Download Infection process of Plectosporium alismatis on host and non

Infection process of
Plectosporium alismatis
on host and non-host
species in the
Alismataceae
Introduction
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Australia- aquatic weeds
Family Alismataceae- marsh herbs
Species are
Alisma plantago-aquatica
 Domasonium minus
 Alisma lanceolatum
 Sagittaria montevidensis
 Sagittaria graminea
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Species treated with herbicides
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Lead to resistance in Alimatacea
In 1994, identified a pathogen on Alimataceae
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Called Plectosporium alismatis
Some symptoms
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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
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P. alismates was a proposed mycoherbicide
Known hosts are:
A.plantago-aqutica
 A. lanceolatum
 D. minus
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Objectives of study
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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
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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
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Transfer to LBA
Incubate
After sporulation- spores harvested
Inoculate
Incubate
Harvest
Obtain spore suspension solution
Pathogenicity Study
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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
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Leaf discs of A.plantago-aquatica, S. monteuidensis
and S. graminea
Inoculate with spore suspension of RH97
Incubate
Control dishes
Light Microscopy
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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
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Avoid clusters
What is considered germinated
Appressoria
Test for melanisation
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PAS reagent
Fluorescence Microscopy
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6 leaf discs/ species- removed at 6 intervalsfixed and cleared
Wash leaves
Buffer
Staining
Washing
Mounting in glycerol
examination
Electron microscopy
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Several leaf discs / species- inoculated
After 24-36 hours of incubation
Examination of penetration sites
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Scotch tape
Non-inoculated also examined
Results
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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
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Conidia of P. alismatis
Dicellular
 Germination results in unbranched germ tube to
form club-shaped appressorium
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S. montevidensis
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Multiple germ tubes
Germ tube elongation
No directional growth observed
Variety of locations for appressoria
development
Stomata
S.graminea
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Necrosis in epidermal cells
‘haloes’
Suggest papilla
Melanised appressoria not observed
A. plantago-aquatica and S.
Graminea
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Penetration after 24h
Smooth haloes
Depressions- suggest stress
Size of penetrations
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Successful invasion
Removal of inoculum
symptoms
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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
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First critical study of infection process
3 targets for weed control
 A. plantago-aquatica least conductive
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P.alismatis attached to both susceptible and
resistant species at a similar rate
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What does this mean?
Was the plant at all trying to stop the infection?
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Questions and thoughts