability between the five independent replicates. Twenty-six out of thirty-one carbon sources in the microplates, belonging to six guilds, were utilized by some soybean microbial communities (seven carbohydrates, seven carboxylic acids, five amino acids, two amine/amide, two polymers and three miscellaneous). Substrates with high coordinate score in PC2, which allowed differentiation between treatments, were b-methyl-Dglucoside, i-erythritol, D-xylose, L-threonine, itaconic acid, and

Fig. 3 – DGGE fingerprints of PCR-amplified 16S rRNA gene fragments of the rhizosphere bacterial communities of soybean plants at 60 days after sowing. Control: rhizosphere bacterial community from soybean plants non-inoculated with B. amyloliquefaciens BNM122 strain; BNM122: rhizosphere bacterial community from soybean plants inoculated with B. amyloliquefaciens BNM122 strain; fungicides: rhizosphere bacterial community from soybean plants treated with carbendazim + thiram.

a-D-lactose (Table 3). When particular substrate guilds were analyzed, no significant differences were observed between treatments. However, there was a tendency to a higher use of carbohydrates (P = 0.06) by the rhizosphere bacterial community of soybean plants treated with fungicides and a higher use of amino acids (P = 0.06) by communities of control and inoculated plants. On the other hand, there were no significant differences (P > 0.05) between treatments in richness, evenness and diversity (H) indexes (data not shown).

3.2.3.

Genetic structure of the bacterial community

The DGGE analysis of PCR-amplified 16S rRNA gene fragments was performed in order to investigate differences in the structural composition of the bacterial communities associated with the soybean rhizosphere that resulted from the seed treatment with fungicide or seed inoculation with strain BNM122. The results of PCR–DGGE fingerprinting and cluster analysis based on these profiles using Pearson/UPGMA are shown in Fig. 3. Bacterial community profiles from the rhizosphere of

Table 3 – Carbon substrates utilized by microbial communities in Biolog EcoplatesTM, significantly correlated to PC1 and PC2. PC1 Carbon source D-Galactonic acid g-lactone N-acetyl-D-glucosamine L-Arginine L-Asparagine Phenylethylamine D-Malic acid Putrescine a

PC2 a

r

Carbon source

0.61 0.65 0.61 0.76 0.70 0.75 0.63

b-Methyl-D-glucoside Erythritol D-Xylose L-Threonine Itaconic acid a-D-Lactose

ra 0.61 0.68 0.62 0.84 0.63 0.60

Positive correlation indicates greater response in treatments with higher coordinate scores for the axis, negative correlation indicates greater utilization in treatment with lower coordinate scores for the axis (see Fig. 2).

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Table 4 – Nodulation, mycorrhization and vegetative growth of soybean plants whose seeds were either inoculated with B. amyloliquefaciens BNM122 or treated with two fungicides (carbendazim + thiram). Treatments

Number of nodules per plant

Mycorrhization

Dry weight (mg pl1)

Primary root

Secondary root

Intensity (%)

Abundance (%)

Shoot

Root

21.22 a 20.44 a 17.22 a

17.22 a 16.78 a 13.44 a

20.78 a 14.53 b 8.46 c

5.18 a 1.93 b 0.97 c

12.38 a 11.66 a 9.96 a

6.84 a 6.58 a 6.37 a

Control BNM122 Fungicides

Data are the mean of nine replicates for the number of nodules and dry weight of shoots and roots. Mycorrhization was determined in 8 plants, randomly chosen from 10 independent replicates. Different letters mean significant differences (P < 0.05) between treatments.

plants taken at 60 DAS were highly similar to each other (more than 80%), suggesting the presence of dominating bacterial types associated with the soybean rhizosphere.

4. Plant-microbial symbioses and vegetative growth of soybean plants The impact of seed treatments on the reduction of soil-borne fungal diseases in bacterial and fungal symbioses was assessed by evaluating the number of nodules and the presence of AM fungal structures on soybean roots, respectively. Seed treatments did not affect (P > 0.05) the nodulation of soybean plants by B. japonicum. Table 4 shows that the number of nodules in primary and secondary roots was similar in both control and treated plants. Neither fungicide nor BNM122 application on seeds had a negative effect on vegetative plant growth (Table 4). However, the intensity (M%) and abundance (A%) of structures involved in the symbiosis between soybean roots and AM fungi were reduced in fungicide- and BNM122treated plants in comparison with the values observed in control plants. Intensity was 20.78% for control, 14.53% for BNM122, and 8.46% for fungicide-treated plants and abundance was 5.18%, 1.93% and 0.97% for control, BNM122- and fungicide-treated plants, respectively.

5.

Discussion

The results obtained in this study showed the ability of B. amyloliquefaciens BNM122 strain to colonize seeds and roots when applied as a coating on soybean seeds. The capacity of strain BNM122 to colonize seeds did not show differences between the soybean cultivars used in the assays. On the contrary, Simon et al. (2001), working with inbred tomato lines, found that one line was supportive for a high and another for a low growth of B. cereus W85 in the spermosphere. It has been shown that spermosphere colonization is a key step for the establishment of Bacillus strains on just-emerging roots and also for their biocontrol activity (Reva et al., 2004). Although there are studies about seed and root colonization by Bacillus species and specifically with B. amyloliquefaciens strains (Reva et al., 2004; Pereira et al., 2007), less attention has been paid to colonization of different plant cultivars. However, it is known that changes in plant exudates are dependent on the genetic background of plants, and that these changes may negatively affect the performance of an

introduced bacterium (Rengel, 2002). Strain BNM122 also had the ability to colonize roots of soybean Nidera A4910RG and to maintain, at least under our experimental conditions, a high number of roots up to 30 days after inoculation. Germination and seedling establishment are the first critical steps in the plant life cycle, hence, a MBCA should maintain a high number on seeds and roots to protect seeds and young seedlings against soil-borne pathogenic fungi. In this respect, strain BNM122 fulfills those desirable properties. Furthermore, neither the eco-physiological structure nor the catabolic profiles nor the structure of the bacterial community resident on the soybean rhizosphere showed important changes after soybean seeds inoculation with strain BNM122 at 60 days after the treatment. These results are in agreement with previous studies that have shown that repetitive applications of Pseudomonas putida strain 06909rif/nal on citrus plants have a small impact on soil microbial communities (Steddom et al., 2002). Also, Kokalis-Burelle et al. (2006) established that treatments with Bacillus strains do not adversely affect beneficial indigenous rhizosphere bacteria, although the populations of fungi increase. In contrast, other authors have found that coating soybean seeds with B. cereus strain W85 causes dramatic changes in the bacterial community that develops on the roots (Gilbert et al., 1993). In the same direction, Walsh et al. (2003) have shown that previous biocontrol treatment of sugarbeet seeds with P. fluorescens strain F113 has a residual effect on soil resident red clover rhizobia. These discrepancies may be attributed to differences in bacterial species and strains used, inoculum concentration, applications to seeds or directly to soil, etc. All these results highlight the importance of establishing the conditions in which each experiment is performed before concluding about the effects of microbial inoculants on non-target microorganisms. Seed treatment with thiram and carbendazim had more significant effects on the structure of the culturable bacterial community that colonized soybean rhizosphere than seed treatment with strain BNM122. The shift towards a less diverse bacterial community mainly dominated by r-strategists may be due to the nutrients released by microbial cells sensitive to the fungicides. It is known that an increase in easy ready-to-use carbon sources determines an increase in the abundance of rstrategists and, at the same time, a decrease in K-strategists (Fierer et al., 2007). Also, van Elsas et al. (2007) have found that after soil fumigation with chloroform, the microbial EP index decreases due to temporarily enhanced availability of (carbonaceous) resources from lysed cells, resulting in an altered

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microbial community in which chloroform survivors abound (van Elsas et al., 2007). However, in that work, the increase in bacterial r-strategists was transitory, being evident at 7 days and followed by a decline at 60 days after treatment. In the present study, we determined a more persistent effect that may probably be attributed to both the active compounds and their metabolites. Other authors have also found a long-term (3 months) inhibitory effect of the fungicide mancozeb on aerobic N2-fixers and on nitrifying bacteria (Johnsen et al., 2001). Furthermore, Thirup et al. (2001) have shown that the fungicide fenpropimorph has an effect on culturable bacteria and also inhibits the growth of saprotrophic fungi. Physiological profiles analyzed by PCA showed differences in the positional relationship between community types only in PC2. This fact probably reflects a significant degree of similarity among rhizosphere communities in general, suggesting that both seed treatments had a small residual effect on these microbial communities, at least at 60 DAS. The rhizosphere DGGE profiles taken from plants at 60 DAS also showed very little variation between treatments, indicating that the structure of bacterial community was not affected by the fungicide or the bacterial treatment. On the light of these findings, one possible explanation for our results could be that fungicides induced different physiological states (phenotypes) in a genetically similar bacterial community. DGGE profiles are a display of the numerically dominant ribotypes, thus an additional interpretation could be that minor culturable bacteria not detected by DGGE but capable of metabolizing a specific carbon source were selected in the CLPP system. Discrepancies between data from culturable and nonculturable methods are frequently reported in literature (Thirup et al., 2001; Hadwin et al., 2006; Correa et al., 2007). The small differences we observed between treatments may also be attributed to that we sampled microbial communities at 60 DAS and analyzed one microbial compartment, the rhizosphere. Other authors have found that 45% thiram plus 20% carbendazim dressed on clover seeds either increases or decreases the number of Azotobacter and total bacteria depending on the time of analyses. The number of total bacteria decreases at three DAS but increases at plant flowering, while the number of Azotobacter increases at 14 DAS (Niewiadomska, 2004). Also, more complete information could be obtained if other microbial compartments such as rhizoplane and root tissues (endophytes) were analyzed (Germida and Siciliano, 2001; Mougel et al., 2006). However, our main objective was to establish whether soybean seed treatment with BNM122 has an important effect on soil microbial communities that would discourage its use as a biocontrol agent. We also studied the impact of seed treatments with strain BNM122 or fungicides on the two symbioses that soybean plants establish with B. japonicum and AM fungi. We performed the analyses before soybean plants completed their life cycle because we were interested in evaluating nodulation and mycorrhization, two symbioses whose functional activities and structures are known to decrease when plants undergo reproductive stages (Zhang et al., 1995). During the reproductive stage, there is strong C drain from roots to shoots, and the symbionts in the roots starve. Although nodulation and plant

growth were not significantly affected by the two treatments, there was an important reduction in the AM symbiosis. That negative effect on AM symbiosis may be attributed to the nonselective antifungal action of both strain BNM122 and fungicides. It has been demonstrated that strain BNM122 has an antagonistic action against various fungi that belong to different Divisions (Souto et al., 2004; Bachur, 2002) and also, against some bacteria (Souto, personal communication). On the other hand, thiram and carbendazim have a wide spectrum of action, affecting both phytopathogenic and non-phytopathogenic fungi as well as bacteria (Niewiadomska, 2004; Johnsen et al., 2001). It is worth pointing out that despite the important reduction in mycorrhization there was no decrease in plant growth. These results may be assigned to the normal N and the high P levels in the soil used, although what the results would be in soils under N and P limitations remains an open question. A reduction in nodulation and/or in mycorrhization under more restricted nutrient conditions may have a profound impact on soybean growth and yield. In this study, there were no obvious effects of strain BNM122 on soybean growth. However, other authors have found that co-inoculation of Bacillus strains with B. japonicum increases nodulation, plant growth, and grain yield in soybean crops (Bai et al., 2003). On the contrary, Kozdro´j et al. (2004) found that seed treatments with two Pseudomonas species, which showed antifungal activity, reduced maize shoot and root weights probably due to competence for nutrients. Fortunately, that was not the case with strain BNM122 in our experiment. The relevance of this work resides in the fact that the ecological impact on non-target microorganisms by this bacterial strain was compared with that triggered by the usual treatment for disease control, a comparison that is not frequently found in the literature. Besides, we considered the impact of seed treatments on two soybean symbioses whose reduction may negatively affect crop productivity depending on the levels of the soil nutrients available.

6.

Conclusion

In conclusion, the use of the BNM122 strain to control soil-borne fungal pathogens may be a promising ecological alternative to chemical treatments in order to develop a sustainable agricultural management to either replace or reduce agrochemical abuse in soybean crops in Argentina. Our results provide a basis for further studies with this strain concerning inoculantinduced ecological effects under field conditions and also its interaction with other members of the soil fungal community, for example beneficial antagonistic fungi.

Acknowledgements Authors acknowledge financial support from the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (PICT 0110892), the Centro Argentino-Brasilero de Biotecnologı´a (13AR07BR) and the Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´gicas (PIP 5003).

applied soil ecology 41 (2009) 185–194

references

Andrade, F., Sadras, V., 2002. Bases para el manejo del maı´z, el girasol y la soja. Producciones Gra´ficas Siria, 450 pp. (in Spanish). Bachur, M., 2002. Evaluacio´n de Bacillus sp. como agente de biocontrol de Rhizoctonia solani en soja. Undergraduate thesis. Facultad de Agronomı´a. Universidad de Buenos Aires, 68 pp. (in Spanish). Bai, Y., Zhou, X., Smith, D.L., 2003. Enhanced soybean plant growth resulting from coinoculation of Bacillus strains with Bradyrhizobium japonicum. Crop. Sci. 43, 1774–1781. Bradley, C.A., Hartman, G.L., Nelson, R.L., Mueller, D.S., Pedersen, W.L., 2001. Response of ancestral soybean lines and commercial cultivars to rhizoctonia root and hypocotyl rot. Plant Dis. 85, 1091–1095. Cavaglieri, L.R., Andre´s, L., Iba´n˜ez, M., Etcheverry, M.G., 2005. Rhizobacteria and their potential to control Fusarium verticillioides: effect of maize bacterisation and inoculum density. Antonie van Leeuwenhoek 87, 179–187. Chamber, M.A., Montes, F.J., 1982. Effects of some seeds disinfectants and methods of rhizobial inoculation on soybeans (Glycine max [L.] Merr.). Plant Soil 66, 353–360. Compant, S., Duffy, B., Nowak, J., Cle´ment, C., Barka, E.A., 2005. Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71, 49–51. Correa, O.S., Romero, A.M., Montecchia, M.S., Soria, M.A., 2007. Tomato genotype and Azospirillum inoculation modulate the changes in bacterial communities associated with roots and leaves. J. Appl. Microbiol. 102, 781–786. De Leij, F.A.A.M., Whipps, J.M., Lynch, J.M., 1993. The use of colony development for the characterization of communities in soil and roots. Microb. Ecol. 27, 81–97. Dobranic, J.K., Zak, J.C., 1999. A microtiter plate procedure for evaluating fungal functional diversity. Mycologia 91, 756– 765. Dorrance, A.E., Kleinhenz, M.D., McClure, S.A., Tuttle, N.T., 2003. Temperature, moisture, and seed treatment effects on Rhizoctonia solani root rot of soybean. Plant Dis. 87, 533–538. Emmert, E.A.B., Handelsman, J., 1999. Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiol. Lett. 171, 1–9. Fierer, N., Bradford, M.A., Jackson, R.B., 2007. Towards an ecological classification of soil bacteria. Ecology 88, 1354– 1364. Garcı´a F. 2001. Balance de fo´sforo en los suelos de la regio´n pampeana. Informaciones Agrono´micas del Cono Sur, 9:1-3. INPOFOS Cono Sur (in Spanish). Gilbert, G.S., Parke, J.L., Clayton, M.K., Handelsman, J., 1993. Effects of an introduced bacterium on bacterial communities on roots. Ecology 74, 840–854. Germida, J.J., Siciliano, S.D., 2001. Taxonomic diversity of bacteria associated with the roots of modern, recent and ancient wheat cultivars. Biol. Fertil. Soils 33, 410–415. Go´mez, E., Garland, J., Conti, M., 2004. Reproducibility in the response of soil bacterial community-level physiological profiles from a land use intensification gradient. Appl. Soil Ecol. 26, 21–30. Gomes, N.C.M., Kosheleva, I.A., Abraham, W.R., Smalla, K., 2005. Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community. FEMS Microbiol. Ecol. 54, 21–33. Hadwin, A.K.M., Del Rio, L.F., Pinto, L.J., Painter, M., Routledge, R., Moore, M.M., 2006. Microbial communities in wetlands of the Athabasca oil sands: genetic and metabolic characterization. FEMS Microb. Ecol. 55, 68–78.

193

Heuer, H., Krsek, M., Baker, P., Smalla, K., Wellington, E.M.H., 1997. Analysis of actinomycete communities by specific amplification of genes enconding 16S rRNA and gelelectrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 63, 3233–3241. Insam, H., 1997. A new set of substrates proposed for community characterization in environmental samples. In: Insam, H., Rangger, A. (Eds.), Microbial Communities. Functional Versus Structural Approaches. Springer Verlag, Heidelberg, pp. 260–261. Johnsen, K., Jacobsen, C., Torsvik, V., Sørensen, J., 2001. Pesticide effects on bacterial diversity in agricultural soils-a review. Biol. Fertil. Soils 33, 443–453. Kokalis-Burelle, N., Kloepper, J.W., Reddy, M.S., 2006. Plant growth-promoting rhizobacteria as transplant amendments and their effects on indigenous rhizosphere microorganisms. Appl. Soil Ecol. 31, 91–100. Kozdro´j, J., Trevors, J.T., van Elsas, J.D., 2004. Influence of introduced potential biocontrol agents on maize seedling growth and bacterial community structure in the rhizosphere. Soil Biol. Biochem. 36, 1775–1784. Mallik, M.A.B., Tesfai, K., 1985. Pesticidal effect on soybean– rhizobia symbiosis. Plant Soil 85, 33–41. Mougel, C., Offre, P., Ranjard, L., Corberand, T., Gamalero, E., Lemanceau, P., 2006. Dynamic of the genetic structure of bacterial and fungal communities at different developmental stages of Medicago truncatula Gaertn. cv. Jemalomg line J5. New Phytol. 170, 165–175. Niewiadomska, A., 2004. Effect of carbendazim, imazetapir and thiram on nitrogenase activity, the number of microorganisms in soil and yield of red clover (Trifolium pratense L.). Pol. J. Environ. Stud. 13, 403–410. Pereira, P., Nesci, A., Etcheverry, M., 2007. Efficacy of bacterial seed treatments for the control of Fusarium verticillioides in maize. Biocontrol 42, 281–287. Perticari, A., Arias, N., Baigorri, H., De Battista, J., Lett, L., Montecchia, M., Pacheco Basurco, J., Simonella, A., Toresani, S., Ventimiglia, L., Vicentini, R. 2003. Inoculacio´n y fijacio´n biolo´gica de nitro´geno en el cultivo de soja. Capı´tulo 7. El libro de la soja. SEMA, pp. 69–76 (in Spanish). Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesiculararbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br. Mycol. Soc. 55, 158–160. Prı´ncipe, A., Alvarez, F., Castro, M.G., Zachi, L., Fischer, S.E., Mori, G.B., Jofre´, E., 2007. Biocontrol and PGPR features in native strains isolated from saline soils of Argentina. Curr. Microbiol. 55, 314–322. Ramos, M.L.G., Ribeiro Jr., W.Q., 1993. Effect of fungicides on survival of Rhizobium on seeds and the nodulation of bean (Phaseolus vulgaris L.). Plant Soil 152, 145–150. Rengel, Z., 2002. Genetic control of root exudation. Plant Soil 245, 59–70. Reva, O.N., Dixelius, C., Meijer, J., Priest, F.G., 2004. Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis. FEMS Microbiol. Ecol. 48, 249–259. SAGPyA, 2001. Datos de produccio´n de la Secretarı´a de Agricultura, Ganaderı´a y Pesca de Argentina (in Spanish). Satorre, E. 2003. El libro de la soja. SEMA. 264 pp. (in Spanish). Schreiner, R.P., Mihara, K.L., McDaniel, H., Bethlenfalvay, G.J., 1997. Mycorrhizal fungi influence plant soil functions and interactions. Plant Soil 188, 199–209. Simon, H.M., Dodsworth, J.A., Guenthner, B., Handelsman, J., Goodman, R.M., 2001. Influence of tomato genotype on growth of inoculated and indigenous bacteria in the spermosphere. Appl. Environ. Microbiol. 67, 514–530. Sneath, P.H., Sokal, R.R., 1973. Numerical Taxonomy. W.H. Freeman and Co., San Francisco.

194

applied soil ecology 41 (2009) 185–194

Souto, G.I., Correa, O.S., Montecchia, M.S., Kerber, N.L., Pucheu, N.L., Bachur, M., Garcia, A.F., 2004. Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds. J. Appl. Microbiol. 97, 1247–1256. Steddom, K., Becker, O., Menge, J.A., 2002. Repetitive applications of the biocontrol agent Pseudomonas putida 06909-rif/nal and effects on populations of Phytophthora parasitica in Citrus Orchards. Phytopathology 92, 850–856. Tesfai, K., Mallik, M.A.B., 1986. Effect of fungicide application on soybean-rhizobia symbiosis and isolation of fungicideresistant strains of Rhizobium japonicum. Bull. Environ. Contam. Toxicol. 36, 819–826. Thirup, L., Johnsen, K., Torsvik, V., Spliid, N.H., Jacobsen, C.S., 2001. Effects of fenpropimorph on bacteria and fungi during decomposition of barley roots. Soil Biol. Biochem. 33, 1517–1524. Trouvelot, A., Kough, J.L., Gianinazzi-Pearson, V., 1986. Mesure du taux de mycorhization VA d’un systeme radiculaire. Recherche de methodes d’estimation ayant une

signification fonctionnelle. In: Gianinazzi-Pearson, V., Gianinazzi, S. (Eds.), Physiological and Genetical Aspects of Mycorrhizae. INRA, Paris, pp. 217–221. van Elsas, J.D., Hill, P., Chronakova, A., Grekova, M., Topalova, Y., Elhottova, D., Kristufek, V., 2007. Survival of genetically marked Escherichia coli O157:H7 in soil as affected by soil microbial community shifts. ISME J. 1, 204–214. Viglizzo, F., Pordomingo, M. G., Castro, F., Lectora, A., 2002. La sustentabilidad ambiental del agro pampeado. Ediciones INTA (in Spanish). Walsh, U.F., Moe¨nne-Loccoz, Y., Tichy, H.V., Gardner, A., Corkery, D.M., Lorkhe, S., O’Gara, F., 2003. Residual impact of the biocontrol inoculant Pseudomonas fluorescens F113 on the resident population of rhizobia nodulating a red clover rotation crop. Microb. Ecol. 45, 145–155. Zhang, F., Hamel, C., Kianmehr, H., Smith, D.L., 1995. Root-zone temperature and soybean [Glycine max (L.) Merr.] vesiculararbuscular mycorrhizae: development and interactions with the nitrogen fixing symbiosis. Environ. Exp. Bot. 35, 287–298.