The presence of the arbuscular mycorrhizal fungus Glomus intraradices influences enzymatic activities of the root pathogen Aphanomyces euteiches in pea roots

 Fungal enzyme activities were quantified in an interaction study between the fungus Glomus intraradices and the pea pathogen Aphanomyces euteiches. Fungal and host enzymes were separated by polyacrylamide gel electrophoresis and the activity of A. euteiches–specific glucose-6-phosphate dehydrogenase (Gd), phosphoglucomutase and peptidase (PEP) enzymes were quantified by densitometry. The activity of A. euteiches–specific enzymes increased until 14 days after inoculation with A. euteiches, and then decreased. The plants preinoculated with G. intraradices showed no symptoms of severe root rot even though the pathogen was present and active in these plants. Thus, plants preinoculated with G. intraradices were more tolerant of infection with A. euteiches than non-mycorrhizal plants. This effect was evident even though the A. euteiches infection levels of mycorrhizal and non-mycorrhizal plants were the same. A. euteiches enzyme activities in the mycorrhizal plants were different to those in non-mycorrhizal plants. The peaks of PEP and Gd enzyme activity of A. euteiches were lower and the development of A. euteiches PEP activity was later in the mycorrhizal plants than in the non-mycorrhizal plants. Accepted: 14 November 1996     

Proteomic Profiling Unravels Insights into the Molecular Background Underlying Increased <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>-Tolerance of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity. Requests concerning seeds from the Medicago truncatula lines F83.005-5 and F83.005-9 should be addressed to Jean-Marie Prospéri, INRA-SGAP Laboratory, Laboratoire de Ressources Génétiques et d’Amélioration des Luzernes méditerranéennes, Mauguio, France, jean-marie.prosperi@ensam.inra.fr.     

Quantifying the relationship between disease severity and the amount of Aphanomyces euteiches detected in roots of alfalfa and pea with a real-time PCR assay

A real-time PCR assay was used to quantify the relationship in alfalfa and pea between disease severity and the amount of Aphanomyces euteiches detected in roots. The study included isolates of race 1 and race 2 of the alfalfa pathovar of A. euteiches and an isolate obtained from diseased pea. Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were positive ( ? 0.57) and significant (P  0.0007) for individual alfalfa plants, bulked alfalfa plant samples, and individual pea plants. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. The results clearly demonstrate that resistance to A. euteiches in both alfalfa and pea is characterized by a reduction in pathogen colonization relative to levels observed for susceptible reactions. The assay was very specific for A. euteiches, producing very linear assays with DNA extracted from pathogen isolates obtained from alfalfa, pea, and bean. Possible applications of the assay in conjunction with other real-time PCR assays specific to other legume pathogens are discussed in relation to simultaneous disease screening for multiple plant pathogens and the study of microbial population dynamics in mixed plant infections.     

Root exudates of mycorrhizal tomato plants exhibit a different effect on microconidia germination of Fusarium oxysporum f. sp. lycopersici than root exudates from non-mycorrhizal tomato plants

The effect of root exudates from mycorrhizal and non-mycorrhizal tomato plants on microconidia germination of the tomato pathogen Fusarium oxysporum f. sp. lycopersici was tested. Microconidia germination was enhanced in the presence of root exudates from mycorrhizal tomato plants. The more tomato plants were colonized by the arbuscular mycorrhizal fungus Glomus mosseae, the more microconidia germination was increased, indicating that alterations of the exudation pattern depended on the degree of root AM colonization. Moreover, alterations of the exudation pattern of mycorrhizal plants are not only local, but also systemic. Testing the exudates from plants with a high and a low P level revealed that the alterations of the root exudates from mycorrhizal plants, resulting in a changed effect on microconidia germination, are not due to an improved P status of mycorrhizal plants.     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

Effect of arbuscular mycorrhizal fungi and Pseudomonas fluorescens on root-rot and wilt,growth and yield of Coleus forskohlii

Root-rot and wilt caused by Fusarium chlamydosporum affects the cultivation of Coleus forskohlii, a medicinal plant grown for its roots, which contain a pharmaceutically important compound called forskolin. In this study, management of this disease under low and high inoculum levels was assessed with four arbuscular mycorrhizal (AM) fungi and a strain of Pseudomonas fluorescens. The AM fungus Glomus fasciculatum and P. fluorescens were the most effective treatments that reduced the severity of root-rot and wilt of C. forskohlii by 56–65% and 61–66%, respectively, under lower and higher levels of pathogen F. chlamydosporum. G. fasciculatum increased the dry shoot and root weight by 108–241% and 92–204%, respectively, while in plants treated with P. fluorescens, an increase of 97–223% and 97–172% in dry shoot and root weight, respectively, was observed. Although P. fluorescens was effective, it gave higher root yields only under lower inoculum level of the pathogen. G. fasciculatum performed equally well under both lower and higher inoculum levels. Increase in yields with both the biocontrol agents was accompanied by increase in P uptake (230–303%) and in K uptake (270–335%). The forskolin content of the roots was significantly increased (14–21%) by G. fasciculatum, P. fluorescens or G. mosseae under lower inoculum level of pathogen.     

Mycorrhizal colonization and drought stress affect Δ13C in CO2‐labeled lettuce plants

We studied the role of different arbuscular‐mycorrhizal (AM) fungi on lettuce (Lactuca sativa L.) plant carbon metabolism under drought stress. Plants were grown in pots maintained at two levels of soil moisture and labeled during photosynthesis with CO₂. P‐fertilized plants were used as a non‐mycorrhizal control. Well‐watered mycorrhizal plants showed similar growth to that of P‐fertilized plants. The level of mycorrhizal root infection was not significantly affected by fungal species or by water treatment. In contrast, important differences in Δ¹³C between P‐fertilized and AM plants were found in shoot and root tissues as a consequence of both water limitation and fungal presence. Δ¹³C in shoots and roots increased in non‐mycorrhizal treatment as compared with the well‐watered plants, whereas this parameter decreased significantly in mycorrhizal plants. Photosynthetic activity was increased in AM plants in well‐watered and droughted plants. G. deserticola was the most beneficial endophyte for water use efficiency in both water treatments. Transpiration rate was not affected by any of the treatments. On the basis of total C in plant tissues, in AM plants the newly fixed C seemed to be preferentially utilized for fungal activity rather than being stored in roots.     

Effect of root exudates of mycorrhizal tomato plants on microconidia germination of Fusarium oxysporum f. sp. lycopersici

The effect of root exudates from mycorrhizal and non-mycorrhizal tomato plants on microconidia germination of the tomato pathogen Fusarium oxysporum f. sp. lycopersici was tested. Microconidia germination was enhanced in the presence of root exudates from mycorrhizal tomato plants. Tomato plants were colonised by the arbuscular mycorrhizal fungus Glomus fasciculatum, indicating that alterations of the exudation pattern depended on the degree of root AM colonisation. Testing the exudates from plants with a high and a low P level revealed that the alterations of the root exudates from mycorrhizal plants, resulting in a changed effect on microconidia germination, are not due to an improved P status of mycorrhizal plants.     

Effects of arbuscular mycorrhiza (AM) on health ofLinum usitatissimum L. infected by fungal pathogens

Effects of arbuscular mycorrhizal (AM) symbiosis on health ofLinum usitatissimum infected by fungal pathogens were investigated exemplarily. Physiological and biochemical analyses were done to explain the mechanisms underlying the AM effects. AM plants showed increased resistance against the wilt pathogen (Fusarium oxysporum f. sp.lini), the level of this effects depended on the plant cultivars which all showed the same level of root colonization by arbuscular mycorrhizal fungi (AMF). In contrary to that, AM plants were highly susceptible against the shoot pathogenOidium lini, but they suffered less than non-AM plants in terms of shoot fresh weight, CO₂ assimilation and content of sucrose in shoot apex. This indicates that AM not only activates resistance mechanisms but also can induce tolerance against pathogens. The concentration of phytohormones such as auxin- and gibberellin-like substances were increased in shoots of AM plants. In roots the ethylene production was increased, too. Furthermore the content and composition of free sterols were highly altered in leaves of AM plants. Root infection by AMF caused an increased respiratory activity and a reduced degree of DNA methylation, but both modifications only occurred in infected root parts indicating an increasing gene activity. The presented results suggest that nearly all parts of a plant are influenced by AM but not in the same manner. In the case of mildewed linseed the effect of AM on plant health was impressing, it indicates that AM has an ability to induce tolerance.     

Arbuscular mycorrhizal fungi and biological control of Verticillium-wilted cotton plants

The interaction of arbuscular mycorrhizal fungi (Glomus etunicatum, Glomus intraradices, and Glomus versiforme) with a wilt-causing soil-borne pathogen, Verticillium dahliae, was studied in cotton. It was found that establishment by arbuscular mycorrhizal fungi reduced disease index. In diseased cotton plants colonised by G. etunicatum, the disease index was less than other diseased mycorrhizal and non-mycorrhizal ones. In diseased cotton plants, chlorophyll content was lower than others. Three Glomus species significantly increased content of sugar and protein in shoot and root. Pathogen-infected plants had higher proline concentration in shoot and root than healthy plants. On the other hand, the increased content of proline as stress sensor showed that Verticillium accelerates senescence and reduces yield. These results suggest that the beneficial effects of mycorrhiza can alleviate the pathogenesis effects of V. dahliae partly, and also there is a competitive interaction between the pathogenic and symbiotic fungi.     

The mobilisation and fate of soil and rock phosphate in the rhizosphere of ectomycorrhizal Pinus radiata seedlings in an Allophanic soil

A pot experiment was conducted to investigate the uptake of Zn from experimentally contaminated calcareous soil of low nutrient status by maize inoculated with the arbuscular mycorrhizal (AM) fungus Glomus caledonium. EDTA was applied to the soil to mobilize Zn and thus maximize plant Zn uptake. The highest plant dry matter (DM) yields were obtained with a moderate Zn addition level of 300 mg kg^?1. Plant growth was enhanced by mycorrhizal colonization when no Zn was added and under the highest Zn addition level of 600 mg kg^?1, while application of EDTA to the soil generally inhibited plant growth. EDTA application also increased plant Zn concentration, and Zn accumulation in the roots increased with increasing EDTA addition level. The effects of inoculation with G. caledonium on plant Zn uptake varied with Zn addition level. When no Zn was added, Zn translocation from roots to shoots was enhanced by mycorrhizal colonization. In contrast, when Zn was added to the soil, mycorrhizal colonization resulted in lower shoot Zn concentrations in mycorrhizal plants. The P nutrition of the maize was greatly affected by AM inoculation, with mycorrhizal plants showing higher P concentrations and P uptake. The results indicate that application of EDTA mobilized soil Zn, leading to increased Zn accumulation by the roots and subsequent plant toxicity and growth inhibition. Mycorrhizal colonization alleviated both Zn deficiency and Zn contamination, and also increased host plant growth by influencing mineral nutrition. However, neither EDTA application nor arbuscular mycorrhiza stimulated Zn translocation from roots to shoots or metal phytoextraction under the experimental conditions. The results are discussed in relation to the environmental risk associated with chelate-enhanced phytoextraction and the potential role of arbuscular mycorrhiza in soil remediation.     

Oxidative enzymes activities of mycorrhizal inoculated pepper plant infected with phytophthora infestans

The activities of polyphenol oxidase, peroxidase and catalase were investigated in Glomus etunicatum an arbuscular mycorrhizal inoculated pepper plant infected with Phytophthora infestans. Pepper plant inoculated with pathogen alone, simultaneously inoculated with mycorrhizal and pathogen or dually inoculated with pathogen before mycorrhizal had increased level of the oxidative enzymes activities especially at 4 weeks after planting. Of the three oxidases investigated catalase had the highest level of activities in all the treatments while the level of peroxidase was the lowest. The results showed that pathogen alone, build up the level of the oxidases in the inoculated pepper plant while inoculation of the pepper plant with AM mycorrhizal either simultaneously or dually with pathogen lower the activity of the oxidases indicating a passive protective effect of mycorrhizal.     

Flavonoids in roots of white clover: interaction of arbuscular mycorrhizal fungi and a pathogenic fungus

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.     

Responsiveness of Carob (Ceratonia siliqua L.) Plants to Arbuscular Mycorrhizal Symbiosis Under Different Phosphate Fertilization Levels

This experiment was carried out in pots in a greenhouse to evaluate the effects of arbuscular mycorrhizal fungi (Funneliformis mosseae, Rhizophagus intraradices and Rhizophagus fasciculatus) on carob plant performance under different levels of phosphate fertilization. Non-mycorrhizal (NMyc) and mycorrhizal (Myc) carob plants were subjected to three levels of phosphate fertilization, L1 (0 mg P kg⁻¹ soil), L2 (25 mg P kg⁻¹ soil) and L3 (100 mg P kg⁻¹ soil). Results showed that under L1 and L2 P-fertilization levels, arbuscular mycorrhizal symbiosis significantly improved growth and biomass production of carob plants. Moreover, mineral nutrient (P, K, Na and Ca) acquisition, photosynthetic activity (F_v/F_m), stomatal conductance, total chlorophyll content, and soluble sugar accumulation were also strongly improved in Myc plants in comparison with NMyc ones. Under L1 P-fertilization level, Myc plants showed strongly increased acid phosphatase activity in roots and in the rhizospheric soil than NMyc plants. Furthermore, Myc plants maintained high membrane integrity (over 80%) and low hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents, associated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (G-POD), and catalase (CAT) compared to NMyc plants. However, high phosphorus input (L3) negatively affected root colonization and mycorrhizal plant performance. Thus, carob plants associated with Funneliformis mosseae performed best under phosphorus deficiency and were the least sensitive to the variations of phosphorus input levels.

     

Studies of iron transport by arbuscular mycorrhizal hyphae from soil to peanut and sorghum plants

 The influence of an arbuscular mycorrhizal (AM) fungus on phosphorus (P) and iron (Fe) uptake of peanut (Arachis hypogea L.) and sorghum (Sorghum bicolor L.) plants was studied in a pot experiment under controlled environmental conditions. The plants were grown for 10 weeks in pots containing sterilised calcareous soil with two levels of Fe supply. The soil was inoculated with rhizosphere microorganisms only or with rhizosphere microorganisms together with an AM fungus (Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe). An additional small soil compartment accessible to hyphae but not roots was added to each pot after 6 weeks of plant growth. Radiolabelled P and Fe were supplied to the hyphae compartment 2 weeks after addition of this compartment. After a further 2 weeks, plants were harvested and shoots were analysed for radiolabelled elements. In both plant species, P uptake from the labelled soil increased significantly more in shoots of mycorrhizal plants than non-mycorrhizal plants, thus confirming the well-known activity of the fungus in P uptake. Mycorrhizal inoculation had no significant influence on the concentration of labelled Fe in shoots of peanut plants. In contrast, ⁵⁹Fe increased in shoots of mycorrhizal sorghum plants. The uptake of Fe from labelled soil by sorghum was particularly high under conditions producing a low Fe nutritional status of the plants. These results are preliminary evidence that hyphae of an arbuscular mycorrhizal fungus can mobilise and/or take up Fe from soil and translocate it to the plant. Accepted: 6 March 1998     

Effects of NaCl and Mycorrhizal Fungi on Antioxidative Enzymes in Soybean

The effects of different concentrations of NaCl on the activities of antioxidative enzymes in the shoots and roots of soybean (Glycine max [L.] Merr cv. Pershing) inoculated or not with an arbuscular mycorrhizal fungus, Glomus etunicatum Becker & Gerdemann, were studied. Furthermore, the effect of salt acclimated mycorrhizal fungi on the antioxidative enzymes in soybean plants grown under salt stress (100 mM NaCl) was investigated. Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased in the shoots of both mycorrhizal (M) and nonmycorrhizal (NM) plants grown under NaCl salinity. Salinity increased SOD activity in the roots of M and NM plants, but had no effect on CAT and polyphenol oxidase activities in the roots. M plants had greater SOD, POD and ascorbate peroxidase activity under salinity. Under salt stress, soybean plants inoculated with salt pre-treated mycorrhizal fungi showed increased SOD and POD activity in shoots, relative to those inoculated with the non pre-treated fungi.     

Effects of liming and mycorrhizal colonization on soil phosphate depletion and phosphate uptake by maize (Zea mays L.) and soybean (Glycine max L.) grown in two tropical acid soils

The effects of liming and inoculation with the arbuscular mycorrhizal fungus, Glomus intraradices Schenck and Smith on the uptake of phosphate (P) by maize (Zea mays L.) and soybean (Glycine max [L.] Merr.) and on depletion of inorganic phosphate fractions in rhizosphere soil (Al-P, Fe-P, and Ca-P) were studied in flat plastic containers using two acid soils, an Oxisol and an Ultisol, from Indonesia. The bulk soil pH was adjusted in both soils to 4.7, 5.6, and 6.4 by liming with different amounts of CaCO₃.In both soils, liming increased shoot dry weight, total root length, and mycorrhizal colonization of roots in the two plant species. Mycorrhizal inoculation significantly increased root dry weight in some cases, but much more markedly increased shoot dry weight and P concentration in shoot and roots, and also the calculated P uptake per unit root length. In the rhizosphere soil of mycorrhizal and non-mycorrhizal plants, the depletion of Al-P, Fe-P, and Ca-P depended in some cases on the soil pH. At all pH levels, the extent of P depletion in the rhizosphere soil was greater in mycorrhizal than in non-mycorrhizal plants. Despite these quantitative differences in exploitation of soil P, mycorrhizal roots used the same inorganic P sources as non-mycorrhizal roots. These results do not suggest that mycorrhizal roots have specific properties for P solubilization. Rather, the efficient P uptake from soil solution by the roots determines the effectiveness of the use of the different soil P sources. The results indicate also that both liming and mycorrhizal colonization are important for enhancing P uptake and plant growth in tropical acid soils.     

Uptake of 32P from labelled organic matter,by mycorrhizal and non-mycorrhizal subterranean clover (Trifolium subterraneum L.)

An experiment was designed to study whether hyphae and colonized roots of arbuscular mycorrhiza have more direct access to P in organic matter than roots of non-mycorrhizal plants. Soil supplied with 0, 15 or 45 mg P kg^–1 was uniformly mixed with ³²P-labelled organic matter at four levels (0, 1, 2 and 5 g kg^–1) and inoculated with a mycorrhizal fungus or left uninoculated. Pots were incubated at 60% of field capacity for one week prior to sowing of clover, and plants were harvested after a growth period of 23 days. Mycorrhizal colonization increased shoot dry weight, P concentration and ³²P uptake at all P levels. Specific activity in plants was consistently higher than in corresponding soil. This indicates that the added ³²P never reached an equilibrium with inorganic P in the soil. P mineralized from organic matter thus had a residence time in the soil solution sh ort enought to partially avoid isotopic exchange and adsorption. Mycorrhizal colonization influenced specific activity of ³²P in plants from three of the nine combinations of P and labelled organic matter: At the lowest level of P the specific activity was highest in non-mycorrhizal plants, and at the intermediate level of P there was one treatment where mycorrhizal plants had the highest specific activity. These differences are discussed. Plant dry weight and P concentration did not respond to addition of organic matter, though soil extracts consistently contained higher amounts of inorganic P as a result of organic matter addition. The results suggest that mycorrhizal plants at an early growth stage utilize a substantially higher amount of P released from organic matter than non-mycorrhizal plants. This mycorrhizal advantage does not seem to be related to a mycorrhizal influence on mineralization.