- Maintain nematode cultures on several North American isolates of A. areolatum
Releases
- Releases D. siricidicola to a warmer part of the season in the north eastern U.S. and release the nematodes in the fall.

- Select trees for inoculation primarily by the presence of resin beads

- Examined their crowns if the trees met the criterion

- Choose trees with red or brown needles also accept suppressed trees with green needles

- Trees without needle or with sirex emergence holes are rejected

- Felled the trees and inoculated them with nematodes

- Sealed the cut bole ends with a green wood end sealer
Inoculation
- Harvest nematodes from culture flasks

- Washed mass-reared nematodes from flasks with tap water, counted and placed them in breathable baggies in aliquots for transport to the field

- Mixed one of baggie of polyacrylamide gel and water

- Whisked the mixture until the water was absorbed and decanted it into plastic squeeze bottles
Sample collection
- Collect bolt samples from inoculated trees within March-May
- Cut 68cm bolts from each tree from within the inoculation zone:
   1. At the top (�7.5 m above the base)
   2. At the bottom (�0.5 m)
   3. At the center (�4.0 m)
- Remaining is chipped to kill S. noctilio larvae and D. siricidicola
- Transport the bolts to the quarantine lab, seal cut ends and place in     screened barrels for adult emergence.
- collect the emerging insects and held them at -20 degree Celsius begore siricid identification and dissection
- All siricids like parasitic Hymenoptera and Cerambycidae dissected to assess possible infection by nematodes.
Analysis
-   The population size of S. noctilio was expressed as numbers of insects per m3 of wood (i.e., to account for size differences among trees)
 Sirex diagnosis and nematode collection
- All emerging wasps were dissected
-  . Specimens of both sexes were dissected with a minuten pin at a magnification of 45
-  Ovaries and eggs were examined in females and test in male
-  417 male and 143 female S. noctilio from 2008 to 2012 for DNA analysis
- Woodwasps for DNA analysis were preserved in 95% ethanol in 10 mL scintillation vials and stored at �20 C.
Molecular identification of nematode strains
· DNA extraction
- Samples of nematodes were transferred to 180 ml tissue lysis buffer 
-  Stored at 4 C until DNA isolation and extraction (using QIAamp DNA Micro Kit (Qiagen)) were performed
-  Tubes containing nematodes were maintained in a water bath at 56 C overnight for tissue lysis. 
- DNA is eluted

· PCR and molecular analysis of D. siridicola
- Amplify the internally transcribed mtCO1 sequence CO- F1 and CO- R1) in thermal cycler
-   The samples were then purified using a QIAQuick PCR Purification Kit  

· Restriction analysis
-  Strain identification of nematodes was assessed qualitatively using a combination of restriction enzyme digests 
-  The first method was based on the use of Aci1 to cut the mtCO1 sequence using the polymorphism 
-   The second test employed the enzyme BsrG1 to differentiate between Kamona and D. proximus Bedding
- Incubated overnight at 37 C and then subjected to 60V electrophoresis on a 1.5% (w/v) ethidium bromide-stained agarose gel 
- Visualized under UV illumination after 1 h and 40 min