Microcosm method to assess survival of recombinant bacteria associated with plants and herbivorous insects

A microcosm method was developed to investigate survial and fate of genetically engineered bacteria associated with plant surfaces and a plant-feeding insect, the variegated cutworm,Peridroma saucia. Larvae on radish plants in microcosms were sprayed with nonrecombinantPseudomonas cepacia and a recombinant strain ofP. cepacia carrying the transmissible plasmid R388::Tn1721. Leaf, whole insect, foregut, and frass samples were periodically assayed over a 48-h period to enumerate total bacteria andP. cepacia strains. Immediately after spraying,P. cepacia comprised about 20%–30% of the total population on leaves, which was 2×10⁷ cfu/g of leaf. Approximately 4×10⁷ total cfu were recovered from each gram of whole insect, when theP. cepacia strains averaged about 3×10⁵ cfu/g. After 2 days, the total epiphytic population had increased approximately fourfold, while theP. cepacia strains had decreased to 2%–30% of their initial numbers. After 24 and 48 h, eachP. cepacia strain numbered between 10⁴ and 10⁵ cfu/g of insect in foregut samples, whereas none was detectable in frass. Plasmid transfer fromP. cepacia R388::Tn1721 to the nonrecombinant recipientP. cepacia strain was not observed.     

B. thuringiensis is a Poor Colonist of Leaf Surfaces

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10⁶ cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10⁷ cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.     

Impact of commonly used agrochemicals on bacterial diversity in cultivated soils

The effects of three selected agrochemicals on bacterial diversity in cultivated soil have been studied. The selected agrochemicals are Cerox (an insecticide), Ceresate and Paraquat (both herbicides). The effect on bacterial population was studied by looking at the total heterotrophic bacteria presence and the effect of the agrochemicals on some selected soil microbes. The soil type used was loamy with pH of 6.0–7.0. The soil was placed in opaque pots and bambara bean (Vigna subterranean) seeds cultivated in them. The agrochemicals were applied two weeks after germination of seeds at concentrations based on manufacturer’s recommendation. Plant growth was assessed by weekly measurement of plant height, foliage appearance and number of nodules formed after one month. The results indicated that the diversity index (Di) among the bacteria populations in untreated soil and that of Cerox-treated soils were high with mean diversity index above 0.95. Mean Di for Ceresate-treated soil was 0.88, and that for Paraquattreated soil was 0.85 indicating low bacterial populations in these treatment-type soils. The study also showed that application of the agrochemicals caused reduction in the number of total heterotrophic bacteria population sizes in the soil. Ceresate caused 82.50% reduction in bacteria number from a mean of 40 × 10⁵ cfu g⁻¹ of soil sample to 70 × 10⁴ cfu g⁻¹. Paraquat-treated soil showed 92.86% reduction, from a mean of 56 × 10⁵ cfu g⁻¹ to 40 × 10⁴ cfu g⁻¹. Application of Cerox to the soil did not have any remarkable reduction in bacterial population number. Total viable cell count studies using Congo red yeast-extract mannitol agar indicated reduction in the number of Rhizobium spp. after application of the agrochemicals. Mean number of Rhizobium population numbers per gram of soil was 180 × 10⁴ for the untreated soil. Cerox-treated soil recorded mean number of 138 × 10⁴ rhizobial cfu g⁻¹ of soil, a 23.33% reduction. Ceresate- and Paraquat-treated soils recorded 20 × 10⁴ and 12 × 10⁴ cfu g⁻¹ of soil, respectively, representing 88.89% and 93.33% reduction in Rhizobium population numbers. Correspondingly, the mean number of nodules per plant was 44 for the growth in untreated soil, 30 for the plant in the Cerox-treated soil, 8 for the plant in Paraquat-treated soil and 3 for the plant in Ceresate-treated soil. The study has confirmed detrimental effect of insecticide on bacterial populations in the soil. Total heterotrophic counts, rhizobial counts as well as the number of nodules of all samples taken from the chemically treated soils were all low as compared to values obtained for the untreated soil. However, the effect of the insecticide was minimal in all cases as compared to the effects of the herbicides on the soil fauna. Indiscriminate use of agrochemicals on farms can therefore affect soil flora and subsequently food production.     

Endophytic bacterial flora in the stem tissue of a tropical maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) genotype: isolation,identification and enumeration

The genotypic diversity of indigenous bacterial endophytes within stem of tropical maize (Zea mays L.) was determined in field and greenhouse experiments. Strains were isolated from stem tissues of a tropical maize cultivar (PEHM-1) by trituration and surface disinfestation and their population dynamics was determined. Endophytes were found in most of the growing season at populations ranging from 1.36–6.12 × 10⁵ colony-forming units per gram fresh weight (c.f.u./gm fw) of stem. Analysis of these bacterial endophytes using Gas Chromatography—Fatty Acid Methyl Ester (GC-FAME) led to the identification of Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens as the relatively more predominant group of bacterial species residing in maize stem. When the maize seedlings grown in a greenhouse were inoculated with these four isolates individually, their population densities decreased (1.6–3.1 × 10⁵ c.f.u./gm fw of stem) as compared to the field-grown maize (1.8–3.8 × 10⁵ c.f.u./gm fw of stem). The highest persistence, however, was recovered in the case of B. subtilis with a population density of 3.1 × 10⁵ c.f.u./gm fw of stem tissue on 28 days after emergence (DAE). This is the first report on population dynamics of bacterial endophytes from tropical maize and the results establish that symptomless populations of bacteria exist in the maize stem.     

Structure and Colonization Dynamics of Epiphytic Bacterial Communities and of Selected Component Strains on Tomato (Lycopersicon esculentum) Leaves

The sizes and compositions of bacterial populations found on leaves of greenhouse and field grown tomato plants were studied by dilution plating, fatty acid methyl ester analysis (FAME), and BIOLOG plates of isolates in pure cultures. In the greenhouse, overhead-irrigated plants sustained higher microbial populations (up to 10⁵ cfu g⁻¹) than soil-irrigated plants (10³ cfu g⁻¹). Strains isolated from overhead-irrigated plants grown in a vegetable garden (n=216) and from greenhouse-grown plants (n=114) were subjected to FAME analysis. Similarly, strains from soil-irrigated field-grown plants (n=83) were identified using BIOLOG plates. In each case, populations were dominated by a few genera. When concentrated phyllosphere washes (CPW) were sprayed on greenhouse-grown, soil-irrigated plants, leaf bacterial populations of more than 10⁵ CFU g⁻¹ were sustained for 4 days; sterile buffer-sprayed leaves sustained less than 10⁴ CFU g⁻¹. No significant enrichment of any strain isolated from the sprayed leaves could be detected by FAME identification of randomly selected colonies. However, when recurring leaf saprophytic species (both Gram-positive and Gram-negative) isolated from these experiments and from plants grown outdoors were tested for epiphytic colonization under stressful conditions, all could still be detected at various levels up to 4 days after inoculation, indicating differential epiphytic fitness. The non-epiphytic bacteriaEscherichia coli andAzospirillum brasilense disappeared from the leaf surface within the same experimental period.     

An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus,by means of seed inoculants

Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudomonas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P. betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYVV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the colonization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5×10⁶ CFU g^–1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations.     

Long-Term Field Release of Bioluminescent Sinorhizobium meliloti Strains to Assess the Influence of a recA Mutation on the Strains' Survival

A field release experiment was carried out to study the fate of the isogenic, firefly luciferase (luc) gene-tagged Sinorhizobium meliloti strains L1 (RecA⁻) and L33 (RecA⁺) in the environment. Both strains were released at concentrations of approximately 10⁶ cfu g⁻¹ soil in replicate and randomized field plots, which had been sown with alfalfa (Medicago sativa). The survival of both strains during the following 7 years could be subdivided into three phases: a sharp decline for more than two orders of magnitude within the first 4 months (phase I), followed by fluctuations around an average number of 10⁴ cfu g⁻¹ soil for nearly 4 years (phase II), and a further decline to approximately 60 cfu g⁻¹ (phase III). At most sampling dates, no significant differences in the survival of both strains were detected, indicating that the recA gene function was dispensable under these environmental conditions. During the field inoculation, both strains were dispersed accidentally by wind in small numbers to noninoculated field plots. Strain L33 established at a concentration of more than 10³ cfu g⁻¹ soil with subsequent seasonal fluctuations. Although strain L1 must have been disseminated to a similar extent, it could never be recovered from noninoculated field plots, indicating that the recA mutation interfered with the strain's capability to establish there. At the beginning of the field experiment, an indigenous alfalfa-nodulating population was below the limit of detection. In the following years, however, an indigenous population arose, which finally outcompeted both strains for saprophytic growth and alfalfa nodulation. RecA⁻ strain L1 was outcompeted for alfalfa nodulation slightly faster than its RecA⁺ counterpart L33. The diversity of the indigenous population was characterized by employing the Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction fingerprint method. Typing of 2731 root nodule isolates revealed a total of 38 fingerprint groups. More than 80% of the isolates could be grouped into six dominant fingerprint groups, indicating that a few dominant bacterial strain types had outcompeted the released strains.     

Long-term survival of the plant-growth-promoting bacteria Azospirillum brasilense and Pseudomonas fluorescens in dry alginate inoculant

Two plant-growth-promoting bacteria, Azospirillum brasilense Cd and Pseudomonas fluorescens 313, immobilized in 1983 in two types of alginate-bead inoculant (with and without skim-milk supplement) and later dried and stored at ambient temperature for 14 years, were recovered in 1996. The population in each type of bead had decreased, yet significant numbers survived (10⁵–10⁶ cfu/g beads). Population numbers depended on the bead type and the three independent bacterial counting methods: the conventional plate-count method, indirect enzyme-linked immunosorbent assay and the limited-enrichment technique. Both bacterial species retained several of their original physiological features. When inoculated onto wheat plants, both species colonized and produced plant-growth effects equal to those of the contemporary strain from a culture collection or to their own 1983 records. This study showed that bacteria can survive in alginate inoculant over long periods. Received: 1 May 1998 / Received revision: 24 August 1998 / Accepted: 3 September 1998     

Biological Control of Rice Blast byPseudomonas fluorescensStrainPf7–14: Evaluation of a Marker Gene and Formulations

Pseudomonas fluorescensstrainPf7–14 was evaluated for biological control of rice blast in field experiments. StrainPf7–14 was formulated in methylcellulose:talc (1:4) and applied to IR50 rice (Oryza sativa) seeds as a seed treatment and as foliar sprays in seedbed and field experiments. When applied as a seed treatment followed by three foliar applications, the strain provided a 68.5% suppression of rice blast in the seedbed experiment and a 59.6% suppression in the field experiment. The persistence and migration ofPf7–14 on the rice plant was studied with the aid oflacZYgenes inserted into the bacterium. In greenhouse experiments,Pf7–14gal was detected on rice roots at 10⁶to 10⁵cfu/g of root tissue for 110 days, the duration of the rice crop. Migration of the strain from the seeds to the leaves occurred only until the seedlings were 16 days old. WhenPf7–14 was applied to the rice plants by foliar sprays, 10⁴cfu of the bacterium per gram of leaf tissue was detected for the next 40 days. The limited migration of the bacterial biocontrol agent emphasizes the need for multiple foliar applications of the bacterium to sustain the bacterial population for effective suppression of rice blast.     

Utility of some nitrogen-fixing microorganisms in the phyllosphere of crop plants

Summary The utility of spraying some known N₂-fixing microorganisms on rice leaves grown both in N-less sand culture and under field conditions was examined. The effect was compared with that of spraying a phyllosphere N₂-fixing isolate of Klebsiella, KUPBR₂, and application of nitrogenous fertilizers. All the growth parameters studied including dry weight and N-content were enhanced. Under field conditions number of tillers was increased by 26% withKlebsiella pneumoniae M5al and by 65% with Aphanothece. The dry weight of the plants was enhanced by 61–119%. The yield per 10 m² was almost doubled with Aphanothece, Beijerinckia 8007,Mycobacterium flavum, K. pneumoniae M5al and KUPBR₂. The increases observed withStreptomyces sp. G12 though less spectacular was significant at 1% level with respect to several growth parameters.K. pneumoniae M5al,M. flavum andStreptomyces sp. G12 exhibited nitrogenase activity both in laboratory culture and in association with rice plants.     

Quantification and Modeling of Plasmid Mobilization on Seeds and Roots

Mobilization frequencies of the nonconjugative plasmid pMON5003 were quantified using Escherichia coli TB1(pRK2013) as donor of a helper plasmid, E. coli M182 (pMON5003) as donor of the nonconjugative plasmid, and Pseudomonas fluorescens as recipient. Initial mating experiments were conducted in nutrient and minimal salts media and pea seed exudates. Mobilization rates were higher during early stationary growth of donors, helpers, and recipients. Numbers of transconjugants were higher in biparental matings when donors contained both conjugative and nonconjugative plasmids, versus tri-parental matings. A mathematical model was developed to predict a nonconjugative plasmid transfer rate parameter (δ), estimating the proportion of conjugative matings in which a plasmid is mobilized. Values of δ ranged from 8 × 10⁻³ to 7.9 × 10⁻¹. Transfer frequencies for pMON5003 from E. coli to P. fluorescens on pea seeds and roots were determined. Transconjugants (P. fluorescens 2-79 (pMON5003)) were isolated from seeds, roots, and soil, but mobilization frequencies were lower than in liquid media.     

Life cycle and population dynamics of a protective insect symbiont reveal severe bottlenecks during vertical transmission

Insects engage in mutualistic relationships with a wide variety of microorganisms that are usually transmitted vertically to the next generation. During transmission, the symbiont populations often suffer significant bottlenecks that may entail major genetic and genomic consequences. Here we investigated the life-cycle and the severity of transmission bottlenecks in a symbiotic system with an unusual way of post-hatch vertical transmission by using quantitative PCRs and morphological 3D-reconstructions. European beewolves (Philanthus triangulum, Hymenoptera: Crabronidae) harbor symbiotic bacteria (‘Candidatus Streptomyces philanthi’) in specialized antennal gland reservoirs and secrete them into their subterranean brood cells. The symbionts are later taken up by the beewolf larva and incorporated into the cocoon material to provide protection against pathogenic microorganisms. Even after months of hibernation, the symbiont population on the cocoon is estimated to encompass around 1.4 × 10⁵ cells. However, our results indicate that only few of these bacterial cells (about 9.7 × 10²) are taken up from the cocoon by the emerging female. The symbiont population subsequently undergoes logistic growth within the antennal gland reservoirs and reaches a maximum of about 1.5 × 10⁷ cells 3–4 days after emergence. The maximum specific growth rate is estimated to be 0.084–0.105 h⁻¹. With a total reduction in cell numbers of about 6.7 × 10⁻⁵ during vertical transmission, the symbiont population experiences one of the most severe bottlenecks known for any symbiotic system to date. This extreme bottleneck may have significantly affected the evolution of the beewolf-Streptomyces symbiosis by increased genetic drift, an accumulation of mildly deleterious mutations and genome erosion.     

Grazing of nonindigenous bacteria by nano-sized protozoa in a natural coastal system

Mesocosms (4.5 m³) situated in a closed bay area were used to investigate the effect of protozoan predation on nonindigenous bacteria. Pseudomonas fluorescens strain Agl was released into mesocosms as a single inoculum of 1 × 10⁵ cells ml^–1 (final concentration) or as four inocula (same concentration each) at intervals of 3 days. Mesocosms that had received growth media corresponding to the inoculum served as controls. Numbers of P. fluorescens Ag1 decreased rapidly whether released as single or multiple inocula. Direct estimation of protozoan predation using fluorescently labeled P. fluorescens from log phase and starved cultures, respectively, revealed that natural populations of heterotrophic nanoflagellates consumed substantial amounts of the nonindigenous bacterial strain. The volume of fluorescently labeled cells prepared from starved cells was 68% of log phase cell volume, but the individual clearance of the small cells was five to seven times higher than that of the log phase bacteria. The natural populations of nanoflagellates consumed 34–62% of P. fluorescens Ag1 daily if starved bacteria were offered as food, and 3–13% if the cells were in the logarithmic growth phase. This suggests that the effect of protozoan predation on nonindigenous bacterial strains is substantial because cultured bacteria are likely to starve in natural environments. The addition of P. fluorescens Ag1 and the growth medium enhanced the abundance of natural bacteria, chlorophyll a, heterotrophic nanoflagellates, and ciliates, but it did not improve the growth conditions for the released strain. The effects on the indigenous populations were more pronounced after addition of fresh medium than following inoculation with cells, which possibly was due to the lower nutrient content of spent medium. However, these results, based on direct estimation of protozoan predation on log phase and starved nonindigenous bacteria, point to the conclusion that mortality induced by bacterivorous predators is the key factor determining removal of nonindigenous bacteria introduced in natural aquatic systems. Correspondence to: K. Christoffersen.     

Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Post treatment of groundwater denitrification fluidized bed reactor effluents to achieve drinking water quality

Post treatment of effluents from heterotrophic groundwater denitrification fluidized bed reactors (FBR) designed to achieve drinking water quality has been investigated. The denitrification process adds to the dissolved organic compounds, biomass and bacteria in the effluent. They are also lacking dissolved oxygen. Effluents from the process were treated in combined post treatment processes based on either a trickling filter and sedimentation unit (‘TF combination’) or contact flocculation (‘CF combination’). Both processes were followed by sand filtration, granular activated carbon (GAC) and chlorination. Results regarding total suspended solids (TSS) and turbidity removal showed an advantage to the ‘CF combination’, and the target turbidity (NTU <1) was always achieved when the alum dose was 10 or 20 mg l⁻¹. Backwash of the sand filter and GAC column was required after 27 h of operation (average value). An average total reduction in dissolved organic carbon (DOC) of 40% was observed with a final DOC of 3.5–4 mg L⁻¹. Most of the removal of the DOC occurred in the sand filter (28%), while the GAC contribution was smaller (18%). No regrowth potential was observed using the Werner method when a pure culture of Pseudomonas fluorescens P17 was used as inoculum in samples of chlorinated effluent (post chlorination). When a mixed culture of indigenous bacteria was used as inoculum, a high regrowth potential was observed. Installing an additional chlorination unit before the sand filter column (pre and post chlorination) resulted in effluent with no regrowth potential for both Pseudomonas fluorescens P17 and indigenous bacteria. Received 17 October 1997/ Accepted in revised form 29 May 1998     

Comparison of conditions for mycelial growth of <Emphasis Type="Italic">Lepista sordida</Emphasis> causing fairy rings on <Emphasis Type="Italic">Zoysia matrella</Emphasis> turf to those on <Emphasis Type="Italic">Agrostis palustris</Emphasis> turf

Symptoms of fairy rings caused by Lepista sordida have been reported on Zoysiagrass (Zoysia spp.) turf maintained at fairway height (2 cm), but not on bentgrass (Agrostis spp.) maintained at putting green height (0.5 cm). The mycelia of this fungus inhabit primarily the upper 0–2 cm layer of the soil extending into the thatch. To compare conditions for the mycelial growth in Z. matrella turf to those in A. palustris turf, we examined the effects of nutrients, temperature, water potential, and pH in the field as well as in the laboratory. Greater growth of the mycelia was observed in medium that included hot water extracts from soil of the 0–1 cm zone in Z. matrella turf compared to that from A. palustris. The upper soil layer in Z. matrella turf contained more organic matter from clippings than that in A. palustris. The temperature and water potential of the 0–2 cm soil zone in Z. matrella turf were also more favorable for the mycelial growth. The soil pH values of this zone in Z. matrella turf were less favorable compared to A. palustris but within the range for accelerating mycelial growth. Part of this study was presented orally at the 46th meeting of the Mycological Society of Japan in 2002     

Indoor and outdoor airborne fungal propagule concentrations in Mexico City

Thirty homes of asthmatic adults located in Mexico City were examined to determine the predominant culturable fungi and the changes in their airborne concentrations. Fungi were cultured and identified microscopically from air samples collected in naturally ventilated homes, during both wet (July–August) and cool dry (November–December) seasons, and from settled dust from the same homes. Airborne dust from indoor yielded 99–4950 cfu m⁻³, and settled dust 10²–10⁶ cfu g⁻¹ on DG18 agar. The indoor geometric mean concentration of airborne fungi during the cool dry season was 460 cfu m⁻³ while in the wet season it was 141 cfu m⁻³. Similarly, numbers of airborne fungal propagules out of doors decreased 60% between the dry and wet season. In general, the total fungal concentrations in indoor air were less than 10³ cfu m⁻³ and a large proportion of them was collected in Stage-2 of the Andersen sampler. Moreover, the ratio between indoor and outdoor concentrations was <3:1. Five of the 30 sampled homes yielded >500 cfu m⁻³ of one genus, with up to 1493Cladosporium cfu m⁻³ or 2549Penicillium cfu m⁻³. Also, these two genera were predominant in both airborne and settled dust, and their concentrations were greater indoors than out, indicating a possible indoor source of fungal propagules. The predominant species wereCladosporium herbarum, Penicillium aurantiogriseum andP. chrysogenum. These results suggest that exposure to large concentrations of fungi occurs indoors and is associated with both seasons of the year and with particular home characteristics.     

Effect of Xanthobacter,isolated and characterized from rice roots,on growth of wetland rice

With an autotrophic, N-free medium, Xanthobacter populations were isolated from the roots of wetland rice grown under field conditions. Xanthobacter populations ranged from 3.2×10⁴ to 5.1×10⁵ colony-forming units (cfu) g^-1 of root and averaged 47-fold higher on the root or rhizoplane than in the neighbouring nonrhizosphere. Characterization studies indicated dissimilarities in carbon utilization and motility among the isolated Xanthobacter strains and other recognized Xanthobacter species. Under gnotobiotic conditions, the population of one isolate, Xanthobacter sp. JW-KR1, increased from 10⁵ to 10⁷ cfu plant^-1 1 d after inoculation when a rice plant was present, but declined to numbers below the limit of detection (<10⁴ cfu assembly^-1) after 3 d in the absence of a plant. Scanning electron microscopy revealed Xanthobacter as pleomorphic forms on the rhizoplane. To assess the effect of Xanthobacter on plant growth, rice plants were grown under greenhouse conditions in plant assemblies containing sand and half-strength Hoagland's nutrient solution with and without nitrogen. Plants were either inoculated with 10⁵ cfu Xanthobacter g^-1 of sand or left uninoculated. After 40 d, plants without nitrogen showed no significant differences in top or root dry weight, plant height, root length, or number of tillers or leaves, whether the plants were inoculated or uninoculated. However, when nitrogen was added, inoculated plants had a significantly larger top dry weight (15%) and number of leaves (19%) than uninoculated plants. Under conditions of added and no added nitrogen, acetylene reduction assays showed Xanthobacter sp. JW-KR1 produced <0.1 (below detection limit) and 7 nmol C₂H₄ plant^-1 h^-1, respectively. Under the conditions studied, the results suggest that both Xanthobacter and wetland rice derive some benefits from their association.     

A specific marker,pat, for studying the fate of introduced bacteria and their DNA in soil using a combination of detection techniques

A specific eucaryotic DNA marker from Solanum tuberosum cv Bintje (688 bp patatin cDNA fragment) was cloned into the unique HindIII-site of plasmid RP4. RP4:: pat was transferred from Escherichia coli to Pseudomonas fluorescens R2f by filter mating.Homology to pat was not detected in the microbial population of Ede loamy sand soil, nor in that of the rhizosphere of wheat growing in this soil, as evidenced by colony filter hybridization. More sensitive molecular detection techniques like most-probable-number recovery/hybridization analysis, and analysis of total community DNA from soil by polymerase chain reaction (PCR) amplification did not reveal the presence of the pat sequence either. P. fluorescens R2f (RP4:: pat), introduced into sterile soil extract microcosms, initially showed poor survival and plasmid loss, after which the introduced populations grew and stabilized at a level of about Log₁₀ 7 cfu per mL. Between 25 and 50% of the population maintained the plasmid, as evidenced by filter hybridization of colonies from non-selective agar plates using the pat fragment as probe.Introduced R2f (RP4:: pat) could be recovered from soil microcosms using selective plating followed by colony hybridization and MPN recovery/hybridization with the pat probe. The presence of the pat marker always coincided with the presence of the resistance genes on RP4:: pat, indicating pat was an adequate marker of the presence of this plasmid. In addition, it adequately described the population dynamics of the introduced strain in soil, since no loss of the plasmid occurred.Hybridization to pat was also useful to show transfer of plasmid RP4:: pat to a recipient strain in soil; transfer to indigenous bacteria was not detected.Analysis by slot-blot hybridization of total community DNA extracted from inoculated soils indicated about Log₁₀ 6 cfu per g of dry soil were still detectable. Application of the PCR on this DNA indicated pat was detectable at least at a level of Log₁₀ 4 immunofluorescence-detectable cells per g of dry soil. Thus extraction of total community DNA followed by PCR permitted the detection of genetically engineered microorganisms present in soil as non-culturable cells.     

Growth rates and nutrition status of an open and a closed population of Rhododendron ferrugineum L . in the northwestern Alps (France)

 This study evaluates the utility of cell compounds as indicators of nutrition status of plant populations. An overview of the soluble free amino-acids, carbohydrates and P-compounds in the two year classes of leaves from Rhododendron ferrugineum populations showing variable biological performances, was drawn up using carbon-13 and phosphorus-31 nuclear magnetic resonance spectroscopy (NMR). The results showed differences between the age of leaves and the degree of population closure. The open population, which has the smallest growth rate and net primary productivity, had generally lower levels of amino-acids but higher levels of carbohydrates and P-compounds. Neither the amounts of mineral nitrogen produced by the soils nor the total nitrogen concentrations in leaves differed significantly. Still we hypothesize that the nitrogen availability could be largely responsible for the variations observed between the populations, as phosphorus analyses showed that the two populations did not suffer significant P deficiencies. Differences in vitality of R. ferrugineum may be explained by the fact that in an open population R. ferrugineum is forced into a situation of sharing nutrients with other species while in a closed population it is the sole species to exploit a specific pool of nutrients. Finally we observed that the ¹³C- and ³¹P-NMR approach is more suitable for studying the nutrition status of plant populations under field conditions than the determination of the total amounts of different elementary nutrients. Received: 2 May 1995 / Accepted: 22 February 1996