• 12 week testing period

• Two experiments

  • Both started in temperature controlled glasshouses in UK before field trial in south-east Spain

  • Open pot cultures

  • Experiment 1

    • Plants were harvested at Weeks 3, 6, 9

  • Experiment 2

    • Plants were harvested at Weeks 4, 8, 12

• Results were intended to quantify the root colonization/plant growth

  • Reflection of the glomus spp. working to create mycorrhizas

  • Statistics to quantify included

    • Standard deviation % frequency of colonization (%F)

    • Intensity of colonization (%M)

    • Arbuscule intensity (%A)

  • Inoculum of AM fungi

    • Inoculum of AM fungi was from colonized plants grown in open pot-cultures in a temperature-controlled glasshouse

    • Inoculum contained an attapulgite clay growth medium (Oil-Dri, Ltd., UK), which had chopped colonized roots from the host plants and spores of AM fungi

    • Six fungi were tested in two experiments; expt 1: Glomus deserticola BEG73, Glomus fistulosum BEG3, Glomus geosporum BEG11, Glomus microaggregatum BEG56 and Gigaspora margarita BEG34; expt 2: Glomus deserticola BEG73, Glomus fistulosum BEG3, Glomus geosporum BEG11, Glomus microaggregatum BEG56 and Glomus mosseae BEG25

    • (TISSERANT, B., BRENAC, V., REQUENA, N., JEFFRIES, P. and DODD, J.C. (1998)

  • Plant and growth substrates

    • All seedlings were thoroughly sterilized

    • The growth medium from above, was matched to soil conditions of the field test site

    • 50 grams of inoculum of different individual AM fungi or a non-mycorrhizal root growth substrate inoculum was placed as a layer in each pot and the Terragreen mixture used to fill the pot

    • Three seedlings of either Anthyllis cytisoides, Allium porrum or Thymus vulgaris were placed in each pot in experiment 1, and six seedlings per pot in experiment 2

    • (TISSERANT, B., BRENAC, V., REQUENA, N., JEFFRIES, P. and DODD, J.C. (1998)

  • Bradyrhizobial inoculum

    • Two equal groups of Anthyllis cytisoides,

      • One was inoculated with Bradyrhizobium sp. isolated from nodules of Anthyllis cytisoides growing in the Sierra de Filabres field site, and the other was not

      • The bacteria were grown in yeast extract mannitol (YEM) broth in 250-ml flasks containing 100 ml of medium on a rotary shaker at 28 °C for 1 week

      • After this the culture was centrifuged and the pellet resuspended in sterile water

      • 3 ml of the solution was applied per pot as inoculum for seedlings of Anthyllis cytisoides at planting in both experiments 1 and 2

    • (TISSERANT, B., BRENAC, V., REQUENA, N., JEFFRIES, P. and DODD, J.C. (1998)

  • Glasshouse growth conditions

    • Experiments occurred in the temperate section, temperature controlled

    • Pots watered every 2 days

    • Weekly irrigation with commercial fertilizer

    • Supplementary lighting

• Data collection

  • Harvests

  • Protein extraction

  • Spore and extraradical mycelium extracts

  • Gel electrophoresis

  • Enzyme staining

• Field Trial

  • Used Anthyllis cytisoides seedlings colonized by Glomus microaggregatum BEG56

  • 150 plants were transplanted into separate 1-l pots with 1 kg of steam-sterilized soil from the Sierra de Filabres field site and maintained in a glasshouse

  • (TISSERANT, B., BRENAC, V., REQUENA, N., JEFFRIES, P. and DODD, J.C. (1998)

  • Tested disturbed and undisturbed conditions

• Root colonization/plant growth-

  • Experiment 1: Based on visual estimation of presence of external mycelium (Table 1)

    • Anthyllis cytisoides- less external mycelium around their roots for all the species of AM fungi used compared with Allium porrum and Thymus vulgaris plants after 3, 6 and 9 week of growth

    • Allium porrum and Thymus vulgaris colonized by either Glomus microaggregatum, Glomus deserticola or Glomus fistulosum had the first production of an external mycelial network while plants colonized by Glomus geosporum had the longest production

    • Gigaspora margarita- No external mycelium was observed

    • In experiment 2 %F > %M but the same general trends emerged

    • Either could be used to detect AM fungi

    • The percentage of root length colonized by different AM fungi (%F and %M) in the roots of Anthyllis cytisoides remained low (17% for %F and 14% for %M) throughout the experiment compared with Allium porrum and Thymus vulgaris

    • Allium porrum had the highest levels of colonization (%F and %M) across all species of AM fungi throughout the experiment

    • Thymus vulgaris root length colonized (%F and %M) for all AM fungi in roots of increased slightly between weeks 4 and 8 but decreased after 12 weeks (Table 2)

    • The arbuscular intensity (%A) in colonized roots differed between plants

      • Allium porrum roots- highest values (Table 2)

The trend of better growth in mycorrhizal plants was observed

• Bradyrhizobial modulation-

  • Experiment 1 few nodules were detected on the roots of Anthyllis cytisoides inoculated by Bradyrhizobium sp.

  • Larger numbers of nodules detected in experiment 2 on Anthyllis cytisoides seedlings 8 and 12 weeks after inoculation

• Isozyme analysis-

  • Enzymes stained and the AM fungi which could be detected consistently in roots of the three host plants at the respective harvests

  • Bands on gels were designated as MSIs when, for a particular AM fungus, they were consistently found in the colonized roots of the host but could not be detected in extracts from non-colonized plants

  • Allium porrum and Thymus vulgaris with Glomus deserticola BEG73 mycorrhizas detected on gel stained because of two MSIs at all harvests in both experiments