PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium

Bacteria attached to the rumen epithelium (or epimural community) are not well characterised and their role in rumen functioning is not totally understood. There is just one published report of a clone library from one cow that suggests that this epimural community differs from the bacteria associated with the rumen digestive contents. However, this time-consuming approach is not adapted for examining microbial population changes in groups of animals. In in vivo studies, when samples from several animals have to be analysed simultaneously, a simpler technique has to be used. In this study, a genetic fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterise the structure of the bacterial population attached to the rumen epithelium. This community was compared with that present in the solid and liquid phases of rumen content under two contrasting diets. Rumen samples were obtained from four forage-fed and four high-concentrate-fed (80 : 20, wheat grain : hay) 5-month-old lambs. After slaughter, samples from five epithelial sites and the solid and liquid digesta phases were taken for DNA extraction and analysis. Bacterial communities were profiled by PCR-DGGE using bacterial-specific 16S rDNA primers. Analysis of the fingerprint revealed that the epithelial community differed from those of rumen content in both diets. As expected, the nature of the feed influenced the bacterial communities from the solid and liquid rumen phases but no diet effect was observed in the rumen epithelial profiles suggesting a strong host effect on this bacterial population. Additionally, no differences were observed among the five epithelial sampling sites taken from each animal. The profile of the bacterial population attached to the rumen epithelium presented a high inter-animal variation, whether this difference has an influence in the function of this community remains to be determined.     

Microbial population dynamics in laboratory-scale solid waste bioreactors in the presence or absence of biosolids

Aims:  Decomposition of solid waste is microbially mediated, yet little is known about the associated structure and temporal changes in prokaryotic communities. Bioreactors were used to simulate landfill conditions and archaeal and bacterial community development in leachate was examined over 8 months.
Methods and Results:  Municipal solid waste (MSW) was deposited in laboratory bioreactors with or without biosolids and combustion residues (ash). The near-neutral pH fell about half a log by day 25, but recovered to ∼7·0 by day 50. Cell concentrations in bioreactors containing only MSW were significantly higher than those from co-disposal bioreactors. Archaeal and bacterial community structure was analysed by denaturing gradient gel electrophoresis targeting 16S rRNA genes, showing temporal population shifts for both domains. mcrA sequences retrieved from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales .
Conclusion:  Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and distinct temporal trends. The solid waste filled bioreactors allowed simulation of solid waste decomposition in landfills while also reducing the variables.
Significance and Impact of the Study:  This study advances the basic understanding of changes in microbial community structure during solid waste decomposition, which may ultimately improve the efficiency of solid waste management.     

A comparison of bacteria and benthic invertebrates as indicators of ecological health in streams

1. We set out to evaluate the reliability of bacterial communities as an indicator of freshwater ecological health.
2. Samples of epilithic biofilm were taken over a 1-year period from four streams, each impacted by varying degrees of human modification. The bacteria within each sample were characterised using a whole community DNA fingerprinting technique (automated ribosomal intergenic spacer analysis). Spatial and temporal differences in community structure between samples were visualised using multi-dimensional scaling and quantified using permutational multivariate anova . Macrobenthic invertebrates, which are commonly used as indicators of stream ecological health, were also sampled for comparison.
3. Multivariate analysis revealed a clear gradient in macroinvertebrate community structure between sites exposed to increased human impact. Bacterial communities, however, could only distinguish the most impacted site from the remainder.
4. Additional research is required to increase the sensitivity of bacterial community analyses before endorsing their use as an indicator of freshwater ecological health.     

Bacterial Succession on the Leaf Surface: A Novel System for Studying Successional Dynamics

Succession is a widely studied process in plant and animal systems, but succession in microbial communities has received relatively little attention despite the ubiquity of microorganisms in natural habitats. One important microbial habitat is the phyllosphere, or leaf surface, which harbors large, diverse populations of bacteria and offers unique opportunities for the study of succession and temporal community assembly patterns. To explore bacterial community successional patterns, we sampled phyllosphere communities on cottonwood (Populus deltoides) trees multiple times across the growing season, from leaf emergence to leaf fall. Bacterial community composition was highly variable throughout the growing season; leaves sampled as little as a week apart were found to harbor significantly different communities, and the temporal variability on a given tree exceeded the variability in community composition between individual trees sampled on a given day. The bacterial communities clearly clustered into early-, mid-, and late-season clusters, with early- and late-season communities being more similar to each other than to the mid-season communities, and these patterns appeared consistent from year to year. Although we observed clear and predictable changes in bacterial community composition during the course of the growing season, changes in phyllosphere bacterial diversity were less predictable. We examined the species–time relationship, a measure of species turnover rate, and found that the relationship was fundamentally similar to that observed in plant and invertebrate communities, just on a shorter time scale. The temporal dynamics we observed suggest that although phyllosphere bacterial communities have high levels of phylogenetic diversity and rapid turnover rates, these communities follow predictable successional patterns from season to season.     

Temporal variability in the diversity and composition of stream bacterioplankton communities

Bacterioplankton in freshwater streams play a critical role in stream nutrient cycling. Despite their ecological importance, the temporal variability in the structure of stream bacterioplankton communities remains understudied. We investigated the composition and temporal variability of stream bacterial communities and the influence of physicochemical parameters on these communities. We used barcoded pyrosequencing to survey bacterial communities in 107 streamwater samples collected from four locations in the Colorado Rocky Mountains from September 2008 to November 2009. The four sampled locations harboured distinct communities yet, at each sampling location, there was pronounced temporal variability in both community composition and alpha diversity levels. These temporal shifts in bacterioplankton community structure were not seasonal; rather, their diversity and composition appeared to be driven by intermittent changes in various streamwater biogeochemical conditions. Bacterial communities varied independently of time, as indicated by the observation that communities in samples collected close together in time were no more similar than those collected months apart. The temporal turnover in community composition was higher than observed in most previously studied microbial, plant or animal communities, highlighting the importance of stochastic processes and disturbance events in structuring these communities over time. Detailed temporal sampling is important if the objective is to monitor microbial community dynamics in pulsed ecosystems like streams.     

The influence of urea feeding on the bacterial and archaeal community in the forestomach of collared peccary (Artiodactyla, Tayassuidae)

Aims:  This study was carried out to test whether bacterial and archaeal populations, and products of fermentation in each compartment of collared peccary stomach, vary significantly with urea feeding. Bacteria and archaeal population variation among the four stomach compartments were also compared.
Methods and Results:  Archaeal and bacterial communities in the forestomach of four individuals per treatment – peccaries fed diets with and without urea – were analysed at molecular level using PCR followed by denaturing gradient gel electrophoresis. Volatile fatty acids profiles in the three different compartments of the forestomach were also compared. The bacterial community composition varied considerably among each compartment and with urea provision, but no variation was observed between archaeal populations. Differences in bacterial communities between treatments – with and without urea – were greater than amongst stomach compartments. The acetate: propionate proportion decreased with urea provision in diet. Some differences in bacterial but not archaeal community composition were observed in each compartment of the collared peccary forestomach.
Conclusions:  There are some differences in bacterial but not archaeal populations in each compartment of collared peccary stomach. Use of urea in the diet of peccary can substantially modify the profile of volatile fatty acids released in its forestomach, but does not influence the archaeal community composition. Urea has an important effect on bacterial population DGGE profile present in the peccary's forestomach.
Significance and Impact of the Study:  These results demonstrate the ability of the collared peccary to use urea as source of nonprotein nitrogen, and confirm a hypothesis that the collared peccary has a digestive physiology more similar to ruminant than nonruminant animals.     

Microbiome analysis of dairy cows fed pasture or total mixed ration diets

Understanding rumen microbial ecology is essential for the development of feed systems designed to improve livestock productivity, health and for methane mitigation strategies from cattle. Although rumen microbial communities have been studied previously, few studies have applied next-generation sequencing technologies to that ecosystem. The aim of this study was to characterize changes in microbial community structure arising from feeding dairy cows two widely used diets: pasture and total mixed ration (TMR). Bacterial, archaeal and protozoal communities were characterized by terminal restriction fragment length polymorphism of the amplified SSU rRNA gene and statistical analysis showed that bacterial and archaeal communities were significantly affected by diet, whereas no effect was observed for the protozoal community. Deep amplicon sequencing of the 16S rRNA gene revealed significant differences in the bacterial communities between the diets and between rumen solid and liquid content. At the family level, some important groups of rumen bacteria were clearly associated with specific diets, including the higher abundance of the Fibrobacteraceae in TMR solid samples and members of the propionate-producing Veillonelaceae in pasture samples. This study will be relevant to the study of rumen microbial ecology and livestock feed management.     

High reproducibility of ammonia-oxidizing bacterial communities in parallel sequential batch reactors

Aims:  To investigate whether the ammonia-oxidizing bacterial (AOB) communities of replicate nitrifying bioreactors (i) co-evolve or diverge over time and (ii) are stable or dynamic during periods of complete nitrification.
Methods and Results:  Three sequential batch reactors (SBR) were inoculated with sludge from a municipal wastewater treatment plant, fed with ammonium-enriched tap water and operated in parallel for 134 days. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) demonstrated co-evolvement of the AOB communities over time. During start-up, temporary decreases in nitrification were noticed, and the AOB community rate of change values (Δ _{t (week)}) were medium to high (12–22%). During the adjacent period of complete nitrification, low AOB community dynamics were observed (Δ _{t (week)} < 5%). Further pragmatic processing of the DGGE profiles revealed a high range-weighted richness and a medium functional organization of the AOB communities.
Conclusions:  After a start-up period, high functional stability and low dynamics of the AOB communities were observed. Deterministic rather than stochastic driving forces led to AOB community co-evolvement in the replicate SBR.
Significance and Impact of the Study:  Replicates in identical set-ups are reproducible, and pragmatic processing of DGGE patterns is a straightforward tool to score and compare the functionality of the bacterial communities.     

Exploring the bovine rumen bacterial community from birth to adulthood

The mammalian gut microbiota is essential in shaping many of its host''s functional attributes. One such microbiota resides in the bovine digestive tract in a compartment termed as the rumen. The rumen microbiota is necessary for the proper physiological development of the rumen and for the animal''s ability to digest and convert plant mass into food products, making it highly significant to humans. The establishment of this microbial population and the changes occurring with the host''s age are important for understanding this key microbial community. Despite its importance, little information about colonization of the microbial populations in newborn animals, and the gradual changes occurring thereafter, exists. Here, we characterized the overall bovine ruminal bacterial populations of five age groups, from 1-day-old calves to 2-year-old cows. We describe the changes occurring in the rumen ecosystem after birth, reflected by a decline in aerobic and facultative anaerobic taxa and an increase in anaerobic ones. Some rumen bacteria that are essential for mature rumen function could be detected as early as 1 day after birth, long before the rumen is active or even before ingestion of plant material occurs. The diversity and within-group similarity increased with age, suggesting a more diverse but homogeneous and specific mature community, compared with the more heterogeneous and less diverse primary community. In addition, a convergence toward a mature bacterial arrangement with age was observed. These findings have also been reported for human gut microbiota, suggesting that similar forces drive the establishment of gut microbiotas in these two distinct mammalian digestive systems.     

Structural and functional responses of sewage microbial communities used for the treatment of a complex mixture of volatile organic compounds (VOCs)

Aims:  The aim of this work was to assess the impact of the applied mass loading on the selection of an efficient microbial community able to degrade a complex mixture of volatile organic compounds (VOCs).
Methods and Results:  Two reactors were used and were supplied with a gaseous effluent containing 11 VOCs with different concentrations. The response of the microflora was monitored as a function of time: biodegradation activity, bacterial density and diversity. The results showed that the applied mass loading seems to have an impact on the functioning and the genetic structure of the bacterial community.
Conclusions:  A high mass loading seems to induce a low efficient functioning in terms of elimination efficiency and a simplification of the genetic structure of the total bacterial community with the apparition of a dominant microflora. A low mass loading seems to favour a better functioning and allows to keep a healthier bacterial diversity.
Significance and Impact of the Study:  In the treatment processes of gaseous effluents, it would be judicious to define the functioning parameters of the process to keep the diversity of important functional bacterial groups. These results provide also useful information about changes in microbial communities following natural or anthropogenic alterations in different ecosystems.     

Impact of subacute ruminal acidosis (SARA) adaptation and recovery on the density and diversity of bacteria in the rumen of dairy cows

Subacute ruminal acidosis (SARA) is characterized by ruminal pH depression and microbial perturbation. The impact of SARA adaptation and recovery on rumen bacterial density and diversity was investigated following high-grain feeding. Four ruminally cannulated dairy cows were fed a hay diet, transitioned to a 65% grain diet for 3 weeks, and returned to the hay diet for 3 weeks. Rumen fluid, rumen solids, and feces were sampled during weeks 0 (hay), 1 and 3 (high grain), and 4 and 6 (hay). SARA was diagnosed during week 1, with a pH below 5.6 for 4.6±1.4 h. Bacterial density was significantly lower in the rumen solids with high grain (P=0.047). Rumen fluid clone libraries from weeks 0, 3, and 6 were assessed at the 98% level and 154 operational taxonomic units were resolved. Week 3 diversity significantly differed from week 0, and community structure differed from weeks 0 and 6 (P<0.0001). Clones belonging to the phylum Firmicutes predominated. Compared with the hay diet, the high-grain diet contained clones from Selenomonas ruminantium and Succiniclasticum ruminis, but lacked Eubacterium spp. SARA adaptation was found to significantly alter bacterial density, diversity, and community structure, warranting further investigation into the role bacteria play in SARA adaptation.     

Investigating the relationship between raw milk bacterial composition, as described by intergenic transcribed spacer–PCR fingerprinting, and pasture altitude

Aims:  To assess the bacterial biodiversity level in bovine raw milk used to produce Fontina, a Protected Designation of Origin cheese manufactured at high-altitude pastures and in valleys of Valle d'Aosta region (North-western Italian Alps) without any starters. To study the relation between microbial composition and pasture altitude, in order to distinguish high-altitude milk against valley and lowland milk.
Methods and Results:  The microflora from milks sampled at different alpine pasture, valley and lowland farms were fingerprinted by PCR of the 16S–23S intergenic transcribed spacers (ITS-PCR). The resulting band patterns were analysed by generalized multivariate statistical techniques to handle discrete (band presence–absence) and continuous (altitude) information. The fingerprints featured numerous bands and marked variability indicating complex, differentiated bacterial communities. Alpine pasture milks were distinguished from lowland ones by cluster analysis, while this technique less clearly discriminated alpine pasture and valley samples. Generalized principal component analysis and clustering-after-ordination enabled a more effective distinction of alpine pasture, valley and lowland samples.
Conclusions:  Alpine raw milks for Fontina production contain highly diverse bacterial communities, the composition of which is related to the altitude of the pasture where milk was produced.
Significance and Impact of the Study:  This research may provide analytical support to the important issue represented by the authentication of the geographical origin of alpine milk productions.     

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle

Aims: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. Methods and Results: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate–forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T‐RFLP and automated ribosomal intergenic spacer analysis) and real‐time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0·009) in high concentrate–forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. Conclusions: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. Significance and Impact of the Study: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.     

The Microbial Community in the Feces of the Giant Panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>) as Determined by PCR-TGGE Profiling and Clone Library Analysis

Despite having a typical carnivorous digestive tract, the giant panda has a diet consisting exclusively of bamboo, a low-efficiency food source. Given this paradox, we sought to investigate if the giant panda digestive tract is inhabited by organisms indicative of high cellulose diet or their gastrointestinal tract anatomy. The diversity and dynamics of the predominant bacteria in the fecal flora of two adult (male and female) and one young (male) giant panda reared in two different zoos over a 2-year period was studied using 16S rDNA-based approaches. The temperature gradient gel electrophoresis (TGGE) profiles of the 16S rDNA V3 region of the three individuals were highly similar. The structure of their fecal flora remained relatively stable over the 2-year period. Both the most predominant band in TGGE patterns shared by the three pandas and the biggest operational taxonomic unit (OTU) in the clone library were phylogenetically related to Escherichia coli. Gram-negative, facultative bacteria constituted almost 60% of the whole community in the clone library. All the OTUs were related to previously described phylotypes known to reside in the intestine or rumen. The results of our study indicate that the predominant bacterial populations in the intestine of the three pandas were markedly different from that of herbivores. The unbalanced intestinal community structure may play a role in the inefficient digestion of bamboo by the giant pandas.     

The host species affects the microbial community in the goat rumen

AIMS: This study was carried out to determine whether bacterial and ciliate populations in goat rumen vary significantly between different goat species living in the same environment. METHODS AND RESULTS: Bacterial and ciliate communities in the rumen of three goat species were analysed at the molecular level using denaturing gradient gel electrophoresis. The microbial community varied considerably among goats living in the same environment. Interspecies variation in the bacterial population was noticeably greater than intraspecies variation. In contrast, there was considerable variation in the ciliate population among goats within the same species, and intraspecies similarities were no greater than those observed across species. CONCLUSIONS: Because environmental factors and diets were identical for all goats, differences in bacterial populations reflect species-specific differences in rumen microbes. SIGNIFICANCE AND IMPACT OF THE STUDY: Factors related to the host species have an important effect on determining the bacterial composition in the goat rumen.     

Monitoring the bacterial community during fermentation of sunki, an unsalted, fermented vegetable traditional to the Kiso area of Japan

Aims:  To investigate the microbial community in sunki , an indigenous, unsalted, fermented vegetable, made from the leaves of red beet.
Methods and Results:  Fermenting samples were collected at 1- to 2-day intervals from four houses and investigated by culture-dependent and culture-independent techniques. PCR-Denaturing-Gradient-Gel-Electrophoresis profiles indicated that the bacterial community was stable and Lactobacillus delbrueckii , Lact. fermentum and Lact. plantarum were dominant during the fermentation. This result agreed well with that obtained by the culturing technique. Moulds, yeasts or bacteria other than lactic acid bacteria (LAB) were not detected.
Conclusions:  The bacterial community was stable throughout the fermentation, and Lact. delbrueckii , Lact. fermentum and Lact. plantarum were dominant. The acidic pH and lactic acid produced by LAB probably preserve the sunki from spoilage.
Significance and Impact of the Study:  This is the first report on the use of both culture-dependent and culture-independent techniques to study the bacterial community in sunki . A combination of culture-dependent and culture-independent techniques is necessary for the analysis of complex microbial communities.     

Changes in microbial community structure, methanogenesis and rumen fermentation in response to saponin-rich fractions from different plant materials

Aims:  Investigation of the effects of saponin-rich fractions on rumen fermentation, methane production and the microbial community.
Methods and Results:  Saponins were extracted from Carduus , Sesbania and Knautia leaves and fenugreek seeds. Two levels of saponin-rich fractions with a substrate were incubated using the Hohenheim gas method. Methane was measured using an infrared-based methane analyser and microbial communities using quantitative PCR. On addition of saponin-rich fractions, methane and short-chain fatty acid production was not affected. The protozoal counts decreased by 10–39%. Sesbania saponins decreased methanogen population by 78%. Decrease in ruminal fungal population (20–60%) and increase in Fibrobacter succinogenes (21–45%) and Ruminococcus flavefaciens (23–40%) were observed.
Conclusions:  The saponins evaluated possessed anti-protozoal activity; however, this activity did not lead to methane reduction. Fenugreek saponins seemed to have potential for increasing rumen efficiency. The saponins altered the microbial community towards proliferation of fibre-degrading bacteria and inhibition of fungal population.
Significance and Impact of the Study:  The uni-directional relationship between protozoal numbers and methanogenesis, as affected by saponins, is not obligatory. All saponins might not hold promise for decreasing methane production from ruminants.     

A Cross-Taxon Analysis of Insect-Associated Bacterial Diversity

Although it is well known that plants and animals harbor microbial symbionts that can influence host traits, the factors regulating the structure of these microbial communities often remain largely undetermined. This is particularly true for insect-associated microbial communities, as few cross-taxon comparisons have been conducted to date. To address this knowledge gap and determine how host phylogeny and ecology affect insect-associated microbial communities, we collected 137 insect specimens representing 39 species, 28 families, and 8 orders, and characterized the bacterial communities associated with each specimen via 16S rRNA gene sequencing. Bacterial taxa within the phylum Proteobacteria were dominant in nearly all insects sampled. On average, the insect-associated bacterial communities were not very diverse, with individuals typically harboring fewer than 8 bacterial phylotypes. Bacterial communities also tended to be dominated by a single phylotype; on average, the most abundant phylotype represented 54.7% of community membership. Bacterial communities were significantly more similar among closely related insects than among less-related insects, a pattern driven by within-species community similarity but detected at every level of insect taxonomy tested. Diet was a poor predictor of bacterial community composition. Individual insect species harbored remarkably unique communities: the distribution of 69.0% of bacterial phylotypes was limited to unique insect species, whereas only 5.7% of phylotypes were detected in more than five insect species. Together these results suggest that host characteristics strongly regulate the colonization and assembly of bacterial communities across insect lineages, patterns that are driven either by co-evolution between insects and their symbionts or by closely related insects sharing conserved traits that directly select for similar bacterial communities.     

Effect of disodium fumarate on ruminal metabolism and rumen bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA

The aim of the present study was to examine the effects of feeding diets with addition of disodium fumarate (DF) to goats on ruminal metabolism and changes of rumen bacterial communities. Four cannulated goats were used in a 4 × 4 Latin square design. The results showed that ruminal pH increased linearly (P<0.01)as the amount of DF added increased, while lactate production decreased linearly (P<0.01). DF addition did not affect the production of acetate, propionate, butyrate, TVFA and NH₃-N. The effect of DF on the changes in rumen bacterial-community structure of goats was analyzed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in rumen bacterial community structure were determined based on the Shannon index of diversity for pairwise comparison of the DGGE fingerprints and revealed significant changes in rumen microbiota after DF addition. As compared with those fed with the control diet, goats fed on the diets with DF addition showed a higher bacterial diversity. The sequences of seven amplicons in total 11 clones showed less than 97% similarity with those of previously identified or unidentified bacteria, suggesting that most bacteria in the gastrointestinal tract have not been cultured or identified. Amplicons related to Succinivibrio dextrinosolvens species were found in most DGGE fingerprints derived from goats on the diet containing DF, but not in goats on the control diet. These results demonstrated the ability of DF to improve the metabolism of rumen lactate fermentation and to influence the bacterial composition of the rumen in goats.     

Impact of two bacterial biocontrol agents on bacterial and fungal culturable groups associated with the roots of field-grown maize

Aims:  To assess the impact of Bacillus amyloliquefaciens and Microbacterium oleovorans on bacterial and fungal groups associated to the roots of field-grown maize.
Methods and Results:  Identification and count of bacterial and fungal culturable populations associated to the roots of maize seedlings, changes in culturable community structure according to the richness and diversity indexes concept and shifts in microbial activity through analysis of cellulolytic, ammonification and nitrification potentials were determined, in relation to kernel treatment with biological control agents. Following the treatment of maize kernels with B. amyloliquefaciens at 10⁷ CFU ml⁻¹, an increase in bacterial diversity was observed at the rhizoplane of resultant seedlings. Bacterial richness was significantly increased at the root inner tissues of seedlings treated with Mic. oleovorans . Fusarium , Aspergillus , Penicillium and Trichoderma were the main fungal genera isolated and there population sizes were unequally affected by the addition of biocontrol agents.
Conclusions:  Numbers and types of isolated bacteria and fungi changed in response to the addition of biocontrol agents, while microbial activity remained unchanged with respect to control.
Significance and Impact of the Study:  This study provides an insight of the effects of proven biocontrol agents on micro-organisms naturally associated to the target crop.