Arbuscular mycorrhizal contribution to heavy metal uptake by maize (Zea mays L.) in pot culture with contaminated soil

In two pot-culture experiments with maize in a silty loam (P2 soil) contaminated by atmospheric deposition from a metal smelter, root colonization with indigenous or introduced arbuscular mycorrhizal (AM) fungi and their influence on plant metal uptake (Cd, Zn, Cu, Pb, Mn) were investigated. Soil was -irradiated for the nonmycorrhizal control. In experiment 1, nonirradiated soil provided the mycorrhizal treatment, whereas in experiment 2 the irradiated soil was inoculated with spores of a fungal culture from P2 soil or a laboratory reference culture, Glomus mosseae. Light intensity was considerably higher in experiment 2 and resulted in a fourfold higher shoot and tenfold higher root biomass. Under the conditions of experiment 1, biomass was significantly higher and Cd, Cu, Zn and Mn concentrations significantly lower in the mycorrhizal plants than in the nonmycorrhizal plants, suggesting a protection against metal toxicity. In contrast, in experiment 2, biomass did not differ between treatments and only Cu root concentration was decreased with G. mosseae-inoculated plants, whereas Cu shoot concentration was significantly increased with the indigenous P2 fungal culture. The latter achieved a significantly higher root colonization than G. mosseae (31.7 and 19.1%, respectively) suggesting its higher metal tolerance. Zn shoot concentration was higher in both mycorrhizal treatments and Pb concentrations, particularly in the roots, also tended to increase with mycorrhizal colonization. Cd concentrations were not altered between treatments. Cu and Zn, but not Pb and Cd root-shoot translocation increased with mycorrhizal colonization. The results show that the influence of AM on plant metal uptake depends on plant growth conditions, on the fungal partner and on the metal, and cannot be generalized. It is suggested that metal-tolerant mycorrhizal inoculants might be considered for soil reclamation, since under adverse conditions AM may be more important for plant metal resistance. Under the optimized conditions of normal agricultural practice, however, AM colonization even may increase plant metal absorption from polluted soils.     

Field response of wheat to arbuscular mycorrhizal fungi and drought stress

Mycorrhizal plants often have greater tolerance to drought than nonmycorrhizal plants. This study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi inoculation on growth, grain yield and mineral acquisition of two winter wheat (Triticum aestivum L.) cultivars grown in the field under well-watered and water-stressed conditions. Wheat seeds were planted in furrows after treatment with or without the AM fungi Glomus mosseae or G. etunicatum. Roots were sampled at four growth stages (leaf, tillering, heading and grain-filling) to quantify AM fungi. There was negligible AM fungi colonization during winter months following seeding (leaf sampling in February), when soil temperature was low. During the spring, AM fungi colonization increased gradually. Mycorrhizal colonization was higher in well-watered plants colonized with AM fungi isolates than water-stressed plants. Plants inoculated with G. etunicatum generally had higher colonization than plants colonized with G. mosseae under both soil moisture conditions. Biomass and grain yields were higher in mycorrhizal than nonmycorrhizal plots irrespective of soil moisture, and G. etunicatum inoculated plants generally had higher biomass and grain yields than those colonized by G. mosseae under either soil moisture condition. The mycorrhizal plants had higher shoot P and Fe concentrations than nonmycorrhizal plants at all samplings regardless of soil moisture conditions. The improved growth, yield and nutrient uptake in wheat plants reported here demonstrate the potential of mycorrhizal inoculation to reduce the effects of drought stress on wheat grown under field conditions in semiarid areas of the world.     

Differences in the effects of three arbuscular mycorrhizal fungal strains on P and Pb accumulation by maize plants

The effect of arbuscular mycorrhizal (AM) fungi on the accumulation and transport of lead was studied in a pot experiment on maize plants grown in anthropogenically-polluted substrate. The plants remained uninoculated or were inoculated with different Glomus intraradices isolates, either indigenous to the polluted substrate used or reference from non-polluted soil. A considerably lower tolerance to the conditions of polluted substrate was observed for the reference isolate that showed significantly lower frequency of root colonisation as well as arbuscule and vesicule abundance. Plants inoculated with the reference isolate also had significantly lower shoot P concentrations than plants inoculated with the isolate from polluted substrate. Nevertheless, inoculation with either indigenous or reference G. intraradices isolate resulted in higher shoot and root biomass and inoculated plants showed lower Pb concentrations in their shoots than uninoculated plants, regardless of differences in root colonisation. Root biomass of maize plants was divided according to AM-induced colouration into brightly yellow segments intensively colonised by AM fungus and non-colonised or only slightly colonised whitish ones. Intensively colonised segments of the isolate from polluted substrate contained significantly higher concentrations of phosphorus and lead than non-colonised ones, which suggest significant participation of fungal structures in element accumulation. Responsible Editor: Peter Christie.     

Influence of temperature on colonization of spring barleys by vesicular arbuscular mycorrhizal fungi

The effects of three soil temperatures on growth of spring barleys (Hordeum vulgare L.) and on their root colonization by vesicular arbuscular mycorrhizal (VAM) fungi from agricultural soils in Montana (USA) or Syria at different inoculum concentrations were tested in soil incubators in the greenhouse. The number of mycorrhizal plants as well as the proportion and intensity of roots colonized increased with higher soil temperatures. VAM fungi from Montana, primarily Glomus macrocarpum, were cold tolerant at 11°C while those from Syria, primarily G. hoi, were heat tolerant at 26°C. Inoculum potential of Montana VAM fungi was higher than Syrian VAM fungi in cool soils. Harmal, selected from Syrian barley land races, had the highest colonization by mycorrhizal fungi of the cultivars tested.Journal Series Paper: J-2532 Montana Agricultural Experiment Station.     

Response of cassava to VA mycorrhizal inoculation and phosphorus application in greenhouse and field experiments

Cassava (Manihot esculenta Crantz) was grown in the greenhouse and in the field at different levels of phosphorus applied, with or without inoculation with VA mycorrhiza in sterilized or unsterilized soil. When grown in a sterilized soil to which eight levels of P had been applied the non-inoculated plants required the application of 3200 kg P ha⁻¹ to reach near-maximum yield of plant dry matter (DM) at 3 months. Inoculated plants, however, showed only a minor response to applied P. Mycorrhizal inoculation in the P check increased top growth over 80 fold and total P uptake over 100 fold. Relating dry matter produced to the available P concentration in the soil (Bray II), a critical level of 15 ppm P was obtained for mycorrhizal and 190 ppm P for non-mycorrhizal plants. This indicates that the determination of critical levels of P in the soil is highly dependent on the degree of mycorrhizal infection of the root system. In a second greenhouse trial with two sterilized and non-sterilized soils it was found that in both sterilized soils, inoculation was most effective at intermediate levels of applied P resulting in a 15–30 fold increase in DM at 100 kg P ha⁻¹. In the unsterilized soil inoculation had no significant effect in the quilichao soil, but increased DM over 3 fold in the Carimagua soil, indicating that the latter had a native mycorrhizal population less effective than the former. When cassava was grown in the field in plots with 11 levels of P applied, uninoculated plants grown in sterilized soil remained extremely P deficient for 4–5 months after which they recuperated through mycorrhizal infection from unsterilized borders or subsoil. Still, after 11 months inoculation had increased root yields by 40%. In the non-sterilized soil inoculation had no significant effect as the introduced strain was equally as effective as the native mycorrhizal population. These trials indicate that cassava is extremely dependent on an effective mycorrhizal association for normal growth in low-P soils, but that in most natural soils this association is rapidly established and inoculation of cassava in the field can only be effective in soils with a low quantity and quality of native mycorrhiza. In that case, plants should be inoculated with highly effective strains.     

Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (Single Stranded Conformation Polymorphism)

PCR amplification of a region of the large subunit ribosomal DNA sequence with Glomus specific primers was used to detect arbuscular mycorrhizal fungi in root tissue of four plant species. The primers were specific to Glomus mosseae, Glomus caledonium, Glomus geosporum, Glomus coronatum, Glomus fragilistratum and Glomus constrictum, and did not recognise sequences from Glomus claroideum. Sequence differences between isolates were detected by Single Stranded Conformation Polymorphisms (SSCPs) in polyacrylamide gels under non-denaturing conditions. Isolates of G. mosseae, G. caledonium and G. coronatum could be separated by their SSCP patterns, while three isolates of G. geosporumshowed no variation. Specific SSCP patterns from isolates of G. mosseae and G. caledonium allowed detection of both fungi in the same root segment. Sequence differences leading to variations in SSCP patterns were confirmed by direct sequencing. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chitinase in roots of mycorrhizal Allium porrum: regulation and localization

Chitinase (EC 3.2.1.14) activity was measured in roots of Allium prorrum L. (leek) during development of a vesicular-arbuscular mycorrhizal symbiosis with Glomus versiforme (Karst.) Berch. During the early stages of infection, between 10 and 20 d after inoculation, the specific activity of chitinase was higher in mycorrhizal roots than in the uninfected controls. However, 60–90 d after inoculation, when the symbiosis was fully established, the mycorrhizal roots contained much less chitinase than control roots. Chitinase was purified from A. porrum roots. An antiserum against beanleaf chitinase was found to cross-react specifically with chitinase in the extracts from non-mycorrhizal and mycorrhizal A. porrum roots. This antiserum was used for the immunocytochemical localization of the enzyme with fluorescent and gold-labelled probes. Chitinase was localized in the vacuoles and in the extracellular spaces of non-mycorrhizal and mycorrhizal roots. There was no immunolabelling on the fungal cell walls in the intercellular or the intracellular phases. It is concluded that the chitin in the fungal walls is inaccessible to plant chitinase. This casts doubts on the possible involvement of this hydrolase in the development of the mycorrhizal fungus. However, fungal penetration does appear to cause a typical defense response in the first stages that is later depressed.     

Contribution of the arbuscular mycorrhizal symbiosis to heavy metal phytoremediation

High concentrations of heavy metals (HM) in the soil have detrimental effects on ecosystems and are a risk to human health as they can enter the food chain via agricultural products or contaminated drinking water. Phytoremediation, a sustainable and inexpensive technology based on the removal of pollutants from the environment by plants, is becoming an increasingly important objective in plant research. However, as phytoremediation is a slow process, improvement of efficiency and thus increased stabilization or removal of HMs from soils is an important goal. Arbuscular mycorrhizal (AM) fungi provide an attractive system to advance plant-based environmental clean-up. During symbiotic interaction the hyphal network functionally extends the root system of their hosts. Thus, plants in symbiosis with AM fungi have the potential to take up HM from an enlarged soil volume. In this review, we summarize current knowledge about the contribution of the AM symbiosis to phytoremediation of heavy metals.     

Root exudation from Leucaena leucocephala in relation to mycorrhizal colonization

Lower amounts of root eduxates (13 mg/g dry root) emerged from leucaena plants inoculated with the mycorrhizal fungus, Glomus fasciculatum, than uninoculated plants (21 mg/g dry root). Mycorrhizal plants exuded less K⁺, P_i and sugars (mainly glucose) but more protein, nitrogen, phenolics and gibberellins than uninoculated plants. Glycine, alanine, cysteine, arginine, tryptophan and valine occurred only in the root exudates of the former. Uninoculated plants exuded more of a root-elongation inhibitory substance than the uninoculated ones.R.J. Mada and D.J. Bagyaraj are with the Department of Agricultural Microbiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560065, India.     

Growth response of Atriplex nummularia to inoculation with arbuscular mycorrhizal fungi at different salinity levels

Chenopods are generally regarded as non-host plants for mycorrhizal fungi and are believed not to benefit from colonization by mycorrhizal fungi. Perennial Atriplex nummularia Lindl., growing under field conditions, showed a relatively high level of colonization by mycorrhizal fungi (10–30% of root length colonized) in spring and summer. Accordingly, two glasshouse experiments were designed to assess the effects of inoculation with mycorrhizal fungi (with a single species or a mixture of different species) on growth, nutrient uptake, and rhizosphere bacterial community composition of A. nummularia at high and low salinity levels (2.2 and 12 dSm^–1). Only low and patchy colonization by mycorrhizal fungi (1–2 of root length colonized) was detected in inoculated plants under glasshouse conditions which was unaffected by salinity. Despite the low colonization, inoculation increased plant growth and affected nutrient uptake at both salinity levels. The effects were higher at an early stage of plant development (6weeks) than at a later stage (9–10 weeks). Salinity affected the bacterial community composition in the rhizosphere as examined by ribosomal intergenic spacer amplification (RISA) of 16S rDNA, digitization of the band patterns and multivariate analysis. The effects of inoculation with mycorrhizal fungi on growth of A. nummularia may be attributed to (i) direct effects of mycorrhizal fungi on plant nutrient uptake and/or (ii) indirect effects via mycorrhizal-induced changes in the bacterial community composition.     

The effect of season on VA mycorrhiza of the almond tree and of phosphate fertilization and species of endophyte on its mycorrhizal dependency

Summary Prunus dulcis (Miller), a tree which is able to develop in low fertility soils, forms VA mycorrhiza. Under glasshouse conditions the growth and P concentration in the leaf tissue of non-mycorrhizal plants, given a customary agronomic dose of P-fertilizer, were lower than those of mycorrhizal plants. The relative mycorrhizal dependency¹⁴ values of the almond tree were higher when a mixture of locally isolated (mainlyGlomus fasciculatus) was used as inoculum. These indigenous endophytes were more tolerant of added fertilizers thanGlomus mosseae taken from the pot-culture collection.The amount of VA infection and the number of Endogonaceae spores in the rhizosphere of almond trees growing in the field steadily increase from winter (the flowering season of this crop) until summer or early autumn.     

Violets of the section Melanium, their colonization by arbuscular mycorrhizal fungi and their occurrence on heavy metal heaps

Violets of the sections Melanium were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Heartsease (Viola tricolor) from several heavy metal soils was AMF-positive at many sites but not at extreme biomes. The zinc violets Viola lutea ssp. westfalica (blue zinc violet) and ssp. calaminaria (yellow zinc violet) were always AMF-positive on heavy metal soils as their natural habitats. As shown for the blue form, zinc violets germinate independently of AMF and can be grown in non-polluted garden soils. Thus the zinc violets are obligatorily neither mycotrophs nor metalophytes. The alpine V. lutea, likely ancestor of the zinc violets, was at best poorly colonized by AMF. As determined by atomic absorption spectrometry, the contents of Zn and Pb were lower in AMF colonized plants than in the heavy metal soils from where the samples had been taken. AMF might prevent the uptake of toxic levels of heavy metals into the plant organs. Dithizone staining indicated a differential deposition of heavy metals in tissues of heartsease. Leaf hairs were particularly rich in heavy metals, indicating that part of the excess of heavy metals is sequestered into these cells.     

Hyperaccumulators, arbuscular mycorrhizal fungi and stress of heavy metals

Use of plants, with hyperaccumulating ability or in association with soil microbes including the symbiotic fungi, arbuscular mycorrhiza (AM), are among the most common biological methods of treating heavy metals in soil. Both hyperaccumulating plants and AM fungi have some unique abilities, which make them suitable to treat heavy metals. Hyperaccumulator plants have some genes, being expressed at the time of heavy metal pollution, and can accordingly localize high concentration of heavy metals to their tissues, without showing the toxicity symptoms. A key solution to the issue of heavy metal pollution may be the proper integration of hyperaccumulator plants and AM fungi. The interactions between the soil microbes and the host plant can also be important for the treatment of soils polluted with heavy metals.     

Ectomycorrhizal and arbuscular mycorrhizal colonization of two species of floodplain willows

To understand the relationships between the distribution of Chosenia arbutifolia and Salix sachalinensis and their mycorrhizal colonization, changes in the quality and types of ectomycorrhizas and arbuscular mycorrhizas of the seedlings of two species were studied at five different sites with different soil conditions in the floodplain of the Satsunai River, Hokkaido. High ectomycorrhizal and low arbuscular mycorrhizal colonization were found in roots of both plants. Ectomycorrhizal colonization of S. sachalinensis in wet sandy or muddy soil conditions was at the same level as that in dry gravelly sites. In contrast, ectomycorrhizal colonization of C. arbutifolia seedlings was lower from wet sandy sites than that from dry gravelly sites. In all study sites, the same three morphological types of ectomycorrhizas were dominant.     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

Heavy metal uptake by arbuscular mycorrhizas of Elsholtzia splendens and the potential for phytoremediation of contaminated soil

A pot culture experiment and a field experiment were carried out separately to study heavy metal (HM) uptake from soil contaminated with Cu, Zn, Pb and Cd by Elsholtzia splendens Nakai ex F. Maekawa inoculated with arbuscular mycorrhizal (AM) fungi and the potential for phytoremediation. The HM-contaminated soil in the pot experiment was collected from the field experiment site. Two AM fungal inocula, MI containing only one AM fungal strain, Glomus caledonium 90036, and M II consisting of Gigaspora margarita ZJ37, Gigaspora decipens ZJ38, Scutellospora gilmori ZJ39, Acaulospora spp. andGlomus spp., were applied to the soil under unsterilized conditions. In the pot experiment, the plants were harvested after 24 weeks of growth. Mycorrhizal colonization rate, plant dry weight (DW) and P, Cu, Zn, Pb, Cd concentrations were determined. MI-treated plants had higher mycorrhizal colonization rates than MII-treated plants. Both MI and MII increased shoot and root DW, and MII was more effective than MI. In shoots, the highest P, Cu, Zn and Pb concentrations were all observed in the plants treated with MII, while MI decreased Zn and Pb concentrations and increased P but did not alter Cu, and Cd concentrations were not affected by either of two inocula. In roots, MII increased P, Zn, Pb concentrations but did not alter Cu and Cd, and MI did not affect P, Cu, Zn, Pb, Cd concentrations. Cu, Zn, Pb, Cd uptake into shoots and roots all increased in MII-treated plants, while in MI-treated plants, Cu and Zn uptake into shoots and Cu, Zn, Pb, Cd into roots increased but Pb and Cd uptake into shoots decreased. In general, MII was more effective than MI in promoting plant growth and HM uptake. The field experiment following the pot experiment was carried out to investigate the effects of MII under field conditions. The 45-day-old nonmycorrhizal and MII-colonized seedlings of E. splendens were transplanted to HM-contaminated plots and harvested after 5 months. MII-inoculation increased shoot DW and shoot P, Cu, Zn, Pb concentrations significantly but did not alter shoot Cd concentrations, which led to higher uptake of Cu, Zn, Pb, Cd by E. splendens shoots. These results indicate that the AM fungal consortium represented by MII can benefit phytoextraction of HMs and therefore play a role in phytoremediation of HM-contaminated soils.     

Trade-offs between arbuscular mycorrhizal fungal competitive ability and host growth promotion in Plantago lanceolata

In this study we tested for trade-offs between the benefit arbuscular mycorrhizal (AM) fungi provide for hosts and their competitive ability in host roots, and whether this potential trade-off shifts in the presence of a plant stress (herbivory). We used three species of AM fungi previously determined to vary in host growth promotion and spore production in association with host plants. We found that these AM fungal species competed for root space, and the best competitor, Scutellospora calospora, was the worst mutualist. In addition, the worst competitor, Glomus white, was the best mutualist. Competition proved to have stronger effects on fungal infection patterns than herbivory, and competitive dominance was not altered by herbivory. We found a similar pattern in a previous test of competition among AM fungi, and we discuss the implications of these results for the persistence of the mutualism and feedbacks between AM fungi and their plant hosts.     

Colonization of pennycresses (Thlaspi spp.) of the Brassicaceae by arbuscular mycorrhizal fungi

Members of the Brassicaceae are generally believed to be non-mycorrhizal. Pennycress (Thlaspi) species of this family from diverse locations in Slovenia, Austria, Italy and Germany were examined for their colonisation by arbuscular mycorrhizal fungi (AMF). Meadow species (T. praecox, T. caerulescens and T. montanum) were sparsely but distinctly colonised, as indicated by the occurrence of intraradical hyphae, vesicles, coils, and occasionally arbuscules. Species from other locations were poorly colonised, but arbuscules were not discernible. The genus Thlaspi comprises several heavy metal hyperaccumulating species (T. caerulescens, T. goesingense, T. calaminare, T. cepaeifolium). All samples collected from heavy metal soils were at best poorly colonized. Thus the chance is small to find a "hypersystem" in phytoremediation consisting of an AM fungus which prevents the uptake of the major part of the heavy metals and of a Thlaspi species which effectively deposits the residual heavy metals inevitably taken up into its vacuoles. In two different PCR approaches, fungal DNA was amplified from most of the Thlaspi roots examined, even from those with a very low incidence of AMF colonization. Sequencing of the 28S- and 18S-rDNA PCR-products revealed that different Thlaspi field samples were colonized by Glomus intraradices and thus by a common AM fungus. However, none of the sequences obtained was identical to any other found in the present study or deposited in the databanks, which might indicate that a species continuum exists in the G. intraradices clade. An effective colonization of Thlaspi by AMF could not be established in greenhouse experiments. Although the data show that Thlaspi can be colonized by AMF, it is doubtful whether an effective symbiosis with the mutual exchange of metabolites is formed by both partners.     

The response of mycorrhizal colonization to elevated CO2 and climate change in Pascopyrum smithii and Bouteloua gracilis

Large intact soil cores of nearly pure stands of Pascopyrum smithii (western wheatgrass, C₃) and Bouteloua gracilis (blue grama, C₄) were extracted from the Central Plains Experimental Range in northeastern Colorado, USA and transferred to controlled environment chambers. Cores were exposed to a variety of water, temperature and CO₂ regimes for a total of four annual growth cycles. Root subsamples were harvested after the completion of the second and fourth growth cycles at a time corresponding to late winter, and were examined microscopically for the presence of mycorrhizae. After two growth cycles in the growth chambers, 54% of the root length was colonized in P. smithii, compared to 35% in blue grama. Field control plants had significantly lower colonization. Elevation of CO₂ increased mycorrhizal colonization in B. gracilis by 46% but had no effect in P. smithii. Temperatures 4° C higher than normal decreased colonization in P. smithii by 15%. Increased annual precipitation decreased colonization in both species. Simulated climate change conditions of elevated CO₂, elevated temperature and lowered precipitation decreased colonization in P. smithii but had less effect on B. gracilis. After four growth cycles in P. smithii, trends of treatments remained similar, but overall colonization rate decreased.     

Quantification of mycelial development of arbuscular mycorrhizal fungi using image analysis

Quantitative and reproducible information concerning the development of the extraradical mycelium of arbuscular mycorrhizal fungi (AMF) is lacking due to the difficulties in extracting, identifying and estimating hyphal lengths. In this study, using a rhizobox growth system, the lengths of hyphae of AMF estimated using an image analysis system were not significantly different from data obtained by a trained observer using a modified grid-line intersect method. The assessment of lengths of hyphae on membrane filters or slides was, however, much quicker using image analysis, and allowed the complete sample to be quantified, unlike the grid-line method where a limited number of fields of view are assessed. The image analysis procedure is objective, observer-independent and less laborious than the manual method of assessment. Of the four different methods of sample preparation compared, membrane filter methods were found to be the most appropriate for quantitative sampling from three non-soil substrates. Glomus monosporum (UKC M3) produced twice as much extraradical mycelium and hyphal length per centimetre of colonised root than G. geosporum (BEG 11) on both leek and linseed in a durite sand at final harvest (63 days). Both AMF also produced more hyphal length per centimetre of colonised root on linseed than on leek. The spatial distribution of both AMF, however, was similar in durite sand and no correlation with levels of NaHCO₃-extractable phosphorus was noted. In a third experiment, with G. manihotis (UKC INDO-1) colonising a tropical forage legume, Pueraria phaseoloides, in two other growth substrates, a different pattern of development of the extra-radical mycelium was observed. Because of a higher content of particulate matter, which collected on the membrane filters, the extraction technique had to be modified to give optimal performance of the image analysis system.