Lila N. Salhi, David Bernard-Perron, Suha Jabaji, B. Franz Lang Microorganisms with biofertilizing and biocontrol properties in cranberries Lila N. Salhi, David Bernard-Perron, Suha Jabaji, B. Franz Lang

Overview What are plant endophytes? Benefits of endophytes for plants Only few endophytes are known Are there more? what are they good for? Result: endophyte diversity is completely underestimated Which ones are beneficial? Conclusions : Exiting future opportunities

Why microorganisms? Every child knows (?): bacteria dissolve minerals (P, K) and make them available via plant roots. Microbial community is involved in: Stimulation of plant growth Improvement of soil fertility Holguin et al., 1999). Ciencia y Desarrollo 25 (No.144): 26-35

Plant growth promoting microorganisms Colonize plant roots (rhizosphere and endophytes). Dominate in competition with other microbes. Promote plant growth by: Wide spectrum of hormonal stimulation factors Biofertilization (nutrient solubilization, nitrogen fixation) Pathogen-control (antibiotics, fungicides, insecticides …) Rothballer et al., 2003; Gamalero et al., 2004, Persello-Cartieauxet al., 2003

Are endophytes always plant growth promoting? No! Potential interaction types (fluid, sometimes transitional): Neutral : No effect Mutualistic : beneficial Pathogenic : harmful Zhu et al, 2012

Fungal endophytes of cranberries: little is known Ericoid mycorrhiza Dark, septate endophytes Growth inside plant cells. Hyphae don't extend into soil Heterogeneous group Darkly-pigmented Septate hyphae Rhizoscyphus ericae Oidiodendron maius Phialocephala fortinii Smith and Read, 2008; Jumpponen and Trappe, 1998

… and bacterial endophytes: even less is known Pseudomonas syringae Gram negative with polar flagella Usually a pathogen, but … Successful in controlling Cottonball disease (Monilinia oxycocci) Pseudomonas sp voland et al, 1999

Questions and Hypotheses A much more diverse universe of endophytes? Which isolates in our microbial collection are beneficial? How to implant them sustainably with plants ?

1. Isolation from plant tissue 3. DNA Sequencing and Blast (NCBI) Molecular identification of microorganisms 1. Isolation from plant tissue ITS ITS1-F BMBC-F ITS4-R 5.8S ITS1 ITS2 28S 18S 600 nt 750 nt IGS ETS rRNA gene in fungi Leave Stem Root 2. DNA Extraction and PCR tRNA 534-R 27-F 507 nt 5S 23S 16S rRNA gene in bacteria 3. DNA Sequencing and Blast (NCBI) BLAST Muyzer et al. (1993) ; White et al. 1990; blast.ncbi.nlm.nih.gov

Halo representing mineral solubilization Checking of microorganism properties Phosphate solubilization Nitrogen fixation Biocontrol NBRIP medium Insoluble phosphate Nitrogen free medium PDA medium Halo representing mineral solubilization Bacterial growth Fungal or bacterial growth inhibition

2 – Beneficial Proprieties Results 1 – Microbial Diversity 2 – Beneficial Proprieties

137 microorganisms isolated Unexpected endophyte diversity: the tip of an iceberg? Roots 65 Fungi, 36 Bacteria 137 microorganisms isolated Trimmatostroma betulinum Not identified Bacteria : Bacillus sp. Paenibacillus sp Pantoea sp Fungi Others Stems Leaves Roots

Stems 16 Fungi, 8 Bacteria Bacteria: Fungi: Others Stems Leaves Alternaria sp. Nemania serpens Fungi: Paenibacillus sp. Bacteria: Others Stems Leaves Roots

Leaves 8 Bacteria Bacteria: Others Stems Leaves Roots Bacillus subtilis Bacteria: Others Stems Leaves Roots

Phosphate solubilization Which endophytes may be beneficial? Phosphate solubilization Nitrogen fixation Bacillus subtilis Burkholderia sp. Rahnella aquatilis Rhodococcus qingshengii Burkholderia sp. Rahnella aquatilis Panteoa sp. Cohnella ginsengisoli Rhizobium sp.

Penicillium janthinellum Biocontrol Pseudomonas sp. Penicillium janthinellum Fungal-fungal interaction Bacterial-fungal interaction

Conclusions Unexpectedly large endophyte diversity in cranberry tissues Different types of microbes in roots and aerial plant parts. Some endophytes have plant growth promoting properties. Others have strong fungicide properties. What Next?

Thanks ! This work was possible thanks to financial support by Le ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec