FBIP: Fungal Root Endophytes of Selected Erica species

Study Extent

Eastern Cape, Makana

Sampling

Assessment of fungal root endophyte diversity includes culturable isolates and unculturable fungi as well. DNA was extracted from surface sterilised and crushed root material and submitted for 454 sequencing at the GS Facility at Rhodes University. The sequences were compared with internal transcribed spacer (ITS) sequences available in the GenBank and UNITE databases by BLASTn search and the closest matches with homology greater than 95 % were included in the clustal alignment and phylogenetic analysis.Erica seeds were obtained from Kirstenbosch Botanical Gardens.

Method steps

1. This is a current PhD project being undertaken by Ms Christine Bizabani. Ms Bizabani registered in 2012 and is due to complete at the end of 2014. Progress to date includes selection and collection or root material from Erica caffra, E. demissa, E. chamissonis, isolation and maintenance of culturable fungal root endophytes. Over 50 isolates have been morphologically and molecularly identified and have been sent to the National Fungal Culture Collection (see attached spreadsheet). Fungal isolates from E. glumifora, E cerinthoides and E. nemirosa are in process. An Honours student will contribute to a portion of the project in 2014. Assessment of fungal endophyte root biodiversity through DNA pyrosequencing and determination of phylogenetic relationships. Timeframe Jan 2014 - Oct 2014, Ms Bizabani (PhD Student). Timeframe Feb 2014- Oct 2014: An Honours student will be given one or two of the collected Erica species to work on in 2014. The student has not be identified. Assessment of fungal root endophyte diversity not only includes culturable isolates but unculturable fungi as well. DNA will be extracted from surface sterilised and crushed root material and submitted for 454 sequencing at the GS Facility at Rhodes University. The sequences will be compared with internal transcribed spacer (ITS) sequences available in the GenBank and UNITE databases by BLASTn search and the closest matches with homology greater than 95 % will be included in the clustal alignment and phylogenetic analysis. The sequences will be aligned using Clustal X. Phylogenetic analysis will be done using Molecular Evolutionary Genetics Analysis (MEGA) by the Neighbor-joining method. The robustness of the phylogeny will be analyzed by bootstrap analysis using 1000 iterations. The branches corresponding to partitions in less than 50 % bootstrap replicates will be collapsed. Positions containing gaps and missing data will be eliminated from the data set. This analysis will be conducted with the assistance of Dr Marieka Gryzenhout at the University of the Free State. Resynthesis and confirmation of endophyte status. Timeframe June 2013 - July 2014, Ms Bizabani (PhD Student) Commercial Erica seeds obtatined from Kirstenbosch will be used to test for ericoid mycorrhiza formation of selected isolates. The seeds will be surface sterilized with 3% bleach containing 100 μl l-1 Tween 20 for 1 min. The seeds will then be subsequently washed in sterile Milli-Q water twice. After sterilization the seeds will be pre treated prior to germination by soaking in Milli-Q water overnight or applying a smoke treatment. Seeds will be germinated on moist sterile filter paper and will be kept at 22 ⁰C during germination with 16 h light period and 8 h dark period. The germinated seedlings will then be transferred to sterile 150 ml square petri dishes measuring 120 mm x 15 mm containing 20 g of autoclaved vermiculite medium. Prior to seedling transfer the medium will be watered with 3 ml Sterile Milli-Q water and 0.5 ml of nutrient solution containing 15.0 μM KH2PO4, 17.0 μM MgSO4, 8.0 x 10-3 μM H3BO3, 4.0 x 10-3 μM MnCl2.4H2O, 3.0 x 10-4 μM ZnSO4.H2O, 8.0 x 10-5 μM CuSO4 and 8.0 x 10-6 μM (NH4) MO7O24.4H2O. Nine seedlings will be placed in each petri dish. After 3 weeks the root zones of the seedlings will be inoculated individually with 500 μl mycelial slurry of a particular isolated. There will be 3 replicate petri dishes (27 seedlings) for each treatment and uninoculated controls. If necessary Vaccinni corymbosium (Blueberry) micropropagated plantlets will be obtained through Amatola Berries in Stutterheim these will be inoculated in a pot trial. After 10-12 weeks roots from seedlings or plantlets will be excised and rinsed prior to clearing, staining and microscopic examination for mycorrhizal structures. Due to the length of time required for these resynthesis tests this will not form part of an Honours project. Ecological characterization of fungal isolates. Timeframe Sept 2013 - November 2014. Ms Bizabani (PhD Student) Timeframe Feb 2014- Oct 2014: A selection of isolates will assigned to an Honours student in 2014. The student has not be identified. Ecological activity of fungal endophytes will be assessed to determine biological control activity against other soil borne fungal pathogens, siderophore production, nitrogen and phosphate assimilation, phosphate solubilisation, lignocellulolytic activity and tolerance to metal toxicity. Antimicrobial screening will be conducted in dual culture between pathogens (Fusarium and Phytophthora) and endophytic isolates. Pathogen growth will be monitored daily. The endophytic isolates will also be tested for siderophore production which is a iron chelator using a Chrome Azurol S agar plate assay. The ability of the isolates to utilize organically bound nitrogen and phosphate will be assessed in a liquid basal medium amended with various organic (such as glutamine, arginine, BSA, glycerophosphate and RNA). Medium will be incubated for 7-10 days and filtered mycelium will be oven dried and biomass will be recorded. Phosphate solubilisation will be assessed on bromophenol blue medium containing insoluble Ca phosphate as the only P source. Development of yellow halo will indicate the presence of organic acids which contribute to the release of P. Linocellulolytic activity will be assessed using a basal medium amended with cellulose azure dye. A release of azure dye ndicates cellulose degradation. Tolerance to zinc, copper and cadmium will also be assessed in media amended with different concentrations of the metals. Fungal growth will be measure at regular intervals over an 8 week period.

Some specimen identified to Species level

1. Fungi
   rank: kingdom

Eastern Cape, Makana