Persistence of toxins and cells of Bacillus thuringiensis subsp. kurstaki introduced in sprays to Sardinia soils

Sprays of commercial insecticidal preparations of the bacterium, Bacillus thuringiensis subsp. kurstaki (Btk), usually a mixture of cells, spores and parasporal crystals, have been used for the last 10 yr in Sardinia (Italy) to protect cork oak forests against the gypsy moth (Lymantria dispar L.). Until now, the protective antilepidopteran efficacies of each of the various spray treatments rather than their effects on the environment have been evaluated. Consequently, the persistence of Btk and its toxin, released in sprays (FORAY 48B^®), in soils of cork oak stands, located in Orotelli, Tempio Pausania and Calangianus (Sardinia), were investigated. In the Calangianus soil, the numbers of Btk remained essentially constant for 28 months (the longest time studied) after spraying, indicating that Btk was able to compete with the indigenous microbial community; the toxin was detected 28 months after spraying by immunological assay, but at a reduced concentration; and the larvicidal activity decreased essentially linearly to 14 months and then decreased markedly between 14 and 28 months. In the Tempio Pausania and Orotelli soils, cells of Btk were detected, whereas the toxin was not detected by immunological and larvicidal assays, 52 and 88 months (the longest times studied) after spraying, respectively. The numbers of Btk cells detected were probably too low to account for the presence of the toxin in all of the soils studied, as there was no correlation between numbers of Btk and toxin detected by immunological assays (correlation coefficient of −0.66) in the Calangianus soil. Our results indicated that Btk and its toxin introduced into soils in sprays can persist for long periods (at least 88 months for Btk and at least 28 months for its toxin).     

Effects of physicochemical interactions and microbial activity on the persistence of Cry1Aa Bt (Bacillus thuringiensis) toxin in soil

Genetically modified crops, that produce Cry insecticidal crystal proteins (Cry) from Bacillus thuringiensis (Bt), release these toxins into soils through root exudates and upon decomposition of residues. The fate of these toxins in soil has not yet been clearly elucidated. Persistence can be influenced by biotic (degradation by microorganisms) and abiotic factors (physicochemical interactions with soil components, especially adsorption). The aim of this study was to follow the fate of Cry1Aa Bt toxin in contrasting soils subjected to different treatments to enhance or inhibit microbial activity, in order to establish the importance of biotic and abiotic processes for the fate of Bt toxin. The toxin was efficiently extracted from each soil using an alkaline buffer containing a protein, bovine serum albumin, and a nonionic surfactant, Tween 20. The marked decline of extractable toxin after incubation of weeks to months was soil-dependent. The decrease of extractable toxin with incubation time was not related to microbial degradation but mainly to physicochemical interactions with the surfaces that may decrease immunochemical detectability or enhance protein fixation. Hydrophobic interactions may play an important role in determining the interaction of the toxin with surfaces.     

Microbial utilisation of two proteins adsorbed to a vertisol clay fraction: toxin from Bacillus thuringiensis subsp. tenebrionis and bovine serum albumin

Clay minerals have been shown to reduce the extent and rate of biodegradation of several compounds. Here, we investigated the ability of soil clays to protect proteins from biodegradation: the insecticidal protein from Bacillus thuringiensis subsp. tenebrionis (Btt toxin) and Bovine Serum Albumin (BSA). The two proteins adsorbed in large amounts (up to 0.24 g BSA g⁻¹ clay and 0.74 g Btt toxin g⁻¹ clay) and irreversibly to smectite clay particles from a vertisol. We measured the growth of a soil inoculum in the presence of each of proteins as the sole source of carbon. When clay was present in the medium, microbial growth was directly proportional to the amount of free protein (i.e. nonadsorbed). Hence, the two proteins were unavailable when adsorbed to clay. The clay had little influence on the ability of microorganisms to hydrolyse a soluble substrate. The inhibitory effect of clays on utilisation of BSA and Btt toxin was interpreted as being the result of the adsorption of the proteins to clay, which rendered the proteins unavailable for microbial utilisation.     

Physiological characteristics (SDH and ALP activities) of arbuscular mycorrhizal colonization as affected by Bacillus thuringiensis inoculation under two phosphorus levels

The effect of Bacillus thuringiensis (B.t.) inoculation on plant growth and on the intra- and extraradical mycorrhizal development of lettuce roots colonized by Glomus mosseae or Glomus intraradices was examined in an inert, soil-less substrate. Histochemical determination of succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities which indicate active fungal metabolism was carried out at two phosphorus (P) levels. The presence of B.t. increased extra- and intraradical colonization [measured as frequency (%F), intensity (%I) and percentage of arbuscules (%A)] for both arbuscular mycorrhizal fungi (AMF) rather than plant growth or nutrition regardless P level. Under the lowest level of P fertilization, B.t. enhanced to a similar extent the extra- and intraradical development of both endophytes, but the proportion of fungal tissue showing SDH or ALP was increased in G. intraradices-colonized plants. [SDH: 458% (M) and 512% (A); ALP: 358% (M) and 300% (A)]. P supply decreased G. intraradices colonization to a higher extent than G. mosseae. Nevertheless, the totality of G. intraradices structures developed in P-amended medium showed intraradical o extraradical activity, while in G. mosseae-colonized roots, SDH and ALP activities highly decreased relative to fungal tissue determined by TB staining as affected by P. Our results show that bacterial inoculation compensates the negative effect of P on the intraradical fungal growth and vitality. P amendment reduced in a higher extent G. intraradices infection intensity (non-vital and vital staining) and G. mosseae activity (ALP staining). Thus, big differences in the proportion of SDH-active infection showing ALP activity in mycelium developed by each endophyte were noted at the highest P level. Physiological plant parameters such as photosynthetic activity did not explain specific changes on each arbuscular-mycorrhizal fungus as affected by P or B.t. inoculation. The increased extraradical mycelium development and metabolic fungal activity as a result of B.t. inoculation positively affected N and P plant content and photosynthetic rate in G. intraradices-colonized plants under the lowest P conditions. In general, the increased metabolically active fungal biomass in co-inoculated plants was irrespective of P level and was not related to the P plant uptake from the inert soil-less substrate. These results show the bacterial effect increasing the physiological and metabolic status of AM endophytes, which not only confirms but also extends previous findings on arbuscular mycorrhizae-bacteria interactions. The present study emphasizes the ecological and practical importance of rhizosphere free-living bacteria as mycorrhizae-helper microorganisms.     

Adsorption and desorption of monomeric Bt (Bacillus thuringiensis) Cry1Aa toxin on montmorillonite and kaolinite

Genetically modified crops, which produce pesticidal proteins from Bacillus thuringiensis, release the toxins into soils through root exudates and upon decomposition of crop residues. Although the phenomena of gene transfer and emergence of resistance have been well documented, the fate of these toxins in soil has not yet been clearly elucidated. The aim of this study was to elucidate the adsorption and the desorbability of the Cry1Aa Bt insecticidal protein in contact with two sodium-saturated clays: montmorillonite and kaolinite. Because the toxin is released into soil in small quantities, it was assumed that it will be in a monomeric state in solution until it oligomerized on cell membranes. The originality of this study was to focus on the monomeric form of the protein. Specific sample conditions were required to avoid polymerisation. A pH above 6.5 and an ionic strength of at least 150 mM (NaCl) were necessary to keep the protein in solution and in a monomeric state. The adsorption isotherms obtained were of the L-type (low affinity) for both clays and fitted the Langmuir equation. The adsorption maximum of the toxin, calculated by the Langmuir nonlinear regression, decreased with increasing pH from 6.5, which was close to the isoelectric point, to 9. At pH 6.5, the calculated adsorption was 1.7 g g⁻¹ on montmorillonite and 0.04 g g⁻¹ on kaolinite. Desorbability measurements showed that a small fraction of toxin could be desorbed by water (up to 14%) and more by alkaline pH buffers (36 ± 7%), indicating that it was not tightly bound. Numerous surfactants were evaluated and the toxin was found to be easily desorbed from both clays when using zwitterionic and nonionic surfactants such as CHAPS, Triton-X-100, and Tween 20. This finding has important implications for the optimization of detection methods for Bt toxin in soil.     

Promotion of plant growth and in situ degradation of phenol by an engineered Pseudomonas fluorescens strain in different contaminated environments

We show that Pseudomonas fluorescens strain P13, a plant growth-promoting bacterium, enhanced the growth of corn in uncontaminated soil but not in contaminated soil, perhaps because of its inability to reduce phytotoxicity. Another bacterial strain, Pseudomonas aeruginosa strain SZH16, showed in situ phenol-degrading activity and contained a plasmid loaded with a gene encoding for catechol 2, 3-dioxygenase, an important enzyme in the degradation pathway of aromatic compounds. We implanted this biodegradation ability into strain P13, using horizontal gene transfer techniques using strain SZH16 as the donor and P13 as the recipient, to generate a phenol-degrading transconjugant which obtained the effective plasmid from strain SZH16. Introduction of the transconjugant P13 strain into an artificially phenol-spiked soil promoted the growth of corn and in situ phenol degradation, and the increase in plant biomass correlated with the decrease in soil phenol content. Furthermore, the transconjugant P13 strain was also found to stimulate corn growth and reduce phenol concentration in water containing phenol and in historically contaminated field soils, indicating that the transconjugant strain could promote plant growth in both contaminated and uncontaminated environments. The transconjugant P13 strain was more efficient than either strain P13 or SZH16, and shows how plant growth-promoting bacteria which show no, or only limited, ability to degrade organic pollutants may be modified. This technique is attractive for many environmental remediation and agronomic applications.     

Effects of nutrients and surfactants on pyrene mineralization and Mycobacterium spp. populations in contaminated soil

The impact of nutrient (KNO₃ and KH₂PO₄) or surfactant (Triton X-100 and Tergitol NP-10) amendments on the mineralization of ¹⁴C-pyrene and the indigenous Mycobacterium spp. community structure in a petroleum-contaminated soil were determined. All soil amendments enhanced pyrene mineralization with nutrients being slightly more effective than surfactants. 16S rRNA genes were PCR-amplified using Mycobacterium spp.-specific primers, separated by temperature gradient gel electrophoresis (TGGE), and prominent bands sequenced to compare the mycobacteria communities. The soil sample with the highest level of mineralization had no detectable changes in the community structure. Disappearance of a specific phylotype occurred in soils with lower mineralization rates. Phylogenetic analysis of sequenced TGGE bands indicated existence of novel strains.     

Association between Burkholderia species and arbuscular mycorrhizal fungus spores in soil

Burkholderia pseudomallei, the bacterial cause of the potentially fatal infection known as melioidosis, has a facultative intracellular lifestyle. The intracellular presence of B. pseudomallei in various eukaryotes including arbuscular mycorrhizal fungus (AMF) spores can be demonstrated in vitro. AMF spores were isolated from soils in a melioidosis-endemic area. B. pseudomallei and other Burkholderia spp. DNA was detected in these AMF spore samples, confirming an AMF spore-Burkholderia spp. association in soils which did not yield Burkholderia spp. by culture. This association may explain the environmental persistence, difficulty of recovery and dispersal of Burkholderia spp. in specific environments.     

The regeneration status of the endangered Acer opalus subsp. granatense throughout its geographical distribution in the Iberian Peninsula

Acer opalus subsp. granatense is an endemic endangered tree with a wide but fragmented distribution in the Mediterranean mountains. The persistence of its small populations often depends on just a few adults, and consequently is highly vulnerable to factors limiting recruitment. In this paper, we examined the regeneration status of this maple in 16 populations throughout its whole geographical distribution in the Iberian Peninsula. Age and size structures were analysed as indicators of the viability of the species. Additionally, we studied the effects of herbivory by ungulates and the role of shrubs as nurse plants in maple regeneration dynamic. Our results show that A. opalus subsp. granatense has active recruitment throughout its range in the Iberian Peninsula. Shrubs served as the main microsites for recruitment, not only for early establishment but also for long-term survival. However, ungulates heavily damaged maple saplings in all locations and microhabitats. A direct consequence of herbivory is the uncoupling of age and size structures, saplings becoming older but not higher, possibly affecting population turnover in the long term. We suggest that the conservation of the small populations of Acer opalus subsp. granatense heavily depends on the control of herbivory pressure throughout the maple distribution area.     

Transgenic Bt plants decompose less in soil than non-Bt plants

Bt plants are plants that have been genetically modified to express the insecticidal proteins (e.g. Cry1Ab, Cry1Ac, Cry3A) from subspecies of the bacterium, Bacillus thuringiensis (Bt), to kill lepidopteran pests that feed on corn, rice, tobacco, canola, and cotton and coleopteran pests that feed on potato. The biomass of these transgenic Bt plants (Bt+) was decomposed less in soil than the biomass of their near-isogenic non-Bt plant counterparts (Bt−). Soil was amended with 0.5, 1, or 2% (wt wt⁻¹) ground, dried (50 °C) leaves or stems of Bt corn plants; with 0.5% (wt wt⁻¹) ground, dried biomass of Bt rice, tobacco, canola, cotton, and potato plants; with biomass of the near-isogenic plants without the respective cry genes; or not amended. The gross metabolic activity of the soil was determined by CO₂ evolution. The amounts of C evolved as CO₂ were significantly lower from soil microcosms amended with biomass of Bt plants than of non-Bt plants. This difference occurred with stems and leaves from two hybrids of Bt corn, one of which had a higher C:N ratio than its near-isogenic non-Bt counterpart and the other which had essentially the same C:N ratio, even when glucose, nitrogen (NH₄NO₃), or glucose plus nitrogen were added with the biomass. The C:N ratios of the other Bt plants (including two other hybrids of Bt corn) and their near-isogenic non-Bt counterparts were also not related to their relative biodegradation. Bt corn had a significantly higher lignin content than near-isogenic non-Bt corn. However, the lignin content of the other Bt plants, which was significantly lower than that of both Bt and non-Bt corn, was generally not statistically significantly different, although 10-66% higher, from that of their respective non-Bt near-isolines. The numbers of culturable bacteria and fungi and the activity of representative enzymes involved in the degradation of plant biomass were not significantly different between soil amended with biomass of Bt or non-Bt corn. The degradation of the biomass of all Bt plants in the absence of soil but inoculated with a microbial suspension from the same soil was also significantly less than that of their respective inoculated non-Bt plants. The addition of streptomycin, cycloheximide, or both to the soil suspension did not alter the relative degradation of Bt+ and Bt− biomass, suggesting that differences in the soil microbiota were not responsible for the differential decomposition of Bt+ and Bt− biomass. All samples of soil amended with biomass of Bt plants were immunologically positive for the respective Cry proteins and toxic to the larvae of the tobacco hornworm (Manduca sexta), which was used as a representative lepidopteran in insect bioassays (no insecticidal assay was done for the Cry3A protein from potato). The ecological and environmental relevance of these findings is not clear.     

Host-parasite soil communities and environmental constraints: Modelling of soil functions involved in interactions between plant-parasitic nematodes and Pasteuria penetrans

Nematodes belonging to the genus Meloidogyne are the most ubiquitous and widespread plant-parasitic nematodes. They occur worldwide, are polyphagous and can parasitize most cultivated plants leading to reduced crop yields. They are especially harmful in developing countries because of the lack of suitable and feasible management strategies. Among all the control practices (chemicals, physical techniques, cultural practices, resistance), the use of natural enemies as biological control agents is the most recently developed. Pasteuria penetrans which is an obligate Gram-positive, endospore-forming bacterium, is perhaps the most promising plant-parasitic nematode biocontrol agent. Despite much research conducted on prey-predator interactions (host-parasite specificity, mechanisms of attachment, field efficacy), the influence of the soil environment on host-parasite interactions is poorly understood even when the soil appears to be the key factor. Beyond common studies on the influence of climatic conditions on the attachment of endospores of P. penetrans to nematodes, more knowledge about the systemic interactions between plants, soil water dynamics, soil texture and structure, and other biota on the parasitism of nematodes by P. penetrans would improve their utilization as biological control agents. The aim of this review is to analyze the literature dealing with the influence of the soil on nematode - P. penetrans interactions in order to suggest a helpful conceptual model based on partitioning the Pasteuria population in sub-populations according to their soil habitat (dispersible and non-dispersible aggregates, microporosity, macroporosity), not all of them being available for attachment and infection on nematodes. Such concerns should be taken into account by epidemiologists for improving biological management strategies based on the use of this bacterium.     

Effects of physical and chemical properties of soils on adsorption of the insecticidal protein (Cry1Ab) from Bacillus thuringiensis at Cry1Ab protein concentrations relevant for experimental field sites

The adsorption of the insecticidal Cry1Ab protein of Bacillus thuringiensis (Bt) on Na-montmorillonite (M-Na) and soil clay fractions was studied. The aim of this study was not to find the adsorption capacity of the soils from the experimental field site, where Bt corn (MON810) was cultivated, but rather to characterize the adsorption behavior of the Cry1Ab protein at concentrations typically found at experimental field sites. In kinetic experiments, the Cry1Ab protein adsorbed rapidly (<60 min) on M-Na. As the concentration of M-Na was varied and the added Cry1Ab protein concentration was kept constant (20 and 45 ng ml⁻¹), the adsorption per unit weight of Cry1Ab protein decreased with increasing concentrations of M-Na. Adsorption of Cry1Ab protein on M-Na decreased as the pH value of the suspension increased. All adsorption isotherms could be described mathematically by a linear regression with the parameter k, the distribution coefficient, being the slope of the regression line. Although their mineralogical composition was nearly identical, the soil clay fractions showed different k values. The different k values were correlated with the physical and chemical properties of the soil clay fractions, such as the organic carbon content, the specific external surface area, and the electrokinetic charge of the external surfaces of the clays, as well as with the external surface charge density. An increase in the amount of soil organic matter, as well as an increase in the electrokinetic external surface charge of the soil clays, decreased the distribution coefficient k. An increase of the specific external surface areas of the soil clays resulted in a higher distribution coefficient k.Less than 10% of adsorbed Cry1Ab protein was reversibly adsorbed on the soil clays and, thus, desorbed. The desorption efficiency of distilled water was higher than that of a solution of CaCl₂ (2.25 mmol) and of dissolved organic carbon (50 mg C l⁻¹).     

Induction of toluene degradation and growth promotion in corn and wheat by horizontal gene transfer within endophytic bacteria

Some experiments involving important crop plants, corn and wheat, were carried out to characterize the agronomic and environmental application of Burkholderia cepacia strain FX2 able to degrade toluene and containing plasmids with the gene encoding for catechol 2, 3-dioxygenase (C23O), a key enzyme in the degradation pathway of monocyclic aromatic compounds. The inoculation of corn and wheat with FX2 led to the promotion of plant growth and reduction in evapotranspiration of toluene into the air. Endophytic bacteria able to grow on toluene as the only source of carbon and containing a C23O gene were found in the plants inoculated with FX2 but not in their non-inoculated controls. Compared to control plants, a greater number of toluene-degrading, phosphate-solubilizing and siderophore-producing endophytes were found in inoculated plants. Furthermore, a direct correlation occurred between plant biomass responses and the magnitude of C23O-containing endophytes. Phylogenetic tree comparison, plasmid analysis and filter mating assays showed that the C23O gene was transferred horizontally from FX2 to the natural endophytic bacteria of corn and wheat. Horizontal gene transfer among endophytic bacteria might contribute to pollutant degradation, growth promotion and potential for disease suppression in corn and wheat.     

Rhizobia nodulating African Acacia spp. and Sesbania sesban trees in southern Ethiopian soils are metabolically and genomically diverse

The diversity of 110 rhizobial strains isolated from Acacia abyssinica, A. seyal, A. tortilis, Faidherbia albida, Sesbania sesban, Phaseolus vulgaris, and Vigna unguiculata grown in soils across diverse agro-ecological zones in southern Ethiopia was assessed using the Biolog™ system and amplified fragment length polymorphism (AFLP) fingerprinting technique. By cluster analysis of the metabolic and genomic fingerprints, the test strains were grouped into 13 Biolog and 11 AFLP clusters. Twenty-two strains in the Biolog method and 15 strains in the AFLP analysis were linked to eight and four reference species, respectively, out of the 28 included in the study. Most of the test strains (more than 80% of 110) were not related to any of the reference species by both methods. Forty-six test strains (42% of 110) were grouped into seven corresponding Biolog and AFLP clusters, suggesting that these groups represented the same strains, or in some cases clonal descendants of the same organisms. In contrast to the strains from S. sesban, isolates from Acacia spp. were represented in several Biolog and AFLP clusters indicating the promiscuous nature of the latter and widespread occurrence of compatible rhizobia in most of the soil sampling locations. The results showed that indigenous rhizobia nodulating native woody species in Ethiopian soils constituted metabolically and genomically diverse groups that are not linked to reference species.     

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.     

Transformation of C-labelled lignin and humic substances in forest soil by the saprobic basidiomycetes Gymnopus erythropus and Hypholoma fasciculare

Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are involved in the transformation of lignin, as well as humic and fulvic acids. The aim of this work was to evaluate their importance in lignin transformation in forest litter. Two litter decomposing basidiomycete species differing in their abilities to degrade lignin - Hypholoma fasciculare, and Gymnopus erythropus - were cultured on sterile or non-sterile oak litter and their transformation of a ¹⁴C-labelled synthetic lignin (dehydrogenation polymer ¹⁴C-DHP) was compared with that of the indigenous litter microflora. Both in sterile and non-sterile litter, colonisation by basidiomycetes led to higher titres of lignocellulose-degrading enzymes, in particular of laccase and Mn-peroxidase (MnP). The titres of the latter were 6 to 40-fold increased in the presence of basidiomycetes compared to non-sterile litter. During 10 weeks, G. erythropus mineralised over 31% of ¹⁴C-DHP in sterile litter and 23% in non-sterile litter compared to 14% in the non-sterile control. Lignin mineralization by H. fasciculare was comparable to the non-sterile control, 12% in sterile litter and 16% in the non-sterile litter. The largest part of ¹⁴C from ¹⁴C-DHP was transformed into humic compounds during litter treatment with both fungi as well as in the control. In addition to the fast lignin mineralization, microcosms containing G. erythropus also showed a lower final content of unaltered lignin and 23-28% of the lignin was converted into water-soluble compounds with relatively low molecular mass (<5 kDa). Both G. erythropus and H. fasciculare were also able to further mineralise humic compounds. During a 10-week fungal treatment of an artificial ¹⁴C-humic acid (¹⁴C-HA) supplemented to the natural humic material of a forest soil, the fungi mineralised 42% and 19% of the labelled material, respectively, under sterile conditions. The ¹⁴C-HA mineralization by introduced basidiomycetes in microcosms containing non-sterile humic material, however, did not significantly differ from that of a non-sterile control and was around 12%. Altogether the results show that saprobic basidiomycetes can considerably differ in their rates of lignin and humic substance conversion. Furthermore, lignin degradation in forest soil can rather slow down by interspecific competition than it is accelerated by cooperation of different microorganisms occupying specific nutritional niches. Therefore, the overall contribution of saprobic basidiomycetes depends on their particular eco-physiological status and the competitive pressure, and may be often lower than initially expected. Significant lignin transformation including partial mineralization is seemingly not exclusively dependent on exceptional high titres of ligninolytic enzymes but also on so far unknown factors. Higher endocellulase production and subsequent weight loss was found in microcosms where saprobic basidiomycetes were combined with indigenous microbes. Potentially, lignin degradation by the basidiomycetes may have increased cellulose availability to the indigenous microbes.     

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.     

Comparison of g23 gene sequence diversity between Novosphingobium and Sphingomonas phages and phage communities in the floodwater of a Japanese paddy field

Our previous study indicated that the diversity of the major capsid gene (g23) of T4-type bacteriophages (phages) of Novosphingobium and Sphingomonas strains isolated from the floodwater of a Japanese paddy field is comparable to those of the clones obtained from other Japanese paddy fields. For more strict comparison of the diversity, this study examined g23 sequences between Novosphingobium and Sphingomonas phages and phage communities in the identical floodwater of a Japanese paddy field. The clones were obtained by applying g23-specific primers to DNA extracted from the floodwaters. Many 23 clones in the floodwater were grouped into the same clusters of Paddy Groups I-VI with g23 genes of Novosphingobium/Sphingomonas phages with some clones belonging to an additional cluster. In addition, the remaining clones belonged to the clusters of marine clones and T4-type enterophages. These findings indicate that the g23 genes in the floodwater are more diversified than those of Novosphingobium/Sphingomonas phages including g23 genes closely related to the genes of enterophages and marine origins.