Evaluation of different Coniothyrium minitans inoculum sources and application rates on apothecial production and infection of Sclerotinia sclerotiorum sclerotia

The effects of three Coniothyrium minitans isolates (Conio, IVT1 and Contans^®), applied to soil as conidial suspensions or as maizemeal-perlite (MP) inocula (Conio), on apothecial production and infection of Sclerotinia sclerotiorum sclerotia were assessed in two soil pot bioassays and two novel box bioassays in the glasshouse at different times of the year. C. minitans isolate Conio applied as either MP or ground MP at full rate (10⁶-10⁷ cfu cm⁻³ soil) consistently decreased the carpogenic germination, recovery and viability of sclerotia and increased C. minitans infection of the sclerotia of S. sclerotiorum by in comparison with either MP or conidial suspension treatments applied at lower rates (10³-10⁴ cfu cm⁻³ soil). Additionally, when applied at the same rate, MP inoculum of C. minitans was consistently more effective at reducing carpogenic germination than a conidial suspension. The effect of MP and ground MP at full rate on carpogenic germination was expressed relatively early as those sclerotia recovered before apothecia appeared on the soil surface already had reduced numbers of apothecial initials. In general, there were few differences between the isolates of C. minitans applied as conidial suspensions. Box bioassays carried out at different times of the year indicated that temperature and soil moisture influenced both apothecial production and mycoparasitism. Inoculum concentration of C. minitans and time of application appear to be important factors in reducting apothecial production by S. sclerotiorum.     

Survival of Coniothyrium minitans associated with sclerotia of Sclerotinia sclerotiorum in soil

The development and survival of the mycoparasite Coniothyrium minitans associated with sclerotia of the plant pathogen Sclerotinia sclerotiorum was studied in pasteurised and non-sterile (untreated) soil. Using scanning electron microscopy, developing pycnidia were first seen within the sclerotial medulla at 7 days post-inoculation with the mycoparasite in pasteurised soil. However, by 14 days post-inoculation, pycnidia had developed fully in both pasteurised and non-pasteurised treatments, and conidial droplets were exuded onto the outer surface of the infected sclerotia. Thirty days post-inoculation, irrespective of soil treatment, the majority of the sclerotial medulla had been converted to pycnidia, with the sclerotial rind remaining largely intact. The pycnidia and dried intact droplets were still observed 6 months post-inoculation with C. minitans, although the conidia on the outer surface of the dried droplets had largely collapsed by this stage. Germinability studies at 10 months post-inoculation showed that approximately 13% of the conidia in dried droplets were still viable. This work shows the potential for infected sclerotia of S. sclerotiorum to provide a unique reservoir for the survival of C. minitans.     

Variation in oxalic acid production and mycelial compatibility within field populations of Sclerotinia sclerotiorum

This study was aimed at detecting mycelial compatibility groups and variations in oxalic acid production in Sclerotinia sclerotiorum. For this purpose, 121 isolates of this plant pathogen recovered from lettuce, soybean and sunflower field crops, and grouped in 46 MCGs were tested for their ability to release oxalic acid and other organic acids to the medium. Oxalic acid production on liquid media was measured spectrophotometrically and release of organic acids was estimated by isolate abilities to discolour solid media amended with bromophenol blue. There were significant differences among MCGs in both oxalic acid and organic acids releasing, ranging the mean production of oxalic acid between 18 and 110 μg oxalic acid mg⁻¹ dry wt. When isolates were grouped by their hosts, those obtained from soybean presented the highest release of oxalic acid (71 μg oxalic acid mg⁻¹ dry wt), while those from sunflower showed the highest release of other acids to the medium. Solid medium discoloration was not correlated with oxalic acid concentration in liquid medium (Spearman R=−0.085; P=0.126).     

Control of apothecia of Sclerotinia sclerotiorum by soil amendment with S-H mixture or Perlka in bean, canola and wheat fields

Ascospores of Sclerotinia sclerotiorum produced from apothecia are the primary source of inoculum for causing diseases such as white mold of common bean, pod rot of pea, stem blight of canola and head rot of sunflower and safflower in the Canadian prairies. A field study was conducted for 4 years to determine efficacy of control of production of apothecia from carpogenically germinated sclerotia of S. sclerotiorum by soil amendment with Perlka^® (calcium cyanamide) and S-H mixture (a formulated compound). Results of the 4-year experiments showed that amendment of soil with Perlka^® at low (30 g/m²) or high (60 g/m²) rate was effective in reducing carpogenic germination of sclerotia and production of apothecia under the canopy of host crops (common bean and canola) and a non-host crop (wheat). In the experiments of 1988, for example, the numbers of apothecia produced in the treatments of Perlka^®-low rate (30 g/m²), Perlka^®-high rate (60 g/m²) and untreated control were 42, 46, and 182 apothecia/plot (m²), respectively, for bean; 89, 42, and 318 apothecia/plot (m²), respectively, for canola; and 146, 143, and 412 apothecia/plot (m²), respectively, for wheat. However, soil amendment of S-H mixture at low (30 g/m²) or high (60 g/m²) rate was ineffective in reducing carpogenic germination of sclerotia and production of apothecia for all the 4 years of testing in all three crops. The ineffectiveness of S-H mixture and the practicality of Perlka^® for control of Sclerotinia diseases of crops grown under Canadian prairie conditions are discussed.     

Identification and use of potential bacterial organic antifungal volatiles in biocontrol

Bacteria, isolated from canola and soybean plants, produced antifungal organic volatile compounds. These compounds inhibited sclerotia and ascospore germination, and mycelial growth of Sclerotinia sclerotiorum, in vitro and in soil tests. Ascospore germination in cavity slides was inhibited 54-90% by the volatile producers. When mycelial plugs or the sclerotia, exposed to these volatiles, were transferred to fresh agar plates, the pathogen could not grow, indicating the fungicidal nature of the volatiles. Head space volatiles, produced by bacteria, were trapped with activated charcoal, by passing nitrogen continuously over shake cultures for 48 h. The compounds were eluted from the charcoal with methylene chloride and identified using Gas Chromatography-Mass Spectrometry (GC-MS). The volatile compounds included aldehydes, alcohols, ketones and sulfides. Of the 23 compounds assayed for antifungal activity in divided Petri plates, with filter-disks soaked with these compounds (100 and 150 μl), only six compounds completely inhibited mycelial growth or sclerotia formation, suggesting their potential role in biological control. The compounds are benzothiazole, cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl 1-hexanol, and nonanal. Volatiles may play an important role in the inhibition of sclerotial activity, limiting ascospore production, and reducing disease levels. Studies are under way to understand this phenomenon under field conditions. This is the first report on the identification and use of bacterial antifungal organic volatiles in biocontrol.     

Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum, thus providing a potential means of biological disease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (−50 kPa, −1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at −1000 kPa, over the 40-day period, compared to only 60% at −50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.     

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.     

Factors influencing survival and long-term population viability of New Zealand long-tailed bats (Chalinolobus tuberculatus): Implications for conservation

A population viability analysis is important for the management of endangered populations and requires the estimation of survival parameters. The long-tailed bat (Chalinolobus tuberculatus) is one of only two native terrestrial mammals currently found in New Zealand and is classed as vulnerable. Its viability in temperate beech (Nothofagus) forest, Eglinton Valley, Fiordland, New Zealand was estimated using mark-recapture data collected between 1993 and 2003 using the Program MARK. Survival was estimated based on a total of 5286 captures representing 1026 individuals. Overall annual survival varied between 0.34 and 0.83 but varied significantly among three sub-populations and with sex and age. Females generally had a higher survival rate compared to males; and adults had higher survival relative to juveniles. Survival of all bats was lower in years when the number of introduced mammalian predators was high and when the winter temperature was warmer than average. High numbers of introduced predators occurred during three of the 10 years in the study. Climate change may mean that the conditions that promote high predator numbers may occur more frequently. A preliminary population viability analysis using a projection matrix on the overall adult female population showed an average 5% decline per year (λ = 0.95). Increased predator control targeting a range of predators is required in years when their numbers are high in order to halt the decline of this population of long-tailed bats. Population estimates using minimum number alive estimates supported the population estimates derived from Program MARK and a population viability analysis using matrices.     

Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: importance of bacterial secondary metabolite, 2,4-diacetylpholoroglucinol

The antimicrobial metabolites 2,4-diacetylphloroglucinol (2,4-DAPG) and pyoluteorin contribute to the ability of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogens. P. fluorescens strain CHA0 and its derivatives CHA89 (antibiotics-deficient) and CHA0/pME3424 (antibiotics overproducing) were investigated as potential biocontrol agents against Meloidogyne javanica the root-knot nematode. Exposure of root-knot nematode to culture filtrates of P. fluorescens under in vitro conditions significantly reduced egg hatch and caused substantial mortality of M. javanica juveniles. Nutrient broth yeast extract (NBY) medium amended with 2% (w/v) glucose or 1 mM EDTA markedly repressed hatch inhibition activity of the strain CHA0 but not that of CHA0/pME3424 or CHA89. On the other hand, NBY medium amended with glucose significantly enhanced nematicidal activity of the strain CHA0/pME3424. Neither glucose nor EDTA had an influence on the nematicidal activity of the strains CHA0 and CHA89. Under in vitro conditions, antibiotic overproducing strain CHA0/pME3424 and CHA0 expressed phl‘-’lacZ reporter gene but strain CHA89 did not. Expression of the reporter gene reflects actual production of DAPG. In general, CHA0/pME3424 expressed reporter gene to a greater extent compared to its wild type counterpart CHA0. Regardless of the bacterial strains, reporter gene expression was markedly enhanced when NBY medium was amended with glucose but EDTA had no such effect. A positive correlation between the degree of juvenile mortality and extent of phl‘-’lacZ reporter gene expression was also observed in vitro. Strain CHA0 produced zones of 4-6 mm on MM medium containing gelatin while strain CHA0/pME3424 and CHA89 did not. When MM medium containing gelatin was amended with 2% glucose of 1 mM EDTA size of haloes produced by the strain CHA0 reduced to 2 mm. Under glasshouse conditions aqueous cell suspension of the strains CHA0 or CHA0/pME3424 at various inoculum levels (10⁷, 10⁸ or 10⁹ cfu ml⁻¹) significantly reduced root-knot development. CHA89 caused significant reduction in galling when applied at 10⁹ cfu ml⁻¹. To better understand the mechanism of nematode suppression, split root bioassay was performed. Split-root experiments, that guarantee a spatial separation of inducing agent and a challenging pathogen, showed that soil treatment of one half of the root system with cell suspension of CHA0 or CHA0/pME3424 resulted in a significant systemic induced resistance leading to reduction of M. javanica infection of tomato roots in the non-baterized nematode treated half. The results clearly suggest that the antibiotic 2,4-DAPG from P. fluorescens CHA0 act as the inducing agents of systemic resistance in tomato roots. Populations of CHA0 and its derivatives declined progressively by 10-fold between first and fourth harvests (0-21 days after inoculation). However, bacterial populations increased at final harvest (28 days after application).     

Growth promoting effects of corn (Zea mays) bacterial isolates under greenhouse and field conditions

Fertilizer costs are a major component of corn production. The use of biofertilizers may be one way of reducing production costs. In this study we present isolation and identification of three plant growth promoting bacteria that were identified as Enterobacter cloacae (CR1), Pseudomonas putida (CR7) and Stenotrophomonas maltophilia (CR3). All bacterial strains produced IAA in the presence of 100 mg l⁻¹ of tryptophan and antifungal metabolites to several soilborne pathogens. S. maltophilia and E. cloacae had broad spectrum activity against most Fusarium species. The only strain that was positive for nitrogen fixation was E. cloacae and it, and P. putida, were also positive for phosphate solubilization. These bacteria and the corn isolate Sphingobacterium canadense CR11, and known plant growth promoting bacterium Burkholderia phytofirmans E24 were used to inoculate corn seed to examine growth promotion of two lines of corn, varieties 39D82 and 39M27 under greenhouse conditions. When grown in sterilized sand varieties 39M27 and 39D82 showed significant increases in total dry weights of root and shoot of 10-20% and 13-28% and 17-32% and 21-31% respectively. Plants of the two varieties grown in soil collected from a corn field had respective increases in dry weights of root and shoot of 10-30% and 12-35% and 11-19% and 10-18%. In sand, a bacterial mixture was highly effective whereas in soil individual bacteria namely P. putida CR7 and E. cloacae CR1 gave the best results with 39M27 and 39D82 respectively. These isolates and another corn isolate, Azospirillum zeae N7, were tested in a sandy soil with a 55 and 110 kg ha⁻¹ of nitrogen fertility at the Delhi research Station of Agriculture and Agri-Food Canada over two years. Although out of seven bacterial treatments, no treatment provided a statistically significant yield increase over control plots but S. canadense CR11 and A. zeae N7 provided statistically significant yield increase as compared to other bacteria. The 110 kg rate of nitrogen provided significant yield increase compared to the 55 kg rate in both years.     

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.     

Suppression of damping-off of cucumber caused by Pythium ultimum with live cells and extracts of Serratia marcescens N4-5

Environmentally friendly control measures are needed for the soil-borne pathogen, Pythium ultimum. This pathogen can cause severe losses to field- and greenhouse-grown cucumber and other cucurbits. Live cells and ethanol extracts of cultures of the bacterium Serratia marcescens N4-5 provided significant suppression of damping-off of cucumber caused by P. ultimum when applied as a seed treatment. Live cells of this bacterium also suppressed damping-off caused by P. ultimum on cantaloupe, muskmelon, and pumpkin. Culture filtrates from strain N4-5 contained chitinase and protease activities while ethanol extracts contained the antibiotic prodigiosin, the surfactant serrawettin W1, and possibly other unidentified surfactants. Production of prodigiosin and serrawettin W1 was temperature-dependent, both compounds being detected in extracts from N4-5 grown at 28 °C but not in extracts from N4-5 grown at 37 °C. Ethanol extracts from strain N4-5 grown at 28 °C inhibited germination of sporangia and mycelial growth by P. ultimum in in vitro experiments. There was no in vitro inhibition of P. ultimum associated with ethanol extracts of strain N4-5 grown at 37 °C. Prodigiosin, purified from two consecutive thin-layer chromatography runs using different solvent systems, inhibited germination of sporangia and mycelial growth of P. ultimum. Another unidentified compound(s) also inhibited germination of sporangia but did not inhibit mycelial growth. There was no in vitro inhibition associated with serrawettin W1. These results demonstrate that live cells and cell-free extracts of S. marcescens N4-5 are effective for suppression of damping-off of cucumber caused by P. ultimum possibly due in part to the production of the antibiotic prodigiosin.     

Fungal colonization as affected by litter depth and decomposition stage of needle litter

The present study was designated to evaluate the relative effects of litter depth and decomposition stage of needles on fungal colonization of needle litter in field experiments. The experiment was carried out in coniferous temperate forests in central Japan. Needle litter of Chamaecyparis obtusa and Pinus pentaphylla var. himekomatsu at two decomposition stages (recently dead and partly decomposed) were placed into the organic layer at two depths (on the surface of and beneath the litter layer). Fungal colonization of needles after 1 year was examined in terms of hyphal abundance and frequency of fungal species. Total and live hyphal length on needles were affected by the litter depth and (or) the decomposition stage of needles. Length of darkly pigmented hyphae on needles was 1.7-2.6 times greater beneath the litter layer than on the litter surface regardless of the decomposition stage of needles. Length of clamp-bearing hyphae in Pinus pentaphylla was 5.0-5.2 times greater in partly decomposed needles than in recently dead needles regardless of the litter depth. Frequencies of Pestalotiopsis spp. and Cladosporium cladosporioides were higher on recently dead needles than on partly decomposed needles and (or) were higher on the litter surface than beneath the litter layer. Frequencies of Trichoderma, Penicillium, and Umbelopsis species generally were higher on partly decomposed needles than on recently dead needles and were higher beneath the litter layer than on the surface.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.     

The abundance of nitrogen cycle genes amoA and nifH depends on land-uses and soil types in South-Eastern Australia

Nitrogen is a critical nutrient in plant-based primary production systems, therefore measurements of N cycling by microorganisms may add value to agricultural soil monitoring programs. Bacterial-mediated nitrogen cycling was investigated in soils from two broad land-uses (managed and remnant vegetation) across different Soil Orders from three geomorphic zones in Victoria, Australia, by examining the abundance of the genes amoA and nifH using quantitative polymerase chain reaction (qPCR). The aim of the study was to identify parameters influencing bacterial populations possessing the genes nifH and amoA, and examine their distribution at a regional scale across different management treatments. The gene amoA was most abundant in the neutral to slightly alkaline surface soils from Calcarosols in North-West Victoria. There was a highly significant (P < 0.001) interaction between land-use and geomorphic zones in terms of the abundance of amoA. Detection of the gene nifH was site specific with low copy number (less than 100 copies per nanogram of DNA) observed for some strongly acidic surface soil sites in North-East Victoria (Dermosols) and South-West Victoria (Sodosols/Chromosols), while nifH was more abundant in selected Calcarosols of North-West Victoria. The gene amoA was detected across more sites than nifH and was strongly influenced by land-use, with almost consistently greater abundance in managed compared to remnant sites, particularly for North-West and South-West Victoria. The abundance of nifH was not related to land-use, with similar copy numbers observed for both managed and remnant sites at some locations. For the gene nifH, there was no significant interaction between land-use and geomorphic zones, between managed and remnant sites or between the three geomorphic zones. Regression tree analysis revealed a number of likely soil chemical and microbial variables which may act as drivers of gene abundance of amoA and nifH. Variables identified as drivers for amoA included pH, Olsen P, microbial biomass carbon, nitrate and total nitrogen while for nifH the variables were microbial biomass carbon, electrical conductivity, microbial biomass nitrogen, total nitrogen and total potassium. Measures of N cycling genes could be used as an additional indicator of soil health to assess potential ecosystem functions. The spatial scale of the current study demonstrates that a landscape approach may assist soil health monitoring programs by evaluating N cycle gene abundance in the context of the different microbial and chemical conditions related to Soil Order and land-use management.     

Soil texture and irrigation influence the transport and the development of Pasteuria penetrans, a bacterial parasite of root-knot nematodes

The transport of the spores of Pasteuria penetrans was studied in three contrasted textured soils (a sandy, a sandy-clay and a clay soils), cultivated with tomato, inoculated with juveniles of Meloidogyne javanica and watered with 25 or 150 mm day⁻¹. One month after inoculation of the nematodes, 53% of the spores inoculated were leached by water flow in the sandy soil but only 14% in the sandy-clay soil and 0.1% in the clay soil. No nematodes survived in the clay soil, while the population was multiplied both in the sandy and in the sandy-clay soils. But juveniles of M. javanica were more infected by P. penetrans in the sandy-clay soil than in the sandy soil. Comparing different combinations of bare soils containing 1.1-57% of clay showed that the best spore percolation and retention balance occurred in soils amended with 10-30% clay. However, the spore recoveries decreased when the soil was enriched with more than 30% clay. The role of clay particles on the extractability of spores and on their availability to attach to the nematode cuticle in the soil is discussed.     

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.     

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.     

Genetic diversity within Aspergillus flavus strains isolated from peanut-cropped soils in Argentina

The genetic diversity of Aspergillus flavus populations isolated from the peanut-cropped soils in the peanut-growing region at Cordoba Province was evaluated by analysis of vegetative compatibility group (VCG). VCG_s were determined through complementation assays between nitrate-nonutilizing (NNO) mutants. Fifty-six VCG_s were identified from 100 isolates. Twenty-five VCG_s contained two or more isolates and 31 VCG_s contained only a single isolate. In general, there were significant differences among VCG_s in aflatoxin and CPA production. One VCG group included a single atoxigenic strain since it was neither aflatoxin nor cyclopiazonic acid producer. This isolate could be useful as a biological control agent, since it was unable to form a stable heterokaryon in the complementation test with the other isolates. Seven A. flavus isolated from soil were atypical because they simultaneously produced aflatoxins B, G and CPA.     

The bioprotective effect of AM root colonization against the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici in barley depends on the barley variety

The systemic effect of root colonization by the arbuscular mycorrhizal fungus (AMF) Glomus mosseae on the susceptibility of old and modern barley varieties to the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici (Ggt) was studied in a split-root system. Plants were precolonized on one side of the split-root system with the AMF and thereafter the other side of the split-root system was inoculated with the pathogen. At the end of the experiment the level of bioprotection was estimated by quantifying lesioned roots and the determination of the root fresh weight. AM root colonization provided protection in some of the barley genotypes tested, but not in others. This protective effect seemed to vary in the oldest and the most modern barley variety tested.