Rhizobium inoculation of Calopogonium caeruleum

The shade-tolerant cover legume Calopogonium caeruleum is promiscuous in its nodulating habits. In sand culture, symbiotic effectiveness of the strains tested was variable; 6 strains of rhizobia markedly improved shoot yields and 20 increased shoot N content. In pot experiments using cultivated and non-cultivated soils, inoculation gave no significant increase in shoot yields. When grown under rubber in plantation conditions at four localities, shoot dry matter yields, N content and nodulation also were not different from uninoculated plants when sampled for up to 2 yr after planting. This occurred despite the low numbers (< 10 g^?1 soil) of native rhizobia at some sites and an appreciable establishment (> 70% recovery in nodules) by the inoculant strains.     

Improved zinc tolerance in Eucalyptus globulus inoculated with Glomus deserticola and Trametes versicolor or Coriolopsis rigida

The potential of interactions between saprophytic and arbuscular mycorrhizal (AM) fungi to improve Eucalyptus globulus grown in soil contaminated with Zn were investigated. The presence of 100 mg kg ⁻¹ Zn decreased the shoot and root dry weight of E. globulus colonized with Glomus deserticola less than in plants not colonized with AM. Zn also decreased the extent of root length colonization by AM and the AM fungus metabolic activity, measured as succinate dehydrogenase (SDH) activity of the fungal mycelium inside the E. globulus root. The saprophytic fungi Trametes versicolor and Coriolopsis rigida increased the shoot dry weight and the tolerance of E. globulus to Zn when these plants were AM-colonized. Both saprophytic fungi increased the percentage of AM root length colonization and elevated G. deserticola SDH activity in the presence of all Zn concentrations applied to the soil. In the presence of 500 and 1000 mg kg⁻¹ Zn, there were higher metal concentrations in roots and shoots of AM than in non-AM plants; furthermore, both saprophytic fungi increased Zn uptake by E. globulus colonized by G. deserticola. The higher root to shoot metal ratio observed in mycorrhizal E. globulus plants indicates that G. deserticola enhanced Zn uptake and accumulation in the root system, playing a filtering/sequestering role in the presence of Zn. However, saprophytic fungi did not increase the root to shoot Zn ratio in mycorrhizal E. globulus plants. The effect of the saprophytic fungi on the tolerance and the accumulation of Zn in E. globulus was mediated by its effect on the colonization and metabolic activity of the AM fungi.     

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.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

In vitro effects of Fusarium blight-controlling fungicides on pathogens of Poa pratensis

An experimental iprodione fungicide,3-(3,5dichlorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide, controls Fusarium blight of Kentucky bluegrass (Poa pratensis L.), but it also amplifies the proportion of crowns colonized by Fusarium and the number of its propagules in soils. In contrast, the disease, the proportion of infected crowns, and the numbers of propagules in soil are generally suppressed by benomyl, methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate. Triadimefon, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone, also controls the disease but is not stimulatory or inhibitory of fusaria. Iprodione and benomyl were studied for their effects on growth and sporulation of Fusarium acuminatum isolated from diseased crowns; iprodione had no or slightly stimulatory effects, and benomyl greatly suppressed these processes, except in a benomyl-tolerant strain.Toxicities of iprodione and benomyl to 1555 identified Fusarium isolates from Kentucky bluegrass turf were determined, as were the toxicities of iprodione to 23 turfgrass pathogens. Of the Fusarium spp, only F. solani was significantly inhibited by iprodione, whereas all were inhibited by benomyl. Iprodione-sensitive fungi included species of Bipolaris, Corticium, Curvularia, Drechslera, Rhizoctonia, Sclerotinia, and Typhula. Insensitive fungi included Colletotrichum, Fusarium, Gaeumannomyces, and Pythium.Investigations with selective fungicides indicate that the primary causal agent of Fusarium blight is not among the fusaria, and that re-interpretation of the disease and its etiology is necessary.     

Mycoparasitism by Pythium oligandrum and P. acanthicum

Isolates of the reported mycoparasites Pythium oligandrum, P. acanthicum and P. periplocum markedly reduced growth and cellulolysis by Botryotrichum piluliferum, grew rapidly across agar plates precolonized by Phialophora radicicola var radicicola (sensu Deacon) and, where tested (not P. periplocum), were non-pathogenic towards higher plants. Isolates of P. echinulatum, P. mamillatum. P. megalacanthum, P. spinosum, P. ultimum and one isolate of P. acanthicum behaved differently from the mycoparasites and could, themselves, be placed in two groupings in these tests. It is suggested that the ability or otherwise to grow on Phialophora-precolonized agar plates may help to distinguish broad biological groupings within the genus Pythium, but these groupings may cut across conventional taxonomic ones.One isolate of P. acanthicum was tested for its effects on a range of cellulolytic fungi: it reduced their growth to different extents, as did P. oligandrum.Plates of potato-dextrose agar precolonized by Phialophora radicicola were used to isolate selectively P. oligandrum and similar fungi from soils, but the use of hemp seed baits in conjunction with precolonized plates was less selective for these fungi.Straw pieces precolonized by P. oligandrum and buried in soil decomposed at the same rate as virgin straws or those precolonized by P. ultimum or Mucor hiemalis. Subsequently, Stachyholrys atra appeared to colonize straws more frequently from soil, and Fusarium spp. less so, in the presence of P. oligandrum than in its absence. In the laboratory, P. oligandrum was antagonized by Slachyholrys, whereas Fusarium spp. were frequently overgrown by the Pythium.     

Accumulation of specific flavonoids in soybean (Glycine max (L.) Merr.) as a function of the early tripartite symbiosis with arbuscular mycorrhizal fungi and Bradyrhizobium japonicum (Kirchner) Jordan

This study is the first report assessing the effect of soil inoculation on the signalling interaction of Bradyrhizobium japonicum, arbuscular mycorrhizal fungi (AMF) and soybean plants throughout the early stages of colonisation that lead to the tripartite symbiosis. In a study using soil disturbance to produce contrasting indigenous AMF treatments, the flavonoids daidzein, genistein and coumestrol were identified as possible signals for regulating the establishment of the tripartite symbiosis. However, it was unclear whether soil disturbance induced changes in flavonoid root accumulation other than through changing the potential for AMF colonization. In this study, soil treatments comprising all possible combinations of AMF and B. japonicum were established to test whether (1) modifications in root flavonoid accumulation depend on the potential for AMF colonization, and (2) synthesis and accumulation of flavonoids in the roots change over time as a function of the early plant-microbial interactions that lead to the tripartite symbiosis. The study was comprised of two phases. First, maize was grown over 3-week periods to promote the development of the AM fungus Glomus clarum. Second, the interaction between soybean, G. clarum and B. japonicum was evaluated at 6, 10, 14 and 40 days after plant emergence. Root colonization by G. clarum had a positive effect on nodulation 14 days after emergence, producing, 30% more nodules which were 40% heavier than those on roots solely inoculated with B. japonicum. The tripartite symbiosis resulted in 23% more N₂ being fixed than did the simpler symbiosis between soybean and B. japonicum. The presence of both symbionts changed accumulation of flavonoids in roots. Daidzein and coumestrol increased with plant growth. However, development of the tripartite symbiosis caused a decrease in coumestrol; accumulation of daidzein, the most abundant flavonoid, was reduced in the presence of AMF.     

Translocations of eight species of burrow-nesting seabirds (genera Pterodroma, Pelecanoides, Pachyptila and Puffinus: Family Procellariidae)

Development of seabird translocation techniques is required to meet species recovery objectives, to improve conservation status, and to restore ecological processes. During 1997-2008 we undertook translocation trials on eight petrel species of four genera within the New Zealand region: common diving petrel (Pelecanoides urinatrix), fairy prion (Pachyptila turtur), grey-faced petrel (Pterodroma macroptera gouldi), Pycroft’s petrel (Pterodroma pycrofti), Chatham petrel (Pterodroma axillaris), Chatham Island taiko (Magenta petrel; Pterodroma magentae), fluttering shearwater (Puffinus gavia), and Hutton’s shearwater (Puffinus huttoni). A total of 1791 chicks within 5 weeks of fledging were moved up to 240 km, placed in artificial burrows and hand-fed until they fledged. Of these, 1546 fledged, and so far at least 68 have returned as adults to the translocation sites. Most birds were crop-fed a puree based on tinned sardines and fresh water. This diet worked well for all species regardless of their typical natural diet (planktonic crustaceans, squid, or fish) with all species fledging above or close to mean natural fledging weights.     

The incidence of Gaeumannomyces graminis var. Tritici and Phialophora radicicola var. Graminicola on wheat grown after different cropping sequences

The effects of three treatment cropping sequences (fallow, lucerne or grass-clover ley) on the incidence of Gaeumannomyces graminis var. tritici and Phialophora radicicola var. graminicola were measured in a field experiment. Increases in G. g. tritici population in the soils of the first wheat crop and the incidence of take-all in the second wheat crop were greater after fallow or lucerne than after grass-clover. These differential increases were not associated with differences in survival of G. g. tritici during the treatment cropping but were correlated negatively with the population of P.r. graminicola in the soil. After the third wheat crop the P. r. graminicola population after grass-clover had decreased and take-all was as prevalent as after fallow or lucerne.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

Parasitism of sclerotia of Sclerotium rolfsii by trichoderma harzianum

The ability of Trichoderma harzianum isolate 203 to attack the soil-borne plant pathogen Sclerotium rolfsii is apparently connected with the production by the isolates of chitinase and β-(1,3)-glucanase inside the attacked sclerotia during parasitism.SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytoplasmic contents. It is assumed that T. harzianum utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.     

Hyperparasitism of oospores of Phytophthora megasperma var. sojae

Hyperparasites of oospores of Phytophthora megasperma Drechs. var. sojae Hildb. were present in each of 15 field soils tested. Maximum numbers of oospores parasitized ranged from 42.5 to 87.5% for flooded soils, and from 25.5 to 73.0% for soils adjusted to 50% water holding capacity; the mean for all soils was 51.5%. The frequency of hyperparasitism was not correlated with the disease potential soils for Phytophthora root-rot of soybean as determined in seedling tests on flooded soil samples. Of eight isolated hyperparasitic fungi tested in steamed soil, the most efficient parasites were Hyphochytrium catenoides, Humicola fuscoatra, and Pythium monospermum, each of which parasitized at least 76% of oospores during 3 weeks. Hyphae were not parasitized by any of the eight fungi. Parasitism by H. catenoides in sterilized soil increased as soil temperature increased from 16° to 28°C. Parasitism by P. monospermum was maximum at 20°–24°C. Oospores of P. meyasperma var. sojae race 7 were more resistant to infection by hyperparasites than were oospores of races 1 and 3. Oospores produced in culture were slightly more susceptible to hyperparasitism in soils than were oospores produced in soybean seedlings.     

Influence oF Pseudomonas putida on nodulation of Phaseolus vulgaris

The effect of Pseudomonas putida (isolate M17) on Rhizobium phaseoli nodulation of the common bean, Phaseolus vulgaris, was investigated under field and greenhouse conditions. The results indicated that P. putida markedly increased nodulation compared to R. phaseoli controls. Furthermore, 2-ketogluconic acid, a phosphate-solubiliring compound, was detected in P. putida M17. This could imply an increased P supply to roots of P. vutgaris, which may function to increase nodules. Bean yields and shoot fresh weight were not significantly altered by the addition of P. putida M17.     

Hydrolytic enzyme activities in maize (Zea mays) and sorghum (Sorghum bicolor) roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices

Two strains of Gluconacetobacter diazotrophicus (Pal 5, UAP5541) and the arbuscular mycorrhizal fungus Glomus intraradices increased both the shoot and root dry weight of sorghum 45 days after inoculation, whereas they had no effect on the shoot and root dry weight of maize. Co-inoculation (Gluconacetobacter diazotrophicus plus Glomus mosseae) did not increase the shoot and root dry weight of either plant. There was a synergistic effect of Gluconacetobacter diazotrophicus on root colonization of maize by Glomus intraradices, whereas an antagonistic interaction was observed in the sorghum root where the number of Gluconacetobacter diazotrophicus and the colonization by Glomus intraradices were reduced. Plant roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices, either separately or together, significantly increased root endoglucanase, endopolymethylgalacturonase and endoxyloglucanase activities. The increase varied according to the plant. For example, in comparison with non-inoculated plants, there were higher endoglucanase (+328%), endopolymethylgalacturonase (+180%) and endoxyloglucanase (+125%) activities in 45-day old co-inoculated maize, but not in 45-day old sorghum. The possibility is discussed that hydrolytic enzyme activities were increased as a result of inoculation with Gluconacetobacter diazotrophicus, considering this to be one of the mechanisms by which these bacteria may increase root colonization by AM fungi.     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.     

Effects of Lumbricus terrestris, Allolobophora chlorotica and Eisenia fetida on microbial community dynamics in oil-contaminated soil

Oil spills are one of the most common types of soil pollution. Bioremediation has become an attractive alternative to physicochemical methods of remediation, where feasible. Earthworms have been shown to stimulate the degradation of petroleum hydrocarbons in soil, and it was hypothesized that the role of earthworms in remediation lies in the enhancement of an oil degrading microbial community. The aim of this study was to characterize microbial activity and community dynamics in oil-contaminated soil incubated with or without earthworms. Three earthworm species (Eisenia fetida, Allolobophora chlorotica and Lumbricus terrestris) were incubated in crude oil polluted soil (ca. 10,000 mg/kg total petroleum hydrocarbons (TPH)) and a reference soil for 28 d. Control treatments with manual mixing and/or cattle dung amendment were also included. In the oil-contaminated soil, respiration and concentration of microbial biomass was significantly enhanced by earthworm amendment, and TPH concentrations decreased significantly. These effects were less evident in treatments with A. chlorotica, possibly due to a difference in behavior, since individuals of this endogeic species were found in a state of inactivity (aestivation). Microbial community dynamics were described by phospholipid fatty acid (PLFA) analyses. After 28 d, similar shifts in the soil PLFA composition were observed in the oil-contaminated soil irrespective of worm species. Fungal:bacterial ratios were increased in the presence of worms, but also by addition of dung as a food source, indicating a non-specific effect of metabolizable substrates. In contrast, the fatty acids 17:1ω8 (=Δ9-heptadecenoic acid) and 20:4ω6c (arachidonic acid) were specifically stimulated by the presence of earthworms in the oil-contaminated soil. The results showed that earthworms can contribute positively to bioremediation of oil-contaminated soil, but that the effect may be species-dependent.     

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.     

Competition between species of Rhizobium for nodulation of Glycine max

Glycine max cv. Malayan is a promiscuously nodulating cultivar which formed nodules with 6 out of 9 strains of Rhizobium spp of diverse origin and all strains of R. japonicum tested. No generalizations can be made as to the probability of strains isolated from a particular host being infective on Malayan as only some isolated from Centrosema pubescens, and Cajanus cajan were able to form nodules. In competition with R. japonicum at 30°C all 20 strains of Rhizobium spp isolated from Malayan grown in Nigeria formed fewer than 50% of the nodules and 14 strains fewer than 25%. Competition was influenced by root temperature. Three strains of Rhizobium spp were poor competitors with R. japonicum between 24° and 33°C but at 36°C they formed more nodules (74–88%) than R. japonicum. Another strain of Rhizobium spp formed the majority of the nodules between 27° and 36°C whereas R. japonicum formed the most at 24°C.     

Reduction of sclerotial inoculum of Sclerotinia sclerotiorum with Coniothyrium minitans

Aspects of the biology of C. minitans and its potential for control of S. sclerotiorum were investigated.Temperatures below 7°C resulted in comparatively slow rates of germination and infection of sclerotia by C. minitans. The optimum temperature for germination, growth, infection of sclerotia, and destructive parasitism by C. minitans was 20°C. The optimum relative humidity for germination, growth and infection by C. minitans was above 95%.Autumn inoculations with suspensions of conidia, pycnidia and mycelium of C. minitans in the field resulted in negligible numbers of sclerotia remaining viable after 1 month. With culture-grown sclerotia 2 months were required for a similar reduction of sclerotial viability. In the absence of C. minitans mulching had no significant effect on sclerotial viability. In the presence of C. minitans mulching did, however, influence the viability and infection by C. minitans of culture-grown sclerotia. Populations of field sclerotia also differed from culture-grown sclerotia in that they harboured an internal population of microorganisms, which included C. minitans, and had a lower level of viability at the commencement of the treatments.A winter application of C. minitans did not result in significant infection of sclerotia nor in a reduction in viability of sclerotia. This failure is believed to have resulted from low temperatures and dry conditions.     

Distribution of Pseudomonas populations harboring phlD or hcnAB biocontrol genes is related to depth in vineyard soils

The abundance and population structure of pseudomonads in soils collected from long-(1006 years) and short-(54 years) term grapevine monocultures in Switzerland were examined across five soil horizons within the 1.20-1.35 m range. Soil samples were baited with grapevine, and rhizosphere pseudomonads containing the biocontrol genes phlD (2,4-diacetylphloroglucinol synthesis) and/or hcnAB (hydrogen cyanide synthesis) were analyzed by MPN-PCR. The numbers of total, phlD⁺ and hcnAB⁺ pseudomonads decreased with depth by 1.5-2 log (short-term monoculture) and 3-3.5 log (long-term monoculture). In addition, the percentages of phlD⁺ (except in short-term monoculture) and hcnAB⁺ pseudomonads were also lower in deeper horizons. RFLP-profiling of phlD⁺ and hcnAB⁺ pseudomonads revealed three phlD and twelve hcnAB alleles overall, but the number of alleles for both decreased in relation to depth. The only phlD allele found in deeper horizons was also found in topsoil, whereas one hcnAB allele (k) found in deeper horizons in long-term monoculture was absent in the topsoil. This suggests that certain Pseudomonas ecotypes are adapted to specific depths. Four hcnAB alleles enabled discrimination between monocultures. We conclude that soil depth is a factor selecting phlD and hcnAB genotypes, and that the allelic diversity of the two biocontrol genes decreases with depth.