Confirmed Nursery Protocol - aphis

Sample Processing
• Bottles containing water samples and leaf baits are placed on their sides, and
incubated for 3 days at 18 – 22°C in the dark.
• Baits should then be removed, rinsed in distilled water, and blotted dry. Leaf
pieces should be processed immediately for detection while whole leaves should be
placed in moist chambers for up to 14 days to allow lesion development.
• Leaf baits are placed into selective PARPH-V8 medium for isolation of P.
ramorum and tested utilizing nested or real-time PCR.
• Transfer the isolation plates immediately to a state or federally-approved
processing laboratory via overnight courier, or
• Maintain the agar plates at 20°C in the dark for at least three days. Because P.
ramorum grows slower than most other species of Phytophthora commonly found in
water, leaving the filters on the agar plates for a three day incubation period is critical for
recovery of this species.
• Check the plates at regular intervals under low magnification-using an inverted or
dissecting microscope-for colonies with typical morphological characters of P. ramorum
(e.g., coralloid hyphae, semi-papillate sporangia, and large chlamydospores).
• Contact laboratory personnel if P. ramorum is suspected from any samples. See
Appendix 3 for shipping details in the culture and morphology protocol at
http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/downloads/pdf_files/cultur
eprotocol6-07.pdf
PAR(PH)-V8 Selective Medium: For Phytophthora species. Adapted from Jeffers &
Martin, 1986; Ferguson & Jeffers, 1999.
Amount Per:
Concentration
(PPM)
Ingredient 1.0 Liter 0.5 Liter
Basal Medium
Clarified V8 Concentrate 50 ml 25 ml
Distilled Water 950 ml 475 ml
Difco Bacto Agar 15 g 7.5g
Amendments
Delvocid (50% pimaricin) 10 mg = 0.01 g 5 mg 5
Sodium Ampicillin 250 mg = 0.25 g 125 mg ~250
Rifamycin-SV (sodium salt) 10 mg = 0.01 g 5 mg ~10
Terraclor [75% PCNB]* 66.7 mg = 0.0667 33.4 mg 50
Hymexazol* 50 mg = 0.05 25 mg 50
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