Effects of soil pH on the biodegradation of chlorpyrifos and isolation of a chlorpyrifos-degrading bacterium

We examined the role of microorganisms in the degradation of the organophosphate insecticide chlorpyrifos in soils from the United Kingdom and Australia. The kinetics of degradation in five United Kingdom soils varying in pH from 4.7 to 8.4 suggested that dissipation of chlorpyrifos was mediated by the cometabolic activities of the soil microorganisms. Repeated application of chlorpyrifos to these soils did not result in the development of a microbial population with an enhanced ability to degrade the pesticide. A robust bacterial population that utilized chlorpyrifos as a source of carbon was detected in an Australian soil. The enhanced ability to degrade chlorpyrifos in the Australian soil was successfully transferred to the five United Kingdom soils. Only soils with a pH of >/=6.7 were able to maintain this degrading ability 90 days after inoculation. Transfer and proliferation of degrading microorganisms from the Australian soil to the United Kingdom soils was monitored by molecular fingerprinting of bacterial 16S rRNA genes by PCR-denaturing gradient gel electrophoresis (DGGE). Two bands were found to be associated with enhanced degradation of chlorpyrifos. Band 1 had sequence similarity to enterics and their relatives, while band 2 had sequence similarity to strains of Pseudomonas. Liquid enrichment culture using the Australian soil as the source of the inoculum led to the isolation of a chlorpyrifos-degrading bacterium. This strain had a 16S rRNA gene with a sequence identical to that of band 1 in the DGGE profile of the Australian soil. DNA probing indicated that genes similar to known organophosphate-degrading (opd) genes were present in the United Kingdom soils. However, no DNA hybridization signal was detected for the Australian soil or the isolated degrader. This indicates that unrelated genes were present in both the Australian soil and the chlorpyrifos-degrading isolate. These results are consistent with our observations that degradation of chlorpyrifos in these systems was unusual, as it was growth linked and involved complete mineralization. As the 16S rRNA gene of the isolate matched a visible DGGE band from the Australian soil, the isolate is likely to be both prominent and involved in the degradation of chlorpyrifos in this soil.     

Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating

A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A gamma-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in gamma-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.     

Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.     

In vitro soil receptivity assays to egg-parasitic nematophagous fungi

Soil application of nematophagous fungi for the biological control of plant-parasitic nematodes often fails, and in many cases it has been difficult to reisolate the agent delivered to the soil. A reason for these results could be the inability of the fungi to proliferate in soil. We used a soil–membrane technique to study the capacity of several isolates of the nematophagous fungi Pochonia chlamydosporia and Paecilomyces lilacinus to grow and establish in sterilized and nonsterilized sandy soils from SE Spain and Western Australia. Growth of all fungi tested was inhibited in nonsterilized soil, although there was intraspecific variability in sensitivity among isolates of the same species. With respect to hyphal density, P. chlamydosporia isolate 5 (from Italy) was the least inhibited in nonsterilized soil from both sites. Relative growth analyses confirmed this result for soil from SE Spain, while with this method, P. chlamydosporia isolate 4624 (from Australia) appeared to be least inhibited in the Australian soil. The results indicate that a soil can be more receptive to its indigenous isolates than to nonindigenous isolates. Apparently, soil microbiota can determine the ability of nematophagous fungi to proliferate in soil.     

Use of membrane filters for selective isolation of actinomycetes from soil

Abstract A method using membrane filters of appropriate pore size, to selectively isolate actinomycetes from a mixed population of soil microorganisms, is described.
The method is based on the ability of actinomycetes to propagate and pass through the pores of filters while bacteria and fungi are retained on the membrane surface.     

Application of actinomycetes to soil to ameliorate water repellency

AIMS: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. METHODS AND RESULTS: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. CONCLUSIONS: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.     

Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil

AIMS: To devise and evaluate a method for selective isolation of the less abundant actinomycetes, Nocardia spp. in soil. METHODS AND RESULTS: This newly developed method is based on differentiating Nocardia from other actinomycete taxa by centrifugation. A water suspension of air-dried soil is centrifuged through a gradient consisting of 10, 20, 30, 40 and 50% sucrose at 240 x g for 30 min. The 20% sucrose layer, which is enriched with Nocardia spp., is then diluted and plated on humic acid-vitamin agar supplemented with antibacterial agents. The proposed method consistently achieved selective isolation of Nocardia spp. in all 14 soil samples tested, which accounted for 5-89% of the total microbial population recovered. Tentative taxonomic characterization based on a restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal DNA suggested that many of the soil isolates could belong to N. asteroides, N. salmonicida or N. uniformis. CONCLUSIONS: Differential centrifugation can successfully and efficiently isolate soil Nocardia populations that are suppressed by conventional dilution plating approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: The development and application of new methodologies with which to isolate less-explored actinomycete taxa is important for improving our knowledge about their taxonomy, ecology and industrial applications.     

Detection and cultivation of soil verrucomicrobia

Only one isolate each of the class "Spartobacteria" (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class "Spartobacteria" and were related to "Chthoniobacter flavus." Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class "Spartobacteria" were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class "Spartobacteria" were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class "Spartobacteria" was inhibited by other culturable bacteria.     

Autoradiographic method to screen for soil microorganisms which accumulate zinc

An autoradiographic method was developed to screen for and isolate soil microorganisms which accumulate zinc (Zn). Diluted soil samples (Rubicon fine sand, Entic Haplorthods [pH 5.9]) were plated on soil extract-glucose agar containing radioactive 65Zn. After 7 days of incubation, individual colonies which accumulated sufficient 65Zn could be detected by autoradiography. These colonies were isolated and confirmed as Zn accumulators in pure culture by using the autoradiographic plate technique. Most Zn accumulators were filamentous fungi, identified as Penicillium janthinellum, Aspergillus fumigatus, and Paecilomyces sp. Isolates of Penicillium janthinellum were the most common Zn accumulators. The most abundant Zn-accumulating bacteria were Bacillus spp. The validity of the autoradiographic plate technique to differentiate soil microbes which accumulate Zn was examined independently by energy dispersive X-ray analysis in a scanning electron microscope. This method confirmed that fungal isolates which gave positive autoradiographic responses in the plate assay bioaccumulated more Zn in their biomass than fungal isolates from the same soil sample which gave negative autoradiographic responses. Thus, this technique can be applied to specifically screen for and isolate microbes from the environment which bioaccumulate Zn.     

Aerobic biotransformation of 2,4-dinitroanisole in soil and soil Bacillus sp.

2,4-Dinitroanisole (DNAN) is a low sensitive melt-cast chemical being tested by the Military Industry as a replacement for 2,4,6-trinitrotoluene (TNT) in explosive formulations. Little is known about the fate of DNAN and its transformation products in the natural environment. Here we report aerobic biotransformation of DNAN in artificially contaminated soil microcosms. DNAN was completely transformed in 8 days in soil slurries supplemented with carbon and nitrogen sources. DNAN was completely transformed in 34 days in slurries supplemented with carbons alone and persisted in unamended microcosms. A strain of Bacillus (named 13G) that transformed DNAN by co-metabolism was isolated from the soil. HPLC and LC–MS analyses of cell-free and resting cell assays of Bacillus 13G with DNAN showed the formation of 2-amino-4-nitroanisole as the major end-product via the intermediary formation of the arylnitroso (ArNO) and arylhydroxylamino (ArNHOH) derivatives, indicating regioselective reduction of the ortho-nitro group. A series of secondary reactions involving ArNO and ArNHOH gave the corresponding azoxy- and azo-dimers. Acetylated and demethylated products were identified. Overall, this paper provides the evidence of fast DNAN transformation by the indigenous microbial populations of an amended soil with no history of contamination with explosives and a first insight into the aerobic metabolism of DNAN by the soil isolate Bacillus 13G.     

Temporal change in culturable phenanthrene degraders in response to long-term exposure to phenanthrene in a soil column system

Widespread environmental contamination by polycyclic aromatic hydrocarbons (PAH) has led to increased interest in the use of natural attenuation as a clean-up strategy. However, few bioremediation studies have investigated the behaviour of the indigenous PAH-degrading community after long-term exposure to a PAH. In this study, a column packed with sandy loam soil was exposed to a solution saturated with phenanthrene ( approximately 1.2 mg l-1) for a 6-month period to examine the temporal response of the indigenous phenanthrene-degrading community. Initial soil, effluent, and final soil samples were collected and analysed for phenanthrene concentration and culturable phenanthrene degraders. Phenanthrene-degrading isolates were grouped by colony morphology. For each unique group, 16S rDNA polymerase chain reaction was performed, and then sequencing analysis was used to identify the isolate at the genus level. Twenty-five phenanthrene-degrading isolates, potentially representing 19 genera, were obtained from this analysis. Of these, eight genera have not been reported previously to degrade phenanthrene, including Afipia, Janthinobacterium, Leptothrix, Massilia, Methylobacterium, Rhizobium, Sinorhizobium and Thiobacillus. Results indicate that the dominant phenanthrene-degrading population changed over the course of this 6-month experiment. Specifically, the isolates obtained initially from the soil were not subsequently found in either effluent samples or the soil at the end of the experiment. Furthermore, several isolates that were found in the soil at the end of the experiment were not observed in the soil initially or in the effluent samples. This study confirms earlier findings indicating that a diverse community participates in phenanthrene degradation in the environment, and also suggests that the composition of this community is temporally variable.     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Aerobic and anaerobic growth of rifampin-resistant denitrifying bacteria in soil

The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth followed by a gradual decline in numbers. After 28 days, both isolates maintained viable populations of between 10(4) and 10(5) CFU g dry soil-1 under both denitrifying and aerobic conditions. One of the isolates consistently grew better under denitrifying conditions, and the other isolate consistently grew better under aerobic conditions. The relative pattern of denitrifying versus aerobic growth for each organism was not affected by the addition of glucose. The growth yields of the two isolates varied with soil type, but the relative pattern of denitrifying versus aerobic growth was consistent in three soils with greatly different properties. Five of nine isolates introduced into Bruno soil at low population densities (approximately 10(5) CFU g dry soil-1) exhibited better growth after 2 days under denitrifying conditions. It was not possible to predict the prevalence of the denitrifying or aerobic mode of growth in nonsterile soil from the growth characteristics of the isolates in pure cultures or sterile soil.     

Autoradiographic method to screen for soil microorganisms which accumulate zinc.

An autoradiographic method was developed to screen for and isolate soil microorganisms which accumulate zinc (Zn). Diluted soil samples (Rubicon fine sand, Entic Haplorthods [pH 5.9]) were plated on soil extract-glucose agar containing radioactive 65Zn. After 7 days of incubation, individual colonies which accumulated sufficient 65Zn could be detected by autoradiography. These colonies were isolated and confirmed as Zn accumulators in pure culture by using the autoradiographic plate technique. Most Zn accumulators were filamentous fungi, identified as Penicillium janthinellum, Aspergillus fumigatus, and Paecilomyces sp. Isolates of Penicillium janthinellum were the most common Zn accumulators. The most abundant Zn-accumulating bacteria were Bacillus spp. The validity of the autoradiographic plate technique to differentiate soil microbes which accumulate Zn was examined independently by energy dispersive X-ray analysis in a scanning electron microscope. This method confirmed that fungal isolates which gave positive autoradiographic responses in the plate assay bioaccumulated more Zn in their biomass than fungal isolates from the same soil sample which gave negative autoradiographic responses. Thus, this technique can be applied to specifically screen for and isolate microbes from the environment which bioaccumulate Zn.     

The isolation of Mycobacterium avium complex from soil, water, and dusts

Previously, it was difficult to isolate the Mycobacterium avium complex from soil, water, and dusts, because rapidly growing mycobacteria always overgrew slowly growing ones. We used Ogawa egg medium containing both ethambutol and ofloxacin, which inhibit the nonpathogenic slowly growing mycobacteria and most rapidly growing mycobacteria, respectively, as an aid to screen for pathogenic slowly growing mycobacteria; we could thereby isolate a number of the M. avium complex and M. scrofulaceum strains from soil, water, and dusts in this country.     

Aerobic and anaerobic growth of rifampin-resistant denitrifying bacteria in soil.

The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth followed by a gradual decline in numbers. After 28 days, both isolates maintained viable populations of between 10(4) and 10(5) CFU g dry soil-1 under both denitrifying and aerobic conditions. One of the isolates consistently grew better under denitrifying conditions, and the other isolate consistently grew better under aerobic conditions. The relative pattern of denitrifying versus aerobic growth for each organism was not affected by the addition of glucose. The growth yields of the two isolates varied with soil type, but the relative pattern of denitrifying versus aerobic growth was consistent in three soils with greatly different properties. Five of nine isolates introduced into Bruno soil at low population densities (approximately 10(5) CFU g dry soil-1) exhibited better growth after 2 days under denitrifying conditions. It was not possible to predict the prevalence of the denitrifying or aerobic mode of growth in nonsterile soil from the growth characteristics of the isolates in pure cultures or sterile soil.     

Relationship between spatial and genetic distance in Agrobacterium spp. in 1 cubic centimeter of soil

The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place. Agrobacterium spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 microm in diameter were sampled with spatial coordinates in 1 cm(3) of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (10(3) agrobacteria g(-1)), a high genetic diversity was maintained. In two independent microsamples (500 microm in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis.     

The ectomycorrhizal symbiosis between Lactarius deliciosus and Pinus sylvestris in forest soil samples: symbiotic efficiency and development on roots of a rDNA internal transcribed spacer-selected isolate of L. deliciosus

The effect on plant growth of pre-inoculation of Pinus sylvestris with the ectomycorrhizal (ECM) edible basidiomycete Lactarius deliciosus (isolate D45) under controlled conditions, and the development on roots of this basidiomycete, were investigated in gamma-irradiated and unsterilized containers containing different forest soil cores or a perlite-vermiculite mixture. Five months after planting, L. deliciosus mycorrhizal plants exhibited greater growth than the non-mycorrhizal ones in all soil types, i.e. up to a 325% increase in shoot height in the sterilized soils. The experiment demonstrated the dependency of P. sylvestris seedlings upon ECM symbiosis for their survival in gamma-irradiated, microbiologically disturbed soil samples. Furthermore, in two soils, the growth of L. deliciosus-inoculated seedlings was greater in the sterilized soil samples than in the non-sterilized ones, i.e. 46% and 132% increase in shoot height under sterilized soil conditions. In containers randomly sampled from each soil type, the degree of root colonization by the inoculated isolate, calculated as the number of mycorrhizal root tips divided by the total number of root tips x100, ranged from 80% to 35%. Within the short term, the inoculated isolate developed rapidly on roots, dominated, and hampered ectomycorrhiza formation by various unidentified (but not Lactarius) resident ECM fungi in unsterilized soil types. Results indicate that the ECM species L. deliciosus is worth investigating to ascertain if other isolates benefit pine growth like the isolate D45, and are therefore also attractive candidates for forestry applications in the Mediterranean area.     

天祝高寒草地植被、土壤及土壤微生物时间动态的比较

对天祝高寒草地21a前（1982年）、后（2003年）植被状况、土壤理化性质、土壤三大类微生物（细菌、放线菌和真菌）和各生理群微生物（硝化细菌、好气性固氮菌和好气性纤维素分解菌）及不同退化程度（围栏内、围栏外和鼠丘地）草地土壤微生物数量变化特点进行了对比研究。结果表明：（1）与1982年相比,目前该区天然草地植被总盖度、主要优良牧草种类、产草量等显著下降,草地植被退化明显;（2）草地土壤pH升高,土壤含水量、有机质、氮、磷含量均下降,草地土壤理化性质劣于1982年;（3）目前该区天然草地土壤三大类微生物数量及各生理群微生物数量变化十分明显,1982年土壤细菌、放线菌和真菌及微生物总数分别是2003年的153．6、5．5、4．1倍和151．2倍;土壤硝化细菌、好气性固氮菌和好气性纤维素分解菌数量分别是2003年的5．7、43．3倍和94．4倍;（4）轻度退化草地（围栏内）土壤各类微生物数量明显高于严重退化草地（围栏外、鼠丘地）,其数量前者一般为后者的1．5—4．5倍。     

Exploration of soil bacterial communities for their potential as bioresource

Soil is a repository of diverse microorganisms, which has frequently been used to isolate and exploit microbes for industrial, environmental and agricultural applications. Knowledge about the structure and dynamics of bacterial communities in soil has been limited as only a small fraction of bacterial diversity is accessible to culture methods. Traditional enrichment techniques and the pure culture approach for microbiological studies have offered only a narrow portal for examining the soil microbial flora due to their limited selectivity. Therefore, the morphological and nutritional criteria used to describe bacterial community failed to provide a natural taxonomic order according to evolutionary relationship. Molecular methods under an emerging discipline of biology "molecular microbial ecology" are now helping in getting these constraints removed to some extent. Nucleic acid extraction from soil is the first crucial step in the application of most of the molecular techniques, which have largely been dominated by diverse variations of PCR. Due to its rapidity, sensitivity and specificity, PCR-based finger printing techniques have proved extremely useful in assessing the changes in microbial community structure. Such techniques can yield complex community profiles and can also provide useful phylogenetic information. Fluorescent in situ hybridization (FISH) can be used to evaluate the distribution and function of bacterial population in situ. DNA microarray techniques have also been developed and being frequently used for the evaluation of ecological role and phylogenetic affiliations of bacterial populations in the soil.