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Restoration of environmental diversity by effective ecosymbiont monitoring

Objective

To isolate and characterize symbiotic microorganisms from a
desertified Mediterranean ecosystem and to develop macromolecular
methods for their identification. To also select effective
cultures from existing collections and to employ these methods
for their characterisation, screening and to monitor the fate of
the introduced microbial inoculants within a mixed population of
microsymbionts.


The site for field work is a desertified ecosystem in Almeria, SE
Spain (230mm annual rainfall). Arbuscular-mycorrhizal fungi
(AMF), Rhizobium bacteria and plant-growth promoting bacteria
(PGPR) will be isolated from the roots of shrub legumes. The
diversity of AMF at the site will be assessed through the year.
Pot cultures will be established and characterized on spore
morphology, anatomy of colonisation, and isoenzyme profiles.
Bacteria will be characterized by conventional molecular
techniques. Ecological characteristics of the AMF will be
studied in laboratory microcosms. Their reproductive and
spreading strategy will be evaluated by measuring the levels of
colonization relative to production of extraradical mycelium, and
metabolic activity of mycelium using image analysis.

Greenhouse studies will compare indigenous AMF populations in
desertified soils with exotic isolates. Inoculant AMF in tandem
with effective rhizobial strains will also be assessed for
stimulation of nitrogen fixation using 15N methodologies, and N
transfer will also be ascertained by isotopic dilution
techniques. Bacterial isolates will be marked genetically for
resistance to antibiotics via selection of spontaneous mutants to
detect them in field soils.

Immunological, electrophoretic and other methods will be
developed to compare the spread of introduced and indigenous AMF
using rhizoboxes where hyphae can be separated from roots.
Isozyme profiles of AMF will be screened for potential to monitor
which AMF is present. Competition between isolates will also be
studied using monoclonal antibodies (Ab) using hyphae as
immunogens to gather data on the spread of AMF by measuring the
proportion of the total extramatrical mycelium belonging to
individual AMF. Antibodies will be screened against the other
AMF isolates in order to find Ab able to differentiate AMF. In
surveys of mixed populations in rhizoboxes, the mycelium will be
extracted and assayed using fluorescent stain-conjugated Ab or in
an ELISA format utilizing Ab coupled to enzymes which catalyze a
specific quantitative colour reaction. The immunofluorescent
staining of the AMF will allow the observation and identification
of hyphae in situ, this will be combined with activity staining
using an image analysis system to quantify the differences in
fluorescence.

The selected organisms will be used as inoculants to enhance the
establishment of three pioneer shrubs in a desertified
Mediterranean ecosystem, thus accelerating its sustainable
rehabilitation. The inoculant microsymbionts will be made
available for the target desertified ecosystem. Small-scale
field plots of pioneer legumes inoculated with the recommended
microsymbionts will be established and monitored for successful
establishment.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

University of Kent at Canterbury
Address

CT2 7NJ Canterbury
United Kingdom

Participants (3)

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Spain
Address
1,Profesor Albareda 1
18008 Granada
FORESTRY COMMISSION RESEARCH AGENCY
United Kingdom
Address
Northern Research Station
EH25 9SY Roslin-midlothian
TECHNICAL UNIVERSITY OF MUNICH
Germany
Address
Alte Akademie 12
85350 Freising