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Alvin
Adlina
Hannah

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Biological control of
Sirex noctilio
(Hymenoptera: Siricidae)
in the north eastern United States using an exotic parasitic nematode

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The European woodwasp, Sirex noctilio L., is a killer of Pinus species in the southern hemisphere. It was first discovered outside its' native range of Europe, North Africa, and the Middle East in New Zealand . S. noctilio found a perfect environment in which to prosper, with abundant monocultures of its host and without a guild of natural enemies. The Australian Congress of Scientific and Industrial Research Organization (CSIRO), along with the Commonwealth Institute of Biological Control, mounted a worldwide campaign in the 1960s and 1970s to explore for its natural enemies. They found out the most successful natural enemy proved to be a nematode, Deladenus siricidicola Bedding.

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Sites
- Southward of Hills Creek State Park, Pennsylvania
Nematode and fungus culture
- Use nematode culture that mass produces D. siricidicola which is kamona strain
- Culture in lab on a fast-growing isolate of the fungus A. areolatum on potato dextrose agar

- 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

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- The nematode biological control system developed by CSIRO hasn’t worked well in the U.S. A possible solution is to undertake further exploration in Europe to find a strain of D. siricidicola better adapted to the strain of S. noctilio and environmental conditions in North America.

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Typical biological control agent include parasitoid, pathogen, prey-predator, but Sirex noctilio is a nematode.

Changes in any of the components, as well as other biotic and abiotic factors, may alter the dynamics of the system .

In the CSIRO biological control program for S. noctilio, the same fungus and nematode strains are used from year to year, the infection rate of the nematode is carefully monitored and maintained, and the nematode is applied according to uniform protocols

However, the situation is especially complex and unpredictable in North America due to the diverse native pine forests, multiple strains and species of the symbiotic fungus

Different strains of S. noctilio can be susceptible or resistant to sterilization by the same strain of D. siricidicola

Based on the first studies conducted in 2011 in New York, 44% of their trees contained S. noctilio infected with nematodes and that infection rates averaged 27.9% across all sites

The authors reported that 38% of female woodwasps had nematodes in the hemocoel for the second study, research conducted in southern and central Ontario, Canada.

The feature of the relationship between S. noctilio and D. siricidicola most relevant to biological control is female sterilization.

Successful sterilization is dependent on a critical synchrony between the release of juvenile nematodes from the mother into the host hemocoel and the development of the host eggs .

A spectrum of host effects can result, ranging from a few shrunken, malformed eggs without nematodes to a complement of fully formed egg shells neatly packed with juveniles.

A small increase in temperature (+4 C) could lower S. noctilio egg numbers, induce abnormal egg formation, and decrease larval development and adult size; higher temperatures reduced the rates of infection and sterilization by D. siricidicola.

Sterilization of S. noctilio is highly desirable. Using trap trees and a virulent sterilizing nematode strain is an augmentative biological control strategy that is aimed at establishment and recycling in the environment for some unknown time.

A recent review hypothesized that pine species could influence D. siricidicola and it is now clear, based on several studies, that levels of nematode infection in S. noctilio can be associated with pine species.

We speculate that the relatively higher rates of nematode infection reflect the European origins of S. noctilio and P. sylvestris.

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The numbers of siricids (primarily S. noctilio) that emerged in sample barrels and were dissected ranged widely, from <10 to >700 per site per year .

Trees attacked by S. noctilio and 464 numbers of trees were sample. The percentages of uninfested trees averaged 36.4 ± 19.7%.

The percentages of trees containing nematode-infected S. noctilio averaged just 28.1 ± 20.5%.

Study showed, S. noctilio densities declined steadily overall, with over 1000 woodwasps per m³ in 2007 through as low as 40.0 per m³ in more recent years .

In the later years, 2009–2012, as expected, infection rates in the uninoculated control trees were consistently lower than or about 77–86 81 equal to the nematode-treated trees .

However, the nematode infection rate was significantly higher  for treated trees  than for control trees .

Surprisingly, the DNA analyses revealed that, in spite of the higher infection rates in trees inoculated with Kamonas, most of those infections were not caused by Kamonas .

Infection rates in trees treated with Kamonas were
consistently numerically higher in P. sylvestris than in P. resinosa, but not signifificantly so when testing years separately.