An unseen foe in arthropod conservation efforts: The case of Wolbachia infections in the Karner blue butterfly

Infection by endosymbiotic bacteria is an underappreciated threat to endangered arthropods with serious implications for captive management programs. We examined the nature of Wolbachia infection in the North American endangered Karner blue butterfly, Lycaeides melissa samuelis, as a case study. Screening for Wolbachia across the range of the species confirmed widespread infection in the western half of the Karner blue’s range. Multilocus sequence typing using six genes confirmed that the infection in the western populations is attributable to a single strain of Wolbachia. This strain was also detected in the closely related Melissa blue butterfly, L. m. melissa, the presumed source of the infection. The infection in the Karner blue butterfly was perfectly correlated with the presence of a foreign mitochondrial DNA variant present in the Melissa blue butterfly, consistent with the hypothesis that the mitochondrial introgression was driven by the spread of Wolbachia. A single individual out of 71 screened from the eastern portion of the range of the Karner blue butterfly was also infected, however this infection was attributable to a different strain. Simulation models of the demographic effects of the spread of Wolbachia infection to uninfected populations and metapopulations suggest that such an infection might further reduce already small population sizes and substantially increase the probability of population extirpation. We discuss threats to other endangered arthropods in the light of this case study and make recommendations for minimizing the impact of endosymbiont infections in conservation plans, especially those including captive propagation and augmentation of endangered arthropod populations.     

Species diversity versus phylogenetic diversity: A practical study in the taxonomically difficult genus Dactylorhiza (Orchidaceae)

Setting conservation priorities in taxonomically complex groups such as the orchid genus Dactylorhiza is a difficult task. As an alternative to taxonomic diversity, we used here a molecular phylogenetic analysis and the results of a genetic investigation using plastid microsatellites with an extensive geographic sampling to assess in a more objective way the patterns of diversity within this genus. Although western Europe is thought to be the main diversity centre for the genus due to the large number of species found there, we found higher phylogenetic and genetic diversity as well as higher endemicity in the Caucasus and the Mediterranean Basin, two biodiversity hotspots. Species number seems to be correlated with taxonomic effort, tentatively estimated here by the number of herbaria, and is thus biased and not an appropriate measure of diversity. Our results show that phylogenetic analyses and genetic data obtained with molecular tools can offer an alternative measure of biodiversity that is not sensitive to taxonomic inflation. Conservation of allotetraploid taxa is also discussed, and it is recommended that sites in which polyploids are formed should be conserved rather than any specific allotetraploid taxon.     

Effects of Medicago sativa, Taraxacum officinale and Bromus sterilis on the density and diversity of Collembola in grassy arable fallows of different ages

The density and diversity of Collembola of nine grassy arable fallows of different ages were investigated in a factorial design with the factors ‘plant species’ (legume: Medicago sativa, herb: Taraxacum officinale, grass: Bromus sterilis) and ‘age class’ (2-3, 6-8 and 12-15 years) including the random effect ‘site’ (1-9). In May 2008, four plots were selected randomly at each fallow. Within each plot five M. sativa, T. officinale and B. sterilis plants were extracted with their associated soil using steel cylinders. The material from each plant species was used for extraction of Collembola and for determination of environmental parameters. Thus, the new aspect of the present study compared to other field studies investigating the relationships between plant and Collembolan communities is the focus on the “micro-scale”, investigating the Collembolan communities of the soil associated with single plants.We found that species richness and density of total and euedaphic Collembola were significantly higher in B. sterilis than in T. officinale samples with the M. sativa samples being intermediate. Fine-root feeding euedaphic Collembola particularly benefited from the higher amount of fine roots in B. sterilis samples. We also discovered that the age of the fallows had no significant influence on the number of Collembolan species and the density of the Collembolan groups. Notably, however, species of the epedaphic genera Lepidocyrtus and Sminthurinus were associated with the 12-15 year-old fallows and presumably benefited from the high number of plant species in the old fallows. Finally, canonical correspondence analysis (CCA) indicated that the soil organic matter content and the microbial biomass, both potential food sources for many Collembolan species, were important structuring forces for the Collembolan communities.     

Interaction between the soil yeast Rhodotorula mucilaginosa and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea

The effect of the soil yeast, Rhodotorula mucilaginosa LBA, on Glomus mosseae (BEG n°12) and Gigaspora rosea (BEG n°9) was studied in vitro and in greenhouse trials. Hyphal length of G. mosseae and G. rosea spores increased significantly in the presence of R. mucilaginosa. Exudates from R. mucilaginosa stimulated hyphal growth of G. mosseae and G. rosea spores. Increase in hyphal length of G. mosseae coincided with an increase in R. mucilaginosa exudates. No stimulation of G. rosea hyphal growth was detected when 0.3 and 0.5 ml per petri dish of yeast exudates was applied. Percentage root length colonization by G. mosseae in soybean (Glycine max L. Merill) and by G. rosea in red clover (Trifolium pratense L. cv. Huia) was increased only when the soil yeast was inoculated before G. mosseae or G. rosea was introduced. Beneficial effects of R. mucilaginosa on arbuscular mycorrhizal (AM) colonization were found when the soil yeast was inoculated either as a thin agar slice or as a volume of 5 and 10 ml of an aqueous solution. R. mucilaginosa exudates (20 ml per pots) applied to soil increased significantly the percentage of AM colonization of soybean and red clover.     

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.     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.     

Lyophyllum sp. strain Karsten alleviates pH pressure in acid soil and enhances the survival of Variovorax paradoxus HB44 and other bacteria in the mycosphere

In previous work, Variovorax paradoxus strain HB44, next to Burkholderia terrae BS001 and Dyella japonica BS003, were found to be selected in the mycosphere of the tricholomataceous fungi Laccaria proxima and Lyophyllum sp. strain Karsten in an acid soil denoted G. V. paradoxus HB44 showed poor survival in G bulk soil, irrespective of prior soil sterilization, and this poor survival also occurred for B. terrae BS001 and D. japonica BS003. In contrast, the survival rate of strain HB44 in two other soils, with pH values > 5.5, was significantly raised. Also, significantly enhanced strain HB44 survival in G soil was found if the pH was raised to 5.5 or 6.5, and it was even shown to grow (in the presence of the exogenous carbon source glycerol) at such pH values in the sterile G soil. This behaviour was similar to that of the V. paradoxus type strain. Strikingly, Lyophyllum sp. strain Karsten, when colonizing the sterilized G soil, significantly raised the soil pH from about 4.6 to ≥5.0. The pH raise was dependent on time, hyphal development, as well as on initial soil pH, but was consistent throughout. The modulated soil pH conditions were shown to be permissive for the survival and growth of strain HB44, and this was extended to strains BS001 and BS003. These findings corroborate the hypothesis that L. sp. strain Karsten provides a suitable habitat for acid-sensitive strains like HB44, BS001 and BS003 in its mycosphere in acid soil, which is strongly defined by the establishment of a growth-permissive pH.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

Molecular markers and a sequence deletion in intron 2 of the putative partial homologue of LEAFY reveal geographical structure to genetic diversity in the acutely threatened legume genus Clianthus

Clianthus is an acutely threatened, bird-pollinated genus endemic to New Zealand, represented in the wild by only one population of C. puniceus and 11 populations of C. maximus, each with very few individuals (typically <10 per population). A limited number of named Clianthus cultivars of indeterminate origin are commonly grown as ornamentals. Genomic DNA from individual Clianthus plants was extracted for genetic diversity analysis using a range of molecular markers, including amplified fragment length polymorphism (AFLP). Data were analysed by the unweighted pair-group method with arithmetic averaging (UPGMA), the generation of Neighbor-Joining trees, and analyses of molecular variance (AMOVA). Genetic distance between wild populations of C. maximus was highly correlated with geographical distance between populations. Sequencing of intron 2 of a putative partial homologue of the floral meristem identity gene LEAFY (CmLFY) revealed a 7 bp deletion that was exhibited homozygously in the more northern populations of C. maximus, and in all individuals tested from the sole population of C. puniceus. This deletion was not exhibited in more southern populations of C. maximus. Further, one geographically intermediate population contained some plants that were heterozygous for the deletion. Parallel analyses of cultivated Clianthus genotypes, more than half of which were also homozygous for the 7 bp deletion, showed that these were not representative of the broad, but threatened, diversity remaining in the wild. It is argued that wild populations of C. maximus are unlikely to have arisen from the escape of plants from cultivation. Conservation effort should focus on the protection and study of the extant plants in these wild populations, rather than on the introduction of disturbance regimes to uncover potential seed banks.     

Changes in soil biological activities under reduced soil pH during Thlaspi caerulescens phytoextraction

Phytoextraction of soil Cd and Zn may require reduction in soil pH in order to achieve high metal uptake. Reducing the pH of high metal soil, however, could negatively affect soil ecosystem function and health. The objectives of this study were to characterize the quantitative causal relationship between pH and soil biological activities in two Zn and Cd contaminated soils and to investigate the relationship between metals and soil biological activities under low pH. Soils were adjusted to five or six different pH levels by sulfur addition, followed by salt leaching. Thlaspi caerulescens was grown for 6 months, and both the rhizosphere and non-rhizosphere soil biological activities were tested after harvest. Reducing pH significantly lowered soil alkaline phosphatase activity, arylsulphatase activity, nitrification potential, and respiration. However, acid phosphatase activity was increased with decreasing pH. The relationship between soil biological activities and pH was well characterized by linear or quadratic regression models with R² values ranging from 0.57 to 0.99. In general, the three enzyme activities, nitrification potential, and the ratio of alkaline phosphatase to acid phosphatase activity were very sensitive indicators of soil pH status while soil respiration was not sensitive to pH change. The rhizosphere soil had higher biological activities than non-rhizosphere soil. The negative effects observed in the non-rhizosphere soil were alleviated by the rhizosphere influence. However, rhizosphere soil after 6 months phytoextraction showed lower nitrification potential than non-rhizosphere soil, probably due to substrate limitation in our study.     

Soil feedback effects to the foredune grass Ammophila arenaria by endoparasitic root-feeding nematodes and whole soil communities

In coastal foredunes, the grass Ammophila arenaria develops a soil community that contributes to die-back and replacement by later successional plant species. Root-feeding nematodes and pathogenic soil microorganisms are involved in this negative feedback. Regular burial by wind-blown beach sand results in vigorous growth of A. arenaria, probably because of enabling a temporary escape from negative soil feedback. Here, we examine the role of root-feeding nematodes as compared to the whole soil community in causing negative feedback to A. arenaria. We performed a 3-year sand burial experiment in the field and every year we determined the feedback of different soil communities to plant growth in growth chamber bioassays.In the field, we established A. arenaria in tubes with beach sand, added three endoparasitic root-feeding nematode species (Meloidogyne maritima, Heterodera arenaria and Pratylenchus penetrans) or root zone soil to the plants, and created series of ceased and continued sand burial. During three subsequent years, plant biomass was measured and numbers of nematodes were counted. Every year, bioassays were performed with the field soils and biomass of seed-grown A. arenaria plants was measured to determine the strength of feedback of the established soil communities to the plant.In the field, addition of root zone soil had a negative effect on biomass of buried plants. In the bioassays, addition of root zone soil also reduced the biomass of newly planted seedlings, however, only in the case when the field plants had not been buried with beach sand. Addition of the three endoparasitic root-feeding nematodes did not influence plant biomass in the field and in the bioassays. Our results strongly suggest that the negative feedback to A. arenaria is not due to the combination of the three endoparasitic nematodes, but to other components in the soil community, or their interactions with the nematodes.     

Calcium concentrations of soil affect suppressiveness against Aphanomyces root rot of pea

The potential for field soils to cause Aphanomyces root rot of pea (Pisum sativum) was estimated for a large number of samples from commercial pea fields over a period of 5 years, using a greenhouse bioassay. The aim of the research project was to gain a mechanistic understanding of soil suppressiveness to the disease. Regression analysis showed that of the measured soil variables (Ca, Mg, K, P, pH), soil Ca concentrations had the strongest (negative) correlation with disease prevalence, and also a significant negative correlation with disease severity in samples with confirmed presence of the disease. Greenhouse bioassays using a set of non-infested soils inoculated with artificially produced oospore inoculum of the casual organism Aphanomyces euteiches, showed a similar negative correlation between soil Ca content and disease severity. Disease severity was not consistently affected by soil sterilisation, but was lowered by the addition of two different Ca salts. In contrast, addition of sodium bicarbonate to two soils lowered the content of water-soluble Ca in the soils and increased disease severity. Studies of cultures of A. euteiches exposed to varying Ca concentrations in vitro showed that zoospore production was inhibited at submillimolar concentrations, while mycelial growth was stimulated or unaffected. We conclude that free Ca is a major variable controlling the degree of soil suppressiveness against A. euteiches, and that inhibition of zoospore production from oospores is a possible mechanism.     

Attraction to prey and prey-associated odours by the predatory mite Hypoaspis aculeifer in a soil experimental system

In terms of food searching and trophic interactions among soil organisms volatile signalling plays a crucial role. Still, volatile-based foraging decisions are poorly understood, especially as the dispersal and perception of cues is more restricted within the soil pore space than in simple laboratory approaches where volatiles disperse freely in the air. Focusing on the tritrophic interaction between the soil fungus Trichoderma viride, the fungivorous collembolan Folsomia candida and the predatory mite Hypoaspis aculeifer, we tested the effect of prey and prey-associated volatile signals on the food choice decision of the predatory mite in a laboratory experiment under soil conditions as well as the possible occurrence and relevance of grazing-induced indirect defence signals. The mites showed a clear preference for ungrazed and grazed fungal mycelium. However, we found no evidence of induced indirect defence as ungrazed mycelium was preferred to grazed fungus.     

Field survival of the phytostimulator Azospirillum lipoferum CRT1 and functional impact on maize crop, biodegradation of crop residues, and soil faunal indicators in a context of decreasing nitrogen fertilisation

Heavy nitrogen fertilisation is often implemented in maize cropping systems, but it can have negative environmental effects. Nitrogen-fixing, phytohormone-producing Azospirillum plant growth-promoting rhizobacteria (PGPR) have been proposed as crop inoculants to maintain high yield when decreasing nitrogen fertilisation. In this context, agronomic and ecological effects of the inoculation of maize seeds with the PGPR Azospirillum lipoferum CRT1 were studied in two consecutive years. The inoculant was recovered from maize at 10⁵ CFU g⁻¹ root or higher. Inoculation enhanced root growth and development based on results of root biomass, rooting depth and/or parameters describing root system architecture, and a transient positive effect on shoot height was observed in the first year. Inoculation did not increase yield, but reducing mineral nitrogen fertilisation had only a minor effect on yield. This suggests that the lack of positive effect of the PGPR on yield was due to the fact that the whole field was heavily fertilised in years prior to the start of the experiment. Soil nitrogen levels decreased during the 2 years of the study, and the inoculant had no effect on residual soil nitrogen levels at harvest. Inoculation had no impact on Fusarium symptoms and concentration of the mycotoxin deoxynivalenol in maize kernels, but both were influenced by the interaction between inoculation and nitrogen fertilisation level. Inoculation did not influence meso/macrofaunal soil populations, but had a small but significant effect (smaller than the effect of added nitrogen) on decomposition, nitrogen mineralisation and mesofaunal colonisation of maize leaves (in litter bags). Overall, the ecological impact of seed inoculation with the PGPR A. lipoferum CRT1 was small, and its magnitude was smaller than that of chemical nitrogen fertilisation.     

Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices

Arbuscular mycorrhizal (AM) fungi are key organisms of the soil/plant system, influencing soil fertility and plant nutrition, and contributing to soil aggregation and soil structure stability by the combined action of extraradical hyphae and of an insoluble, hydrophobic proteinaceous substance named glomalin-related soil protein (GRSP). Since the GRSP extraction procedures have recently revealed problems related to co-extracting substances, the relationship between GRSP and AM fungi still remains to be verified. In this work the hypothesis that GRSP concentration is positively correlated with the occurrence of AM fungi was tested by using Medicago sativa plants inoculated with different isolates of Glomus mosseae and Glomus intraradices in a microcosm experiment. Our results show that (i) mycorrhizal establishment produced an increase in GRSP concentration - compared to initial values - in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1-2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP concentration and soil aggregate stability were positively correlated with mycorrhizal root volume and weakly correlated with total root volume; (iv) MWD values of soil aggregates were positively correlated with values of total hyphal length and hyphal density of the AM fungi utilized.The different ability of AM fungal isolates to affect GRSP concentration and to form extensive and dense mycelial networks, which may directly affect soil aggregates stability by hyphal enmeshment of soil particles, suggests the possibility of selecting the most efficient isolates to be utilized for soil quality improvement and land restoration programs.     

A phylogenetic microarray targeting 16S rRNA genes from the bacterial division Acidobacteria reveals a lineage-specific distribution in a soil clay fraction

We designed an oligonucleotide microarray using probe sequences based upon a phylogenetic analysis of 16S rRNA genes recovered from members of the bacterial division Acidobacteria. A total of 42,194 oligonucleotide probes targeting members of the Acidobacteria division at multiple phylogenetic levels were included on a high-density microarray. Positive control hybridizations revealed a linear relationship between hybridization signal and template concentration, and a substantial decrease in non-specific hybridization was achieved through the addition of 2.5 M betaine to the hybridization buffer. A mean hybridization signal value was calculated for each Acidobacteria lineage, with the resultant lineage-specific hybridization data revealing strong predictive value for the positive control hybridizations. The Acidobacteria phylochip was then used to evaluate Acidobacteria rRNA genes from a Wisconsin soil and within a soil clay fraction. The Acidobacteria hybridization profile revealed the predominance of Acidobacteria subdivisions four and six, and also suggested a decrease in the abundance of subdivision six relative to subdivision four in the soil clay fraction. The change in relative abundance of these subdivisions in a soil clay fraction was supported by data from quantitative PCR. These results support the utility of a phylogenetic microarray in revealing changes in microbial population-level distributions in a complex soil microbial assemblage.     

Potential gene exchange between Bacillus thuringiensis subsp. kurstaki and Bacillus spp. in soil in situ

The possible transfer of genes from Bacillus thuringiensis subsp. kurstaki (Btk) to indigenous Bacillus spp. was investigated in soil samples from stands of cork oak in Orotelli (Sardinia, Italy) collected 5 years after spraying of the stands with a commercial insecticidal preparation (FORAY 48B) of Btk. Two colonies with a morphology different from that of Btk were isolated and identified as Bacillus mycoides by morphological and physiological characteristics and by 16S rDNA analysis. Amplification by the polymerase chain reaction (PCR) of the DNA of the two isolated B. mycoides colonies with primers used for the identification of the Btk cry genes showed the presence of a fragment of 238 bp of the cry1Ab9 gene that had a similarity of 100% with the sequence of the cry1Ab9 gene present in GenBank, indicating that the isolates of B. mycoides acquired part of the sequence of this gene from Btk. No cells of Btk or B. mycoides carrying the 238-bp fragment of the cry1Ab9 gene were isolated from samples of unsprayed control soil. However, the isolates of B. mycoides were not able to express the partial Cry1Ab protein. Hybridization with probes for IS231 and the cry1Ab9 gene suggested that the inverted repeated sequence, IS231, was probably involved in the transfer of the 238-bp fragment from Btk to B. mycoides. These results indicate that transfer of genes between introduced Btk and indigenous Bacillus spp. can occur in soil under field conditions.     

Changes in agricultural management drive the diversity of Burkholderia species isolated from soil on PCAT medium

In order to assess the diversity of culturable Burkholderia populations in rhizosphere and bulk soil and to evaluate how different agricultural management regimes and land use history affect this diversity, four treatments were evaluated: permanent grassland; grassland converted into maize monoculture; arable land and arable land converted into grassland. Burkholderia isolates obtained on PCAT medium were grouped in 47 clusters using 16S ribosomal RNA gene based PCR-DGGE combined with BOX genomic fingerprinting (DGGE-BOX). The distribution of the isolates in the DGGE-BOX clusters was used to calculate the Shannon diversity index per treatment. Interestingly, we observed that the Burkholderia diversity was affected by changes in the agricultural management, since the highest diversity was observed in permanent grassland and in continuous arable land. In addition, the diversity tended to be higher in the rhizosphere than in the corresponding bulk soil. The use of species abundance models indicated that rhizosphere communities had more even distributions than communities collected from the bulk soil. Identification of isolates revealed that only 2% of these belonged to the B. cepacia complex and that the majority was assigned to either (1) new Burkholderia species or (2) Burkholderia species that had originally been isolated from soil. Isolates classified as B. hospita, B. caledonica and Burkholderia sp. ‘LMG 22934’ and ‘LMG 22936’ were found mainly in the arable land, while isolates belonging to Burkholderia sp. ‘LMG 22929’ and B. phytofirmans were associated with the grassland area. Another potentially new Burkholderia species, ‘LMG 22932’, was found in both areas, in close association with the maize rhizosphere.