1. Infection process of Plectosporium alismatis on host and non-host species in the Alismataceae

2. Introduction Australia- aquatic weeds Australia- aquatic weeds Family Alismataceae- marsh herbs Family Alismataceae- marsh herbs Species are Species are Alisma plantago-aquatica Alisma plantago-aquatica Domasonium minus Domasonium minus Alisma lanceolatum Alisma lanceolatum Sagittaria montevidensis Sagittaria montevidensis Sagittaria graminea Sagittaria graminea

3. Species treated with herbicides Species treated with herbicides Lead to resistance in Alimatacea Lead to resistance in Alimatacea In 1994, identified a pathogen on Alimataceae In 1994, identified a pathogen on Alimataceae Called Plectosporium alismatis Called Plectosporium alismatis

4. Some symptoms Caused necrotic lesions on leaves, petioles, inflorescence and stalks Caused necrotic lesions on leaves, petioles, inflorescence and stalks Started as lens shaped necrotic spots Started as lens shaped necrotic spots Then formed elongated leasions Then formed elongated leasions Soon suppressed plant growth Soon suppressed plant growth Had a limited host range Had a limited host range

6. P. alismates was a proposed mycoherbicide P. alismates was a proposed mycoherbicide Known hosts are: Known hosts are: A.plantago-aqutica A.plantago-aqutica A. lanceolatum A. lanceolatum D. minus D. minus

7. Objectives of study To study the infection process To study the infection process Watch to see what happened after inoculation to both resistant and susceptible species Watch to see what happened after inoculation to both resistant and susceptible species See why some were resistant See why some were resistant

8. Methods and Materials Obtained 42 isolates of P. alismatis from South- eastern Australia Obtained 42 isolates of P. alismatis from South- eastern Australia Isolated from host on lima bean agar Isolated from host on lima bean agar Put back on host Put back on host Reisolated from single spores Reisolated from single spores

9. Inoculum Production Transfer to LBA Transfer to LBA Incubate Incubate After sporulation- spores harvested After sporulation- spores harvested Inoculate Inoculate Incubate Incubate Harvest Harvest Obtain spore suspension solution Obtain spore suspension solution

10. Pathogenicity Study Before infection Before infection Leaves from healthy plants Leaves from healthy plants Transfer to Petri dish Transfer to Petri dish Inoculation with spore suspension solution Inoculation with spore suspension solution Incubate control dishes Incubate control dishes Replicate experiment 3 times Replicate experiment 3 times Found the isolate RH97 was most pathogenic Found the isolate RH97 was most pathogenic

11. Infection Study Leaf discs of A.plantago-aquatica, S. monteuidensis and S. graminea Leaf discs of A.plantago-aquatica, S. monteuidensis and S. graminea Inoculate with spore suspension of RH97 Inoculate with spore suspension of RH97 Incubate Incubate Control dishes Control dishes

12. Light Microscopy 6 leaf discs/ species 6 leaf discs/ species Removed at intervals 6, 12, 18, 24, 30, 36h Removed at intervals 6, 12, 18, 24, 30, 36h Fixed and cleared Fixed and cleared Examined 100 conidia Examined 100 conidia # of conidia germinated # of conidia germinated # of conidia germinated and formed an appressorium # of conidia germinated and formed an appressorium # of ungerminated conidia # of ungerminated conidia

13. Avoid clusters Avoid clusters What is considered germinated What is considered germinated Appressoria Appressoria Test for melanisation Test for melanisation PAS reagent PAS reagent

14. Fluorescence Microscopy 6 leaf discs/ species- removed at 6 intervals- fixed and cleared 6 leaf discs/ species- removed at 6 intervals- fixed and cleared Wash leaves Wash leaves Buffer Buffer Staining Staining Washing Washing Mounting in glycerol Mounting in glycerol examination examination

15. Electron microscopy Several leaf discs / species- inoculated Several leaf discs / species- inoculated After 24-36 hours of incubation After 24-36 hours of incubation Examination of penetration sites Examination of penetration sites Scotch tape Scotch tape Non-inoculated also examined Non-inoculated also examined

16. Results All 3 species had both conidia, germination and appressoria after 6 h 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 <50% of conidia elongated to form germ tube after 12 h but no appressoria till after 18 h Rates between species – not that different Rates between species – not that different

17. Conidia of P. alismatis Conidia of P. alismatis Dicellular Dicellular Germination results in unbranched germ tube to form club-shaped appressorium Germination results in unbranched germ tube to form club-shaped appressorium

18. S. montevidensis Multiple germ tubes Multiple germ tubes Germ tube elongation Germ tube elongation No directional growth observed No directional growth observed Variety of locations for appressoria development Variety of locations for appressoria development Stomata Stomata

19. S.graminea Necrosis in epidermal cells Necrosis in epidermal cells ‘haloes’ ‘haloes’ Suggest papilla Suggest papilla Melanised appressoria not observed Melanised appressoria not observed

20. A. plantago-aquatica and S. Graminea Penetration after 24h Penetration after 24h Smooth haloes Smooth haloes Depressions- suggest stress Depressions- suggest stress Size of penetrations Size of penetrations

21. Successful invasion Successful invasion Removal of inoculum Removal of inoculum

22. symptoms On A.plantago-aquatica and S.graminea On A.plantago-aquatica and S.graminea 4-6 days 4-6 days Pepper spotting Pepper spotting Brown necrotic spots with yellow haloes Brown necrotic spots with yellow haloes Yellow haloes- toxins Yellow haloes- toxins Lens shaped lesions Lens shaped lesions No evidence observed on leaves of S. montevidensis No evidence observed on leaves of S. montevidensis

23. First critical study of infection process First critical study of infection process 3 targets for weed control 3 targets for weed control A. plantago-aquatica least conductive A. plantago-aquatica least conductive P.alismatis attached to both susceptible and resistant species at a similar rate P.alismatis attached to both susceptible and resistant species at a similar rate What does this mean? What does this mean? Was the plant at all trying to stop the infection? Was the plant at all trying to stop the infection?

26. Questions and thoughts Questions and thoughts