Control of Mn status and infection rate by genotype of both host and pathogen in the wheat take-all interaction

Take-all is a world-wide root-rotting disease of cereals. The causal organism of take-all of wheat is the soil-borne fungus Gaeumannomyces graminis var tritici (Ggt). No resistance to take-all, worthy of inclusion in a plant breeding programme, has been discovered in wheat but the severity of take-all is increased in host plants whose tissues are deficient for manganese (Mn). Take-all of wheat will be decreased by all techniques which lift Mn concentrations in shoots and roots of Mn-deficient hosts to adequate levels. Wheat seedlings were grown in a Mn-deficient calcareous sand in small pots and inoculated with four field isolates of Ggt. Infection by three virulent isolates was increased under conditions which were Mn deficient for the wheat host but infection by a weakly virulent isolate, already low, was further decreased. Only the three virulent isolates caused visible oxidation of Mn in vitro. The sensitivity of Ggt isolates to manganous ions in vitro did not explain the extent of infection they caused on wheat hosts. In a similar experiment four Australian wheat genotypes were grown in the same Mn-deficient calcareous sand and inoculated with one virulent isolate of Ggt. Two genotypes were inefficient at taking up manganese and were very susceptible to take-all, one was very efficient at taking up manganese and was resistant to take-all, and the fourth genotype was intermediate for both characters. All genotypes were equally resistant under Mn-adequate conditions.     

Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat

Endophytic actinobacteria isolated from healthy cereal plants were assessed for their ability to control fungal root pathogens of cereal crops both in vitro and in planta. Thirty eight strains belonging to the genera Streptomyces, Microbispora, Micromonospora, and Nocardioidies were assayed for their ability to produce antifungal compounds in vitro against Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease in wheat, Rhizoctonia solani and Pythium spp. Spores of these strains were applied as coatings to wheat seed, with five replicates (25 plants), and assayed for the control of take-all disease in planta in steamed soil. The biocontrol activity of the 17 most active actinobacterial strains was tested further in a field soil naturally infested with take-all and Rhizoctonia. Sixty-four percent of this group of microorganisms exhibited antifungal activity in vitro, which is not unexpected as actinobacteria are recognized as prolific producers of bioactive secondary metabolites. Seventeen of the actinobacteria displayed statistically significant activity in planta against Ggt in the steamed soil bioassay. The active endophytes included a number of Streptomyces, as well as Microbispora and Nocardioides spp. and were also able to control the development of disease symptoms in treated plants exposed to Ggt and Rhizoctonia in the field soil. The results of this study indicate that endophytic actinobacteria may provide an advantage as biological control agents for use in the field, where others have failed, due to their ability to colonize the internal tissues of the host plant.     

Relationships between take-all,soil conduciveness to the disease,populations of fluorescent pseudomonads and nitrogen fertilizers

Take-all of wheat, caused by Gaeumannomyces graminis var tritici (Ggt), is reduced by ammoniacal fertilizers as compared to nitrate sources. This influence of nitrogen on the disease is only observed on nodal roots at flowering. But soil conduciveness to take-all, as measured in a soil bioassay, is modified earlier. Forty days after nitrogen application at early tillering, the NH₄-treated soil became less conducive than the NO₃-treated one. When nitrogen applications are done at sowing and at tillering, differences in disease propagation between the two soils are enhanced. Results from four years of experimentation show that when the level of natural soil inoculum is high, disease severity is reduced by ammonium, showing an effect on the parasitic phase of Ggt. At a low level of natural inoculum the effect of the source of nitrogen is mainly observed on the percent of infected plants, indicating that the saprophytic and preparasitic phases are affected. Rhizospheric bacterial populations increase from sowing to tillering, but differences on take-all conduciveness after tillering are not correlated with differences in the amounts of aerobic bacteria or fluorescent pseudomonads isolated from soils treated with different sources of nitrogen. Qualitative changes in fluorescent Pseudomonas spp. populations, like in vitro antagonism, are more likely to explain differences in soil conduciveness to take-all than are quantitative changes in this group. Nevertheless, the introduction of Ggt in a cropped soil leads to a greater increase in fluorescent pseudomonads populations than in total aerobic bacteria.The delay between reducing soil conduciveness and reducing disease in the field with ammonium nitrogen fertilization, the qualitative change of fluorescent pseudomonads populations and the role of necroses in rhizobacteria multiplication, provide information leading to our representation of a dynamic model based on the differentiation of the wheat root system into seminal and nodal roots.     

Effect of certain herbicide treatments on pasture composition and inoculum of the take-all fungus

The improvement of pastures by the use of a range of herbicides to eliminate grasses, and their effect on populations of the take-all fungus (Gaeumannomyces graminis vartritici=Ggt) were studied in the field (at Esperance Downs, on the south-coast of Western Australia) from 1982 to 1985. Field trials were conducted to evaluate three herbicide treatments (2,4-D amine+propyzamide; 2,4-D amine+paraquat; paraquat/ diquat) and an unsprayed control. A pot trial involving these treatments with two levels of nitrogen was undertaken to confirm treatment effects observed in the field trial. All herbicide treatments resulted in reduced grass composition of pastures, in both the year of spraying and in the second year of pasture, but reduced dry matter production in the year of spraying. In the year of spraying, however, inoculum ofGgt was reduced (P<0.1) only following the 2,4-D amine+propyzamide treatment and was greater (P<0.1) after 2,4-D amine+paraquat treatment than the unsprayed treatment. Despite reduced grass levels in the herbicide-treated plots in the second year of pasture,Ggt inoculum did not differ between treatments, nor did it after a wheat crop which followed a second year pasture. There was high correlation (P<0.001) between disease levels and dry weights of grasses in the pot trial. There was significantly less (P<0.001) grass in pots treated with herbicides compared to the unsprayed control but no difference (P>0.05) was evident between treatments. Inoculum levels were lower (P<0.05) in the treated pots than the unsprayed control with no evidence of differences among treatments (P>0.05). Nitrogen level had no effect on disease (P>0.05). All herbicide treatments tested reduced grass level and total dry matter, both in the field and in pots. Whereas in the pot trial reduced grass levels resulted in reducedGgt inoculum, in the field such a reduction occurred only with the 2,4-D amine+propyzamide treatment and only in the year of spraying. Herbicide treatments had no effect onGgt inoculum in second year of pasture or crop. Unknown soil and environmental factors in the field precluded a simple relationship between grass level in pasture and subsequent level ofGgt inoculum, and where such a relationship did occur (2,4-D amine+propyzamide treatment) it appeared to be shortlived.     

Control of the take-all fungus byMicrodochium bolleyi,and interactions involvingM. bolleyi,Phialophora graminicola andPericonia macrospinosa on cereal roots

Summary In glasshouse experiments,Microdochium bolleyi (Mb) significantly reduced infection of wheat roots by the take-all fungus,Gaeumannomyces graminis vartritici (Ggt), when inocula were dispersed in soil at ratios of 10∶1 (Mb:Ggt) or more. Spread of take-all lesions up roots from a layer of inoculum also was reduced when Mb was inoculated immediately below the crown. In contrast,Periconia macrospinosa did not control take-all even at an inoculum ratio of 100∶1. M. bolleyi interfered with growth on roots byPhialophora graminicola, a known biocontrol agent of take-all. It is suggested that this phenomenon and control of take-all by these fungi occur by competition for cortical cells that senesce in the normal course of root development.     

Identity and frequency of occurrence ofTrichoderma spp. in roots of wheat and rye-grass in Western Australia and their effect on root rot caused byGaeumannomyces graminis var.tritici

Trichoderma hamatum, T. harzianum andT. koningii were isolated from wheat and rye-grass roots from a field in Western Australia. Frequency of occurrence ofTrichoderma spp. was higher on roots subjected to washing only, for both wheat and rye-grass than the roots which were surface-sterilized with 0.6% or 1.25% NaOCl.Trichoderma spp. were recovered at a higher frequency on PDA amended with lactic acid (pH 4.5) than on PDA alone (pH 5.6) or PDA with streptomycin. In general,Trichoderma spp. were isolated at a higher frequency from roots of wheat than that of rye-grass.T. hamatum occurred at a higher frequency in rye-grass roots than in wheat, whereasT. harzianum was more common in roots of wheat than in rye-grass, especially in seedling and milky ripe stages.T. koningii was recovered at a higher frequency from roots at seedling stage of rye-grass than wheat, the reverse being true at tillering stage.T. koningii was not recovered from roots of either host in any sampling when they were surface sterilized with 1.25% NaOCl.The take-all fungus was isolated from wheat and rye-grass roots more frequently at tillering and stem extension stages than others. It was severely pathogenic to both hosts in sterilized and non-sterilized soil.Addition of lactic acid, HCl or streptomycin to PDA did not affect the growth of theTrichoderma spp. tested, but the growth was slower on Martin's medium than on other media. In generalT. harzianum andT. koningii grow faster thanT. hamatum. The growth of the three species were not different at 20 and 25°C, but at 15°c growing of all species was significantly reduced.Incorporation of lactic acid into PDA prevented the bacterial growth in all treatments. Streptomycin too reduced but to a lesser degree than lactic acid. Surface sterilization with NaOCl decreased the recovery of both bacteria and fungi. T. hamatum andT. koningii reduced the mortality of wheat and rye-grass plants inoculated with the take-all fungus in sterilized and non-sterilized soil, whereT. harzianum did not protect wheat or rye-grass from infection by the take-all fungus.     

Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici

Bacillus subtilis strain Z-14 has biological control activity against the take-all fungus Gaeumannomyces graminis var. tritici (Ggt). In Petri dishes, the crude extract from B. subtilis Z-14 culture filtrate reduced take-all severity in roots of wheat seedlings by 91.3% and potted plants by 69.8% compared to the Ggt-inoculated control. Treatment with the crude extract also significantly (P?<?.05) increased growth of roots’ average length, and fresh weight in comparison with those of the Ggt-inoculated control. B. subtilis Z-14 culture filtrate was relatively thermally stable with 88.2% of the antifungal activity being retained after being heated at 100°C for 30?min. Meanwhile, the antifungal activity remained almost unchanged (>95%) when the culture filtrate was exposed to a pH ranging from 3 to 8, but significantly reduced in basic conditions. This activity was not transferred to the organic solvent phase after treatment with organic extraction agents. B. subtilis Z-14 culture filtrate exhibited a broad spectrum of antifungal activities against various phytopathogenic fungi. Three homologs of iturin A (C14–16) were characterised by liquid chromatography-mass spectroscopy (LC-MS) and electrospray ionisation mass spectrometry/mass spectrometry collision-induced dissociation (ESI-MS/MS CID).     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Plant genotype, micronutrient fertilization and take-all infection influence bacterial populations in the rhizosphere of wheat

The relationship between micronutrient efficiency of four wheat (Triticum aestivum L.) genotypes, tolerance to take-all disease (caused by Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker), and bacterial populations in the rhizosphere was tested in soil fertilized differentially with Zn and Mn. Plant growth was reduced by Mn or Zn deficiency and also by take-all. There was an inverse relationship between micronutrient efficiency of wheat genotypes when grown in deficient soils and the length of take-all lesions on roots (efficient genotypes had shorter lesions than inefficient ones). In comparison to the rhizosphere of control plants of genotypes Aroona and C8MM receiving sufficient Mn and Zn, the total numbers of bacterial cfu (colony forming units) were greater in the rhizosphere of Zn-efficient genotype Aroona under Zn deficiency and in Mn-efficient genotype C8MM under Mn deficiency. These effects were not observed in other genotypes. Take-all decreased the number of bacterial cfu in the rhizosphere of fully-fertilized plants but not of those subjected to either Mn or Zn deficiency. In contrast, the Zn deficiency treatment acted synergistically with take-all to increase the number of fluorescent pseudomonads in the rhizosphere. Although numbers of Mn-oxidising and Mn-reducing bacteria were generally low, take-all disease increased the number of Mn reducers in the rhizosphere of Mn-efficient genotypes Aroona and C8MM. Under Mn-deficiency conditions, the number of Mn reducers in the rhizosphere increased in Aroona but not in C8MM wheat. The results suggest that bacterial microflora may play a role in the expression of Mn and Zn efficiency and tolerance to take-all in some wheat genotypes.     

Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.     

Prediction of take-all disease risk in field soils using a rapid and quantitative DNA soil assay

A rapid, routine DNA-based assay to quantify Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease of cereals, has been developed and used for the prediction of take-all in a wide range of field soils. Based on the correlation of the DNA-based assay and a soil bioassay, the risk of disease development can be estimated. Ggt DNA levels of <30 pg, 30–50 pg and >50 pg in 0.1 g soil organic matter correspond to low, moderate and high levels of the disease, respectively. Limitations in the prediction of take-all, including sampling requirements to obtain representative soil samples from fields and increasing the sensitivity and the accuracy of the DNA assay, are described. The main advantage in using the DNA-based assay, in estimating the amount of Ggt inoculum in soil, is that the levels of Ggt in soil samples can be assessed rapidly and accurately. Farmers can now have soil samples assessed before sowing. The DNA result can be used to predict the potential yield loss and determine the most appropriate management options using decision support software that is currently available. This DNA technology is currently being used commercially to detect and predict take-all. This revised version was published online in June 2006 with corrections to the Cover Date.     

Susceptibility to take‐all of cereal and grass species,and their effects on pathogen inoculum

Two field trials were conducted to investigate different herbage grasses and cereals for their susceptibility to the disease take‐all, for their impact on concentrations of the pathogen, Gaeumannomyces graminis var. tritici (Ggt), in soil and for their effect on development of take‐all in a subsequent wheat crop. In the herbage grass trial, Bromus willdenowii was highly susceptible to Ggt, produced the greatest post‐senescence Ggt concentrations in soil and highest incidence of take‐all in following wheat crop. Lolium perenne, Lolium multiflorum and Festuca arundinacea supported low Ggt soil concentrations and fallow the least. The relationship between susceptibility to Ggt and post‐senescence concentrations in soil differed between pasture grasses and cereals. In a trial in which Ggt was added to half the plots and where wheat, barley, triticale, rye or fallow were compared, the susceptibility of the cereals to take‐all was not clearly linked to post‐harvest soil Ggt concentrations. In particular, triticale and rye had low and negligible take‐all infection respectively, but greater post‐harvest soil Ggt concentrations than barley or wheat. This indicates that low Ggt concentrations on roots may build up during crop senescence on some cereals. Soil Ggt concentrations were greater following harvest in inoculated plots sown to cereals, but in the second year there was more take‐all in the previously non‐inoculated than inoculated plots. Thus, the grass and cereal species differed in susceptibility to take‐all, in their impact on Ggt multiplication and in associated take‐all severity in following wheat crop.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.     

Use of a root assessment tray for the detection of take-all on lateral roots of wheat seedlings

A root assessment tray was designed for the meticulous assessment of take-all on wheat seedling roots from soil bioassays. Subsequently, the detection of lateral root infections (in addition to the more obvious infections on main axes of seminal roots) resulted in increased estimates of propagule numbers of the take-all fungus (Gaeumannomyces graminis var.tritici) for 196 of the 368 soil samples bioassayed in a field study conducted in Western Australia between 1984 and 1986.     

Role of antibiotics and siderophores in biocontrol of take-all disease of wheat

Both antibiotics and siderophores have been implicated in the control of soilborne plant pathogens by fluorescent pseudomonads. In Pseudomonas fluorescens 2–79, which suppresses take-all of wheat, the importance of the antibiotic phenazine-1-carboxylic acid was established with mutants deficient or complemented for antiobiotic production and by isolation of the antibiotic from the roots of wheat colonized by the bacteria. Genetic and biochemical studies of phenazine synthesis have focused on two loci; the first is involved in production of both anthranilic acid and phenazine-1-carboxylic acid, and the second encodes genes involved directly in phenazine synthesis. Because the antibiotic does not account fully for the suppressiveness of strain 2-79, additional mutants were analyzed to evaluate the role of the fluorescent siderophore and of an antifungal factor (Aff, identified as anthranilic acid) that accumulates when iron is limiting. Whereas strains producing only the siderophore conferred little protection against take-all, Aff⁺ strains were suppressive, but much less so than phenazine-producing strains. Iron-regulated nonsiderophore antibiotics may be produced by fluorescent pseudomonads more frequently than previously recognized, and could be partly responsible for beneficial effects that were attributed in the past to fluorescent siderophores.     

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

Natural suppression of take-all disease of wheat in Montana soils

This research was initiated to determine whether soils suppressive to take-all of wheat caused by Gaeumannomyces graminis var. tritici (Ggt) occur in Montana, and to identify the organisms most likely involved in this suppression. From an initial screening of eight soils collected from different wheat growing areas of Montana, two were highly suppressive to take-all. Microbial characterization of these soils indicated that different mechanisms were involved in the suppression. In Larslan soil, mycoparasitism appeared to be the main mechanism. Two different fungi with exceptional ability to reduce the severity of take-all were isolated from this soil. One of these fungi could parasitize the hyphae of Ggt. Field tests with these fungi in Ggt infested soil showed increases of over 100% in both harvestble tillers and grain yield as compared to treatments without these two fungi. In tests with 48 different bacteria and 10 actinomycetes from Larslan soil, none were able to consistently reduce severity of take-all alone, or in mixtures. In Toston soil, antibiosis by actinomycetes and perhaps the involvement of Pseudomonas spp. in production of antibiotics and/or siderophores appeared to be the most likely mechanisms involved in take-all suppression. Increases in shoot dry weight over that in the Ggt infested control using mixtures of pseudomonads and actinomycetes ranged from 25% to 87%. Actinomycetes added individually or in mixtures to soil infested with Ggt consistently reduced the severity of the disease to a greater extent than did mixtures of Pseudomonas spp.     

Establishment of hairy root cultures of Psoralea species

Eight Psoralea species (Leguminosae) were inoculated with Agrobacterium rhizogenes, strains 8196 and 9402. Hairy roots were only induced by strain 9402. Attention was focussed on Psoralea lachnostachys. Transformed roots grew very rapidly in Gamborg B5 liquid medium with a doubling time of the culture of 38 hours. Whatever the culture conditions, the two furanocoumarins usually found in roots of Psoralea plants, psoralen and angelicin, were not detected in cultured transformed and non transformed roots even when some chitosan was added to the medium. However, 669 g.g^–1 dry matter of psoralen and 215 g.g^–1 dry matter of angelicin were found in roots from soil grown plants. A possible translocation of these compounds from the aerial parts to the roots is suggested.Abbreviations B5 medium Gamborg's medium (Flow laboratories's formulation) - NAA Naphthaleneacetic acid     

Development of two endomycorrhizal symbioses on soybean and comparison with phosphorus fertilization

Summary Soybean (Glycine max L. Merr. cv. Amsoy 71) plants were grown in a greenhouse in a soil very low in plant-available P, and plants were harvested 5 times over a 21-week growth period. Soybeans were inoculated with one of two species of VAM fungi or received daily one of three nutrient solutions of different P concentrations (0.0, 0.2, or 1.0mMP). Until week 9, the dry weights, leaf areas and developmental stage of soybeans inoculated withG. fasciculatum orG. mosseae were similar to the 1.0 or 0.2mMP-treated plants, respectively. Phosphorus concentrations were significantly lower in VAM plants at weeks 6 and 9 as compared to non-VAM soybeans given 1.0mMP, suggesting P input in VAM plants was immediately used for new growth. Total P input for VAM plants was linear over 21 weeks, and the average rate of P uptake for these plants was 0.19mg P d⁻¹. Estimated specific P uptake rates (SPUR) for the mycorrhizae (VAM roots) were twice that of the control (0.0mMP) roots. The calculated SPURs forG. fasciculatum andG. mosseae hyphae were 95 and 120μg P g⁻¹ VAM d⁻¹ respectively, a 4 to 5 fold increase over non-inoculated roots, indicating more attention must be paid to P assimilation by VAM fungi in P-fixing substrates. Contribution from the Western Regional Research Center, USDA-ARS (CRIS No. 5325-20580-003).