Isolation from Leaf BaitsIsolation from Leaf Baits

Use a selective mediumUse a selective mediumPP55ARPH-V8ARPH-V8, PARPH, PCH, P, PARPH, PCH, P1010VP, othersVP, others

Use CMA or CV8A as the basal mediumUse CMA or CV8A as the basal mediumavoid PDA—too rich, can be inhibitoryavoid PDA—too rich, can be inhibitory

CV8A has lots of advantagesCV8A has lots of advantagespromotes sporulation directly on isolation promotes sporulation directly on isolation

plateplateAvoid acidified media and streptomycinAvoid acidified media and streptomycin

detrimental to detrimental to PhytophthoraPhytophthora & & PythiumPythium

Isolation from Leaf BaitsIsolation from Leaf BaitsSelective Medium AmendmentsSelective Medium Amendments

Hymexazol—key ingredientHymexazol—key ingredientprevents growth of prevents growth of mostmost species of species of PythiumPythiumadversely affects some species of adversely affects some species of PhytophthoraPhytophthora

Pimaricin = Nystatin = Pimaricin = Nystatin = DelvocidDelvocidanti-fungus antibiotic from actinomyceteanti-fungus antibiotic from actinomycetelight labile—keep out of lightlight labile—keep out of light

Ampicillin [Sodium salt—disolves in water]Ampicillin [Sodium salt—disolves in water]anti-bacterium antibiotic—inhibits gram+ bacteriaanti-bacterium antibiotic—inhibits gram+ bacteria

+ Rifamycin-SV = Rifampin = Rifampicin+ Rifamycin-SV = Rifampin = Rifampicinanti-bacterium antibiotic—inhibits gram- bacteriaanti-bacterium antibiotic—inhibits gram- bacteria

PCNB: fungicide—inhibits many soilborne fungi PCNB: fungicide—inhibits many soilborne fungi

Retrieving Leaf BaitsRetrieving Leaf Baits

Keep samples moist—do not allow to dry Keep samples moist—do not allow to dry outoutuse damp paper towels & plastic bagsuse damp paper towels & plastic bags

Keep samples cool and darkKeep samples cool and darkuse an ice chestuse an ice chestavoid long periods in car trunks!avoid long periods in car trunks!

Isolation from BaitsIsolation from Baits

Wash tissue pieces in running tap waterWash tissue pieces in running tap waterDo not surface disinfestDo not surface disinfest

some propagules may be on the surfacesome propagules may be on the surfaceBlot dry to remove excess moistureBlot dry to remove excess moistureUse lots of small pieces: 5-6 per plateUse lots of small pieces: 5-6 per platePush pieces Push pieces intointo the agar the agar

need good contact between agar and tissueneed good contact between agar and tissueIncubate in dark at cool temp (20 C or Incubate in dark at cool temp (20 C or

less)less)growth in 2-3 days, watch for 7 daysgrowth in 2-3 days, watch for 7 days

Isolation from LeavesIsolation from Leaves

P. ramorumP. ramorum Positive Positive

Isolate StorageIsolate StorageMedium—CMA or V8AMedium—CMA or V8AContainers—glass vials (8 ml)Containers—glass vials (8 ml)

make mini-slants or leave agar flatmake mini-slants or leave agar flatuse plugs in sterile distilled wateruse plugs in sterile distilled water

Use 2 vials/isolate—one dry, one mineral Use 2 vials/isolate—one dry, one mineral oiloil

Temperature—12-15 C best for all speciesTemperature—12-15 C best for all speciesdo do notnot refrigerate—OK for refrigerate—OK for P. ramorumP. ramorum

Duration—several years; up to 6 yearsDuration—several years; up to 6 yearsKeep records—spreadsheet, databaseKeep records—spreadsheet, database