Fungal endophytes enhance wheat heat and drought tolerance in terms of grain yield and second-generation seed viability.

AIMS: We evaluated the impact of fungal endophyte symbiosis on the growth, ecophysiological and reproductive success of wheat exposed to heat and drought. METHODS AND RESULTS: The resistance of pot-grown wheat to heat or drought stress was measured by quantifying efficiency of photosystem II (Fv /Fm), plant height, average seed weight (ASW), total seed weight (TSW), water-use efficiency (WUE) as well as time to 50% germination and percentage germination of second-generation seeds produced under heat stress, drought stress or well-watered conditions. The endophytic fungi tested increased wheat tolerance for drought and heat. Endophyte SMCD 2206 was the most beneficial, followed by SMCD 2210 and 2215. Surprisingly, second-generation seeds produced by drought-stressed wheat colonized by SMCD 2206, 2210 or 2215 had decreased WUE relative to those produced by endophyte-free, drought-stressed plants. However, these seeds germinated more rapidly than those produced by endophyte-free, stressed parental plants. CONCLUSIONS: The tested consortium of endophytes has the potential to improve wheat adaptation to heat and drought. SIGNIFICANCE AND IMPACT OF THE STUDY: The capacity of endophytes to increase wheat tolerance for abiotic stress and to improved germination in endophyte-free second-generation seeds arising from stressed plants could be applicable to agriculture. The mechanisms by which intergenerational endophyte-mediated affects occurs warrant further research.

Field and soil microcosm studies on the survival and conjugation of a Pseudomonas putida strain bearing a recombinant plasmid, pADPTel.

Pseudomonas putida CR30RNS (pADPTel) is an antibiotic-resistant strain with a recombinant plasmid that confers resistance to tellurite and the ability to catabolize atrazine. The survival of this strain as well as its ability to transfer genes for atrazine degradation and tellurite resistance to indigenous soil bacteria were tested in both fallow soil and canola (Brassica napus) rhizosphere by the use of parallel field and laboratory releases. Culturable CR30RNS (pADPTel) were enumerated in field and microcosm soils at 7- to 14-day intervals over 49 d. Strain CR30RNS (pADPTel) survived for up to 7 weeks in microcosm soils at a density of 10(4) CFU/g soil, whereas in field soils the population declined to 10(3) CFU/g soil by the fourth week. In contrast, when CR30RNS (pADPTel) was introduced into the soil as a seed coating of canola (B. napus 'Karoo'), the bacterium established at higher cell densities in the rhizosphere (10(6)-10(5) CFU/g fresh root mass), with no subsequent decrease in numbers. The presence of selective pressure (i.e., atrazine) had no significant effect on the survival of CR30RNS (pADPTel) in either field or microcosm soils. One year postinoculation field sites were examined for the presence of CR30RNS (pADPTel) and no evidence of culturable parental cells was observed when samples were plated onto selective media. However, the atzC and telAB gene segments were amplified from the field soils at that time. Under laboratory conditions, indigenous soil bacteria were capable of receiving and expressing the engineered plasmid construct at frequencies ranging from 1 to 10(-3) transconjugants per donor. However, no plasmid transfer to indigenous soil bacteria was detected in the field or microcosm soils regardless of the presence of canola rhizosphere and (or) the application of atrazine. Our results show that the survival and population size of P. putida CR30RNS (pADPTel) might be sufficient for degradation of environmental pollutants but that the transfer frequency was too low to be detected under the conditions of this study.

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Distribution and Biogeochemical Importance of Bacterial Populations in a Thick Clay-Rich Aquitard System.

To investigate the distribution of microbial biomass, populations and activities within a clay-rich, low hydraulic conductivity (10-11 to 10-12 m s-1) aquitard complex, cores were aseptically obtained from a series of overlying clayey deposits; a fractured till, unfractured till (20-30 ka BP), a disturbed interfacial zone (20-30 ka BP), and a Cretaceous clay aquitard (71-72 Ma BP). The results of confocal microscopy studies, culture methods, molecular approaches, and extractive fatty acid analyses all indicated low bacterial numbers that were non-homogeneously distributed within the sediments. Various primers for catabolic genes were used to amplify extracted DNA. Results indicated the presence of eubacterial 23S rDNA, and the narH gene for nitrate reductase and ribulose-1,5-biphosphate carboxylase (RuBP carboxylase). Although there was no evidence of limitation by electron acceptors or donors, sulfate-reducing bacteria were not detected below the fractured till zone, using PCR, enrichment, or culture techniques. Denaturing gradient gel electrophoresis (DGGE) analyses indicated differences in community composition and abundance between the various geologic units. Results of FAME analyses of sediments yielded detectable extractable fatty acids throughout the aquitard complex. Bacterial activities were demonstrated by measuring mineralization of (14C) glucose. Porewater chemistry and stable isotope data were in keeping with an environment in which extremely slow growing, low populations of bacteria exert little impact. The observations also support the contention that in low permeability sediments bacteria may survive for geologic time periods.

Effects of chemical speciation in growth media on the toxicity of mercury(II).

The toxicity of metals, including mercury, is expressed differently in different media, and the addition of soluble organics to the growth medium can have a significant impact on bioassay results. Although the effect of medium composition on metal toxicity is generally attributed to its effect on metal speciation (i.e., the chemical form in which the metal occurs), the importance of individual metal-ligand species remains largely unclear. Here, we report the results of a study that investigated, both experimentally and from a modeling perspective, the effects of complex soluble organic supplements on the acute toxicity (i.e., 50% inhibitory concentration [IC50]) of mercury to a Pseudomonas fluorescens isolate in chemically well-defined synthetic growth media (M-IIX). The media consisted of a basal inorganic salts medium supplemented with glycerol (0.1%, vol/vol) and a variety of common protein hydrolysates (0.1%, vol/vol), i.e., Difco beef extract (X = B), Casamino Acids (X = C), peptone (X = P), soytone (X = S), tryptone (X = T), and yeast extract (X = Y). These were analyzed to obtain cation, anion, and amino acid profiles and the results were used to compute the aqueous speciation of Hg(II) in the media. Respirometric bioassays were performed and IC50s were calculated. Medium components varied significantly in their effects on the acute toxicity of Hg(II) to the P. fluorescens isolate. IC50s ranged from 1.48 to 14.54 micrograms of Hg ml-1, and the acute toxicity of Hg(II) in the different media decreased in the order M-IIC >> M-IIP > M-IIB >> M-IIT > M-IIS >>> M-IIY.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of metribuzin on the Rhizobium leguminosarum--lentil (Lens culinaris) symbiosis.

The effects of the triazine herbicide metribuzin (Sencor) on the lentil (Lens culinaris Medic.) - Rhizobium leguminosarum biovar viciae symbiosis were studied in Leonard jars and growth pouches. Lentils inoculated with Rhizobium leguminosarum strain 128C54 or 128C84, and noninoculated lentils grown in plant nutrient solution supplemented with 5 mM KNO3, had metribuzin applied to the plants at either 8 or 13 days after planting. When sprayed at 8 days, metribuzin had a significant (p less than or equal to 0.05) negative effect on plant weight, number of nodules, taproot growth, and acetylene reduction activity. Five to 10 days after spraying, the plants began to recover from the inhibitory effects. When spraying was delayed to 13 days after planting, metribuzin had little effect on plant growth. The R. leguminosarum strain used as inoculant affected the degree of inhibition of lentil growth and the rate of plant recovery. Less than 0.2% of foliarly applied metribuzin was translocated to the root. Thus the detrimental effects of metribuzin application to lentils were mainly due to direct effects on the plant, which resulted in indirect effects on nodulation and nitrogen fixation.

Biotoxicity of mercury as influenced by mercury(II) speciation.

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.

Transduction of Escherichia coli in soil.

Bacteriophage P1-mediated generalized transduction of Escherichia coli K-12 was assessed in nonsterile soil. Auxotrophic recipient cells (thr- leu- thi- rpsL) were incubated in a sandy and a silty clay loam soil, and the transducing phage lysates from prototrophic strains carrying transposon 10(Tn10) in either purE or aroL regions were added. At intervals, the bacterial populations derived from the soils were plated on selective-differential media to enumerate prototrophic (thr+, leu+, or Tcr) transductants. Of 100 bacterial isolates obtained on the selective-differential media, 58 (14 thr+; 11, leu+; 33 Tcr) were confirmed E. coli transductants. The frequency of transduction in soil was ca. 10(-6). These data demonstrate the potential use of bacteriophage P1 to genetically manipulate E. coli in situ.

Persistence of Nosema locustae Spores in Soil as Determined by Fluorescence Microscopy.

Nosema locustae, a protozoan parasite of grasshoppers, is used as a bioinsecticide. In the present study, the persistence of N. locustae spores in soil and the interaction of these spores with the indigenous soil microflora were examined with various forms of microscopy and staining. Fluorescence microscopy was found to be better than phase-contrast or bright-field microscopy for detecting and viewing spores in soil. Fluorescein isothiocyanate was a better fluorescent stain than acridine orange or fluorescein diacetate; water-soluble aniline blue did not stain spores. The eight bright-field microscopy stains tested (phenolic erythrosin, phenolic rose bengal, malachite green, crystal violet, safranin, Congo red, methyl red, and eosin B) were not satisfactory, as spore staining characteristics were either poor or masked by overstained soil debris. A procedure was developed which allowed spores to be extracted from soil with a peptone-phosphate buffer, recovered on a membrane filter, and stained with fluorescein isothiocyanate for microscopic counting. This procedure was used to assess the persistence of N. locustae spores in field and laboratory soils. The number of N. locustae spores in a laboratory model soil system persisted at a high level for over 8 weeks when the soil was incubated at 5 degrees C but exhibited a 1,000-fold decrease after 1 week of incubation at 27 degrees C. Persistence was related to the temperature-dependent activity of the indigenous soil microflora, which, on the basis of microscopic observations, appeared to prey on N. locustae spores. N. locustae spores were detected in an N. locustae-treated field soil at a low level consistent with the level for laboratory soil incubated at 27 degrees C, and they persisted at this level for over 2 months. No spores were detected on vegetation from this field or in the soil from an adjacent, nontreated control field. N. locustae-like spores were also detected in soil from nontreated fields supporting large grasshopper populations.

Ensifer adhaerens Predatory Activity Against Other Bacteria in Soil, as Monitored by Indirect Phage Analysis.

An indirect phage analysis procedure was used to detect and follow the activity of the bacterial predator Ensifer adhaerens in situ in natural soil. The soil was percolated with an aqueous suspension of washed bacterial host cells so that the E. adhaerens cells naturally present in the soil would multiply in response to the host cells. The natural phage development which ensued against these multiplying E. adhaerens cells in the soil was then monitored by noting plaques which developed when the percolation fluid was plated with laboratory strains of E. adhaerens on laboratory media. The activities of the other members of the predation system that includes E. adhaerens (Streptomyces sp. strain 34 and a myxobacter) could not be monitored directly by phage analysis because phage were not found for them. Indirect monitoring was possible, however, because they were susceptible to attack by E. adhaerens. In general, the results were in agreement with previous observations by other methods of the predation sequence. E. adhaerens attacked Micrococcus luteus, Streptomyces sp. strain 34, and the myxobacter but did not attack several other possible species of hosts. It also did not respond to percolation of the soil with various nutrient solutions. E. adhaerens phage activity was not present in half of the soils percolated with M. luteus cells. This seemed to reflect too great a phage-host specificity for the technique as regards these soils, because E. adhaerens-like bacteria other than the strains used for plaquing were present in at least some of these soils. Although E. adhaerens did not attack Escherichia coli or Pseudomonas aeruginosa in soil, there was an overproduction of E. adhaerens phage if these bacteria were percolated simultaneously with M. luteus cells. The possibility is discussed that this represents an activation by M. luteus (or by a heat-extractable factor from it) of other bacterial predators that attack E. coli or P. aeruginosa and that these predators subsequently are themselves attacked by E. adhaerens.

Isolation of arthrobacter bacteriophage from soil.

Soil was percolated with water and various nutrient solutions, and then the percolates were analyzed for bacteriophages which produced plaques on various Arthrobacter strains. The water percolates did not contain detectable phage. In contrast, phages for A. globiformis strains ATCC 8010 and 4336, and for several recent Arthrobacter species soil isolates, were easily detected in nutrient broth, soil extract, and cation-complete medium percolates. These percolates did not contain phage that produced plaques on A. oxydans and a recent Arthrobacter species soil isolate. Percolation with a selective nicotine-salts solution was required for demonstrating phage for these bacteria. None of the percolates contained phage for five additional named Arthrobacter species. In addition, phages were not detected for A. crystallopoietes in a 2-hydroxypyridine percolate of soil. Based on their lytic spectra, the phage isolates from this soil were relatively host specific.

Myceloid growth of Arthrobacter globiformis and other Arthrobacter species.

Transitory myceloid growth occurs in certain complex media with Arthrobacter globiformis strain ATCC 8010. This type of growth, however, was not observed in a medium which contained an array of metal ions but did not contain agents able to complex metal ions. Addition of metal-complexing agents to this medium caused an interruption in the life cycle of strain 8010 so that growth occurred only as the myceloid form. It appeared that manganese was the critical metal that was removed by the metal-complexing agents. During growth, the myceloid cells started to fragment, but wall septation was incomplete. A. globiformis strain ATCC 4336 and several other Arthrobacter species and soil isolates, but not Arthrobacter crystallopoietes, responded to metal-complexing agents as did strain 8010. Biotin and vitamin B12 were not involved in this myceloid growth.

his-Linked hydrogen sulfide locus of Salmonella typhimurium and its expression in Escherichia coli.

A his-linked H2S locus of Salmonella typhimurium has been further defined by direct isolation of H2S mutants. Expression of this locus in Escherichia coli has been demonstrated.