Differential Utilization of Carbon Substrates by Bacteria and Fungi in Tundra Soil

Little is known about the contribution of bacteria and fungi to decomposition of different carbon compounds in arctic soils, which are an important carbon store and possibly vulnerable to climate warming. Soil samples from a subarctic tundra heath were incubated with ¹³C-labeled glucose, acetic acid, glycine, starch, and vanillin, and the incorporation of ¹³C into different phospholipid fatty acids (PLFA; indicative of growth) and neutral lipid fatty acids (NLFA; indicative of fungal storage) was measured after 1 and 7 days. The use of ¹³C-labeled substrates allowed the addition of substrates at concentrations low enough not to affect the total amount of PLFA. The label of glucose and acetic acid was rapidly incorporated into the PLFA in a pattern largely corresponding to the fatty acid concentration profile, while glycine and especially starch were mainly taken up by bacteria and not fungi, showing that different groups of the microbial community were responsible for substrate utilization. The ¹³C-incorporation from the complex substrates (starch and vanillin) increased over time. There was significant allocation of ¹³C into the fungal NLFA, except for starch. For glucose, acetic acid, and glycine, the allocation decreased over time, indicating use of the storage products, whereas for vanillin incorporation into fungal NLFA increased during the incubation. In addition to providing information on functioning of the microbial communities in an arctic soil, our study showed that the combination of PLFA and NLFA analyses yields additional information on the dynamics of substrate degradation.Bacteria and fungi comprise more than 90% of the soil microbial biomass and are the main agents for decomposition of organic matter in soil. Until recently it was thought that these two organism groups could be lumped together in this respect, and total microbial biomass or total activity (respiration) was often the only variable included in soil microbiology studies of decomposition and soil organic matter turnover (39). However, there is increasing evidence suggesting that whether decomposition is performed by bacteria or fungi, thereby channeling energy through the bacterial or the fungal food web, has profound effects on the ecosystem. Such effects can have direct influence on the higher trophic levels in the food web (30) or indirect effects on nutrient mineralization rates (14) and nutrient transfer (19, 20), and they can even determine the extent of carbon sequestration in the soil (37). The situation becomes even more complex when the impact of changes in climate, nitrogen availability, and litter input on the balance between bacteria and fungi is taken into account. The Arctic region has been identified as an area that will be especially vulnerable to these changes (3).Little is known about the contribution of bacteria and fungi to the utilization of plant-derived carbon substrates in arctic soils. Differentiation of the bacterial and fungal contributions to decomposition has hitherto relied to a large extent on changes in bacterial and fungal biomasses, for example, by analysis of patterns of phospholipid fatty acids (PLFA) (40). PLFA are components of the cell membrane, and some of the PLFA extracted from the soil are characteristic for a certain microbial group in the environment. However, for changes in PLFA concentrations after the addition of substrates to be detected, substrates often have to be added at unrealistically large amounts. Even then only small changes in the PLFA concentrations will often be detected (35).One way of overcoming these problems is to follow the incorporation of ¹³C label from added substrates into specific fatty acids (8, 17). This approach adds a new dimension—metabolic function—to the study of soil microbial communities without the need of cultivation. It also increases the sensitivity in tracing responses of organism groups to different substrates as the addition of substrates at low and more realistic concentrations with high specific ¹³C label will induce large changes in the ¹³C concentration of the PLFA without changing the total amount of PLFA.Carbon-13 labeling has been used to follow uptake of recent photosynthates (11, 13, 27), pure substrates (10, 12, 32, 33, 41), and complex labeled plant material (28, 41, 43, 44) into PLFA although seldom in arctic soils. However, microorganisms incorporate carbon not only into phospholipids (indicating growth) but also into storage products, for example, when a nutrient other than carbon is limiting growth or under growth-restricting conditions. Thus, with excess carbon both bacteria and fungi will store carbon for later need, for example, as polyhydroxyalkanoate or glycogen (bacteria) and triacylglycerols (fungi). Thus, neutral lipid fatty acids (NLFA) of fungal origin can be used to indicate storage in fungi (4). Degraded PLFA, resulting in diacylglycerols, will also end up in the corresponding NLFA fraction, and NLFA has thus been suggested as an indicator of recently dead bacterial biomass (42). Therefore, the NLFA/PLFA ratio serves two purposes: for fungal lipids a higher NLFA/PLFA ratio would indicate allocation of lipids to energy storage while for bacterial lipids it would indicate turnover of this bacterial group. However, the latter will probably be of minor importance during short incubations. As far as we know, no studies on soil microorganisms have used incorporation of ¹³C from substrates to indicate both effects on growth (incorporation into PLFA) and storage (incorporation into NLFA).We assessed the uptake of ¹³C-labeled substrates into lipid biomarkers of different microbial groups in a laboratory incubation experiment using soil from an arctic tundra heath. The selected substrates represented carbon sources present in soil. Glucose, acetic acid, and glycine are simple compounds common in plant root exudates, and glycine is also a nitrogen source. Starch is a very common polysaccharide in plant residues. Vanillin is a common product of lignin depolymerization (18) containing a phenol ring and is often used as a model substance to indicate lignin degradation. Starch and vanillin are therefore examples of more complex substrates and are supposedly more difficult to decompose. We followed the incorporation of the label into different PLFA and NLFA over time. We hypothesized that ¹³C from the simple compounds would be more rapidly incorporated into microbial PLFA than ¹³C from the more complex substrates (more rapid growth), and thus we expected ¹³C emanating from the complex substrates to increase in concentration in the PLFA and NLFA over time. We also hypothesized that bacteria would be better than fungi in utilizing simple compounds while the label from the more complex substrates would preferentially be incorporated into PLFA, indicating fungi (6, 29). We also expected ¹³C from the C-rich substrates to be incorporated into NLFA (fungal storage) to a larger extent than C from glycine, which also serves as a nitrogen source (4). However, with time the carbon in storage structures would decrease as it would be used for growth or maintenance energy.     

Potential of Siderophore Production by Bacteria Isolated from Heavy Metal: Polluted and Rhizosphere Soils

Recently, heavy metals have been shown to have a stimulating effect on siderophore biosynthesis in various bacteria. In addition, several studies have found that siderophore production is greater in bacteria isolated from soil near plant roots. The aim of this study was to compare the production of siderophores by bacterial strains isolated from heavy metal-contaminated and uncontaminated soils. Chrome azurol sulphonate was used to detect siderophore secretion by several bacterial strains isolated from heavy metal-contaminated and rhizosphere-uncontaminated soils with both a qualitative disc diffusion method and a quantitative ultraviolet spectrophotometric method. Siderophore production by rhizosphere bacteria was significantly greater than by bacteria isolated from contaminated soil. The Pearson’s correlation test indicated a positive correlation between the amount of siderophore produced by bacteria isolated from the rhizosphere using the quantitative and qualitative detection methods and the amount of heavy metal in the soil. However, a significant negative correlation was observed between the amount of siderophore produced by bacteria isolated from heavy metal-contaminated soil and the amount of heavy metal (r value of ?0.775, P < 0.001).     

Siderophore Utilization by Bradyrhizobium japonicum

Bradyrhizobium japonicum USDA 110 and 61A152 can utilize the hydroxamate-type siderophores ferrichrome and rhodotorulate, in addition to ferric citrate, to overcome iron starvation. These strains can also utilize the pyoverdin-type siderophore pseudobactin St3. The ability to utilize another organism's siderophores may confer a selective advantage in the rhizosphere.     

高产铁载体假单胞菌的筛选及其对铁氧化物的利用

筛选高产铁载体的微生物,研究铁载体的抑菌作用和对不溶性未定型铁氧化物(poorly crystalline iron hydroxides,PCIH)的利用。CAS法筛选高产铁载体菌株,采用琼脂扩散法和生长抑制测定铁载体的抑菌作用,利用16S r RNA基因序列比对鉴定分离菌株,并根据分离菌株的生长情况,确定铁载体对不溶性PCIH的利用。从土壤样品中共筛选到172株产铁载体的菌株,高产铁载体菌株13株,其中仅有菌株Z158的发酵液对金黄色葡萄球菌、藤黄微球菌、普通变形杆菌和副溶血性弧菌具有抑菌作用,抑菌率分别为51.3%、50.2%、37.1%和28.0%。比对该菌株的16S r RNA基因序列,确定Z158属于Pseudomonas aeruginosa。当不溶性的PCIH作为唯一可利用的铁源时,菌株Z158培养24 h的生物量比无铁条件下提高了46.1%。铜绿假单胞菌Z158分泌的铁载体能够抑制病原菌的生长,同时还能获取不溶性未定型铁氧化物PCIH中的铁元素。     

Utilization of Iron Gallate and Other Organic Iron Complexes by Bacteria from Water Supplies

The degradation of four soluble organic iron compounds by bacteria isolated from surface waters and the precipitation of iron from these complexes by the isolates was studied. All eight isolates brought about the precipitation of iron when grown on ferric ammonium citrate agar. Three isolates were able to degrade ferric malonate, and three others degraded ferric malate with iron precipitation. Only three isolates, two strains of Pseudomonas and one of Moraxella, were able to degrade gallic acid when this was supplied as the sole carbon source. One strain of Pseudomonas was found to be active in degrading ferric gallate. Electron microscopy of cells of this bacterium after growth in ferric gallate as the sole carbon source yielded results indicating uniform deposition of the iron on or in the bacterial cells. Seven of the isolates could degrade the iron gallate complex if supplied with additional carbon in the form of yeast extract.     

Siderophore Production by Pathogenic Mucorales and Uptake of Deferoxamine B

Clinical reports have established that mucormycosis, mainly caused by Rhizopus spp., frequently occurs in patients treated with deferoxamine B (DFO, Desferal^®) which is misappropriated by these fungi. Siderophore production by twenty mucoralean isolates was therefore investigated using a commercial iron-depleted culture medium. Siderophore production was detected for most of the isolates. Our experiments confirmed that feroxamine B (iron chelate of DFO) promoted in vitro growth of Rhizopus arrhizus. Electrophoretic analysis of somatic extracts revealed iron-regulated proteins of 60 and 32 kDa which were lacking in iron-depleted culture conditions. Using a fluorescent derivative of deferoxamine B, we showed by fluorescence microscopy the entry of the siderophore within the fungal cells, thus suggesting a shuttle mechanism encompassing the uptake of the entire siderophore-ion complex into the cell. This useful tool renders possible a better understanding of iron metabolism in Mucorales which could lead to the development of new diagnostic method or new antifungal therapy using siderophores as imaging contrast agents or active drug vectors.     

Siderophore Production and Iron Reduction by Pseudomonas mendocina in Response to Iron Deprivation

In aerobic environments microorganisms are faced with a discrepancy of ~10 orders of magnitude between the available Fe (~10^-17M) and their metabolic requirement for it (~10^-7M). In contrast to facultative anaerobic environments, where dissimilatory iron-reducing bacteria (DIRB) are often abundant, few studies have detailed microbial interactions with Fe(III) (hydr)oxides in aerobic environments. To better understand acquisition of Fe from Fe(III) (hydr)oxides, we investigated the production of siderophore and Fe(III) reduction by a strict aerobe in the presence of synthetic hematite as a source of Fe. Pseudomonas mendocina grew best when Fewas supplied as FeEDTA (~1.8x10⁸ colony-forming units [CFU] ml^-1), grew abundantly when Fe was supplied as hematite (~1.2x10⁸ CFU ml^-1), and grew poorly when Fe was withheld from the medium (~5.5x10⁷ CFU ml^-1). As expected, negligible siderophore was produced per cell when Fe was supplied as FeEDTA and more siderophore was produced in the hematite flasks than in the controls. Thus, growth of P. mendocina and the production of siderophore in the presence of hematite present compelling evidence that siderophore was produced as a mechanism to acquire Fe from hematite. For the Fe reduction experiments, Fe reduction by components of the supernatant fluid was induced weakly when Fe was supplied as hematite or as FeEDTA, but much more when the cells were cultured under extreme Fe deprivation. In fact, 16 times as much Fe reduction occurred in the controls as in the presence of either of the FeEDTA or hematite amendments. Our results, which contravene the long-held assumptions that Fe acquisition was facilitated solely by siderophores, provides a new perspective regarding microbial interactions with Fe bearing minerals.     

Siderophore Production and Induction of Iron-regulated Proteins by a Microorganism from Rhizosphere of Barley

This study provides new information on the Fe uptake system capable of supporting growth of the organism. Pseudomonas fluorescens isolated from the rhizosphere of barley, a gramineous plant, produced a siderophore under iron-limiting conditions. Its chemical structure was identified as pyochelin, on the basis of ¹H and ¹³C NMR data of a stable methyl ester derivative. The same iron-limiting conditions induced a new set of outer membrane proteins (75 and 55 kDa), consistent with a siderophore-mediated iron-uptake system.     

Growth, Fe3+ Reductase Activity, and Siderophore Production by Paenibacillus polymyxa SQR-21 Under Differential Iron Conditions

An experiment was planned to evaluate the behavior of Paenibacillus polymyxa SQR-21 under differential iron availability. P. polymyxa was grown under three differential iron conditions (0, 2, 20 μM). Iron starvation slowed bacterial growth and at all iron levels, pH of liquid culture was decreased, but maximum decrease was observed at highest iron level. Cell surface ferrireductase activity decreased as culture aged, while extracellular Fe³⁺-reducing activity constantly increased. Hydroxamates type siderophores production was increased with the decrease in iron levels. Numerous cellular proteins were expressed by P. polymyxa in the range of 5–140 kDa and several of them showed conspicuous differential iron regulation. P. polymyxa seems to have more than one type of iron acquisition mechanism including gradual release of organic acids, cell surface ferrireductases, extracellular reductants, and secretion of low molecular weight hydroxamates chelators. This article is the first to report the kinetic study of P. polymyxa under differential iron availability. The information provided here gives initial information about the iron uptake mechanism of P. polymyxa.     

Iron Demand by Thermophilic and Mesophilic Bacteria Isolated from an Antarctic Geothermal Soil

The thermophilic bacterial strain MP4 assigned to a new species, likely of the genus Alicyclobacillus, was isolated from geothermal soils on the NW slope of Mount Melbourne, Antarctica. These soils have high iron concentrations and the strain MP4 requires iron additions for growth. Four mesophilic bacterial strains Paenibacillus validus MP5, MP8, and MP10, and P. apiarius MP7, isolated from the same site, need iron supply for growth depending on the medium. Growth temperature of thermophilic strain ranges from 42 to 70 °C, and that one of mesophiles from 25 to 44 °C. Thermophilic and mesophilic strains shared microenvironments with temperature of 42–44 °C and showed optima of pH values ranging from 5.5 to 6.0. The thermophilic strain MP4 reached values of 10⁶ CFU ml⁻¹ in aqueous soil extract from the NW slope of Mt. Melbourne, and 10⁵ CFU ml⁻¹ in water extracts from other geothermal Antarctic areas (Mt. Rittmann and Cryptogam Ridge). Growth of thermophilic bacteria in aqueous extracts of the NW slope of Mount Melbourne soils caused a reduction of 50% of soluble iron content, which was recovered in bacterial biomass. These results suggest a possible involvement of the thermophilic strain MP4 in iron bioavailability in these geothermal soils.     

The Soil Type Affects Both the Differential Accumulation of Iron between Wild-type and Ferritin Over-expressor Tobacco Plants and the Sensitivity of their Rhizosphere Bacterioflora to Iron Stress

Transgenic tobacco P6 over-expressing ferritin is known to activate iron transport systems and to have increased iron content. Iron phytoextraction by this transgene is then expected to be higher than that of the wild-type (WT). In the present study, the possibility to modify iron availability for bacteria via the cultivation of the transgene P6 was explored by comparing the sensitivity to iron stress of bacteria isolated from the rhizosphere of the two plant genotypes (WT and P6). This sensitivity was evaluated by measuring the bacterial density when plated on a solid media depleted (supplemented with 8-hydroxiquinoline) or not (supplemented with Fe-8-hydroxyquinoline) in iron. The experimental conditions favorable to the differential iron accumulation between the wild-type and transgenic tobacco were identified. The two plant genotypes were grown in three soils (Hervau, Thory and Oudun) chosen for their differences in iron content, and the plants were yielded at three stages (vegetative, floral bud and flowering). The highest differential accumulation of iron in favor of the over-expressing transgene was found in the plants at the floral bud stage when cultivated in the Oudun and Thory soils. Since at that stage, the plant growth was significantly higher in the Oudun soil, the phytoextraction of iron was the highest in this soil. At the floral bud stage, bacteria isolated from the rhizosphere of the transgene cultivated in the Oudun and Thory soils appeared to be less susceptible to iron stress than those from the wild-type. Bacterial density recovered on agar medium depleted in iron was significantly the highest in the rhizosphere of the transgene cultivated in the Oudun soil. Altogether, these data indicate that the over-expressing ferritin transgenic plants, that accumulate and extract more iron from the rhizosphere than the wild-type plants, select in their rhizosphere bacteria less susceptible to iron stress compared to those selected by the wild-type plants.     

Iron Uptake and Translocation by Macrocystis pyrifera

Parameters of iron uptake have been determined for blade tissue of Macrocystis pyrifera (L.) C. Ag. These include the effects of iron concentration, light, various inhibitors, and blade type. All experiments were conducted in the defined artificial seawater Aquil. Iron uptake is light independent, energy dependent, and dependent on the reduction from Fe³⁺ to Fe²⁺. Iron is concentrated in the sieve tube exudate; exudate analysis revealed the presence of other micronutrients. Iron and other micronutrient translocation is discussed.     

接种促生菌对花生根际土壤微生物及营养元素的影响

植物根际促生菌是一类可促进植物生长的有益细菌,有效的根际促生菌剂可以减少化肥施用。以束村氏菌属(Tsukamurella sp.)P9、伯克霍尔德氏菌属(Burkholderia sp.)P10、以及P9和P10混合菌液作为接种菌株,研究促生菌对花生生长、植株及土壤营养、根际土壤微生物类群及功能的影响。30 d盆栽实验结果表明,接种组的花生鲜重、株高及根长均显著提高;根际土壤细菌总数、固氮菌和溶磷菌数均明显高于未接种组;氮循环功能菌群数量有不同程度提高,土壤蔗糖酶、脲酶及过氧化氢酶均高于对照;土壤碱解氮及速效钾显著提高,植株营养指标有所提升,尤以P10接种效果更优。本研究初步结论表明2株促生菌通过活化土壤微生物、提高植株的有效营养元素含量,促进了花生的生长。     

Proline Uptake and Utilization by Chlorella pyrenoidosa

Conditions for proline uptake and utilization by Chlorella pyrenoidosa Chick are described. Proline is taken up by growing cultures during late log phase growth after depletion of glucose from the medium. However, proline uptake by stationary phase cultures requires the presence of glucose in the medium. The results are consistent with the interpretation that some carbohydrate is required for proline uptake but proline uptake is inhibited by the accumulation of intracellular carbohydrates.     

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.     

Siderophore Production by Microorganisms Isolated From a Podzol Soil Profile

Siderophore-producing bacteria/actinobacteria and fungi were isolated from O- (organic), E- (eluvial), B- (upper illuvial), and C- (parent material) horizons of podzol soil. Siderophores were isolated and hydroxamate type siderophores were detected and quantitated by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. The molecular identification of siderophore-producing isolates showed that there was a high diversity of fungal and bacterial/actinobacterial species throughout the soil profile. The isolated bacteria/actinobacteria showed different abilities in the production of ferrioxamines (E, B, G and D). Moreover, the isolated fungal species showed great variety in the production of ferrichromes, coprogens and fusarinines.     

Glutathione-S-Transferase Activity and Metabolism of Glutathione Conjugates by Rhizosphere Bacteria

Glutathione-S-transferase (GST) activity was determined in 36 species of rhizosphere bacteria with the substrate 1-chloro-2,4-dinitrobenzene (CDNB) and in 18 strains with the herbicide alachlor. Highest levels of CDNB-GST activity (60 to 222 nmol (middot) h(sup-1) (middot) mg(sup-1)) were found in gram-negative bacteria: Enterobacter cloacae, Citrobacter diversus, Klebsiella planticola, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, and Xanthomonas campestris. There was very low CDNB-GST activity in the gram-positive strains. Rapid metabolism of CDNB-glutathione conjugates, attributable to high levels of (gamma)-glutamyltranspeptidase, also occurred in the gram-negative bacteria, especially pseudomonads. Alachlor-GST activity detected in cell extracts and whole-cell suspensions of some strains of the families Enterobacteriaceae and Pseudomonaceae was 50- to 100-fold lower than CDNB-GST activity (0.5 to 2.5 nmol (middot) h(sup-1) (middot) mg(sup-1)) and was, for the most part, constitutive. The glutathione-alachlor conjugate was rarely detected. Cysteineglycine and/or cysteine conjugates were the major products of alachlor-GST metabolism. Whole-cell suspensions of certain Pseudomonas spp. dechlorinated from 20 to 75% of 100 (mu)M alachlor in 24 h. Results indicate that rhizosphere bacteria, especially fluorescent pseudomonads, may play an important role in the degradation of xenobiotics such as alachlor via GST-mediated reactions.     

Importance of Organosulfur Utilization for Survival of Pseudomonas putida in Soil and Rhizosphere

The sulfur present in both agricultural and uncultivated soils is largely in the form of sulfonates and sulfate esters and not as free, bioavailable inorganic sulfate. Desulfurization of the former compounds in vitro has previously been studied in Pseudomonas putida, a common rhizosphere inhabitant. Survival of P. putida strains was now investigated in three sulfur-deficient Danish soils which were found to contain 60 to 70% of their sulfur in sulfonate or sulfate ester form, as determined by X-ray near-edge spectroscopy. The soil fitness of P. putida S-313 was compared with that of isogenic strains with mutations in the sftR and asfA genes (required for in vitro desulfurization of sulfate esters and arylsulfonates, respectively) and in the ssu locus (required in vitro for the desulfurization of both sulfonates and sulfate esters). asfA or sftR mutants showed significantly reduced survival compared to the parent strain in bulk soil that had been enriched with carbon and nitrogen to mimic rhizosphere conditions, but this reduced survival was not observed in the absence of these additives. In a tomato rhizosphere grown in compost, survival of sftR and ssu mutants was reduced relative to the parent strain. The results demonstrate that the ability to desulfurize sulfonates and sulfate esters is critical for survival of bacteria in the rhizosphere but less so in bulk soils outside the influence of plant roots, where carbon is the limiting nutrient for growth.     

Effect of Nematodes on Rhizosphere Colonization by Seed-Applied Bacteria

There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available. However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere. Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots. Transport by other soil biota is likely to occur, but this area has not been well studied. We hypothesized that interactions with soil nematodes may enhance colonization. To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur. Seed-applied Pseudomonas fluorescens SBW25 was capable of rhizosphere colonization at matric potentials of −10 and −40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes. Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.