Infection process Infection process of of Plectosporium Plectosporium alismatisalismatis on host on host

and non-host and non-host species in the species in the AlismataceaeAlismataceae

IntroductionIntroduction

Australia- aquatic weedsAustralia- aquatic weeds Family Family AlismataceaeAlismataceae- marsh herbs- marsh herbs Species areSpecies are

Alisma plantago-aquaticaAlisma plantago-aquatica Domasonium minusDomasonium minus Alisma lanceolatumAlisma lanceolatum Sagittaria montevidensisSagittaria montevidensis Sagittaria gramineaSagittaria graminea

Species treated with herbicidesSpecies treated with herbicides Lead to resistance in Lead to resistance in AlimataceaAlimatacea

In 1994, identified a pathogen on In 1994, identified a pathogen on AlimataceaeAlimataceae Called Called Plectosporium alismatisPlectosporium alismatis

Some symptoms Some symptoms

Caused necrotic lesions on leaves, Caused necrotic lesions on leaves, petioles, inflorescence and stalkspetioles, inflorescence and stalks

Started as lens shaped necrotic Started as lens shaped necrotic spotsspots

Then formed elongated leasionsThen formed elongated leasions Soon suppressed plant growthSoon suppressed plant growth Had a limited host rangeHad a limited host range

P. alismatesP. alismates was a proposed was a proposed mycoherbicidemycoherbicide

Known hosts are:Known hosts are: A.plantago-aquticaA.plantago-aqutica A. lanceolatumA. lanceolatum D. minusD. minus

Objectives of studyObjectives of study

To study the infection processTo study the infection process Watch to see what happened after Watch to see what happened after

inoculation to both resistant and inoculation to both resistant and susceptible speciessusceptible species

See why some were resistantSee why some were resistant

Methods and MaterialsMethods and Materials

Obtained 42 isolates of Obtained 42 isolates of P. alismatisP. alismatis from South- eastern Australiafrom South- eastern Australia

Isolated from host on lima bean agarIsolated from host on lima bean agar Put back on hostPut back on host Reisolated from single sporesReisolated from single spores

Inoculum ProductionInoculum Production

Transfer to LBATransfer to LBA IncubateIncubate After sporulation- spores harvestedAfter sporulation- spores harvested InoculateInoculate IncubateIncubate Harvest Harvest Obtain spore suspension solutionObtain spore suspension solution

Pathogenicity StudyPathogenicity Study

Before infectionBefore infection Leaves from healthy plantsLeaves from healthy plants Transfer to Petri dishTransfer to Petri dish Inoculation with spore suspension Inoculation with spore suspension

solutionsolution Incubate control dishesIncubate control dishes Replicate experiment 3 timesReplicate experiment 3 times Found the isolate RH97 was most Found the isolate RH97 was most

pathogenicpathogenic

Infection StudyInfection Study

Leaf discs of Leaf discs of A.plantago-aquatica, S. A.plantago-aquatica, S. monteuidensismonteuidensis and and S. gramineaS. graminea

Inoculate with spore suspension of Inoculate with spore suspension of RH97RH97

Incubate Incubate Control dishesControl dishes

Light MicroscopyLight Microscopy

6 leaf discs/ species6 leaf discs/ species Removed at intervals 6, 12, 18, 24, Removed at intervals 6, 12, 18, 24,

30, 36h30, 36h Fixed and clearedFixed and cleared Examined 100 conidiaExamined 100 conidia

# of conidia germinated# of conidia germinated # of conidia germinated and formed an # of conidia germinated and formed an

appressoriumappressorium # of ungerminated conidia# of ungerminated conidia

Avoid clustersAvoid clusters What is considered germinatedWhat is considered germinated AppressoriaAppressoria Test for melanisationTest for melanisation

PAS reagentPAS reagent

Fluorescence MicroscopyFluorescence Microscopy

6 leaf discs/ species- removed at 6 6 leaf discs/ species- removed at 6 intervals-fixed and clearedintervals-fixed and cleared

Wash leavesWash leaves BufferBuffer Staining Staining Washing Washing Mounting in glycerolMounting in glycerol examinationexamination

Electron microscopyElectron microscopy

Several leaf discs / species- Several leaf discs / species- inoculatedinoculated

After 24-36 hours of incubationAfter 24-36 hours of incubation Examination of penetration sitesExamination of penetration sites

Scotch tapeScotch tape Non-inoculated also examinedNon-inoculated also examined

ResultsResults

All 3 species had both conidia, All 3 species had both conidia, germination and appressoria after 6 germination and appressoria after 6 hh

<50% of conidia elongated to form <50% of conidia elongated to form germ tube after 12 h but no germ tube after 12 h but no appressoria till after 18 happressoria till after 18 h

Rates between species – not that Rates between species – not that differentdifferent

Conidia of Conidia of P. alismatisP. alismatis DicellularDicellular Germination results in unbranched Germination results in unbranched

germ tube to form club-shaped germ tube to form club-shaped appressoriumappressorium

S. montevidensisS. montevidensis

Multiple germ tubesMultiple germ tubes Germ tube elongationGerm tube elongation No directional growth observedNo directional growth observed Variety of locations for appressoria Variety of locations for appressoria

developmentdevelopment StomataStomata

S.gramineaS.graminea

Necrosis in epidermal cellsNecrosis in epidermal cells ‘‘haloes’haloes’ Suggest papillaSuggest papilla Melanised appressoria not observedMelanised appressoria not observed

A. plantago-aquatica A. plantago-aquatica andand S. S. GramineaGraminea

Penetration after 24hPenetration after 24h Smooth haloesSmooth haloes Depressions- suggest stressDepressions- suggest stress Size of penetrationsSize of penetrations

Successful invasionSuccessful invasion Removal of inoculumRemoval of inoculum

symptomssymptoms On On A.plantago-aquatica A.plantago-aquatica

andand S.graminea S.graminea 4-6 days4-6 days Pepper spotting Pepper spotting Brown necrotic spots Brown necrotic spots

with yellow haloeswith yellow haloes Yellow haloes- toxinsYellow haloes- toxins Lens shaped lesionsLens shaped lesions No evidence observed No evidence observed

on leaves of on leaves of S. S. montevidensismontevidensis

First critical study of infection process First critical study of infection process 3 targets for weed control3 targets for weed control A. plantago-aquaticaA. plantago-aquatica least conductive least conductive

P.alismatis P.alismatis attached to both attached to both susceptible and resistant species at a susceptible and resistant species at a similar ratesimilar rate What does this mean?What does this mean?

Was the plant at all trying to stop the Was the plant at all trying to stop the infection?infection?

Questions and thoughtsQuestions and thoughts