Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

Reduction of sclerotial inoculum of Sclerotinia sclerotiorum with Coniothyrium minitans

Aspects of the biology of C. minitans and its potential for control of S. sclerotiorum were investigated.Temperatures below 7°C resulted in comparatively slow rates of germination and infection of sclerotia by C. minitans. The optimum temperature for germination, growth, infection of sclerotia, and destructive parasitism by C. minitans was 20°C. The optimum relative humidity for germination, growth and infection by C. minitans was above 95%.Autumn inoculations with suspensions of conidia, pycnidia and mycelium of C. minitans in the field resulted in negligible numbers of sclerotia remaining viable after 1 month. With culture-grown sclerotia 2 months were required for a similar reduction of sclerotial viability. In the absence of C. minitans mulching had no significant effect on sclerotial viability. In the presence of C. minitans mulching did, however, influence the viability and infection by C. minitans of culture-grown sclerotia. Populations of field sclerotia also differed from culture-grown sclerotia in that they harboured an internal population of microorganisms, which included C. minitans, and had a lower level of viability at the commencement of the treatments.A winter application of C. minitans did not result in significant infection of sclerotia nor in a reduction in viability of sclerotia. This failure is believed to have resulted from low temperatures and dry conditions.     

Control of apothecia of Sclerotinia sclerotiorum by soil amendment with S-H mixture or Perlka in bean, canola and wheat fields

Ascospores of Sclerotinia sclerotiorum produced from apothecia are the primary source of inoculum for causing diseases such as white mold of common bean, pod rot of pea, stem blight of canola and head rot of sunflower and safflower in the Canadian prairies. A field study was conducted for 4 years to determine efficacy of control of production of apothecia from carpogenically germinated sclerotia of S. sclerotiorum by soil amendment with Perlka^® (calcium cyanamide) and S-H mixture (a formulated compound). Results of the 4-year experiments showed that amendment of soil with Perlka^® at low (30 g/m²) or high (60 g/m²) rate was effective in reducing carpogenic germination of sclerotia and production of apothecia under the canopy of host crops (common bean and canola) and a non-host crop (wheat). In the experiments of 1988, for example, the numbers of apothecia produced in the treatments of Perlka^®-low rate (30 g/m²), Perlka^®-high rate (60 g/m²) and untreated control were 42, 46, and 182 apothecia/plot (m²), respectively, for bean; 89, 42, and 318 apothecia/plot (m²), respectively, for canola; and 146, 143, and 412 apothecia/plot (m²), respectively, for wheat. However, soil amendment of S-H mixture at low (30 g/m²) or high (60 g/m²) rate was ineffective in reducing carpogenic germination of sclerotia and production of apothecia for all the 4 years of testing in all three crops. The ineffectiveness of S-H mixture and the practicality of Perlka^® for control of Sclerotinia diseases of crops grown under Canadian prairie conditions are discussed.     

Identification and use of potential bacterial organic antifungal volatiles in biocontrol

Bacteria, isolated from canola and soybean plants, produced antifungal organic volatile compounds. These compounds inhibited sclerotia and ascospore germination, and mycelial growth of Sclerotinia sclerotiorum, in vitro and in soil tests. Ascospore germination in cavity slides was inhibited 54-90% by the volatile producers. When mycelial plugs or the sclerotia, exposed to these volatiles, were transferred to fresh agar plates, the pathogen could not grow, indicating the fungicidal nature of the volatiles. Head space volatiles, produced by bacteria, were trapped with activated charcoal, by passing nitrogen continuously over shake cultures for 48 h. The compounds were eluted from the charcoal with methylene chloride and identified using Gas Chromatography-Mass Spectrometry (GC-MS). The volatile compounds included aldehydes, alcohols, ketones and sulfides. Of the 23 compounds assayed for antifungal activity in divided Petri plates, with filter-disks soaked with these compounds (100 and 150 μl), only six compounds completely inhibited mycelial growth or sclerotia formation, suggesting their potential role in biological control. The compounds are benzothiazole, cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl 1-hexanol, and nonanal. Volatiles may play an important role in the inhibition of sclerotial activity, limiting ascospore production, and reducing disease levels. Studies are under way to understand this phenomenon under field conditions. This is the first report on the identification and use of bacterial antifungal organic volatiles in biocontrol.     

Survival of Escherichia coli in soil with modified microbial community composition

Understanding the survival and persistence of Escherichia coli in soil with different microbial composition is essential for the accuracy of water quality assessment and microbial source tracking. This microcosm experiment was conducted to investigate the survival pattern of three E. coli strains (originated from soil, dog feces and human feces, separately) in soil with modified microbial community composition. Bile salt No. 3 (BS3) of progressively increased density (0.05%, 0.15%, 0.30% and 0.50%) was added into sandy loam soils and incubated for 90 days. Laboratory cultured E. coli were then inoculated into soil and incubated for another 150 days to monitor their survival pattern. Change of bacterial community diversity by BS3 was detected by both cultivation based and cultivation independent (PCR-Denaturing Gradient Gel Electrophoresis) methods. In general, progressively increased BS3 concentration resulted in decreased CFU counts both at 10 days and 90 days incubation. DGGE analysis indicated only a slight change in bacterial community composition at 10 days but a significant change at 90 days. Cluster analysis suggested that BS3 treatment grouped separately from controls. Survival of E. coli in soil was significantly influenced by the complexity of the microbial community, as die-off rate of E. coli progressively declined with the reduction of microbial community diversity. Differential survival of E. coli under different soil microbial stress highlights the importance of incorporating biotic factors in predictive models for water quality management and microbial source tracking study.     

Impact of the biocontrol agent Trichoderma atroviride SC1 on soil microbial communities of a vineyard in northern Italy

The fungus Trichoderma atroviride SC1 is an experimental biocontrol agent (BCA) that is active against the fungus Armillaria mellea. Following the application of Trichoderma to the surface soil of a vineyard, we used a highly specific real-time PCR, previously validated for the analysis of soil microcosms, to monitor the populations of this fungus at different soil depths over several months. The quantification obtained using this molecular method was highly correlated with laboratory assays of colony-forming units. The native communities of bacteria and fungi in the soil were analyzed using automated ribosomal intergenic spacer analysis (ARISA), and transient changes were observed following the application of T. atroviride SC1 conidia. A principal component of variance analysis demonstrated that the introduction of T. atroviride SC1 had an effect on the soil microflora during the first two weeks following inoculation. However, at later dates, environmental conditions had a higher influence on the surveyed communities than the BCA application, as confirmed through the use of the Shannon index of biodiversity. Soil depth had a strong influence on the composition and biodiversity of fungal communities.     

Microbial populations involved in the suppression of Rhizoctonia solani AG1-1B by lignin incorporation in soil

Rhizoctonia solani causes worldwide losses in numerous crops. Sclerotia of R. solani remain viable for several years in soil and are an important source of primary infection. In this study the effect of soil incorporation of Kraft pine lignin, a side product of the paper industry, on viability of R. solani AG1-1B sclerotia was investigated. The efficacy of lignin was assessed in a sandy loam (Oppuurs) and a silt loam soil (Leest) collected from commercial fields in Belgium. Evaluating sclerotial viability after 4 weeks incubation in the two soils amended with 1% (w/w) Kraft pine lignin demonstrated a soil-dependent effect. In Leest soil the addition of lignin resulted in a significantly reduced sclerotial viability, together with an increased mycoparasitism by Trichoderma spp.; in Oppuurs soil, on the other hand, only a slight and insignificant reduction in sclerotial viability was observed. Based on phospholipid fatty acid analysis, different changes in microbial community structure upon lignin amendment were detected in the two soils. Both amended soils showed a significant increase in Gram negative bacteria. In Leest soil this increase was accompanied with a significantly higher increase in fungi and actinomycetes compared with Oppuurs soil. In addition, Kraft pine lignin resulted in both soils in a small but significant increase in manganese peroxidase activity and this increase tended to be higher in Leest soil. Manganese peroxidase produced by lignin-degrading basidiomycetes has previously been shown to degrade melanin, which protects the sclerotia against biotic and abiotic stress. We hypothesize that lignin-degrading fungi increased the susceptibility of the sclerotia to sclerotial antagonists such as Trichoderma, Gram negative bacteria and actinomycetes. Clearly, the effect observed here did not rely on the stimulation of one microbial group, but is the result of an interaction of different groups.     

Soil-borne strain IC14 of Serratia plymuthica with multiple mechanisms of antifungal activity provides biocontrol of Botrytis cinerea and Sclerotinia sclerotiorum diseases

Plant-associated strain IC14 of the Gram-negative bacterium Serratia plymuthica isolated from soil around melon roots was shown to suppress a wide range of phytopathogenic fungi in vitro. Foliar application of strain IC14 protected cucumber against Botrytis cinerea gray mold and Sclerotinia sclerotiorum white mold diseases of leaves under greenhouse conditions, reducing disease incidence by 76 and 84%, respectively. The strain possessed chitinolytic and proteolytic activities, produced the antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] and siderophores, and secreted the plant growth hormone indole-3-acetic acid. An endochitinase with an apparent molecular mass of 58 kDa, was estimated to be the main secreted chitinolytic enzyme. Two mutants, one with increased chitinolytic activity and the second deficient in chitinolytic activity, were obtained by miniTn5-insertion mutagenesis. Neither mutant differed appreciably from the parental strain in the production of other antifungal compounds or in suppression of B. cinerea and S. sclerotiorum on plates or in the greenhouse, suggesting that chitinolytic activity is less essential for biocontrol of these pathogens by strain IC14. The obtained results present novel information concerning the potential of the soil-borne S. plymuthica strains as biocontrol agents of foliar diseases caused by plant pathogenic fungi.     

Diversity and antagonistic potential of Pseudomonas spp. associated to the rhizosphere of maize grown in a subtropical organic farm

Pseudomonas spp. are one of the most important bacteria inhabiting the rhizosphere of diverse crop plants and have been frequently reported as biological control agents (BCAs). In this work, the diversity and antagonistic potential of Pseudomonas spp. in the rhizosphere of maize cultivars Nitroflint and Nitrodent grown at an organic farm in Brazil was studied by means of culture-dependent and -independent methods, respectively. Sampling of rhizosphere soil took place at three different stages of plant development: 20, 40 and 106 days after sowing. A PCR-DGGE strategy was used to generate specific Pseudomonas spp. fingerprints of 16S rRNA genes amplified from total community rhizosphere DNA. Shifts in the relative abundance of dominant populations (i.e. PCR-DGGE ribotypes) along plant development were detected. A few PCR-DGGE ribotypes were shown to display cultivar-dependent relative abundance. No significant differences in diversity measures of DGGE fingerprints were observed for different maize cultivars and sampling times. The characterisation and assessment of the antagonistic potential of a group of 142 fluorescent Pseudomonas isolated from the rhizosphere of both maize cultivars were carried out. Isolates were phenotypically and genotypically characterised and screened for in vitro antagonism towards three phytopathogenic fungi and the phytopathogenic bacterium Ralstonia solanacearum. Anti-fungal activity was displayed by 13 fluorescent isolates while 40 isolates were antagonistic towards R. solanacearum. High genotypic and phenotypic diversity was estimated for antagonistic fluorescent Pseudomonas spp. PCR-DGGE ribotypes displayed by antagonists matched dominant ribotypes of Pseudomonas DGGE fingerprints, suggesting that antagonists may belong to major Pseudomonas populations in the maize rhizosphere. Antagonists differing in their genotypic and phenotypic characteristics shared the same DGGE electrophoretic mobility, indicating that an enormous genotypic and functional diversity might be hidden behind one single DGGE band. Cloning and sequencing was performed for a DGGE double-band which had no corresponding PCR-DGGE ribotypes among the antagonists. Sequences derived from this band were affiliated to Pseudomonas stutzeri and P. alcaligenes 16S rRNA gene sequences. As used in this study, the combination of culture-dependent and -independent methods has proven to be a powerful tool to relate functional and structural diversity of Pseudomonas spp. in the rhizosphere.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

Spatial distribution of the nematode biocontrol agent Pasteuria penetrans as influenced by its soil habitat

Root-knot nematodes belonging to the Meloidogyne genus are ubiquitous plant-parasitic pests, especially on vegetables. The Pasteuria penetrans bacterium is an obligate parasite of nematodes, parasitizing most of the Meloidogyne species. Spatial distributions of Meloidogyne javanica populations infested or not by P. penetrans and of bacterial populations were studied in a vegetable plot naturally infested by these organisms. It was observed that distributions of M. javanica populations, of populations infested by P. penetrans, and of free bacteria populations were not overlapped. Soil factors involved were investigated. Soil texture and water flow in porosity are concerned, as they directly influence the level of the pool of bacteria and then the chances of both organisms to meet. The soil solution has a direct effect on the attachment of the bacterium on the nematode cuticle.     

Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere

The capability of native bacterial strains isolated from Lolium perenne rhizosphere to behave as plant growth promoting bacteria and /or biocontrol agents was investigated. One strain (BNM 0357) over 13 isolates from the root tips of L. perenne resulted proved to be nitrogenase positive (ARA test) and an IAA producer. Conventional tests and the API 20E diagnostic kit indicated that BNM 0357 behaves to the Enterobacteriaceae family and to the Enterobacter genus. Molecular identification by 16S rRNA sequence analysis indicated that BNM 0357 had the highest similarity to Enterobacter ludwigii (EN-119). Isolate BNM 0357 had the capability to solubilize calcium triphosphate and to antagonize Fusarium solani mycelial growth and spore germination. Strain BNM 0357 also showed the ability to improve the development of the root system of L. perenne. This study disclosed features of E. ludwigii BNM 0357 that deserve further studies aimed at confirming its putative importance as a PGPR.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.     

Soil inoculation with the biocontrol agent Clonostachys rosea and the mycorrhizal fungus Glomus intraradices results in mutual inhibition, plant growth promotion and alteration of soil microbial communities

Interactions between the biocontrol fungus Clonostachys rosea IK 726 and a tomato/Glomus intraradices BEG87 symbiosis were examined with and without wheat bran, which served as a food base for C. rosea. In soil without wheat bran amendment, inoculation with C. rosea increased plant growth and altered shoot nutrient content resulting in an increase and decrease in P and N content, respectively. Inoculation with G. intraradices had no effect on plant growth, but increased the shoot P content. Dual inoculation with G. intraradices and C. rosea followed the pattern of C. rosea in terms of plant growth and nutrient content. Wheat bran amendment resulted in marked plant growth depressions, which were counteracted by both inoculants and dual inoculation increased plant growth synergistically. Amendment with wheat bran increased the population density of C. rosea and reduced mycorrhizal fungus colonisation of roots. The inoculants were mutually inhibitory, which was shown by a reduction in root colonisation with G. intraradices in treatments with C. rosea and a reduction in colony-forming units (cfu) of C. rosea in treatments with G. intraradices, irrespective of wheat bran amendment. Moreover, both inoculants markedly influenced soil microbial communities examined with biomarker fatty acids. Inoculation with G. intraradices increased most groups of microorganisms irrespective of wheat bran amendment, whereas the influence of C. rosea on other soil microorganisms was affected by wheat bran amendment. Overall, inoculation with C. rosea increased and decreased most groups of microorganisms without and with wheat bran amendment, respectively. In conclusion, despite mutual inhibition between the two inoculants this interaction did not impair their observed plant growth promotion. Both inoculants also markedly influenced other soil microorganisms, which should be further studied in relation to their plant growth-promoting features.     

Genetic diversity within Aspergillus flavus strains isolated from peanut-cropped soils in Argentina

The genetic diversity of Aspergillus flavus populations isolated from the peanut-cropped soils in the peanut-growing region at Cordoba Province was evaluated by analysis of vegetative compatibility group (VCG). VCG_s were determined through complementation assays between nitrate-nonutilizing (NNO) mutants. Fifty-six VCG_s were identified from 100 isolates. Twenty-five VCG_s contained two or more isolates and 31 VCG_s contained only a single isolate. In general, there were significant differences among VCG_s in aflatoxin and CPA production. One VCG group included a single atoxigenic strain since it was neither aflatoxin nor cyclopiazonic acid producer. This isolate could be useful as a biological control agent, since it was unable to form a stable heterokaryon in the complementation test with the other isolates. Seven A. flavus isolated from soil were atypical because they simultaneously produced aflatoxins B, G and CPA.     

Survival of the western pond turtle (Emys marmorata) in an urban California environment

The western pond turtle Emys (formerly Clemmys) marmorata is declining throughout its range, primarily due to loss of habitat via urbanization and conversion to agriculture. Urban waterways present several important challenges to freshwater turtle populations, but they also present an opportunity to maintain declining species in a ubiquitous habitat that has high public visibility. The arboretum waterway on the University of California, Davis campus is an example of an extensively altered urban habitat that supports a relatively large E. marmorata population. Over the last 6 years, we monitored the turtle population inhabiting the arboretum waterway to determine the demographic health of the population, and the challenges and opportunities that urban environments pose for pond turtles. Since 1993, the naturally existing arboretum pond turtle population has declined by approximately 40% and has shown little natural recruitment. During this time, we also introduced 31 headstarted turtles into the arboretum. Headstarting is the process of raising juveniles in captivity until they have outgrown their period of greatest vulnerability to predators, and then releasing them into the wild. Our headstarting results demonstrate that this contentious strategy is a viable option for adding young turtles to the population, although it does not address the causes of decline. Over the course of our study, we encountered nine species of non-native turtles in the waterway, and these appear to be a serious threat to the native species. As more habitat becomes urbanized, it is increasingly important to understand how freshwater turtles, such as E. marmorata, adapt to urban waterways and the impact of non-native turtles on native turtle species. Our strong feeling is that urban waterways can provide habitat for viable populations of freshwater turtles and showcase them to the public, but both the aquatic and terrestrial habitat must be managed according to the biological requirements of individual species.     

The adverse effects of soil salinization on the growth of Trifolium alexandrinum L. and associated microbial and biochemical properties in a soil from Iran

The aim of this study was to determine the effects of increasing concentrations of salt solutions (including 0.12, 2, 6, and 10 dS m⁻¹) on the growth of berseem clover (Trifolium alexandrinum L.) and related soil microbial activity, biomass and enzyme activities. Results showed that the dry weights of root and shoot decreased with an increase in the concentrations of salt solutions. Soil salinization depressed the microbiological activities including soil respiration and enzyme activities. Substrate-induced respiration was consistently lower in salinized soils, whereas microbial biomass C did not vary among salinity levels. Higher metabolic quotients (qCO₂) and unaffected microbial biomass C at high EC values may indicate that salinity is a stressful factor, inducing either a shift in the microbial community with less catabolic activity or reduced efficiency of substrate utilization. Acid phosphatase and alkaline phosphatase activities decreased with increasing soil salinity. We found significant, positive correlations between the activities of phosphatase enzymes and plant's root mass, suggesting that any decrease in the activities of the two enzymes could be attributed to the reduced root biomass under saline conditions.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.