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.     

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).     

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).     

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.     

Decomposition processes under Bt (Bacillus thuringiensis) maize: Results of a multi-site experiment

The effects of maize expressing the Bacillus thuringiensis Cry1Ab protein (Bt maize) on decomposition processes under three different European climatic conditions were assessed in the field. Farming practices using Bt maize were compared with conventional farming practices using near-isogenic non-Bt maize lines under realistic agricultural practices. The litter-bag method was used to study litter decomposition and nitrogen mineralization dynamics of wheat straw. After 4 months incubation in the field, decomposition and mineralization were mainly influenced by climatic conditions with no negative effect of the Bt toxin on decomposition processes.     

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.     

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.     

Earthworm populations in a northern U.S. Cornbelt soil are not affected by long-term cultivation of Bt maize expressing Cry1Ab and Cry3Bb1 proteins

Earthworms, which play a key role in biogeochemical processes in soil ecosystems, could be negatively affected by the cultivation of transgenic Bt crops. Studies to date have found few effects of Bt maize on earthworm species. If adverse effects occur, they are likely to be chronic or sub-lethal and expressed over large spatial and temporal scales. Our objective in the present study was to investigate potential effects on earthworm populations in soil cultivated with Bt maize in a large multiple-year field study. We surveyed the earthworm populations in 0.16-ha experimental field plots of two varieties of Cry1Ab Bt maize, one variety of Cry3Bb1 Bt maize, and three non-transgenic control varieties cultivated for four years. Four earthworm species were found in our sample: Aporrectodea caliginosa, Aporrectodea trapezoides, Aporrectodea tuberculata (collectively, the A. caliginosa species complex), and Lumbricus terrestris. We found no significant differences in the biomass of juveniles and adults for all four species between Bt and non-Bt maize varieties. From this and previous studies, we conclude that the effects of Cry1Ab and Cry3Bb1 Bt maize on the A. caliginosa species complex and L. terrestris are small. Nonetheless, general conclusions about the effects of Bt maize on earthworm populations are not warranted due to the small number of species tested. In future laboratory studies, earthworm species should be selected according to their association with a Bt crop and the impact of that species to valued soil ecosystem processes.     

Btk蛋白在红壤胶体上的吸附解吸行为及其对杀虫活性的影响

研究了苏云金芽胞杆菌库斯塔克亚种（Btk）两种分子量不同的杀虫蛋白（65 kDa和130 kDa）在红壤胶体上的吸附、解吸和杀虫活性。Btk杀虫蛋白容易被红壤胶体吸附,0.5 h即达到吸附平衡,65 kDa蛋白的吸附速率高于130 kDa蛋白。两种Btk杀虫蛋白在0.1 mol L^-1Tris缓冲液（pH 8）中的等温吸附曲线均符合Langmuir方程（R²>0.992）,65 kDa蛋白的吸附量高于130 kDa蛋白。在10～50℃范围内,温度升高吸附量略有降低。pH 6～8范围内,吸附量随pH升高而下降。两种Bt蛋白在红壤胶体吸附前后对棉铃虫均具有杀虫活性,而且吸附后杀虫活性提高了2～5倍。解吸实验表明,Btk杀虫蛋白与红壤胶体结合比较牢固,吸附态65 kDa蛋白和130 kDa蛋白经3次去离子水解吸,总解吸率分别为37.2%和22.9%,经3次pH8的Tris缓冲液解吸,总解吸率分别为20.7%和14.8%。红外光谱分析表明,两种分子量的Bt蛋白在吸附前后结构没有明显变化。透射电镜分析表明,吸附前后红壤胶体颗粒粒径未发生改变。吸附态Bt蛋白杀虫活性提高且不易被解吸,提示转Bt基因作物释放蛋白可能存在环境风险。     

苏云金芽孢杆菌菌剂对棉花根际土壤细菌数量及多样性的影响

采用稀释平板培养法与PCR-DGGE技术, 以阿维菌素为阳性对照, 水为阴性对照, 研究了大田喷施推荐剂量(0.1 kg·hm^-2)和高剂量(10 kg·hm^-2)苏云金芽孢杆菌(Bt)菌剂对棉花根际土壤细菌种群数量及结构的影响。结果表明, 喷雾处理后1~3 d, 不同处理组土壤细菌数量间无显著性差异, 在3 d时细菌数量均值达到最大, 之后开始下降, 12 d后清水对照、推荐剂量Bt菌剂、高剂量Bt菌剂处理组土壤细菌数量均值维持在4.0×107 CFU·g^-1左右; 推荐剂量Bt菌剂处理样品土壤细菌数量在6 d时显著高于清水对照, 其余时间与清水对照间无显著性差异; 高剂量Bt菌剂处理与清水对照在整个试验期间均无显著性差异; 阿维菌素处理组土壤细菌数量在0~6 d内与清水对照无显著性差异, 而在12~45 d内显著低于其他3个处理组。DGGE图谱显示, Bt菌剂处理对棉花根际土壤17种细菌均无显著抑制作用。聚类分析结果表明, Bt菌剂对土壤细菌群落结构的扰动在12 d后得到恢复。与阴性对照组相比, Bt菌剂对土壤细菌多样性指数无显著影响, 而阳性对照阿维菌素对土壤细菌种群消长和多样性指数有较强的影响。对DGGE图谱中17条电泳条带的序列分析, 证明棉花根际土壤中存在起固氮作用的慢生根瘤菌属细菌和具有污染修复与净化活性的鞘脂菌属、鞘氨醇单胞菌属和红球菌属细菌。Bt菌剂与阿维菌素处理均对这些土壤有益菌群无明显不利影响。总体结果表明, Bt菌剂无论是在正常推荐剂量下还是在较高剂量(推荐剂量的100倍)下使用, 对棉花根际土壤微生态环境产生的冲击都较小, 是一种生态安全性较高的生物农药。     

Soil persistence of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Bt) toxin from transgenic Bt cotton tissues and its effect on soil enzyme activities

A silty loam soil was incubated with the leaves and stems of two transgenic Bacillus thuringiensis (Bt) cotton varieties and nontransgenic Bt cotton to study the soil persistence of the Bt toxin from the decomposing transgenic Bt cotton tissues and its effect on soil enzyme activities. The results showed that after Bt cotton tissue amendment, Bt toxin was introduced into soil upon decomposition; about 50% of the introduced Bt toxin persisted in soil for at least 56 days. No Bt toxin was detected in the nontransgenic Bt cotton-amended soil; the amount of Bt toxin was the highest in the soil treated with the residue with the higher Bt toxin content. Activities of soil urease, acid phosphomonoesterase, invertase, and cellulase were stimulated by the addition of Bt cotton tissues, whereas activity of soil arylsulfatase was inhibited. Probably cotton tissue stimulated microbial activity in soil, and as a consequence, enzyme activities of soil were generally increased. This effect can mask any negative effect of the Bt toxin on microbial activity and thus on enzyme activities.     

Effect of Bacillus subtilis on granite weathering: A laboratory experiment

We performed a comparative experiment to investigate: (1) how the ubiquitous soil bacterium Bacillus subtilis weathers granite; and (2) which granite-forming minerals weather more rapidly via biological processes. Batch system experiments (granite specimen in a 500 ml solution including NaCl, glucose, yeast extract and bacteria B. subtilis at 27 °C) were carried out for 30 days. Granite surfaces were observed by SEM before and after the experiment. B. subtilis had a strong influence on granite weathering by forming pits. There were 2.4 times as many pits and micropores were 2.3 times wider in granite exposed to B. subtilis when compared with bacteria-free samples. B. subtilis appear to preferentially select an optimum place to adhere to the mineral and dissolve essential elements from the mineral to live. Plagioclase was more vulnerable to bacterial weathering than biotite among the granite composing minerals.     

Btk杀虫蛋白在红壤和棕壤中的吸附热力学研究

采用平衡吸附法测定了苏云金芽胞杆菌库斯塔克亚种(Btk)蛋白在红壤和棕壤中的吸附等温线,并依据等温线进行了吸附平衡常数和热力学参数计算。在278K～318K范围内,Bt杀虫蛋白在红壤和棕壤中的吸附等温线符合Langmuir方程(R2>0.994 1),随着吸附温度升高,Bt杀虫蛋白的吸附量和吸附平衡常数下降,而吸附分离因子RL升高。红壤吸附Bt蛋白的RL值在0.218 1～0.580 1之间,棕壤吸附Bt蛋白的RL值在0.361 7～0.754 1之间,均属于优惠吸附。Bt杀虫蛋白在红壤和棕壤中的吸附是一个自发、放热、熵增过程,红壤吸附Bt杀虫蛋白的活化能在13.43～14.78 kJ mol-1之间,棕壤吸附Bt蛋白的活化能在10.89～11.47 kJ mol-1之间。随着温度升高,活化能和吸附自由能绝对值变大,提示土壤对Bt杀虫蛋白的吸附由物理吸附向化学吸附转变。     

转Bt基因棉秸秆还田利用对土壤肥力的影响

随着转基因的快速发展,大量转Bt棉秸秆的合理利用和处理是不可忽视的重要课题之一。为明确Bt棉秸秆还田利用的可行性和安全性,本研究以不同抗虫转Bt基因棉和常规棉花‘泗棉3号’为研究材料,在分别种植1、2年后将秸秆机械粉碎后原位还田,测试土壤中Bt蛋白残留量、土壤酶活性及养分含量的变化,分析Bt棉秸秆原位还田对土壤肥力特性的影响。研究结果表明,秸秆还田40 d后, Bt棉样地土壤中Bt残留蛋白检测值较低,均与非转基因棉样地无显著性差异。棉秸秆还田后,土壤脲酶、蔗糖酶、蛋白酶、多酚氧化酶、过氧化氢酶、碱性磷酸酶活性皆较秸秆还田前增加,但土壤纤维素酶活性较之前降低。棉秸秆还田使土壤中有机质、有效磷、碱解氮、速效钾和全氮等养分含量及pH明显增加,而Bt抗虫棉与常规棉秸秆还田后对土壤肥力的影响不存在显著差异。对土壤综合肥力指数评价结果表明,秸秆还田对土壤肥力提升与Bt棉抗虫水平无关,土壤肥力指数在两年间由Ⅲ级水平上升至Ⅱ级水平。综上, Bt棉花秸秆还田不会造成土壤综合肥力降低,相反能有效提升土壤肥力;同时还田利用措施可对转基因植株有效灭活,与转基因植物秸秆利用和无害化处理要求相契合。生产中用于Bt转基因棉花秸秆利用和处理在一定程度上是安全可行的。     

Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

Growth promotion and increased potassium uptake of cotton and rape by a potassium releasing strain of Bacillus edaphicus

A potassium-releasing bacterial strain Bacillus edaphicus NBT was examined for plant-growth-promoting effects and nutrient uptake on cotton and rape in K-deficient soil in pot experiments. Inoculation with bacterial strain B. edaphicus NBT was found to increase root and shoot growth of cotton and rape. Strain NBT was able to mobilize potassium efficiently in both plants when illite was added to the soil. In cotton and rape growing in soils treated with insoluble potassium and inoculated with strain NBT, the potassium content was increased by 30 and 26%, respectively. Bacterial inoculation also resulted in higher N and P contents of above ground plant components. The bacterial isolate was also able to colonize and develop in the rhizosphere soil of cotton and rape after root inoculation.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

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.     

Effects of repeated metolachlor applications on its persistence in field soil and degradation kinetics in mixed microbial cultures

 The persistence of metolachlor, a soil-applied herbicide, was studied under field conditions involving repeated herbicide applications. The test field received four applications of metolachlor over an 8-month period, which included two cropping seasons. There was a trend for more rapid rates of degradation with increasing numbers of previous treatments, with fifty percent dissipation time (DT₅₀) of metolachlor declining from 18 days in the first spray to 2.5 days in the fourth spray. An effort was made to isolate the microbial population which had become acclimated to the herbicide from this field soil. A fungal community isolated from this soil showed the capacity to degrade up to 99.6% of the metolachlor within a span of 20 days. The bacterial community isolated could also degrade up to 81.5% of the metolachlor. Hence, this study clearly indicated that repeated applications of metolachlor to soil resulted in the generation of an adapted microbial population with an enhanced ability to degrade the applied herbicide. Received: 13 November 1998