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Infection process of Plectosporium alismatis on host and non-host species in the Alismataceae Introduction Australia- aquatic weeds Family Alismataceae- marsh herbs Species are Alisma plantago-aquatica Domasonium minus Alisma lanceolatum Sagittaria montevidensis Sagittaria graminea Species treated with herbicides Lead to resistance in Alimatacea In 1994, identified a pathogen on Alimataceae Called Plectosporium alismatis Some symptoms Caused necrotic lesions on leaves, petioles, inflorescence and stalks Started as lens shaped necrotic spots Then formed elongated leasions Soon suppressed plant growth Had a limited host range P. alismates was a proposed mycoherbicide Known hosts are: A.plantago-aqutica A. lanceolatum D. minus Objectives of study To study the infection process Watch to see what happened after inoculation to both resistant and susceptible species See why some were resistant Methods and Materials Obtained 42 isolates of P. alismatis from Southeastern Australia Isolated from host on lima bean agar Put back on host Reisolated from single spores Inoculum Production Transfer to LBA Incubate After sporulation- spores harvested Inoculate Incubate Harvest Obtain spore suspension solution Pathogenicity Study Before infection Leaves from healthy plants Transfer to Petri dish Inoculation with spore suspension solution Incubate control dishes Replicate experiment 3 times Found the isolate RH97 was most pathogenic Infection Study Leaf discs of A.plantago-aquatica, S. monteuidensis and S. graminea Inoculate with spore suspension of RH97 Incubate Control dishes Light Microscopy 6 leaf discs/ species Removed at intervals 6, 12, 18, 24, 30, 36h Fixed and cleared Examined 100 conidia # of conidia germinated # of conidia germinated and formed an appressorium # of ungerminated conidia Avoid clusters What is considered germinated Appressoria Test for melanisation PAS reagent Fluorescence Microscopy 6 leaf discs/ species- removed at 6 intervalsfixed and cleared Wash leaves Buffer Staining Washing Mounting in glycerol examination Electron microscopy Several leaf discs / species- inoculated After 24-36 hours of incubation Examination of penetration sites Scotch tape Non-inoculated also examined Results All 3 species had both conidia, germination and appressoria after 6 h <50% of conidia elongated to form germ tube after 12 h but no appressoria till after 18 h Rates between species – not that different Conidia of P. alismatis Dicellular Germination results in unbranched germ tube to form club-shaped appressorium S. montevidensis Multiple germ tubes Germ tube elongation No directional growth observed Variety of locations for appressoria development Stomata S.graminea Necrosis in epidermal cells ‘haloes’ Suggest papilla Melanised appressoria not observed A. plantago-aquatica and S. Graminea Penetration after 24h Smooth haloes Depressions- suggest stress Size of penetrations Successful invasion Removal of inoculum symptoms On A.plantago-aquatica and S.graminea 4-6 days Pepper spotting Brown necrotic spots with yellow haloes Yellow haloes- toxins Lens shaped lesions No evidence observed on leaves of S. montevidensis First critical study of infection process 3 targets for weed control A. plantago-aquatica least conductive P.alismatis attached to both susceptible and resistant species at a similar rate What does this mean? Was the plant at all trying to stop the infection? Questions and thoughts