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A Compilation of Results of Sirex noctilio Projects by CPHST Scientists and their University Cooperators 2006-2012
David W. Williams and Victor C. Mastro USDA, APHIS, PPQ, CPHST Otis Laboratory Buzzards Bay, Massachusetts 02542 May 2015
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Contents
Chronology of the Sirex Program – 2004-2013………………………………….. 3
Executive summaries of Sirex noctilio projects………………………………….. 5
List of peer-reviewed publications produced by the Sirex noctilio program…. 14
I. CHEMICAL ECOLOGY……………………………………………………… 16
Katalin Bӧrӧczky; Pennsylvania State University………………………….. 16
Miriam Cooperband; USDA, APHIS, CPHST Otis Laboratory……………. 19
II. SURVEY AND TRAPPING…………………………..………………………. 42
Damon Crook; USDA, APHIS, CPHST Otis Laboratory………………….. 42
Kelley Zylstra; USDA, APHIS, CPHST Syracuse Laboratory...... 64
Joseph Francese; USDA, APHIS, CPHST Otis Laboratory……………….. 97
III. BIOLOGICAL AND NATURAL CONTROL……………………………… 106
David Williams; USDA, APHIS, CPHST Otis Laboratory………………… 106
Ann Hajek; Cornell University……………………………………………… 159
Kelley Zylstra……………………………………………………………….. 194
IV. COMMUNITY ECOLOGY…………………………………………..……… 199
Matthew Ayres; Dartmouth College………………………………………… 199
Kamal Gandhi; University of Georgia……………………………………… 202
V. MODELING…………………………………………………………………… 209
Scott Myers; USDA, APHIS, CPHST Otis Laboratory…………………….. 209
Kamal Gandhi ………………………………………………………………. 210
VI. MOLECULAR GENETICS…………………………………………………. 214
Evan Braswell; USDA, APHIS, CPHST Mission Laboratory……………... 214
Ann Hajek…………………………………………………………………... 218 3
Chronology of the Sirex Program – 2004-2013
Fall 2004 – Sirex noctilio trapped in Fulton, New York, during bark beetle survey.
Spring 2005 – S. noctilio identified by E. Richard Hoebeke of Cornell University.
July 2005 – Dave Williams asked to initiate Sirex biological control program.
September 2005 – Williams traveled to Australia for two weeks to review Sirex program.
Fall 2005 – Beddingia Lab established; Kelley Zylstra started work.
Fall 2005 – Lynn Goldner (NPL) and Leon Bunce appointed to coordinate Sirex program.
Fall 2005 – Environmental Assessment and Biological Assessment written.
January 2006 – Robin Bedding visited and gave nematode short course.
January 2006 – first session on Sirex at Interagency Research Forum on Invasive Species.
January 2006 – Syracuse Lab established with Robin Tait as director.
Summer 2006 – trapping work started around Syracuse.
August 2006 – first imports of the Kamona strain of Beddingia siricidicola from Ecogrow.
September 2006 – Sirex training session at SUNY Oswego.
November 2006 – first controlled release of Kamona strain in central New York.
January 2007 – first mass rearing of Kamona in Beddingia Lab.
January 2007 – Zylstra became new director of the Syracuse Lab.
May 2007 – Sirex Symposium in South Africa, Williams represented the Otis Lab.
Summer 2007 – discovery of “native” strain of Beddingia siricidicola in New York.
Summer 2007-2012 – continued trapping work.
Fall 2007-2012 – continued controlled nematode releases.
April 2009 – Robyn Rose replaced Goldner as NPL for Sirex.
April 2009 – meeting with APHIS and Forest Service in Riverdale to discuss nematode releases.
Spring 2010 – molecular work carried out by Evan Braswell. 4
May 2011 – review of Sirex research in Riverdale; Sirex officially turned over to Forest Service.
July 2013 – Syracuse Lab closed.
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Executive summaries of Sirex noctilio projects
Sirex noctilio invaded Tasmania in 1952 and the Australian mainland in 1961. In response, the Commonwealth Scientific and Industrial Research Organization (CSIRO) rapidly developed a multipronged management plan for combating the pest. To characterize their program, CSIRO used the metaphor of a three legged stool. The legs were monitoring and survey, silvicultural practices, and biological control. CSIRO undertook directed research in these areas, recognizing the need to integrate the approaches. Following their metaphor, the stool needed all three legs to stand. Research was carried out worldwide through cooperation with other Commonwealth organizations. For example, the biological control leg was supported by global exploration for natural enemies over a range of taxa by the Commonwealth Institute of Biological Control in the United Kingdom.
Thus, when S. noctilio invaded North America in 2004, CPHST already had a powerful model to adapt for use in a pest management program for the United States. The CPHST methods development program was ultimately similar to that of CSIRO, but more along the lines of traditional integrated pest management (IPM) as practiced in the U.S. since the 1970s. IPM is based on a strong foundation of biological and ecological knowledge, including our topical areas of chemical ecology, community ecology, and molecular biology. It is implemented by the development of survey tools, including traps and lures, the mass production and release of biological control agents, and the use of models to better understand and predict the risks posed by the target pest through its seasonal dynamics and host relationships. In the following, we present the salient features of the CPHST management program for S. noctilio in brief executive summaries and then expand on the details of research in its various components.
I. CHEMICAL ECOLOGY
Katalin Bӧrӧczky explored two aspects of the chemical ecology of Sirex: a contact sex pheromone and host volatiles of pine species that may attract wood wasps. Previous observations had suggested that many antennal receptors were contact chemoreceptors. Moreover, contact of these receptors with the body of a dead, pinned Sirex female elicited mating behavior in males. Washing of the pinned females with hexane halted the copulatory behavior. Bӧrӧczky undertook a series of experiments to verify the presence of such a pheromone and then identified it. She discovered that it consisted of three components and developed a synthetic version of the natural product. When she tested it on male Sirex, she found that it also elicited copulatory behavior.
Stressed plants are known to emit volatiles and such substances often attract herbivores to a vulnerable host. Bӧrӧczky investigated the effects of girdling pine trees on their production of volatiles and their attractiveness to Sirex. She girdled two chemotypes of Scots pine as well as white pine with an herbicide and monitored the treated trees for the following eleven weeks. 6
Observations included weekly measurements of volatile emissions and counts of trapped Sirex females. In general herbicide treated trees captured more Sirex than the untreated controlled. The Scots pine with the high-carene chemotype attracted the most females to traps, followed by the low-carene chemotype and then the white pine. In all cases, trees that were girdled emitted more volatiles than controls, and the Scots pine chemotypes induced higher emissions than did the white pine. Bӧrӧczky observed qualitative differences in volatiles among the tree types, which may be a factor in their differences in attractiveness to Sirex. Moving to the southeastern United States, Bӧrӧczky is continuing her work on host volatiles, currently comparing loblolly pine, shortleaf pine, longleaf pine, and Virginia pine using the Gas Chromatograph – Electroantennogram Device (GC-EAD) technique.
Miriam Cooperband discovered a sex pheromone that is produced by male Sirex and apparently causes males to aggregate. It is produced only by males older than two days – not by younger males or females – and probably serves to promote male lekking behavior, which is instrumental in the mating process. Cooperband made the discovery using a variety of techniques, including aerations, GC-Mass Spectrometer (MS), GC-EAD, and Y-tube olfactometer and wind tunnel bioassays. The three-part pheromone consisted of one major component and two minor ones. It was found to be attractive to both males and females in wind tunnel bioassays. Field tests of the pheromone were conducted in central New York State and South Africa to evaluate pheromone concentrations, trap design, and trap height above the ground. Unfortunately, results for the second year of this study suffered from an unforeseen problem: the presence of a contaminant that caused the major component of the pheromone to be non-attractive. Cooperband spent much time elucidating the cause of the problem and trying to obtain pure material. Results in the New York sites also were limited by very sparse Sirex populations. The pheromone is currently being evaluated in South Africa for use in mating disruption.
II. SURVEY AND TRAPPING
Chemical lures for Sirex may include pheromones and host kairomones. No long distance pheromones have yet been identified for the species but contact pheromones are a real possibility. Damon Crook has suggested this based on electron microscope investigations of antennal morphology. A study of characteristic mating behavior by males to dead pinned female wasps (see above) also provides evidence for a contact pheromone. Crook, along with cooperators from Pennsylvania State University, also investigated attractiveness of pine volatiles to Sirex. They compared healthy trees with those that had been girdled with herbicides. Overall, the girdled trees were much more attractive than were the healthy ones. Moreover, Scots pine was found to be more attractive than red pine, and red pine, more attractive than white pine in studies during 2006. In later studies, Scots pine was found to be much more attractive than white pine. Back in the lab, studies with a GC-EAD showed several promising volatiles (“peaks”), but 7 they have proved to be hard to identify. Even if they can be identified, it is generally almost impossible to find them in quantity for testing. Beyond the question of lures is the question of the traps that catch attracted wood wasps. Unfortunately, no trap designs to date have been very effective at catching wasps. Crook also looked at the possible attraction of traps of different colors. As a basis for this he used an Electro-Retinogram System, which found varying sensitivities to different wavelengths. However, trap catches were low across all color treatments in the field. Crook suggests that the height of the tree in the canopy may be a factor in attractiveness.
Kelley Zylstra carried out a number of trapping studies in 2006-2007, the most intensive being the “Age of Girdle” study. Trees were girdled with an herbicide on three dates in 2006: May 17, May 31, and June 12. Later, they were felled, bucked, and split, and Sirex were removed from the billets. Comparing three pine species, she found the most insects in white pine from the early date, the most in Scots pine from the middle date, and the most in red pine from the early and middle date. Investigating the height distribution of Sirex among the species, she found the highest numbers of wood wasps in red and Scots pines toward the base of the bole (most at 3-6 m) and the highest in white pine higher up, 9-12 m. In another 2006 study of lures, she found that a trap tree caught significantly more Sirex than any synthetic lures. In a study in the same year in South Africa, β-pinene was found to be significantly more attractive when applied in December (= June in the Northern Hemisphere) than in January (= July).
Zylstra carried out the Age of Girdle study again in 2007. This time the girdling was done in April, June, and July, and a control was left ungirdled. In both Scots and red pines, the June treatment yielded significantly more trap catches than those at other dates. On the other hand, more Sirex emerged from billet samples when trees were girdled in July, which corresponds to the typical wood wasp flight period.
Zylstra undertook height and lure studies in 2009 and 2010. Heights for trap placement were just below the live crown, mid-bole, and about 3 m from the ground. In 2009, more Sirex were trapped below the live crown than at mid-bole, whereas in 2010, more were trapped at the two higher levels than at 3 m.
During 2011 and 2012, Zylstra investigated ways to improve the catch of the standard funnel trap, which is made of black plastic, by developing a trap of clear material. Her hypothesis was that flying wood wasps would avoid opaque traps whereas they would not readily perceive clear traps and hence might be more likely to fly into them and be caught. She compared catches of black versus clear traps in a laboratory setup and in a field experiment. In both tests, the clear traps consistently caught more Sirex, but the differences were not statistically significant.
In 2011, Zylstra revisited six plots that had been sampled in 2007 and remained undisturbed since then. She found very little if any change in the number of adults trapped, which was quite low in both years. Finally, in the same year, she carried out a small survey for Sirex using 8 girdled trap trees in three areas thought to be on the edge of the infestation, including sites in Connecticut, New Jersey, and Pennsylvania. On rearing billet samples from the trap trees, they found siricid adults emerging in only one site ― in northern New Jersey―but they were not identified positively as S. noctilio.
III. BIOLOGICAL AND NATURAL CONTROL
David Williams was charged with evaluating and implementing nematode biological control using the Australian model. Other than simply importing and releasing the Kamona strain of Beddingia siricidicola, this task involved adapting the Australian nematode and program to the very different set of climatological and ecological conditions in the U.S. Such adaptation required several years of field and laboratory experiments to quantify the infection and sterilization rates of the Kamona strain.
The basic field experiment was a “controlled release”, which started with felling Sirex infested pines in the fall and inoculating them with either the nematode in a gel carrier or simply the gel, as a control. After exposure to winter conditions, the trees were sampled in the spring by cutting three billets from the bole and held in screened barrels for adult emergence. Any remaining woody material was chipped to prevent nematodes from escaping into the environment. The rationale to control releases was concern that the highly infective Kamona strain might also infect non-target North American native siricid species. The releases were used to investigate several questions relevant to the USDA program, such as optimal release timing, best use of trap trees, effects of different pine species on the infection rate and, conversely, effects of Sirex on different host species, effects of different fungal strains, and levels of natural control by parasitoids. To address just the last question, Ibalia leucospoides generally exhibited the highest parasitism rates of the parasitoids by far – as high as 20-25% on average – but the rates appeared to be independent of Sirex population density and unlikely to control it alone. The big “surprise” was the discovery in 2007 of another strain of B. siricidicola in New York and Ontario, one which apparently invaded with S. noctilio. The discovery raised a number of questions relative to the biological control program: What is the infection rate of the “native” strain? Does it sterilize the Sirex egg, as does the Kamona strain? Will it mate with Kamonas and possibly produce inferior hybrids? The controlled releases have attempted to answer these questions.
Williams also carried out many experiments in the laboratory, mainly with the goal of optimizing the mass rearing process. Factors affecting the process included temperature, initial inoculum size, length of the growth period, and fungus strain. Much of the data set on mass rearing awaits a rigorous analysis. As an example of the data, three shipment strains of Kamona were grown on the Otis isolate of A. areolatum for 10-12 weeks and for the most part produced a maximum of over 1 million nematodes per flask at 16˚ C. Other lab studies included investigating the survival 9 over time of nematodes in the “breathable baggies” used to carry them to the field and the attempt to inoculate infested billets with nematodes in the lab (which was not successful).
Ann Hajek undertook two projects related to Sirex biological control and funded by Cooperative Agreements with CPHST: (1) Amylostereum, Beddingia (Deladenus) siricidicola and Sirex noctilio in North America and (2) Fungal symbionts and parasitic nematodes associated with Sirex species in North America and Europe.
The symbiotic fungus, A. areolatum, is important as a food for both Sirex and Beddingia , so characterizing its various strains and their growth is important to implementing biological control. Thus, Hajek’s first project focused on the fungus in large part and stressed the collection of fungal isolates in and around New York State. Using molecular techniques, Hajek has identified three genotypic strains of A. areolatum. Because rapid growth of nematodes is thought to be desirable in their production as biocontrol agents, her students carried out a large study to evaluate growth rates of fungus on several types of agar and, in turn, of nematodes. In another experiment, she investigated the possible effect of pine species on nematode growth by adding ground wood of red pine, white pine, and Scots pine to the agar medium. Adding a relatively small amount of wood increased the production of eggs, juveniles and adults in most cases.
Hajek carried out field studies to document the associations of S. noctilio and the North American Sirex species with the fungal species and strains. Concurrently, she surveyed populations of native siricids for nematode infection and found some nematodes, but none positively identified as Beddingia. Her laboratory also dissected non-target species (primarily beetles) emerging from the controlled release studies (see above) and checked them for nematode infection.
Because S. noctilio, A. areolatum, and B. siricidicola all originated in Europe, Hajek’s second project focused on their distribution and association there. In addition, she carried out small surveys in Maine during 2010 to 2012 with the help of collaborators to find Sirex nitidus and S. nigicornis, native species that she discovered could carry A. areolatum. A handful of specimens carrying the fungus were trapped, but unfortunately, none contained nematodes. The European work was essentially a survey, which required identifying foreign collaborators. Hajek had a very productive trip in the 2010 season, visiting collaborators and making collections of many fungal isolates and moderate numbers of nematodes in Hungary, Denmark, and Spain. She was granted an extension of her cooperative agreement in 2011 to continue surveys in Belgium, Spain, and Italy but had a less successful season in terms of finding siricids. Unfortunately, although she had funds remaining, she was not able to extend the CA a second time and the work was discontinued in 2012.
Zylstra conducted a survey for the “native strain” of B. siricidicola during the years 2006 through 2012. The presence of nematodes was determined by dissection of Sirex adults 10 emerging from billet samples and caught on traps. Annual average infection rates (that is, percentages of the total sample that contained nematodes) ranged from 7.7% to 33.6% over the seven years of sampling. In addition, Zylstra examined the Sirex eggs for sterilization, which is assessed by the presence of nematodes inside eggs. She reported that the sterilization rates (that is, the percentage of infected Sirex adults that had eggs containing nematodes) ranged from 6.6% to 45.5%. Zylstra also dissected native siricids caught in survey traps from 2007 to 2012. The species included Urocerus spp., Tremex columba, Sirex nigricornis, and S. edwardsii. In general, the wood wasps were rare and those infected by nematodes even rarer. Sirex species, which were of greatest interest, were only found to be infected by nematodes in one of the six survey years and the average infection rate for the trapped S. nigricornis was just 13.5%.
Zylstra also undertook a geographical analysis of nematode populations. Hypothesizing that the spatial distribution of nematode occurrence might yield some information on the point of origin of the wood wasp-nematode invasion, Zylstra mapped her annual study sites from 2007 to 2011, indicating whether nematodes were present or absent. Unfortunately, the two categories of sites appeared to be randomly scattered, without any obvious spatial discontinuities or trends.
IV. COMMUNITY ECOLOGY
Matthew Ayres carried out a project in cooperation with Alan Sawyer (retired) of the CPHST Otis Lab to investigate various aspects of Sirex ecology. The executive summary that follows was written by Ayres. Note that the chapters mentioned in parenthesis are a series of eight manuscripts devolving from work by himself and his students and associates. They are contained in his full compilation section.
“Overall goals of the work to be undertaken by Dartmouth College were to (1) provide an improved basis for judging how long S. noctilio has been in a region based on examination of dead trees; (2) evaluate patterns of reproductive success of S. noctilio; and (3) identify and characterize ecological factors that will influence whether S. noctilio becomes a notable forest pest in North America.
Detection. We developed and evaluated a protocol for detecting S. noctilio by genetic identification of A. areolatum in logs after departure of Sirex (see Chapter 5). This was possible via culturing of fungi followed by PCR and sequencing of DNA barcode regions. Unfortunately, the technique proved to have very low efficiency because the fungus has limited persistence within trees or logs following departure of the wood wasps. Also, it now seems that A. areolatum can be associated with native Sirex, which reduces the certainty of diagnoses based on the fungal DNA. However, we have developed an alternative protocol that is cheap, easily transferable, and reasonably efficient, based on the size and patterning of persistent emergence holes and resin drips within and among stands in a region (Chapters 3-4). Because the host trees are generally 11 within even-aged stands, counting annual rings within live and dead trees in a stand permits inferences regarding the duration of occupancy by relatively high numbers of wood wasps.
Population ecology and tendencies in population dynamics. Key factors in the population ecology of S. noctilio include: (1) variable fecundity, (2) skewed sex ratios, (3) host suitability, and (4) community interactions with competitors and antagonists (Chapters 1, 2, 6, 8). Fecundity and sex ratio are highly variable but unrelated to local abundance or host suitability. Therefore, there is no destabilizing positive feedback in population dynamics from these sources (and lower risk of outbreaks than with positive feedback). (Chapter 1). Parasitism rates by Ibalia can be very high (good for limiting outbreak risk) but were negatively related to local abundance, implying predator swamping, and a potential for outbreaks from predator release if populations become moderately abundant for any reason. (Chapter 1). Red pine appears to be of comparable nutritional suitability for S. noctilio but produce fewer Sirex progeny, perhaps because they are less attractive to ovipositing adults (Chapter 1).
Amylostereum areolatum is crucial to larval development, especially for digestion of cellulose. Based on the nutritional stoichiometry of S. noctilio, we hypothesize that the community of symbionts also includes some N-fixing bacteria which are crucial for meeting the protein requirements of growing larvae and which are supported by microbially aided digestion of cellulose (Chapter 7).
Native Ophiostomatoid (“bluestain”) fungi, propagated by native bark beetles, appear to be antagonistic to Sirex noctilio larvae (probably during the first 1-2 instars when mutualistic fungus needs to become established near cambium) (Chapter 1). A. areolatum has very slow growth and competes poorly with native Ophiostomatoid fungi (Chapter 2). Thus, native bark beetles and their phoretic mites, which propagate bluestain fungi are likely to provide resistance in American forests to populations of S. noctilio.
Ecology and population controls in native system. To aid in projecting the potential impacts of S. noctilio in North America, we compared patterns of host tree attack in the recently invaded Finger Lakes region of New York State, with those in its native forests of northwestern Spain (Chapter 6). In the forests of both Spain and New York, S. noctilio were largely restricted to suppressed trees (smaller than average diameter) of the same size class that were also dying for other reasons. In both areas, Scots pine (native to Europe) was the species most likely to have attacks in non-suppressed trees and neither population appeared to be limited by suitable host trees. At least so far in the northeastern United States, the ecology of S. noctilio seems to be more like the situation in Europe (limited pestilence) than in the Southern Hemisphere (high pestilence).”
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In 2009, Kamal Gandhi set up sampling plots in three southeastern states ̶ Georgia, Louisiana, and Virginia ̶ with the dual objectives of surveying the native siricid species and their parasitoids and evaluating several types of traps and lures for attracting wood wasps . She used primarily panel traps, 30 in each state, which were unbaited, baited with α- and β-pinenes, or baited with Sirex lure. She also used identically baited Lindgren traps and felled trees of several pine species as trap trees. In all, she trapped just 79 siricids in five species and no parasitoids. In 2010, she installed Lindgren and panel traps with various baits near wood piles in sawmills in Georgia. She caught just 52 native siricids, all S. nigricornis or S. edwardsii. In Louisiana that year, one of the baits was a bag with pine chips and foliage. Interestingly, it caught more wood wasps than the other baits. As in the previous year, no parasitoids were caught. The felled trap trees from 2009 ultimately produced many more wood wasps and parasitoids than did the traps. Gandhi concluded that trap trees cut, bucked, and stacked provided the greatest numbers of emerging Sirex and parasitoids.
V. MODELING
Scott Myers modeled the phenology of Sirex flight based on physiological time measured in degree-days above 6.8˚ C. He based his model on three years of data collected from laboratory rearing of pine billets infested in the lab, laboratory rearing of billets from girdled trees that were infested in the field, and trap catches of Sirex emerging naturally in the field. Degree-day models were developed using linear regression of the data after probit transformation. Model predictions gave a consistent fit to the data from both lab rearing and trap catches, predicting first flight and peak catch accurately. The completed model was used in the NAPPFAST system to develop raster maps for the eastern U.S. depicting geographical ranges of first flight and peak flight.
Kamal Gandhi modeled risk from Sirex in the southeastern U.S. as related to choice of various common pine species for oviposition and activity and its emergence from them. Choice experiments were carried out in 2009 by caging Sirex adults with free access to billets of loblolly, Virginia, and Scots pines and no-choice by caging the pine billets separately. Sirex exhibited clear preferences for the pine species in the choice criteria. They were four times more active on Virginia pine than Scots pine and showed negligible activity on loblolly pine. Similarly, Virginia pine had ten times more exit holes than Scots pine, whereas loblolly pine had no emergence. The experiments in 2010 were carried out using the same methodology but compared more pine species: white, shortleaf, longleaf, slash, as well as Virginia, Scots, and loblolly. Of the pines in the southeastern region, white and Virginia were most highly preferred. Gandhi also analyzed pine samples for their chemical and physical properties. Species more attractive to Sirex generally had lower specific gravity. Resin samples from white pine differed more than the other species in monoterpene content. Finally, a Sirex risk map was developed that included (among other tree map layers) the results of the choice experiments by the 13 following weightings: white (highest), followed by Scots, Virginia, longleaf, loblolly, and shortleaf, and slash (lowest).
Clearly, the results of the modeling efforts by Myers and Gandhi may be used together to evaluate the spatio-temporal aspects of the risk posed by Sirex in southeastern pine stands.
VI. MOLECULAR GENETICS
With the discovery of the North American “native” strain of B. siricidicola, the biological control program using the conspecific Kamona strain faced uncertainty. Because the strains could not be discriminated morphologically, possible differences in efficacy in the infection process and in sterilizing the host could not be evaluated. Evan Braswell, a population geneticist at the CPHST Mission Laboratory, was asked to investigate the two strains genetically and determine whether they could be identified by molecular means. Samples of the Kamona lab strain and numerous populations from New York State were analyzed. DNA extraction from individual nematodes proved to be a challenge because of their minute size and tough cuticles. Sequencing the COI gene of the mitochondrial DNA and comparing the sequences, Braswell found that the nematode strains could be discriminated reliably. Specifically, two sites within his sequences differed. Having determined that the strains could be discriminated, he investigated tools to identify their possible hybrids. He used the technique of Randomly Amplified Polymorphic DNAs (RAPDs), which employ nuclear DNA. Braswell found characteristic electrophoretic bands in these analyses that distinguished the strains definitively. However, he did not have hybrid populations to work with at the time to compare with the pure strains.
Ann Hajek was indispensible in helping to finish the nematode project. She and her students spent many hours in the lab identifying the strains of Deladenus siricidicola found in our controlled release studies in New York and Pennsylvania using molecular techniques and associating the strains with the physiological traits of infectivity and sterilization.
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List of peer-reviewed publications produced by the Sirex noctilio program
K. Böröczky, D. J. Crook, T. H. Jones, J. C. Kenny, K. E. Zylstra, V. C. Mastro, and J. H. Tumlinson. 2009. Monoalkenes as contact sex pheromone components of the wood wasp Sirex noctilio. J. Chemical. Ecology 35: 1202-1211
K., Böröczky, K. E. Zylstra, N. B. McCartney, V. C Mastro, and J. H. Tumlinson. 2012. Volatile profile differences and the associated Sirex noctilio activity in two host tree species in the Northeastern United States. J. Chemical Ecology 38: 213-221.
M. F. Cooperband, K. Böröczky, A. Hartness, T. H. Jones, K. E. Zylstra, J. H. Tumlinson, and V. C. Mastro. 2012. Male-produced pheromone in the European wood wasp, Sirex noctilio. J. Chemical Ecology 38: 52-62.
D. J. Crook, K. Böröczky, K. E. Zylstra, V. C. Mastro, and J. H. Tumlinson. 2012. The chemical ecology of Sirex noctilio. pp. 149-158. In B. Slippers et al. (eds.), The Sirex wood wasp and its fungal symbiont: Research and management of a worldwide invasive pest. Springer-Verlag, New York.
D. J. Crook, L. M. Kerr, and V. C. Mastro. 2008. Sensilla on the antennal flagellum of Sirex noctilio. Annals Entomological Society America 101: 1094-1102.
J. E. Dinkins, J. J., Riggins, K. E. Zylstra, V. C. Mastro, and K. J. K. Gandhi. 2013. Behavioral and colonization preferences of the European wood wasp, Sirex noctilio F. (Hymenoptera: Siricidae) for two southeastern pine (Pinus spp.) species. J. Insect Behavior. (in review).
K. J. Dodds, K. E., Zylstra, G. D., Dubois, and E. R. Hoebeke. 2013. Saproxylic insects associated with herbicide-stressed Pinus resinosa and Pinus sylvestris used as Sirex noctilio trap trees in New York. Environmental Entomology (in press).
A.E. Hajek, C. Nielsen, R.M. Keppler, S.J. Long, L. Castrillo. 2013. Fidelity among Sirex woodwasps and their fungal symbionts. Invertebrate Microbiology 65:753-762.
S. J. Long, D. W. Williams, and A. E. Hajek. 2009. Sirex species and their parasitoids in Pinus sylvestris in eastern North America. Canadian Entomologist 141: 153-157.
E. E. Morris, A. Jimenez, S. J. Long, D. W. Williams, and A. E. Hajek. 2012. Variability in growth of Deladenus siricidicola on strains of the white rot fungus Amylostereum areolatum. BioControl 57: 677-686.
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E. E. Morris, R.M. Kepler, S.J. Long, D.W. Williams, and A. E. Hajek. 2013. Phylogenetic analysis of Deladenus nematodes parasitizing northeastern North American Sirex species. J. Invertebrate Pathology 113: 177-183.
E. E. Morris, A.E. Hajek, E. Zieman, and D. Williams. 2014. Ability of Deladenus siricidicola and Deladenus proximus to feed and reproduce on species and strains of the white rot fungus Amylostereum. Biological Control (in press)
S. W. Myers, K. E. Zylstra, J. A. Francese, D. M. Borchert, and Sian Bailey. 2013. Phenology and flight periodicity of Sirex noctilio in central New York, U.S.A. Agricultural Forest Entomology DOI:10.1111/affe. 12042
C. Nielsen, D. W. Williams, and A. E. Hajek. 2009. Putative source of the invasive Sirex noctilio fungal symbiont, Amylostereum areolatum, in the eastern United States and its association with native siricid wood wasps. Mycological Research 113: 1242-1253.
D. W. Williams, K. E. Zylstra, and V. C. Mastro. 2012. Ecological considerations in using Deladenus (= Beddingia) siricidicola for the biological control of Sirex noctilio in North America, pp. 135-148. In B. Slippers et al. (eds.), The Sirex wood wasp and its fungal symbiont: Research and management of a worldwide invasive pest. Springer-Verlag, New York.
K. E. Zylstra, K. J. Dodds, J. A. Francese, and V. C. Mastro. 2010. Sirex noctilio in North America: the effect of stem-injection timing on the attractiveness and suitability of trap trees. Agricultural Forest Entomology 12: 243–250.
K. E. Zylstra and V. C. Mastro. 2012. Common mortality factors of wood wasp larvae in three northeastern United States host species. J. Insect Science 12 (83).
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I. CHEMICAL ECOLOGY
Monoalkenes as contact sex pheromone components of the woodwasp Sirex noctilio Katalin Bӧrӧczky For full text of publication see Appendix 1.
Abstract A pheromone on the cuticle of females of the woodwasp Sirex noctilio, a recently introduced pest of pines in North America, induces conspecific males to attempt copulation. Dead females washed with hexane did not elicit copulation attempts from males, whereas reappli- cation of a female hexane body wash onto the cuticle of dead females elicited copulation attempts by 65% of males tested. Analysis of the hexane extract revealed saturated and unsaturated hydrocarbons as major components of the female cuticle. Behavior-guided fractionation of the female body wash led to the identification of three components, (Z)-7-heptacosene, (Z)-7-nonacosene, and (Z)-9-nonacosene, of the sex pheromone of S. noctilio that elicited copulatory responses from males.
Volatile profile differences and the associated Sirex noctilio activity in two host tree species in the northeastern United States Katalin Bӧrӧczky For full text of publication see Appendix 1.
Abstract Sirex noctilio females are known to be attracted to stem sections of stressed pine trees for oviposition. The volatile profiles and attractiveness of Eastern white pine and two chemotypes of Scots pine, after stem injection with herbicide, were compared. In general, trap captures on herbicide-treated trees were higher than on controls. The high-carene chemotype of Scots pine captured the highest numbers of females, followed by the low-carene chemotype, and finally the Eastern white pine. Herbicide-treated trees of both species emitted larger quantities of volatiles than the controls. The herbicide treatment induced higher volatile emission rates in the Scots pine chemotypes than in white pine, though there was no difference between the two chemotypes. However, qualitative differences were found between the volatile profiles of the two species as well as between the two Scots pine chemotypes, which could account for the differential attractiveness of the species and chemotypes tested. 17
Host-produced kairomones from southern pine species for detection, monitoring and control of Sirex noctilio
K. Böröczky
Unpublished report 2012 (with F. Hain and C. Schal)
Objectives
The goal of this study was to screen volatile collection and resin samples from four southern pine species available in North Carolina, Pinus taeda (loblolly pine), P. echinata (shortleaf pine), P. palustris (longleaf pine), and P. virginiana (Virginia pine), with the gas chromatogram- electroantennogram (GC-EAD) technique using female Sirex antenna as a detector. Electrophysiologically active compounds identified with this method may be important components of an improved lure for field trapping and monitoring of the woodwasp.
Progress to date
Infested logs were received from the USDA APHIS rearing facility in Syracuse, NY and transported to the NCDA quarantine facility in Cary, NC in May 2011.
To have staggered emergence, half of the logs were placed in barrels and kept at room temperature in a small quarantine room, while the rest were kept at cooler temperatures (12– 15°C) in an incubator within the quarantine area.
Field volatile collection and resin collection took place at a site in Hill Forest on NC State University property.
Four trees of each species were selected and mechanically girdled on June 23 2011.
Volatiles were collected every week for 4 weeks starting on 6/23/11, and finally 8 weeks after girdling on 9/13/11. To collect volatiles we wrapped around a two-foot trunk section of the tree with Teflon sheet to form a chamber. A portable two-pump system was used to push charcoal- filtered ambient air into the chamber and pull the air out through Super-Q and charcoal volatile collection filters. Compounds were eluted from the filters in the laboratory with hexane and dichloromethane, respectively.
Resin was collected on 6/23 and on 7/12. For resin collection two small holes were punched through the bark on opposite sides of each tree without damaging the cambium. Resin samples were stored at -30°C. 18
Pine samples were screened for electrophysiologically active compounds with an Agilent 5890 Series II GC connected to a custom-made EAD. Due to limited number of emerging Sirex females only the Super-Q volatile collection samples were analyzed with GC-EAD.
For chemical identification of compounds, samples were analyzed in an Agilent 6890N GC coupled with an Agilent 5975 inert XL mass spectrometer. Electrophysiologically active compounds were identified based on their Kovats indexes and their mass spectra.
The volatile profiles of the investigated pine species were very similar with α- and β-pinene being the major components of all the volatile blends. Other monoterpene hydrocarbons, such as camphene, sabinene, -3-carene, and limonene occurred in lower concentrations. Samples also contained oxygenated monoterpenes, though only in trace amounts.
Each pine species was tested with both antennae of at least 4 Sirex females. In general, the major pine components elicited moderate electrophysiological responses, whereas some of the minor components consistently elicited a large response.
Future perspectives
We obtained a new batch of infested logs in December 2011.
We will screen resin samples using S. noctilio females that will emerge from the new batch of infested logs. Since these samples contain more material it will be possible to identify electrophysiologially active minor components.
Evaluation of the electrophysiologically-active compounds in a behavioral assay (under laboratory and/or field conditions) is necessary to determine whether they can be used as lure components. Arrangements will be made with Dr. Tumlinson for summer 2012 field assays.
19
Behavior and sensory ecology of Sirex noctilio evidence of a male aggregation pheromone
Miriam Cooperband
Abstract A male aggregation pheromone was identified for the European woodwasp, Sirex noctilio. Males displayed excitatory behaviors when placed in groups, and were attracted to the odors from other males that were 2 or more days old, but not to males that were less than 2 d old. Aerations collected from the head space of males and females of different ages and numbers found that only males more than 2 d old produced large amounts of the major pheromone component which was identified as (Z)-3-Decen-1-ol. Females did not produce this compound, nor did younger males. Using GC-EAD, male and female antennae exposed to this compound produced strong electrophysiological responses. Male antennae also produced strong responses to a minor component in male aerations. A tentative identification of the minor component has been made and still needs to be tested electrophysiologically. Three types of lures with the major component have been evaluated for release rates and a large field trapping study is underway in pine plantations in South Africa to test lures, loaded with different concentrations of the major and suspected minor components, for attraction.
Insects. In the previous year, behavioral observations aided in determining specifications of a behavioral bioassay in which attraction to semiochemicals could be evaluated. In August 2009, custom aeration and Y-tube 50 bioassay equipment were males 40 designed and ordered for use with females Sirex noctilio and they arrived in 30 emerged
December. Meanwhile, in the 20 fall of 2009 infested trees were 10 felled, cut, and placed into barrels Number which later were brought in from 0 567891011121314151617181920212223 the cold in staggered groups in Week order to stagger emergence over a longer period of time. Barrels of 1 logs were gradually brought total)
0.75 indoors from November 2009 to 0.5
March 2010. The cumulative sex (male/
ratio (males/total) of all emerging sex ratio
ratio 0.25 moving average (3 wks) wasps was 0.73. The emergence pattern from staggered log barrels Sex 0 in the quarantine at Otis, and the 5 6 7 8 9 1011121314151617181920212223
Figure 1: Number of male and female Sirex noctilio that emerged 20
sex ratio of emerging wasps over time, are shown in Fig. 1. Wasps were only viable for use in experiments for 7 or 8 days. The window of opportunity in which 10 or more males were available per week to study was limited to roughly a period of 10 weeks, and even less for females. These wasps were needed for volatile collections, behavioral bioassays, and electroantennograms. Therefore, the small number of wasps available greatly limited the number of replicates that were possible to achieve. Generally, older wasps were used for electroantennograms after they were first used in either volatile Choices by Males Offered collections or behavioral Odors From Males of Different Ages bioassays. N Control Male Age 33 Blank 11 11 0‐1 d old Volatile collections and Figure 2: Air delivery system with a volatile bioassays. The air delivery system, volatile collection 31 Blank 5 16 2‐5 d old chambers, and the bioassay * apparatus were operational ‐100% ‐75% ‐50% ‐25% 0% 25% 50% 75% 100% starting in January 2010 (Fig. 2). To test for Figure 3: Sirex noctilio males showed no preference for odors from 0-1 attraction to odors of adult day-old males, but they were attracted to odors from S. noctilio males that were 2-5 days old (Chi Square Test). wasps, males or females were placed in one of the two 4-L volatile collection chambers. Filtered, humidified air was passed through each chamber from the air delivery system at 0.2 liters per min. From each of the two chambers, the air entered each of the two arms of the Y-tube olfactometer. A male or female wasp was placed in the downwind end of the Y-tube olfactometer and observed for 3 min or until it entered one of the upwind arms, whichever came first. Males and females were both tested for attraction to odors from males and females. The age was recorded for every individual used in both odor production and attraction testing. Preliminary dual choice bioassays were conducted in
30 21 (Z)‐3‐Decen‐1‐ol 20 Not Detected
10 9 Aerations 10 55 2 22 000 00001 No. 0 single multiple single multiple single multiple single multiple <2 d old 2+ d old <2 d old 2+ d old males females
Figure 4: Headspace samples were collected from individuals and groups of each sex, and of different ages, and analyzed using GC-MS. The compound (Z)-3-Decen-1-ol was found in abundance in the headspace of males that were 2 or more days old, but was not found in headspace samples of 0-1 day-old males. It was not found in the headspace of any females sampled. 21 the Y-tube olfactometer to test for attraction of males and females to the natural emanations from males or females in order to establish “who attracts whom”. Males were found to be attracted to the natural emanations of other males, evidence of a male aggregation pheromone. Further testing revealed that males were attracted to odors specifically from males that were 2 or more days old (Fig. 3).
Volatile collections were made of individuals and groups of adult male and female Sirex noctilio of different ages. Headspace volatiles of males were collected using SPME fibers, charcoal, and Porapak filters, and were extracted and analyzed using GC mass spectrometry (MS). Males, but not females, were found to emit a large amount of a compound identified, and later verified, as (Z)-3-Decen-1-ol (Fig. 4). It was later found that the Tumlinson laboratory had also recently isolated and identified this compound from male S. noctilio.
Electrophysiology and active compounds. A gas chromatograph-coupled electroantennogram (GC-EAD) Figure 5: Male antennae produced two strong depolarizations, one in system was set up in the response to the major component in male aerations, and one to a quarantine and was operational by minor component that occurs in trace amounts. February 2010. When testing naturally emitted odors for antennal activity using GC-EAD, both male and female antennae produced large antennal responses to this compound. Male antennae also were found to depolarize strongly in response to a minute peak following the major peak, suggesting the presence of a possible minor pheromone component (Fig 5).
Collaborator and chemist, Dr. Tappey Jones (Virginia Military Figure 6: The active compound was synthesized and tested for Institute), synthesized (Z)-3- antennal activity in both males and females. Both male (left) and Decen-1-ol, and the retention female (right) antennae produced strong EAD responses to synthetic times of the natural and synthetic (Z)-3-Decen-1-ol. compound were compared. The synthetic compound was also tested for responses by male and female antennae using GC-EAD. The synthetic version of (Z)-3-Decen-1-ol eluted at the same retention time as the naturally male-produced compound, and also produced strong antennal responses from both male and 22
female antennae, further helping to confirm the chemical identity of the major component (Fig. 6).
Tests for attraction were conducted in the Y-tube olfactometer at two different concentrations, and the lower concentration of 20 ng of (Z)-3-decen-1-ol on filter paper appeared to produce some attraction of males, but by this time in the 2010 season there were dwindling numbers of adults available for testing, and more replicates are needed to confirm these results.
GC-MS work continued July through September 2010 with the assistance of ARS research chemist Dr. Ashot Khrimian in order to identify the minor pheromone component that elutes immediately after the major component and elicits strong antennal responses from males. Dr. Khrimian helped to identify a compound occurring in trace amounts from extracts of male headspace odors collected previously on Porapak. The retention time of the synthetic version of that compound matched that of the suspected minor compound. Further verification of this compound will be conducted with GC-EAD when adult males become available.
Lure development. Lure release rates of (Z)-3- Decen-1-ol were tested in preparation for field testing from June to September 2010. Release rates from polyethylene vials of two different designs (wide and narrow), as well as gray rubber septa, were evaluated in the laboratory as well as in an outdoor greenhouse (Fig. 7). The Figure 7: Three types of lures tested. two types of polyethylene vials were loaded with 3 mg of (Z)-3-Decen-1-ol, placed in a laboratory fume hood, and weighed three times per day. Another set was placed in the greenhouse and weighed three times per week. Gray rubber septa were loaded with different doses of the major component and placed in the greenhouse. Each week for six weeks, a set of rubber septa were sealed in foil pouches and frozen. At the end of the study, all the rubber septa were extracted in hexane and a sample of each extract was evaluated using the GC. The amount of compound remaining in each rubber septum was quantified using peak area and standards, and average release rates were calculated. 23
The two types of polyethylene vials had similar average release rates between 25 and 85 ng/min, with maximum rates of 360 and 120 ng/min in the greenhouse and fume hood, respectively. The majority of the compound was lost after four weeks in the greenhouse. Gray rubber septa loaded with 3 mg behaved similarly. Gray rubber septa were loaded at three orders of magnitude and compared. Release rates after 5 weeks were similar to release rates in the first week of the dose one order of magnitude lower (Fig. 8).
10000 Field testing. GC-EAD had n=1 1000 revealed antennal responses to 30 mg other minor peaks. One of the 100 3 mg 0.3 mg suspected additional 10 components was tentatively 1
ng/min per ng/min per septum identified as chrysanthenone, 01234567 and another dodecane, but these have not been verified. A 500 n=3 400 literature search found studies 3 mg 300 identifying plants for 1 mg 200 0.3 mg which essential oils naturally 100 contain (Z)-3-Decen-1-ol and 0 ng/min per ng/min per septum chrysanthenone. Buchu 01234567 betulina has been previously 100 100 found to contain (Z)-3-Decen-1- 90 80 ol, and Achillea millefolium 80 60 70 (Yarrow) has been found to Hi temp 40 Temp (F) Temp 60 Lo temp contain 50 Hi RH 20 Lo RH chrysanthenone. Several 40 0 Humidity Relative essential oils of these plants 01234567 Week were purchased and tested in the Figure 8: Release rates of different doses of (Z)-3-Decen-1-ol from GC-MS to investigate the gray rubber septa in the greenhouse.
Table 1: Experimental design of a large field study currently underway in presence of these compounds. Two South Africa. Each treatment, marked with an X, is being repeated 30 times were selected, and in July 2010, a (2 locations, 3 blocks, and 5 replicates). pilot field study in Seneca Falls, NY was conducted in which sticky traps Major component dose Controls were hung at approximate heights of Kairomone Suspected minor lure component dose 30 mg 3 mg 0.3 mg 0 mg 2 m and 15 m in Scots pine trees
(Pinus sylvestris), baited with 0 X X X X narrow polyethylene microcentrifuge N tubes containing either (a) 3 mg of 5:100 X X X (Z)-3-Decen-1-ol (n=7), (b) 3 mg of 0 X X X X Y 5:100 X X X 24 a blend of 150:50:5 of B. betulina oil : Yarrow oil : dodecane (n=7), or (c) empty controls (n=4), however, no S. noctilio were caught in any of these traps.
Meanwhile, an international cooperative agreement was established with Dr. Brett Hurley (Univ. of Pretoria, South Africa) to conduct a large replicated field trapping study to test the antennally active major and suspected minor components in areas heavily infested with Sirex. This study is currently taking place. It is designed to test the major component alone and in a blend with the suspected minor component, at three different concentrations, in the presence and absence of a kairomone lure containing host plant odors, and with a positive and negative control (Table 1). The study is being conducted at pine plantations in South Africa, each divided into three blocks and replicated five times.
Future work in FY 2011-2012 will involve: a) felling infested trees in NY that were girdled prior to the Sirex flight in July, and staggering their emergence as in previous years, b) a continuation of volatile collections for further identification of minor compounds that produce antennal responses, c) verification of activity of suspected minor compounds using GC-EAD, d) continued behavioral testing in the Y-tube olfactometer to identify compounds and concentrations that produce attraction, e) examination of the effects of these odors on both males and females, and f) further field testing of new compounds and blends.
25
Behavior and sensory ecology of Sirex noctilio Discovery of a male-produced sex pheromone
Miriam Cooperband
Unpublished report of FY 2010-2011 activities
Abstract A 3-component male-produced sex pheromone was identified for the European woodwasp, Sirex noctilio, using a combination of techniques including aerations, GC-MS, GC- EAD, Y-tube olfactometer and wind tunnel behavioral bioassays. Females did not produce the main component, nor did males younger than 2 d old. The major component and two minor components were identified, and the ratio of the three compounds was determined to produce an attractive synthetic blend. The pheromone, consisting of a ratio of 100:1:1 parts (Z)-3-Decen-1- ol, (Z)-4-Decen-1-ol, and (E,E)-2,4-Decadienal, was found to be attractive to both males and females in a laboratory wind tunnel bioassay. Multiple Y-tube tests revealed that the addition of other suspected compounds reduced attraction, as did omission of the compounds in the blend, or blending the compounds at a different ratio. Prior to positive identification of the two minor components in the laboratory, a 2010 field trapping study in South Africa found that, although the main component in the absence of the correct minor components did not out-compete the kairomone lure, a significant dose-response to the major pheromone component alone existed. Since the discovery of the two minor pheromone components, a) a preliminary field trapping study was conducted near Syracuse, NY to test the pheromone at 3 concentrations against the kairomone lure, b) trap designs were evaluated in a large field wind tunnel in Ithaca, NY, and c) a large trapping study was initiated and is currently underway in Sirex-infested South African pine plantations to field test i) the new pheromone blend at two concentrations with and without the kairomone lure, ii) two trap designs, and iii) trap efficacy at two heights.
Introduction
At the beginning of the 20th century, Sirex noctilio was introduced into New Zealand, and became a serious pest of Pinus radiata plantations following a period of drought in the 1940’s (Coutts 1965). Since then, research has been conducted to understand its complex biology. Although research on the chemical ecology of S. noctilio has resulted in a better understanding of attractive host plant volatiles, few studies have sought to understand how they find their mates.
Aside from the use of girdled trap trees, lures containing host plant volatiles have been the next best tool available to date for detection and delineation trapping. However, those lures only target ovipositing females. In most infestations, females make up less than half of the population and are sometimes outnumbered by males by as much as 14:1 (Hurley et al. 2008), and sex ratios as high as 100:1 males:females have been reported from individual trees (Morgan and Stewart 1966). A lure and trap combination that targets males or both sexes could potentially tap into 26 this untargeted population and may provide a more sensitive tool for early detection and delineation purposes. It could also potentially remove insects from the population prior to the host-seeking stage and remove potential mates. If effective, it could potentially slow the spread of a newly founded population. This report describes work conducted in the last year and progress made in discovering the sex pheromone for S. noctilio, as well as progress towards improved trapping technology. Figure 1: Number of male and female Sirex noctilio that emerged weekly from staggered barrels, and weekly sex ratio Materials & Methods (males/total).
Insects. In the fall, girdled and infested males trees were felled, cut, and placed into 60 females barrels which were brought indoors in 45 emerged
five batches over five months in order to 30
stagger emergence over a long period of 15 time. Logs were gradually brought Number 0 indoors from November 2010 to March 5 10152025 2011. The total sex ratio (males/total) of Week 1 all emerging wasps in 2011 was 0.70. The 2011 emergence pattern from 0.75 ratio staggered log barrels in the quarantine at 0.5
Otis, and the sex ratio of emerging wasps Sex 0.25 sex ratio moving average (3 wks) over time, are shown in Fig. 1. Wasps 0 were only viable for use in experiments 5 10 15 20 25 for 7 or 8 days. To maximize the use of a limited number of wasps, young wasps were used in behavioral bioassays, males over 2 d old were used in aerations, and the oldest wasps were usually used in electrophysiology after they were first used in behavioral bioassays and/or aerations.
Volatiles and bioassays. The air delivery system, volatile collection chambers, Y-tube olfactometer, and wind tunnel bioassay are depicted in Fig. 2. Males, 2-7 d old, were aerated and odors were collected on SPME fibers, as well as charcoal and Porapak traps that were subsequently extracted in solvent.
Gas chromatography coupled with electroantennographic detection (GC-EAD) was conducted using four male antennae in parallel to search for any minor pheromone components. Male GC-EAD responses and spectra of male
Figure 2: Air delivery system with a volatile collection chamber (top), Y-tube olfactometer 27 volatiles were compared to spectra from the male volatiles using gas chromatography coupled with a mass spectrometer (GC-MS) using identical column configurations and temperature programs. Non-polar columns (HP-5) were initially used, and when it appeared that large peaks were covering potentially important smaller peaks, polar columns (Innowax) were installed in an attempt to improve peak separation.
Retention times, peak shapes, and Kovats indeces were used to Table 1: Supected minor match peaks producing antennal responses in the GC-EAD to components tested for male peaks in the GC-MS. The GC-MS was used to tentatively antennal responses (check boxes). identify matching peaks using the NIST database, which Those eliciting the strongest EAD responses (two checks) were tested sometimes gave several possible identifications for a given for attraction in the Y-tube. peak. Some peaks overlapped with others, reducing the confidence in their identification using NIST. Once tentative Compounds Response identifications of active compounds were made, those synthetic α‐Pinene compounds were purchased or synthesized and tested for GC- β‐Pinene (+)‐α‐Longipinene EAD antennal activity and retention times matching those of Nonanal the active peaks of male headspace odors (Table 1). Synthetic Verbenol minor compounds that were suspected to be in the male (1S)‐(‐)‐Verbenone headspace, and also produced strong EAD responses, were Myrtenol then tested for male attraction in the Y-tube olfactometer in (E)‐3‐Decenol (Z)‐5‐Decenol different blends, concentrations, and ratios (Table 2). (Z)‐3‐Decenol * Last year a behavioral bioassay was developed in which male (Z)‐4‐Decenol 9‐Decenol attraction could be tested in the Y-tube olfactometer. That (E,E)‐2,4‐Decadienal bioassay was used successfully to determine that males Nonanoic Acid produce a pheromone attractive to other males. That bioassay was integral in determining the attraction of males to the male-produced pheromone, and the main pheromone component, (Z)-3-decenol. However, females tested in the same Y-tube olfactometer appeared to have difficulty walking and flying, and lacked motivation to respond to natural male odors in the confined space. Consequently, experiments in 2011 focused initially on the preferences of males to synthetic odors using the Y-tube bioassay. Males were tested for attraction to various concentrations and blends of synthetic compounds in order to identify the minor components required for strong attraction. It was later necessary to develop a bioassay in which females also could be tested for attraction to odors in the laboratory. After a synthetic blend was determined to attract males, it then was used to develop a behavioral bioassay that could test females for attraction as well as males.
A laminar flow wind tunnel (120 cm long x 91 cm high x 75 cm wide) was constructed for use inside a walk-in environmental chamber in the quarantine facility at the Otis Laboratory. A female or male S. noctilio released at the downwind end of the wind tunnel could fly to the source of an odor placed somewhere upwind. Two targets, made of 5-cm diam. black disks (cut from black intercept trap panels), were suspended at the upwind end of the tunnel, each one with 28 a rubber septum attached above it. One septum was loaded with the test odor(s) in hexane, and the other with an equivalent amount of hexane alone (Fig. 2). The targets were alternated after every few replicates to avoid any directional bias in the bioassay.
A second, much larger, wind tunnel (360 cm long x 180 cm high x 180 cm high) was constructed for field or greenhouse use in areas where S. noctilio occurs. It consisted of a 1” PVC pipe frame, with laminizers on both upwind and downwind ends, and a semi-transparent plastic sheet attached by Velcro® to form the ceiling and walls. At the upwind end, an expansion chamber was constructed of the same plastic material which connected a fan to the upwind laminizer.
Table 2: Y-tube tests of males to synthetic test compounds (positive) in hexane compared to hexane alone (negative). Three significant choice differences between positive and negative stimuli were found.
Compounds (ug) Test Z3D* E3D Z5D Z4D EE24D Nonanal α‐pinene β‐pinene N %Resp. Chi‐Sq. Choice A 100 10 10 18 44.4% 0.000 B 10 1 1 28 50.0% 2.657 D 100 1 1 32 37.5% 0.335 E 10 33 63.6% 1.202 F 10 1 1 1 17 58.8% 1.646 G 10 1 28 46.4% 3.977 Neg. H 1 24 79.2% 0.476 I 1 1 32 62.5% 0.200 J 100 18 55.6% 1.646 K 0.1 42 66.7% 1.296 L 100 3 26 53.8% 0.287 M 100 10 20 50.0% 0.403 N 100 10 23 43.5% 1.646 O 100 1 44 70.5% 9.857 Pos. P 100 30 19 47.4% 0.111 R 100 100 23 56.5% 0.699 S 100 1 1 22 63.6% 2.657 T 100 1 1 41 73.2% 11.565 Pos. U 1 14 64.3% 1.019 V 100 1 1 1 14 64.3% 2.942 W 1000 17 58.8% 0.403 X 100 0.1 17 64.7% 2.358 Y 100 1 17 58.8% 0.403 Z 100 1 22 72.7% 1.011 AA 100 1 22 54.5% 0.335 AB 100 1 1 1 48 37.5% 0.223 * Major component, Z3D=(Z)‐3‐decenol; E3D=(E)‐3‐decenol; Z5D=(Z)‐5‐decenol; Z4D=(Z)‐4‐decenol;
Trap design. With the development of an attractive synthetic lure, behavior was examined of S. noctilio flying to the lure and entering different devices in the small wind tunnel at the Otis 29
Laboratory with the goal of learning about their trap-approaching behavior, in order to design a more effective trap. Based on these observations, two new trap designs were developed at the Otis Laboratory and sent to cooperator Ann Hajek at Cornell, along with the commonly used black intercept panel trap, for testing in a large field wind tunnel in the summer of 2011. The wind tunnel was constructed at the Otis Laboratory and transported to Cornell University in Ithaca, NY. It was set up inside a greenhouse which was well-ventilated to allow clean outside air to enter the upwind end of the tunnel, and air exiting the tunnel was exhausted out of the greenhouse. The two new trap designs consisted of an upside-down cone-shaped trap, and a clear jar trap. Traps were baited with a 3-component pheromone lure and suspended at the upwind end of the wind tunnel. Two lure concentrations were tested: 1.0 mg and 0.1 mg of the main component, at 100:1:1 of (Z)-3-decenol, (Z)-4-decenol, and (E,E)-2,4-decadienal, respectively. Male and female S. noctilio, reared from infested wood and collected upon emergence, were kept individually in vials. Individuals were released in the downwind end of the wind tunnel and video recorded flying upwind towards a trap. The number of wasps that encountered and entered each of the traps was recorded.
Field studies. Two field studies were conducted in 2011, and one in the end of 2010. The 2010 study was conducted from October to December in South Africa by cooperator Dr. Brett Hurley at University of Pretoria. The first 2011 study was based around the Syracuse laboratory and took place from June to August. The last study (currently underway) from October to December of 2011 is being conducted in South Africa by cooperator Dr. Brett Hurley.
The cooperative agreement established in 2010 with Dr. Brett Hurley was a large replicated field trapping study to test the antennally active major component in areas heavily infested with Sirex. At the time of this study, the identity or importance of any minor components had not been discovered yet. Due to multiple overlapping GC peaks in the area of the second strong antennal response, efforts to identify a minor component were complicated. Concentrated headspace extracts were sent to Dr. Ashot Khrimian (chemist at USDA ARS) for further chemical analysis, and he found that one of the hidden peaks matching the strong antennal response retention time contained nonanoic acid. It was suspected as a possible minor component based on his GC-MS findings as well as the occurrence of nonanoic acid in pheromones of a number of insects, including several members of Hymenoptera (Lloyd et al. 1975; Francke et al. 2000). Unfortunately, at that time, there were no adult S. noctilio available to conduct GC-EAD on nonanoic acid. It was decided that the field study in South Africa would test the major component alone and in a 100:5 blend with nonanoic acid, at three different concentrations, in the presence and absence of a kairomone lure (Alpha Scents) containing S. noctilio host plant odors, and with a positive and negative control. The study was conducted in pine plantations of 10-15 year old Pinus patula in South Africa, each divided into three blocks and replicated five times.
The first 2011 field study was based around the Syracuse laboratory and tested three concentrations of the newly discovered 3-component blend (0.1 mg, 1.0 mg, and 10 mg) against 30 the 8-component kairomone lure which was used successfully in the above-described 2010 South Africa field study. This study consisted of the four treatments at 10 field sites in Oneida, Wayne, and Oswego Counties (relatively small, mostly private properties, each with a stand of Scots pine). Each treatment had 2 traps at each site, for a total of 80 traps (20 replicates each). Traps were deployed from June 27 to July 1, 2011. Lures were replaced in the week of August 1, 2011.
The second 2011 study, in South Africa, was initially going to utilize the best concentration determined from the Syracuse study and focus on questions of trap height and design. However, due to low trap catch in Syracuse for all four treatments, it was necessary to test concentration in South Africa as well. Therefore, the South African study was designed to test the pheromone at two concentrations (1.0 mg and 0.1 mg), with a negative control (no pheromone), each with and without the kairomone, at two heights (3 m and 7 m), using the black intercept panel traps. Additionally, based on results from the Hajek wind tunnel trap design study, the clear jar trap would be tested, at the 7 m height only, with and without the 1 mg pheromone lure, with and without the kairomone lure. The study was to be conducted at four field sites consisting of large pine plantations (Pinus patula) heavily infested with S. noctilio. Each treatment was to be replicated five times per site. However, due to the steep terrain at one site, and the extra time required to hang traps at the 7 m height, it was necessary to drop one of the four field sites from the planned study, so three sites were used for a total of 15 replicates per treatment. Traps were deployed from October 17 to 21, 2011, with a change of lures scheduled to take place after three weeks.
31
Results and Discussion
Male responses to synthetic blends in the Y-tube olfactometer. Table 2 shows experiments conducted on the synthetic blends in the Y-tube to determine which compounds, blends, concentrations, and ratios elicit attraction in males. Although the main component alone did not significantly differ from hexane in these tests at any of the concentrations tested (Fig. 3a), the concentration that produced the least negative response (100 ug) was selected as a starting point
Figure 3: Key results of Y‐tube olfactometer experiments, testing males when offered a choice between the synthetic component(s) in hexane and hexane alone. Bars show percent responding and direction of choice. a) b)
c) d)
for subsequent testing in conjunction with potential minor components. The fact that the main pheromone compound alone was not strongly attractive at any concentration suggests that it was missing at least one required component.
When offered alone, neither the main component (Z)-3-decenol, nor the minor component (Z)-4- decenol produced significant attraction (Fig. 3b, tests J and U). However, the 2-component blend of 100:1 ug (Z)-3-decenol and (Z)-4-decenol was chosen significantly more than the hexane alternative (Fig. 3b, test O), and other ratios of the two compounds failed to attract males 32
(Fig. 3b, tests R, N, and X). The 100:1 ratio of major to minor components was close to the ratio found in extracts of the natural male headspace odors. The sum of the attraction by each of the two compounds separately was less than the attraction of the 2-component blend, indicating that these two compounds act synergistically.
Other candidate minor components with strong antennal responses were also tested in 100:1 binary blends with (Z)-3-decenol, but those blends failed to produce attraction (Fig. 3c, tests Y, Z, and AA). When a 10:1 ug blend of (Z)-3-decenol and nonanal was tested, males selected the hexane significantly more than that blend, suggesting repellency (Fig. 3c, test G).
The addition of one or more minor components to the 2-component attractive blend of 100:1 (Z)- 3-decenol and (Z)-4-decenol was also investigated (Fig. 3d). The addition of 1 ug of either (E)- 3-decenol or nonanal to the attractive 2-component blend completely removed attraction. The addition of 1 ug of (E,E)-2,4-decadienal to the attractive 2-component blend was significant, and produced slightly higher attraction and response rate than the attractive 2-component blend alone (Fig. 3d, test T). The addition of 1 ug of either nonanal or (Z)-5-decenol to this attractive 3- component blend completely removed attraction (Fig. 3d, tests V and AB).
These results establish the first discovery of a 2-component blend of 100:1 ug (Z)-3-decenol and (Z)-4-decenol, and a 3-component blend of 100:1:1 ug (Z)-3-decenol, (Z)-4-decenol and (E,E)- 2,4-decadienal, both attractive to male S. noctilio in a Y-tube olfactometer. These results also demonstrated that nonanal, (E)-3-decenol, or (Z)-5-decenol, when added to the attractive blends, resulted in a loss of attraction.
Figure 4: The 2- or 3-component blend in hexane was offered next to a Synthetic blends negative stimulus (hexane alone) in the wind tunnel. Bars represent the percent of males or females that landed on or touched either target (actual attractive to both sexes in numbers that chose each target are inside each bar). the wind tunnel. The two- and three-compound blends found to be attractive to males in the Y-tube were subsequently tested in the small wind tunnel in the quarantine at the Otis Laboratory. The wind tunnel bioassay allowed for successful testing of both sexes to the new synthetic attractants.
33
Results found that for both sexes a higher percent of wasps flew upwind to the 3-component blend than to the 2-component blend (Fig. 4). Similarly, a higher percent of wasps landed on or touched the target containing the 3-component blend than the 2-component blend. All males that made a choice selected the positive stimulus over the hexane, regardless of whether the 2- or 3- component blend was offered. Females chose the 3-component blend significantly more than the hexane, however, they did not chose the 2-component blend more often than the hexane. Therefore, in order to find the correct target females required the 3-component blend. For both sexes, the proportion of wasps released that chose the 3-component blend was at least double that for the 2-component blend.
Laboratory observations and trap designs. It was noted that differences in behaviors occurred between males and females. Males were less likely to touch or land on a target than females. The target baited with the 3-component lure was intercepted by 28.9% and 55.2% of males and females, respectively (Fig. 4). A similar pattern was observed with the 2-component blend.
Based on these observations, it was hypothesized that males were less likely to land or come into contact with a solid surface than females. This behavioral difference is supported by the differences in their biology, in which females are required to land on trees to oviposit, and not males, and males are thought to fly in leks at the tops of trees to find mates.
Two new traps were designed with the goal of increasing the male capture rate (Fig. 5). The first trap design aimed to exploit the strong phototactic responses of males. This trap had the top of an intercept trap as a “skirt” suspended beneath an inverted clear funnel leading into a clear bottle. The skirt was lined with window screening which extended up an acrylic bridge from the top of the skirt into the funnel and bottle. This skirt trap required wasps to approach the lure at the top of the skirt, land on the skirt, and then switch to phototaxis to climb into the bottle above.
The second trap consisted of two clear plastic square jars on their sides, stacked on top of each other with the section between them cut away. The top jar was uncovered and had a large mouth (9.5 cm diam.), and contained the lure. This design aimed to exploit the fast flight of both sexes by allowing them to follow the odor plume into the trap and then bump into the clear wall of the trap, falling into the bottom jar which could contain propylene glycol.
The two novel trap designs, and a standard black panel intercept trap, were tested by cooperator Dr. Ann Hajek, who was provided with the large walk-in wind tunnel for use in a green house in Ithaca, NY. A total of 450 virgin male and male
Figure 5: Two novel trap designs, the “skirt” trap (left) and the “clear” trap (right). A female S. noctilio landed on top of the cone trap and inside the bottom of the jar trap 34
wasps, reared from infested Scots pine, were individually released and recorded in the wind tunnel. Three trap types were tested, and two concentrations of the new 3-component pheromone lure, 1.0 mg or 0.1 mg. Of those that flew upwind, the number of wasps that were caught in each trap was recorded (Table 3).
The new skirt design did not work for either sex, but when baited with the 1.0 mg pheromone lure, the new clear trap caught more males than the standard intercept trap. When baited with the 1.0 mg lure, females were caught by both the clear trap and the intercept trap at similar frequencies. But with the 0.1 mg lure, the intercept trap caught more females than the clear trap.
Field studies. In the field study Figure 6: Female trap captures using different doses of (Z)-3- conducted by cooperator Dr. Brett decenol from gray rubber septa in South African pine Hurley from October to December, plantations. Treatments in blue had kairomone; treatments in 2010, in South Africa, prior to the red had no kairomone; open diamonds represent treatments in discovery of the minor pheromone which nonanoic acid was present; closed diamonds represent treatments in which nonanoic acid was absent. components, 3409 females and 14 males were caught in 420 black panel 6 Week 1 Week 2 intercept traps, with 14 treatments, over 5 a period of five weeks (Fig. 6). All 4 traps, including unbaited control traps, 3 caught wasps. The fact that unbaited 2 intercept traps caught wasps suggests 1 0 that there is a visual component to the 6 trap that contributes to its success, at Week 3 Week 4 5
least for female S. noctilio. There was 4 no significant effect of nonanoic acid, 3 but the kairomone was significant, and 2 there was a significant interaction 1 between the kairomone and the major 0 6 Week 5 All weeks compound (Table 4). A significant per trap wasps Mean female dose response was found for the major 5 4 compound in the absence of the 3 kairomone. The kairomone alone 2 clearly outperformed (Z)-3-decenol 1 alone or in combination with nonanoic 0 acid at a ratio of 100:5. 00.33 30 00.33 30
35
The field study in Syracuse, NY, from June 27 to September 13, 2011, aimed to evaluate three concentrations of the new 3-component pheromone. The kairomone lure successfully used in South Africa was used as a positive control. All four lure treatments were tested using the same black intercept traps that were successfully used in South Africa, however, resulting capture rates were low. A total of only 17 female S. noctilio were caught in NY: 14 in the kairomone traps, and 3 in the 0.1 mg traps, and captures were clustered in field locations. Thus, no conclusions could be drawn from that study. Therefore, in addition to testing trap height and design, lure concentration is being revisited in the study in progress in South Africa (Fig. 7), taking place froma) October to December, 2011. b) c)
Figure 7: The pine plantation in South Africa where two types of traps were deployed (a) with two concentrations of pheromone, with or without the kairomone, and at two heights. The new clear trap design (b)
Only the first two weeks of data have been received from the South African study so far, and mostly females were caught so far, so only trapping for females will be discussed in this preliminary report. A preliminary look at the data suggests that the 1.0 mg dose of pheromone caught more females than the 0.1 mg dose, both with and without the Figure 8: Preliminary trap catch in South African field study in kairomone. However, the kairomone November 2011 showing average catch for females for both still appears to be catching the most heights combined, for the intercept traps only, baited with 3‐ females, suggesting that there may be component synthetic pheromone lures at two concentrations, important minor components in the with and without the kairomone lure (N=30). pheromone that still need to be 10 characterized. Few males were caught Blank 8 so far, and the prototype clear trap 0.1 mg caught very few wasps as well, 6 1 mg 4 0.1 mg + Kair 1 mg + Kair 2
Females per Trap Kair 0 1 2 3 4 36 suggesting that the trap design still needs improvement. The preliminary data appear to show little difference in female catch between 3 m and 7 m trap heights (Figure 8).
Based on behaviors observed in these studies, and field observations in the early literature (Morgan and Stewart 1966), this pheromone may serve as a lekking pheromone. If so, males swarming around the tops of trees, producing the pheromone, would attract other males and females seeking a mate. Females may fly through the swarm of males and encounter them in- flight, or perhaps females land once a lek is located and produce another pheromone that attracts a male to land and mate. Although this report marks significant progress in characterizing mating behavior of S. noctilio, many questions remain unanswered and more research is needed.
REFERENCES
COUTTS, M. P. 1965. Sirex noctilio and the physiology of Pinus radiata. Some studies of interactions between the insect, the fungus, and the tree in Tasmania. Forestry and Timber Bureau, Australia Bulletin. p 79.
FRANCKE, W., LÜBKE, G., SCHRÖDER, W., RECKZIEGEL, A., IMPERATRIZ-FONSECAB, V., KLEINERTB, A., ENGELSC, E., HARTFELDER, K., RADTKED, R., and ENGELS, W. 2000. Identification of oxygent containing volatiles in cephalic secretions of workers of Brazilian stingless bees. Journal of the Brazilian Chemical Society 11:562-571.
HURLEY, B. P., SLIPPERS, B., CROFT, P. K., HATTING, H. J., VAN DER LINDE, M., MORRIS, A. R., DYER, C., and WINGFIELD, M. J. 2008. Factors influencing parasitism of Sirex noctilio (Hymenoptera: Siricidae) by the nematode Deladenus siricidicola (Nematoda: Neotylenchidae) in summer rainfall areas of South Africa. Biol. Control 45:450-459.
LLOYD, H. A., BLUM, M. S., and DUFFIELD, R. M. 1975. Chemistry of the male mandibular gland secretion of the ant, Camponotus clarithorax. Insect Biochem. 5:489-494.
MORGAN, D. and STEWART, N. C. 1966. The biology and behaviour of the woodwasp Sirex noctilio F. in New Zealand. Tran. Roy. Soc. N. Z., Zool. 7:195-204.
37
Behavior and sensory ecology of Sirex noctilio
Miriam Cooperband
Unpublished Progress Report FY2012
Field study in South Africa. A large field study was initiated in South Africa in October 2011 in which 240 traps were set at three field sites at pine plantations containing Pinus patula. There were 16 treatments, 3 sites, and 5 replicates. The experimental design is described in Table 1. As a result, 2,212 Sirex noctilio wasps were captured over a 6-week trapping period. Most of the wasps that were captured were females, and the new bottle trap design was found to be ineffective. High numbers of wasps were caught when kairomone was present. Initially, it appeared that the presence and concentration of the pheromone had no effect, but it was later discovered that the pheromone contained a contaminant that caused it to be inactive. Height had no effect on females, but slightly more males were caught in the high traps than the low traps. A subsample of captured female wasps were dissected and it was determined that 92.5% were mated. This indicates that the physiological state of most wasps being trapped is that of seeking an oviposition site.
Table 1. Experimental design of field study conducted in South Africa in October-December, 2011.
Treatment Levels
Pheromone lure 3 0; 0.1; 1 mg Plant volatile lure 2 Yes / No traps traps Panel Trap height 2 Low / High Pheromone lure 2 0; 1 mg Plant volatile lure 2 Yes / No Trap height 1 High only Bottle traps
Sirex pheromone. Our discovery of the new Sirex noctilio pheromone was published in the Journal of Chemical Ecology, in January 2012. That study identified the attractive blend of compounds, but one of the minor compounds eluted at the same retention time as the major compound, so although it was required for attraction in the synthetic blend, its presence in the natural emanations was not proven, just suspected. Additional work was conducted in collaboration with Dr. Allard Cossé who developed a method for separating the peaks of Z-3- decenol and Z-4-decenol. As male wasps began to emerge at the Otis Containment Facility, they 38 were aerated and volatile collections were sent to Dr. Cossé for analysis. He was able to confirm the presence of the Z-4-decenol in one of the volatile samples collected on a SPME fiber. Thus, we confirmed it was being produced as part of the male pheromone, something that was until then suspected but not proven.
The analysis and interpretation of the South African field data lead to unexpected and confusing conclusions (later it was discovered why), and work was conducted to investigate the possibility that additional minor components were required and needed to be identified. The resulting chromatographic data from 24 electroantennograms where male antennae were exposed to the natural male-produced pheromone, were combined and averaged to reduce the signal-to-noise ratio and enhance small or inconsistent antennal responses (Fig. 1).
Figure 1. GC (top) of male pheromone, multiple stacked EAD results (middle), and a combined EAD chromatogram of their average (bottom). The numbered vertical red lines represent possible antennal responses that deemed further investigation. The blue vertical lines represent the three known pheromone components to date.
The retention times and Kovat’s indices of 48 suspected antennal responses were evaluated and the corresponding GC-MS of the natural male pheromone was examined in those locations for compounds. Of those, 13 antennal response peaks that were determined to correspond to compounds in the GC-MS were identified and selected for further analysis. These 13 compounds were either purchased or synthesized. The synthetic compounds were then diluted to mixtures containing 100 ng of compound per ul of hexane, and roughly 0.5 ul were injected against male antennae and evaluated for antennal responses. A list of those compounds, and which ones produced responses, is presented in Table 2.
39
Pheromone contamination. Upon discovery of four compounds that produced antennal responses, the next step was to blend those compounds with the pheromone and evaluate the blends for attraction in the wind tunnel, using the same bioassay that allowed the successful identification of the proper pheromone blend last year. However, it was observed very early on that wasps were not responding to the pheromone lure in the same way they had the prior year. Troubleshooting of the wind tunnel bioassay thus ensued, and in case there was contamination of the wind tunnel, it was cleaned, the filters were changed, and the light bulbs were replaced. This did not solve the problem. Meanwhile, a new study in Cornell was starting in which they were continuing their study from the previous year to test pheromone-baited traps in a field wind tunnel. I sent them lures to use and they quickly notified me of problems with attraction to the lures as well. This indicated that something was wrong with the lures.
I analyzed the pheromone material that the lures were made with this year, and I found that it contained a small amount (about 1%) of E-3-decenol, the stereo-isomer of the main component Z-3-decenol. Last year, we evaluated 1E/100Z blends and found that the addition of 1%E was enough to remove all attraction. Therefore, it was concluded that this amount of contamination was the likely cause of the problem. All pheromone material in our freezer was then analyzed and with only a few exceptions, all of it was contaminated with at least 1%E. I informed the chemist, Tappey Jones, of the problem and asked him to synthesize a new high purity batch. After 4 weeks he sent a new batch, but this was contaminated with 3%E, and was therefore not useable. Tappey was about to move his entire lab and was unable to continue working on the problem, so I had to contact possible alternative sources. I contacted Bedoukian, Alfa Aesar, Allard Cossé, Hercon, and Contech. Most of them were unable to help or it was cost prohibitive ($7-10k). However, I was able to develop a plan whereby Contech would produce the purity pheromone we needed at the purity required for a nominal cost ($200), and in exchange we would field test their Flexlure product loaded with the pheromone.
Testing of contaminated lures. Meanwhile, I provided 0.7%E lures (made from the best remaining material I had in the freezer in sufficient quantities for the field study) to the Syracuse lab for field testing. Nothing was caught using those lures, however, the entire study testing kairomone, pheromone, clear, and black intercept traps only caught 4 Sirex noctilio in total, so 40 results were inconclusive. I also provided cooperators Ann Hajek and Mark Sarvary in Cornell with lures containing E at 0.6%, 0.7%, and 0.8% (the best material I had available at the time) for testing and evaluation in the field wind tunnel to determine if these levels of contamination affected attraction. They found that the 0.6%E was attractive to males but not to females. They also found that there was no interest by males or females at 0.7%E or higher.
Since obtaining some new high purity pheromone was not possible in a short amount of time, the summer research plans were re-evaluated. We modified their work plan to focus on the most probable area of success in light of the problems that had developed with the lures, the timing of emergence, and the lack of new lures in time. The new field wind tunnel study in Cornell would examine male and female attraction to traps baited with UV light vs. no light. They would also investigate the difference in capture of males by clear vs. black intercept panel traps baited with the pheromone. The data collection of these studies has completed and data entry and analysis are currently underway.
New field study in South Africa. As a result of the discovery that the study from last year had contaminated lures, the planned follow-up work in South Africa was re-evaluated, and a new work plan was established with Dr. Brett Hurley. Contech produced pheromone with 0.3%E (low enough E content to be attractive to both sexes), and they provided their Flexlures loaded with Sirex pheromone at two doses (2 mg and 8 mg). I made rubber septa lures (1 mg) with the same pheromone blend they used in their Flexlures. Field cages were procured for a caged mating disruption study, and lures and clear intercept traps were sent to South Africa. The two new studies in South Africa will begin this month. The first will be a trapping study which will test eight treatments: the four levels of pheromone lures (0, 1 mg rubber septa, 2 mg Flexlures, and 8 mg Flexlures) and the two levels of kairomone lures (present vs. absent). They will also be testing clear traps with four treatments (blank, pheromone, kairomone, and pheromone + kairomone). They will also be conducted a field cage mating disruption (proof of concept) study to evaluate the likelihood of success of using the Sirex noctilio pheromone for mating disruption.
Sex ratio. Finally, an analysis of the sex ratio data of wasps emerging from field-collected infested logs over the last four years shows a shift from an extreme male-bias during the beginning of the invasion, to a female-bias after the population became more established (Fig. 2). This can be explained by the haplo-diploid sex determination of Sirex noctilio, and a gradual shift in the population from mates being scarce at the beginning of the invasion, to mates being abundant at a later stage of the invasion.
41
Figure 2. The sex ratio of Sirex noctilio over four years changed from an extreme male bias to a female bias.
1 y = ‐0.1139x + 0.9584 0.81 R² = 0.8447 0.8 0.73 0.70
0.6 0.44 0.4 (males/total)
0.2 Ratio
Sex 0 2009 2010 2011 2012 Year
42
II. SURVEY AND TRAPPING Progress on lure and trap development for monitoring Sirex noctilio
Damon J. Crook
Unpublished presentation
Sirex noctilio is a pest of pines worldwide that was identified in trap in New York in February 2005. Stressed pines, which may be produced by girdling, are preferred for attack by females. There is an urgent need to identify attractants ― both pheromones and kairomones ― and to develop an effective trap.
Pheromones
No long distance pheromone has been identified for Sirex. However, a contact pheromone seems quite likely. The antennae are covered in small uniporous sensory pegs (more on males). The antennal structure is suited to contact chemo-reception (Crook et al. 2008 Ann. Entomol. Soc Amer.). Antennae of male and female Sirex have also been examined using SEM and TEM microscopes. The antennal surface is covered with hundreds of small pegs. Each peg has 2 dendrites and a single pore (Fig. 1). Their small size and internal structure suggests that these pegs are contact chemoreceptors. The numbers of these contact receptors increase towards the tip of the antenna starting at 100 on segment 3 (after the pedicel and scape) to over 300 on the tip segment. Females have similar numbers to males. The antennal structure is therefore perfectly suited to detecting contact pheromones. Female antennae usually consist of 19 flagellar segments, including the scape and pedicel, whereas males have 17 flagellar segments. The Sirex contact pheromone has been confirmed using a behavioral assay; it elicits a characteristic mating response in males consisting of five steps (Fig. 2). The chemical composition consists of three components: (Z)-7-heptacosene, (Z)-7-nonacosene, and (Z)-9-nonacosene.
Host kairomones
Are host volatiles useful as lures for Sirex noctilio? We addressed this question through volatile collections and trapping experiment in collaboration with Penn State University in 2006-2008. Herbicide-treated trees can be effectively used as lures because Sirex noctilio detects typical pine terpenes. Figure 3 gives a summary of trapping results from 2006-2009. The objective of this work was to examine quantitative and qualitative differences in the volatiles emitted by herbicide treated and untreated trees and to compare different pine species. The volatile collection method used a portable system. It was non-destructive and clean, making multiple sampling of trees possible. It also enabled multiple analyses in the lab. Herbicide-treated trees were used as 43
Fig. 1. Antennal morphology
Pore
Dendrites 44
Fig. 2. Mating response of a Sirex male to a dead pinned female treated with the contact pheromone
1)1) Attraction Attraction 2) Grasping
3) Mounting
4) Abdomen bending
5) Copulation 45
Fig. 3. Trapping results in 2008 at a New York site
50 2008
Brecheimer site 40 caught
30 noctilio
S.
of
20
Numbers 10
0
Treated Control Treated Control
Scots pine White pine 46 lures in combination with sticky panels and funnel traps. The results were as follows, where > indicates relative attraction. First, herbicide treated >>> untreated. Second, Scots pine > Red pine > White pine (2006). And third, Scots pine >> White pine (2008, 2009). Figures 4, 5, and 6 compare the amount of total volatiles in 2006 through 2008, respectively. Note that emission was estimated for the whole trunk surface (average height of 40’, 10” diameter at the bottom, and 1” at the top). Volatile profiles of Scots pine and red pine in 2006 and of Scots pine and white pine in 2008 are presented in Figs. 7 and 8, respectively.
It must be stressed that the EAD active pine volatiles observed can get more difficult to identify (Fig. 9) and can be difficult to synthesize or buy for lure testing. Furthermore, there are a number of caveats to the use of host volatiles including the following four:
1) Sirex antennae respond to many compounds in tree aerations.
2) Many compounds are hard to identify and unavailable to buy.
3) Aeration composition can vary from tree to tree, so identifying an attractive blend is “challenging”.
4) The lures could be out-competed by surrounding trees.
Given our results, we can draw several conclusions on Sirex trapping to date:
1) α and β Pinene UHR lures do catch some wasps, but in very low numbers.
2) More compounds in a tree mono-terpene blend lure provide slightly more catch.
3) Traps on girdled trees catch more insects.
4) Wasp behavior suggests they are “canopy loving” and not attracted to traps low down in tree stands (only ovipositing females).
5) No trap design is particularly effective (i.e., panel, funnel, crossvane etc.)
Attempts to improve host based lures and develop a better trap
In previous work, Madden (1971) showed that Pinus radiata logs were more attractive to Sirex wasps up to 3 weeks post felling. Simpson and McQuilkin (1976) showed that monoterpene hydrocarbons accounted for 95% of the volatile composition in Pinus radiata but their composition changed relatively little over a 3 week period. Simpson and McQuilkin (1976) showed that oxygenated components such as camphor and pinocamphone increased from trace amounts to 1% of the total volatile blend after a 3 week period. Could these other components be important for Sirex attraction? 47 48 49
50
51
Working with Sirex edwardsii and Sirex nigricornis, William Shepherd and Brian Sullivan (FS Louisiana) reported that EAG results showed a greater sensitivity to these new compounds of interest (Fig. 10). Crook observed antennal responses by Sirex noctilio females and males to the compounds given in Figs. 11 and 12, respectively (N.B. red compounds were suggested by Simpson and McQuilkin 1976). Figure 13 gives results of a Sirex Lure test conducted in 2009. Conclusions of this 2009 “semio” test were as follows.
1) A multi-component tree blend does improve α/β Pinene lures (but only slightly).
2) We need to try blends on a higher trap in ‘open’ canopy.
3) We need to examine southern pine species that are more attractive to Sirex wasps.
4) We need a better trap for testing lures.
Developing a color trap for Sirex noctilio
Objectives of this work were twofold: to identify wavelengths of light to which Sirex noctilio is sensitive using an Electro-Retinogram System (Fig. 14) and to select colors for field testing on funnel traps.
Female Sirex retinal responses to wavelengths across the visible spectrum are shown in Figs. 15 and 16. Female Sirex retinal responses to 5nm increments showed the main peak in sensitivity to be around 495nm. Figures 17 and 18 show retinal responses of male Sirex.
Figure 19 illustrates colors to test on traps. Note that both red and Scots healthy pine needles show a wavelength peak at around 540-545nm. Dark and light 495nm painted funnel traps were tested in South Africa during October-December 2009 (Fig. 20). The remaining colors were tested in New York in 2010 (Fig. 21).
Conclusions from the color trapping studies were as follows:
1) Trap catch over the entire fieldwork test was very low.
2) No color treatment caught significantly more adult wasps than a standard black funnel trap.
3) Future color trapping should examine if colored traps are more effective if placed higher up in the tree canopy.
52
53 54
55 56
Fig. 14. Electro‐Retinogram equipment
57 58 59 60 61 62
Fig. 20. Dark and light 495nm painted funnel traps were tested in South Africa (October‐December 2009)
495 nm funnel Black funnel IPM black panel 63
64
2006 survey studies: Age of Girdle and trapping
Kelley Zylstra
Total Number of Insects Caught On Sticky Panels
650 600 574 550 500 450 400 350 300 251 250 184 200 150 Number Individuals of 76 100 49 59 50 5 7 4 10 5 9 0
e ae ale ale ba ram d Fem e Ibalia i M -M x- i driida C Bark B. Bupres Rhyssa ire y Cler S Sirex- T. colum esson Xiph cr . U. cressoni-FemU
Figure 1. The total number of insects collected from sticky panel traps throughout the summer of 2006 for the Age of Girdle study.
Total Symphyta Caught On Each Tree Species
210 199 200 190 180 170 160 150 140 130 120 Scotts Pine 110 100 Red Pine 90 White Pine 80 70 60
Number of Individuals of Number 50 40 40 30 24 12 16 20 5 9 9 10 0 0 1 4 2 3 0 1 0 2 0 Sirex-Fem Sirex-Male U. U. Xiphydriidae T. columba cressoni- cressoni- Fem Male
Figure 2. The total number of Symphyta caught on each host species for the Age of Girdle study during 2006. 65
Age of Girdle study (2006)
Total Number of Insects Caught: Trap Trees vs. Control Trees
450 414 400
350 300
250 Trap Tree 200 Control Tree 157 143 150 126
Number of Individuals of Number 100 57 47 45 39 50 23 25 0 5 0 Sirex Sirex Male Symphyta Bupr Ceram BB Female Figure 3. The total number of insects (siricids, cerambycids, parasitoids, buprestids, bark beetles) collected from sticky panel traps on control trees vs. trap trees in the Age of Girdle study during 2006.
Number of Symphyta Caught Per Panel on Scots Pine
55 52 Sirex Fem 50 Sirex Male 45 42 Xiphydriidae
40 U. cressoni Fem 33 35 U. cressoni Male 30 T. columba 25 22 23 20 17 15 10
Caught Individuals of Number 10 4 5 5 3 2 3 2 2 3 0 1 111 00001 000 0 Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Not On Panel
Figure 4. The effect of panel height on # of Sirex caught on Scots pine in the Age of Girdle study during 2006. Note: Results for Red and White pine are not shown here as there was no observable difference detected. 66
Age of Girdle study (2006)
Number of Females Collected From Trap Tree Sticky Panels For Each Girdle Period
50 45 46 40
35 30 32 Girdled 5/15/06 25 Girdled 5/29/06 20 Girdled6/12/06
15 17 # of Insects Collected 10 12 10 5 7 4 5 1 0 Scots Red White Host Species Figure 5. The total number of female Sirex collected from each girdle period off of sticky panels on trap trees. Note: Data from this study was inconsistently collected/labeled and may not be representative of real results.
Total Siricid and Parasitoid Larvae Dissected out Split Logs from 'Age of Girdle' Study
140 120
100
80 60 11.7% parasitism rate 0.83% parasitism rate 40
Number of Larvae/Pupae of Number 20 2.5% parasitism rate 0 Total Sirex Dissected # of Ibalia # of Rhyssa # Infected w/ Nemas Total siricids = 120; Total Ibalia = 14; Total Rhyssa = 1; # infected with nematodes = 3 Note: Rhyssines counted here w ere attached to body of larva, number does not include total collected from siricid galleries. Nematodes unidentified.
Figure 6. The total number of siricid larvae pulled out of split logs (trap trees only) from the Age of Girdle Study. 15% of each trap tree was split for larvae. Larvae were then dissected for a parasitoid/nematode survey. Only 2.5% of the larvae were parasitized with an unidentified nematode.
67
Age of Girdle study (2006)
Number of Larvae Dissected From Each Tree Species (Age of Girdle Study)
60
50 40
30
20 10
Larvae/Pupae of Number 0 Sirex spp. Ibalia Rhyssa Red Pine 44 5 1 Scots Pine 53 3 0 White Pine 23 6 0
Note: Ibalia and Rhyssa were dissected out of the larval siricids
Figure 7. The total number of siricid/parasitoid larvae dissected from each host species from the Age of Girdle study (trap trees only).
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30 27
25 Red pine 20 19 19 18 Scots pine White pine 15 15
10 9 8 8 7 66 6 5 4 No. of siricidsNo. of removed from logs
0 0 0 0 0 0-3m 3-6m 6-9m 9-12m 12-15m 15-18m 18-21m
Height of Log Section Figure 8. The total number of siricids removed from split logs representing each of the 7 height groups for the Age of Girdle study.
Age of Girdle study (2006)
60 54
50 Red pine Scots pine 40 White pine
30
21 21 22 20 No. of siricidsNo. of removed fromlogs 10 10 10 8 4 1 0 Red pine Scots pine White pine Red pine Scots pine White pine Red pine Scots pine White pine (n=18) (n=23) (n=13) (n=18) (n=19) (n=18) (n=16) (n=16) (n=17) 5/17/06 5/31/06 6/12/06 Host Girdle Date Figure 9. The total number of siricids removed from split logs taken from trees girdled at three dates from the Age of Girdle Study
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50
44 45 Red pine Scots pine White pine 40
35
30 28
25 22 20 20 18
15 No. of siricids siricids removed of No.
10 7 6 5 4 1 1 0 0 0 00 0 0 (n=7) (n=6) (n=6) (n=5) (n=8) (n=6) (n=6) (n=6) Heinz Fridge Fridge (n=10) (n=12) (n=10) (n=12) (n=12) (n=12) (n=15) (n=19) Blumer Blumer Rathburn Nine Mile Nine Silk Road Silk Powerline Powerline Dinglehole Mexico TPMexico Excavation Frog Hollow Hagenmeyer Hidden Fields Hidden Site (logs split)
Figure 10. Number of siricids removed from split logs taken from trees from each study site used in 2006 for the Age of Girdle Study
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Delimitation survey (2006)
Total Symphyta Collected in Sirex Delimiting Survey
300
250
200
150
100 Total Number Collected 50
0 Sirex noctilio Sirex spp. Other Symphyta
Figure 11. The total number of Symphyta collected during the delimitation survey.
Semiochemical study (South Africa-2006/2007)
Total Number of Female Sirex noctilio Caught Per Treatment
658 642 700
555 600
500
400 327 December January 300
200 176 149
Number of Females Caught Females of Number 89 100 77
0 6-blend a-pinene b-pinene Control Lure Treatments
Figure 12. The total number of female Sirex noctilio caught per lure treatment in South Africa. 71
Semiochemical study (South Africa-2006/2007)
Mean Number of Female Sirex noctilio Caught Per Treatment
90 44 80 43 70 37 60 22 50 December 40 January 30 12 10 20 5 6 10 Mean Number of Females Caught Females of Number Mean 0 6-blend a-pinene b-pinene Control -10 Lure Treatments
Figure 13. The mean number of female Sirex noctilio caught per pure treatment in South Africa. Small bars represent standard deviation. A Tukey-Kramer HSD post-hoc comparison showed beta-pinene was significantly different from the 6-blend for females caught in January (P = 0.0204; F = 3.53).
Semiochemical study (2006)
2006 Semiochemical Lure Comparison -- NY
25
B 20 =7)
n 15
S.E.) ( 10 (+
5 A A A Mean no. siricids no. Mean trap per caught A A A 0 Blank 70% / 30% / Five Nonanal Nonanal Trap Tree** 30% 70% Component* + 70% / 30% Lure
* -- 63% (+)-a-pinene, 30% b-pinene, 3%-limonene, 3 % myrcene, 1% carene ** -- Trap trees were girdled on June 18 and 19, 2006 Figure 14. The mean number of siricids caught per lure treatment in Syracuse, NY (2006). Small bars represent standard error of the mean. 72
Trap design study (2006)
10 9 8 7 6 5 4 3 No. of Siricidae 2 1 0 Log Sante Drainpipe Crossvane Intercept Panel Intercept Lindgren Funnel Lindgren
Wet Traps Sticky
Figure 15. The total number of siricids caught in each different trap for the trap design study. Note: The results of this study are still being confirmed by Kevin Dodds and are not complete yet.
73
Log study (2006)
2006 Log Study -- NY
12 B 10
=6) 8 n 6
4
trap (+ S.E.) ( S.E.) (+ trap A 2 A A A A Mean no. siricids caught per A 0 0-2 Week 2-4 Week 4-6 Week Drainpipe Drainpipe w/ Drainpipe Trap Tree* Post cut Post Cut Post Cut Only 70% / w/ 0-2 30% Week Log
Treatment
* -- Trap trees were girdled on June 19 and 20, 2006
Figure 16. Mean number of siricids caught per trap in the log study (2006) with standard error
Nematode survey-adult Sirex dissections (2006)
Sirex noctilio collected from Darrow Rd. 70
60 4 50 Sirex Infected With Nematodes 40
Sirex Not 30 Infected With
# of Sirex 55 Nematodes
20 3
10 19
0 Females Males
Figure 17. The number of adults dissected that were positive or negative for nematodes during the summer of 2006. Sirex were field-reared from trees cut down at the SUNY Oswego campus site. Trees were held in field cages at Darrow Road. 13.6% of the females were infected by an unidentified nematode, while 6.9% of the males were infected by an unidentified nematode. 74
2007 survey studies: Age of Girdle and trapping
Kelley Zylstra
Objective A repeat of the 2006 study in order to determine the optimal time to create a trap tree using a chemical girdle in order to attract the most siricids to tree for detection and larval development in the trap tree. Treatments were chemically girdled 3 months before flight, 1 month before flight, at flight, and there was a control treatment.
Results
The effect of girdling age on the attractiveness of host trees to S. noctilio. This data represents both red (Pinus resinosa) and Scots (Pinus sylvestris) pine host species. A total of 10 replicates were performed for each host, consisting of 8 Scots pine and 9 red pine sites (some sites had multiple replicates). For both host species, there were significantly more captures in the June girdling treatment (red Pine: P = 0.001, F3, 107 = 5.55; Scots Pine: P = 0.001, F3, 107 = 5.66). Capital letters represent significant differences in trap catch among treatments conducted on red pine, while lower case letters represent differences among treatments conducted on Scots pine.
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Mean Number of S. noctilio Emerged Per Girdling Age Treatment (Red Pine)
55 50 * 45 40
S. noctilioS. 35 Control 30 3 Mo. Girdle 25 1 Mo. Girdle 20 Flight Girdle 15 10
Mean Number of Number Mean 5 0 Time of Girdle Prior to Flight
(P = 0.0001, F 3, 36 = 9.99, Reps = 10)
The effect of girdling age on the number of emerging S. noctilio in a subsample of red pine treatment tree material. There were significantly more S. noctilio emerging from logs sampled from the July girdle than any other treatment (41.1 ± 9.10) (P = 0.0001; F3, 36 = 9.99). Small bars represent SEM. (*) = treatment group is significantly different from the control group.
Mean Number of S. noctilio Emerged Per Girdling Age (Scots Pine)
180 160 * 140 120 Control 100 3 Mo. Girdle 80 1 Mo. Girdle
S. noctilioS. 60 Flight Girdle
Mean Number of Mean Number 40 20 0 Time of Girdle Prior to Flight
(P = 0.0058, F 3, 35 = 4.93, Reps = 10)
The effect of girdling age on the number of emerging S. noctilio in a subsample of Scots pine treatment tree material. There were significantly more S. noctilio emerging from logs sampled
from the July girdle than any other treatment (117.7 ± 40.3) (P = 0.0058; F3, 35 = 4.93). Small bars represent SEM. (*) = treatment group is significantly different from the control group. 76
Table 1. The total number of male and female S. noctilio emerging per replicate (i.e., field site).
Ratio Site Host Females Males M:F 1 Red Pine 17 18 1:1 2 Red Pine 11 18 1.6:1 3 Red Pine 31 36 1.2:1 4 Red Pine 11 60 5.5:1
5 Red Pine 26 53 2:1 6 Red Pine 28 59 2.1:1 7 Red Pine 16 60 3.8:1 8 Red Pine 29 28 1:1 9 Red Pine 23 86 3.7:1 10 Red Pine 40 39 1:1 11 Scot Pine 16 74 4.6:1 12 Scot Pine 159 302 1.9:1
13 Scot Pine 27 80 3:1 14 Scot Pine 14 138 9.9:1 15 Scot Pine 70 363 5.2:1 16 Scot Pine 41 153 3.7:1 17 Scot Pine 0 5 18 Scot Pine 51 162 3.2:1 19 Scot Pine 11 64 5.8:1
20 Scot Pine 3 11 3.7:1
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Mean sex ratio (♂/♀) red pine = 2.29 Scots pine = 4.22 Significantly different at the 0.05 level by a t-test
Figure 2. Size variation of female S. noctilio from Age of Girdle trap collections. Range 11.48 mm – 30.09 mm, mean size 20.76 mm. Photo credit: Ken Dudzik (USDA Forest Service). 78
Mark-Release-Recapture study (USA) Kelley Zylstra
Objectives To determine distance traveled by released, marked, males and females and determine if trap trees attract more Sirex than naturally stressed trees in the same stand. Two traps were placed at the mid-bole level and a third trap was placed right below the canopy.
Results 550 males and 75 females were released. Zero males or females were recaptured, however, 42 non-marked females were caught during the study.
G N N
G G G
N N N G N
G G N RP N G G
N N N G N G G G N G N
RP = Release point; G = Chemically girdled trees; N = Naturally stressed trees
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Total Sirex spp. Caught by Treatment
35
30
25
20 Chemically Girdled Naturally Stressed 15
10 Number of Sirex Caught of Sirex Number 5
0 7/19 7/26 8/3 8/9 Total Date
Total # of Sirex spp. Caught by Each Trap Height on Sticky Panel Traps 25
20
15
10 Total S. noctilioS. Mean 5
0 10-13 ft 16-19 ft Below Crown
-5 Trap Height P < 0.015; F = 4.44 Mid-bole traps caught significantly more than below live crow n trap
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Miscellaneous canopy observations Kelley Zylstra
Bucket truck releases: 7/25/2007: 84ºF, Sunny/Muggy, 4pm.
-Released approximately 20 males at 55ft high in the bucket truck on the edge of a Scots pine stand near trees that were girdled with Banvel® on 7/8/2007. -Almost all flew to the top of the canopies (branches and needles). A few flew away to trees nearby that were not chemically stressed. Most flew right to the trees that were chemically stressed. -All flew up and around the canopy structure (i.e., not through the canopy but around it). -The ones that flew to trees that were not stressed appeared to be flying with the wind or being carried by the wind.
Tree cage study: 8/1/2007: 88ºF, 3-5PM -50 males released (unknown ages) -30 females released (unknown ages) -Almost all went directly to the top of tree tent; no activity after that.
8/2/2007: 95ºF, Sunny, Humid, 9AM-4PM -83 males (unknown ages) released into first tent -14 females (unknown ages) released into first tent -Almost all went to the sides and top of the tent; a couple stayed in the grass and were inactive. -Several were found hanging on twigs and needles in the canopy (outsides and top of canopy). -Two were found dead and predated on near the ground from releases from the previous day. -27 males (<24h old) were released into second tent -3 females (<24h old) were released into second tent -two of the females went straight to ovipositing on the bole -most males inactive down in vegetation -first tent has no undergrowth, tree is approx 21ft. -second tent has thistle undergrowth, tree is 18ft -in tent one, several males were spotted on needles at the top of the crown just resting.
8/3/2007: 90ºF, Sunny, 10:30AM -20 males released, dusted in Saturn Yellow -5 females released, dusted in Saturn Yellow -Released into tent 2, all insects <24hr old -Almost all flew to the sides of the tent; some went up to the crown 81
-1 female went to the bole and began ovipositing -No activity after release, just resting on branches/needles. -No activity in tent 1, almost all insects were found only on sides of tent.
Host-Mortality Study Kelley Zylstra
Objectives To determine the differences in Sirex attack symptoms on the different host species and correlate it with the actual development of Sirex larvae inside of the hosts. Additionally, causes of mortality of larvae in each host species were determined and include types of parasitism from Ibalia, Rhyssa, nematodes, and the number of siricids killed by pathogens.
Results Repeated in 2008 (see 2008 results doc for final data).
Siricid larvae were found in: white pine that had no needles left on the crown and heavy beading. red pine that had brown needles in the crown and medium-heavy beading. Scots pine that had brown or no needles and medium-heavy beading. **No consistent trend with other symptoms like woodpecker activity, bark beetles or cerambycids.
Total % % % % % Siricids Healthy Rhyssine Ibalia Nemas Pathogens
Scots 1435 72.2 8.9 14.8 0.56 3.6
Red 363 55.4 19 19.8 0.28 5.5
White 45 37.8 33.3 8.9 0 20
The Percentage of Healthy Siricids, Parasitoids, and Dead Siricids Removed From 100 Billets Per Each
Species of Host Material (300 Billets Total)
80 70 60 50 Scots Pine 40 Red Pine 30 White 20 10 Results 0of 2008 Sirex noctilio studies % Healthy % Rhyssine % Ibalia % Nemas % Pathogens
KelleySiricids in 100 Billets of Host Material Zylstra
Percent of Healthy, Parasitized, and Dead Parasitized, Healthy, of Percent Scots Pine 72.2 8.9 14.8 0.56 3.6 Red Pine 55.4 19 19.8 0.28 5.5 White 37.8 33.3 8.9 0 20 82
Scots pine/white pine study Kelley Zylstra and Katalin Bӧrӧczky
Objective To determine if S. noctilio prefers to attack one trap tree pine species over another and how host volatiles correlate with trap catches and emergence data.
Results
Mean S. noctilio Caught per Treatment
4 3.11
3 SP Control
Adults SP Girdled 2 WP Control 0.61 0.3 1 WP Girdled M ean (+ S /- E M )
S . n o ctilio 0 0 Treatment and Host Species
P = 0.001; F3, 152 = 19.14
***See Penn State section for volatile profiles and results.
Mating behavior study
Objective To determine where males are in the pine stand and if they can be caught in trap trees with traps placed at the top of the crown.
Results Trap captures for site were poor for females, only one male was caught (on a mid-bole trap, none in canopy). Study should probably be repeated on a better field site because site history probably affected results. 83
Behavior Study
9 8 7 6 5 S. noctilio- Female 4 S. noctilio- Male 3 2
Number of S. of noctilioNumber 1 0 Girdled Canopy Girdled Mid-Bole Control Canopy Control Mid-Bole Trap Trap Trap Trap
Jack pine/pitch pine study
Objective To survey for S. noctilio in jack and pitch pine in the state. There are currently no records of the pest in those tree species in NY but have been identified in jack pine in Canada.
Results No S. noctilio, or any other siricid, was detected in the traps deployed with trap trees. Tree material from the trap trees have been brought in for rearing. 84
Summary of Sirex research results 2007-2010
Trap Height Study Optimal trap heights have been examined in the last couple years, but it has been a variable in part of different study objectives. In 2007 there was a mark-release-recapture study set up on a single field site. There were 28 trees set up in total, 14 were girdled trap trees and 14 were control trees. Each tree had a trap placed just below the live crown, one at mid-bole and one at approx. 10ft. The mid-bole traps caught more Sirex than the canopy traps. In 2008 a single field site was set up for a behavior study, where 10 trees were girdled and 10 were non-girdled controls. This time traps were placed above the crown and at mid-bole. We did not capture enough Sirex at this field site to really do anything with this data other than to make a general observation that there were zero captures above the canopy.
Total # of Sirex spp. Caught by Each Trap Height
25 on Sticky Panel Traps
20
15
10 Caught per Treatment Caught per Total Mean 5
S. noctilio S. noctilio 0 10-13 ft 16-19 ft Below Crown
-5 Trap Height P < 0.015; F = 4.44 Mid-bole traps caught significantly more than below live crown trap
2008 Study 9 8 7 6 Treatment 5 per 4 3 2 Captured
1 0 Girdled Canopy Girdled Mid‐ Control Canopy Control Mid‐ noctilio Trap Bole Trap Trap Bole Trap S.
Control Tree vs. Trap Tree
Total 1 Field Site, 10 trees per treatment 85
In 2009 we did a lure/trap height study with Penn State. Three field sites were set up, 28 trees in total were girdled. Traps were placed right below live canopy and at mid-bole. All traps also had an artificial lure that was attached to the trap. The traps below the live crown caught more S. noctilio than the traps at mid-bole.
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2009 Total S. noctilio Captured High Trap vs. Low Trap PennState Lure Study 150 Captured
100 noctilio 50 S.
0 Total High Trap Low Trap
In 2010 we set up 4 field sites, each had 5 girdled trees replicates per site. Each tree had a trap just below live crown, one at mid-bole, and one at approx. 10 ft. Trap captures were low, but generally speaking, more S. noctilio were caught in traps below the live canopy and mid-bole than in the traps at approximately 10 ft.
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2010 Trap Height
Natives
Ibalia Below Canopy Mid‐Bole S. noctilio 10ft
0 5 10 15 Total Number Caught per Trap Height 4 field sites; 5 tree reps per site
Trap Tree Method Study Objective was to determine the most effective number of trap trees needed, position within the stand, and whether adding aerosol glue to the trap would increase trap captures. 10 site replicates were set up, 5 of those sites received glue on all traps, and the other 5 did not. There were 4 treatments groups at each site, each treatment group had only 1 trap placed at mid-bole: Single girdled tree, on stand edge. Clump of 3 girdled trees, on stand edge. Single girdled tree, within interior of stand. Clump of girdled trees, within interior of stand.
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Trap Tree Methods 20
18 16 Caught
14 12 noctilio
S. 10 Glue of
Treatment 8 Non‐Glue per 6 Number 4
Total 2 0 Single/Interior Single/Edge Clump/Interior Clump/Edge
Fewer S. noctilio were captured in glued traps than non-glued traps. This study needs to be repeated another year to in order to detect statistical significance between treatment groups.
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Summary of Sirex research results 2011-2012
2011 Winter Trap Interaction Trial
Objective Make observations of the flight interactions with the deployment traps currently used in survey of Sirex noctilio in order to improve efficacy of detection efforts.
Methods A chamber was constructed at the satellite laboratory in Syracuse, NY (picture below). The chamber had a front clear plastic window; everything else was framed in with polyethylene. There were two openings at the top of the chamber, equidistant to each other, which allowed for heat lamps to brighten and warm up the chamber. Our standard black multi-funnel lindgren trap was first tested in the chamber. A Trap was placed beneath each light source. Both male and female S. noctilio were released into the chamber from a center point as they became available for use for the study. The initial response was recorded for each individual and after 1 hour the final response was also tallied. Responses included: No response (remain on station) Fly to and land on a wall Fly to and land on the floor Caught in trap variable 1 Caught in trap variable 2 Misc. Response (land on researcher, land on top of trap, etc) Several different traps were then tested in the chamber and based on preliminary observations several prototype traps were designed and also tested.
Results Initial observations found that woodwasps were able to discern the black traps and avoid them (flying up to, then hovering, and then flying around the trap). We decided to test an opaque trap against a black trap (picture left) to see if the silhouette was affecting trap catch. We tested 4 replicates with a total of 51 woodwasps and found 90
improved trap catch with the opaque funnel traps (Figure below).
No Other Funnel Test 2% Response 16%
Black Trap Wall 31% 4%
White Trap Floor 16% 31%
Next we tweaked the opaque funnel trap to a clear funnel trap (picture above) with the idea that in a field setting the clear trap would blend in with the background forest even more, deterring Sirex from avoiding the trap. We ran 8 replicates with a total of 168 insects and found the clear trap was still an improved trap from the black trap in regards to total captures (figure below).
Results from this test led us to try and get up a quick field trial. However, all the clear traps had to be handmade and only a limited amount of replicates were completed by the flight season.
2011 Summer Clear Trap Field Trial
Objective Test the clear funnel trap Other Clear Funnel Test Wall prototype in the 3% 2% field during Sirex No flight to see if Response there is improved detection efficacy. 9% Black Trap 9% Floor 51% Clear Trap 26% 91
Methods There were 9 separate field site locations. Each site had 2 clumps of two trees that were girdled in June. Each girdled clump had 4 traps: a clear funnel trap and a black funnel trap on each tree (see picture below). Traps were checked weekly.
Results There were a total of 49 S. noctilio captured in the black funnel traps and 77 caught in the clear funnel traps. There were a total of 54 siricids (including S. noctilio) in the black funnel traps and 105 siricids (including S. noctilio) in the clear funnel traps. A t-test was used to compare the clear trap to the black trap for both S. noctilio alone and all siricids (including S. noctilio). Even though clear funnel traps out- caught black funnel traps 1.5:1, there was no statistical
difference for trap type for S. noctilio (P = 0.40; F1,90 = 0.70) or all siricids (P = 0.12; F1, 111 = 2.46) (see figure below). 2011 Trap Test Mean S. noctilio + Native Siricids Caught per Trap Type
2 Adult
1.5 of
1 Black Trap Clear Trap 0.5 Captures
Woodwasps 0 Mean S. noctilio SNOC+Natives
2011 Summer Long Term Stand Study
Objective Re-examine field sites from 2007 to compare trap catches from that year to what the infestations are today.
Methods We returned to 8 of the original field sites from 2007 and replicated the June girdle and trap tree methodology from that year. We chose these specific sites because they were the only sites in the years since that we had not returned to and used in other studies. A clump of 3 trees were girdled, one trap was placed on each tree at 20ft (see picture left). Traps were checked bi-weekly, both in 2007 and in 2011.
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Results Data from only 6 of the field sites are presented here because 2007 data for 2 of the sites has not been made available yet. Though we had limited number of replicates for this study, the few sites we were able to compare had picked up the same relative number of S. noctilio in 2011 as in 2007. Only one site had a noticeable increase in trap captures. The site was located in Pompey, NY. In 2007 we had captured 4 S. noctilio in the traps for the June girdled clump, in 2011 we captured 15 (see table below).
2007 Date 2011 Date Sites 7/8 7/23 8/6 8/20 9/4 9/17 7/11 7/25 8/8 8/22 9/5 9/19 Douglaston Manor 0 2 0 0 0 0 0 0 1 0 0 0 Douglaston Manor 0 0 1 0 0 0 0 0 0 0 0 0 Rt. 69 0 0 0 0 0 0 0 0 0 0 0 0 Rt. 48 0 0 0 1 0 0 0 0 1 0 0 0 Phillips 1 3 0 0 0 0 0 1 0 0 0 0 Walbridge 0 4 0 0 0 0 7 5 2 1 0 0
The table below is a description of each site’s stand attributes, Sirex noctilio activity, and emergence ratios as determined in 2007. Total stand Sirex killed Number Host Site Average Trees BA BA % of dead of Sex ratio of pine BA Sirex species number DBH (ha) (m2/ha) (m2/ha) killed killed S. noctilio/site By Sirex trees (ha) (male : female) Doug. Red pine Manor 30.9 894 49.2 0.93 51.7 16 1:01 Doug. Red pine Manor 22.3 1219 37.7 1.6 41.7 81 1.6 : 1 Red pine Rt. 69 23.9 1202 49.5 0 0 0 2:01 Red pine Rt. 48 21.4 1219 48.5 0 0 0 3.8 : 1 Red pine Phillips 27.9 926 51.8 0 0 0 3.7 : 1 Scots pine Walbridge 22.9 1040 38 0.9 16.4 49 4.6 : 1
2011 Survey of Connecticut, New Jersey, and Pennsylvania
Objective Survey states likely to have spreading S. noctilio populations from NY.
Methods We set up girdled trap trees at 3 sites in CT (near the positive trap find), 3 site locations in NJ (one in the southern part of the state in the pine barrens, 2 in the northern part of the state), and 1 site in PA along the Delaware National Water Gap. We placed two traps per each girdled 93
tree. We girdled the trees in early June. We returned to the sites in July to check the traps. We were due to return again in Sept. for another trap check, with a take down in Oct. but circumstances did not permit that yet.
Results There was no positive trap capture during the July trap checking trip. None of the trees in CT or PA showed any symptoms of attack on the trees at that point. Trees at the site in southern NJ had resin beads, but these were probably due to SPB or other bark beetles. Trees in that forest were definitely stressed and will be a prime location for S. noctilio attack if/when it does occur. One of the northern NJ field sites also had several trees with resin beading and brown decaying crowns from naturally stressed trees in the stand where our trap trees are. Unfortunately we were unable to return to these field sites to conduct a final trap check for the season. We are hoping to be able to return in January in order to fell suspect trees and bring back to our laboratory for rearing.
Summary of Sirex research results 2011-2012
Improved Lindgren Funnel Trap Study (2011-2012)
Background One of the main objectives of the program was to determine the most efficient trap type for Sirex noctilio detections. During the winter of 2011 we designed a laboratory observation chamber in which wasps were released and watched as they approached different trap types. Consequently, we found that S. noctilio tended to avoid and fly around the dark silhouetted traps. We designed a clear Lindgren funnel to test against the black Lindgren funnel and found that the woodwasps were more often being trapped in the clear funnels (26%) than the black funnels (9%). With this, we hand made dozens of clear Lindgren funnel traps and set up a field trial during the summer of 2011 and 2012.
Methods In 2011 we had 9 field site replicates and in 2012 we had 10 field site replicates. We were limited by the time consuming nature of the number of traps we could hand 94
make. Each field site replicate consisted of 4 girdled trap trees. A group of two trees would be girdled side by side, and then approximately 50m away another group of two trees would be girdled. A chemical girdle was applied in early June. Each tree contained two traps, one black and one clear Lindgren funnel trap, placed at approximately 20ft high up the bole of the tree. In total, there were 36 traps of each type deployed in 2011 and 40 of each type deployed in 2012 (76 total for the combined 2 years of each trap type).
Results. In 2011, 49 S. noctilio were caught in the black traps and 77 in the clear traps. The clear funnel trap out-caught the black funnel trap 1.5:1. In 2012, 11 S. noctilio were caught in the black traps and 37 were caught in the clear traps, with the clear traps out-catching the black traps 3.4:1. With the two years of field work combined, there were 60 S. noctilio caught in black traps and 114 caught in clear traps. The clear traps out-performed the black lindgrens 1.9:1. While there is improved trap capture observed with the clear funnel trap, statistically we still found no significant difference (t-test, P = 0.24), most likely due to low replication in the field.
Lindgren Trap Type Field Test 2011‐2012
77 2011 49
37 Clear 2012
Year 11 Lindgren Black 2 YRS 114 Lindgren Combined 60
050100150 Total S. noctilio Captured per Trap Type
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Mean and SEM Values for
Lindgren Trap Type Test 2011‐2012
Black Type
Clear Trap
0 0.5 1 1.5 2 Mean S. noctilio Caught Per Trap Type Small bars represent SEM 19 replicates; 76 clear traps vs. 76 black traps P = 0.24 96
Clear IPM Test
Twenty four trees were set up over ten different geographic locations. Each tree had a clear IPM and a black Lindgren funnel trap placed at about 6 ft on the bole. Each trap had a lure attached to it. Traps were checked every week from July through September.
Two S. noctilio were caught in the clear IPM traps and 2 S.noctilio, 4 Urocerus spp., 1 Tremex, and 1 S. nigricornis were caught on the black Lindgren funnels. There are not enough data to draw any conclusions or run any statistical analysis on from this study.
Emergence from Connecticut, New Jersey, and Pennsylvania Survey
Last year (see 2011 report) we created trap trees for survey in CT, PA, and NJ. This winter we returned to those sites and felled trees to bring back samples for emergence. We had not caught any S. noctilio in the detection traps accompanying the trees, however, approximately 10 siricid species emerged from material brought back from Stokes State Forest in Northern NJ. Those siricids resemble male S. noctilio, however an official confirmation of the identification has not been performed yet. I sent notification of the possible new state record to George Nelson (NJ SPUD), Vic Mastro and Dave Lance in an email dated 6/14/2012. I gave the specimens, kept frozen and then preserved in ethanol to Vic Mastro Oct. 22, 2012. 97
Trapping and survey studies 2006-2008
Joseph Francese
Girdle technique study (USA) Joe Francese
Objective To determine the optimal girdling technique to increase S. noctilio trap catch and host tree colonization.
Results
Girdle technique – trapping data
1.2
1.0 Mean S. noctilio 0.8 caught / Trap (n = 0.6 30) 0.4
0.2
0 Control Garlon 3A Banvel Mechanical
(tricoplyr) (dicamba) S. noctilio 6 14 27 8 Other Siricidae 0 3 5 4
p = 0.002, F3, 105 = 5.235
Semiochemical lure study (USA) Joe Francese
Objective To test different artificial lure combinations as attractants for detection traps. Lure treatments included 70/30 alpha/beta pinene, 30/70 alpha/beta pinene, 33/33/33 alpha/beta/carene, 100 % alpha pinene, 100% pinene, 100% carene, and different mix components.
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Results
Mean Number of Sirex noctilio Captured in the Semio Lure Study (Summer 2007)
2.5 Semiochemical Dose Study (USA) Semiochemical Study (South Africa) 2 Semiochemical Study (South America) Mark-Release-Recapture Study (USA) 1.5 Misc. Canopy Observations (USA) Host-Mortality Study (USA) 1 Controlled Nematode Release Study (USA) 0.5 Commodities Studies (USA) PennState Studies (USA)
Mean Number Caught Mean Number 0 Cornell Studies (USA) Control 70/30 30/70Carnegie 33/33/33 Studies 100a (USA) 100b 100c Mix A Mix B -0.5 U. of Georgia Studies (USA) USFSTreatments Studies (USA) (10 replicates performed across 10 field sites)
Semiochemical dose study (USA) Joe Francese
Objective To test different dose combinations of 33/33/33 alpha/beta/carene as an artificial attractant for detection.
Results
Mean Number of Sirex noctilio Captured in Semio Dose Study (Lure = 33/33/33 alpha/beta/carene)
3
2.5 2
1.5
1
0.5 Mean Number Caught Number Mean 0 Control 1x 3x 1/3x -0.5
Treatments (10 replicate performed across 10 field sites) 99
Semiochemical study (South Africa) Joe Francese
Objective Same as above.
Results 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Number of female Number of Sirex waspsper trap.
Lure
Sirex Blend_1 New Sirex Blend 1 full UHR
Sirex Blend_2 New Sirex Blend 1 17ml polybottle
Sirex Blend_3 New Sirex Blend 1 (500 mg/day) a-pinene_1 75% (+) a-pinene in polybottle a-pinene_2 75% (+) a-pinene (625 mg/day) a-pinene_3 75% (+) a-pinene (25 mg/day) a,b-pinene_1 75% (+) a-pinene b-pinene (1:1) half UHR a,b-pinene_2 75% (+) a-pinene b-pinene (1:1) 2x17ml polybottle a,b-pinene_3 75% (+) a-pinene b-pinene (1:1) 50 mg/day
Control Blank trap (no lure)
3-carene 3-carene half UHR
100
Semiochemical study (South Africa-2006/2007)
Total Number of Female Sirex noctilio Caught Per Treatment
658 642 700
555 600
500
400 327 December January 300
200 176 149
Number of Females Caught Females of Number 89 100 77
0 6-blend a-pinene b-pinene Control Lure Treatments
Figure 12. The total number of female Sirex noctilio caught per lure treatment in South Africa.
Semiochemical study (South Africa-2006/2007)
Mean Number of Female Sirex noctilio Caught Per Treatment
90 44 80 43 70 37 60 22 50 December 40 January 30 12 10 20 5 6 10 Mean Number of Females Caught Females of Number Mean 0 6-blend a-pinene b-pinene Control -10 Lure Treatments
Figure 13. The mean number of female Sirex noctilio caught per pure treatment in South Africa. Small bars represent standard deviation. A Tukey-Kramer HSD post-hoc comparison showed beta-pinene was significantly different from the 6-blend for females caught in January (P = 0.0204; F = 3.53).
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Semiochemical study (USA-2006)
2006 Semiochemical Lure Comparison -- NY
25
B 20 =7)
n 15
S.E.) ( 10 (+
5 A A A Mean no. siricids no. Mean trap per caught A A A 0 Blank 70% / 30% / Five Nonanal Nonanal Trap Tree** 30% 70% Component* + 70% / 30% Lure
* -- 63% (+)-a-pinene, 30% b-pinene, 3%-limonene, 3 % myrcene, 1% carene ** -- Trap trees were girdled on June 18 and 19, 2006
Figure 14. The mean number of siricids caught per lure treatment in Syracuse, NY (2006). Small bars represent standard error mean.
102
Mechanical girdle study 2008
Joe Francese
Objective To determine the optimal time for mechanically girdling a tree in order to attract the most S. noctilio to the trap tree.
Results
Mechanical Girdle (Timing) A 4
3.5
3
2.5
caught / trap (n = 10) (n / trap caught 2 B 1.5 S. noctilio 1 B B B 0.5 Mean No. No. Mean
0 6 Weeks 3 Weeks Flight Two Weeks Control
Prior to Flight Post Flight
103
Garlon dose study (USA) 2008 Joe Francese
Objective To determine if Garlon 3A, an herbicide that is commonly used in the U.S. could be an alternative to Dicamba since the later is not registered in the U.S. Additionally, we tested to see which dose would be most effective.
Results
Garlon Dose Study
6 B = 10)
n 5 AB
4 AB 3 caught per trap ( per caught AB
2 S. noctilio
1 A
Mean No. No. Mean 0 Control Banvel Garlon (Label) Garlon 2X Garlon 4X* (Standard)
* -- Garlon 4X does has the same amount of A.I. as the Banvel
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Trap design study (USA) 2008 Joe Francese
Objective To test which trap type is most effective in for capturing S. noctilio. We tested a 12- funnel lindgren, a crossvane, and a mesh panel that had insect glue spread on it.
Results
Trap Design Study
6
A
5 = 12) n
4
AB
3 caught /caught trap (
2
S. noctilio B No.
1
0 Funnel X-Vane Panel
105
Semiochemical Study (USA)
Damon Crook
Objective To test different artificial lures for attractiveness to S. noctilio in order to determine if lure could be added to a trap instead of creating the typical trap tree. We tested 70/30 alpha/beta pinene, manuka oil, phoebe oil, and scots pine oil, as well as a control.
Results
Semiochemical Study (Essential Oil Comparison)
1.4 A A 1.2
1 A
0.8 A caught / trap (n = 10)
0.6 A
S. noctilio S. 0.4
0.2 Mean No. Mean 0 Control a/b pinene Manuka oil Phoebe oil Scots pine oil
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III. BIOLOGICAL AND NATURAL CONTROL Sirex nematodes: controlled releases and lab experiments
Dave Williams
Field experiments
A. Overall Materials and Methods
B. 2006 controlled release
C. 2007 controlled release
D. 2008 controlled release
E. 2009 controlled release
F. 2010 controlled release
G. 2011 controlled release
H. Sirex population trends
I. Parasitism by Ibalia
Lab experiments
A. Mass rearing
1. Effects of temperature
2. Effects of growth time, inoculum size, etc
B. ─ I. Miscellaneous experimental work
J. Nematode survival in breathable baggies
K. Quarantine inoculations of Kamona nematodes into billets containing S. noctilio
L. Quarantine inoculations of Kamona nematodes into billets containing North American Sirex species.
M. Inventory of nematode and fungus cultures 107
Field experiments during 2006 – 2012
A. Overall Materials and Methods
Sites We used study sites in New York, Pennsylvania, and Michigan over the course of the six- year study, and generally changed locations from year to year to avoid overexploitation, as several studies often shared an individual site. We set up 4-6 sites annually in largely unmanaged plantations of Pinus sylvestris (Scots pine) and P. resinosa (red pine) (Table 1). We added a plantation of P. strobus (eastern white pine) in 2009. Locations of the primary sites in New York and Pennsylvania are shown in Fig. 1, where A ̶ F are 2006 ̶ 2010, respectively.
Nematode and fungus cultures: Because we had not yet begun mass rearing nematodes in the fall of 2006, releases in that year used “bulk” nematodes of the so-called “Kamona strain” purchased directly from Ecogrow, the licensed commercial producer in Canberra, Australia. In subsequent years, we mass reared Kamona nematodes at the Otis Laboratory in 500 ml culture flasks on North American isolates of Amylostereum areolatum fungus cultured on an autoclaved mixture of brown rice and wheat berry, according to methods outlined in the SOP by Calder and Bedding (2002). In 2008, we discovered an “indigenous” strain of B. siricidicola, It was already present in our sites and presumably was introduced with S. noctilio. We dubbed it the “North American strain” and reared and released it in 2009. We used two fungus strains (designated “OAA” and “Hajek”) for mass rearing, both of which were isolated from mycangia of S. noctilio females collected in the field in central New York (Table 1).
Releases Initially we thought to time the releases by extrapolation from the Australian experience, which indicated a time frame of November – December. However, considering the large climatic differences between Australia and North America — the former having mild winters and the latter, very cold winters — we decided to move the date forward to a warmer part of the North American season and release in late September (Table 1). This seemed a reasonable compromise to increase the likelihood that Sirex would be in the large larval stages desirable for infection while temperatures would still be high enough for nematodes to establish and begin propagating in the trees. Our first release, in 2006, was delayed until early November by importation and permit issues. Because temperatures were low by that time, we suspect that the Kamona nematodes were unable to establish in trees (Table 1). We used naturally struck trees (i.e., as opposed to trap trees) for all releases to ensure that the trees would be infested. We chose trees for inoculation primarily by the presence of resin beads. If trees met that criterion, we then examined the crowns. We preferred trees with red or brown needles, but also noted and accepted suppressed trees with green needles. We rejected trees without needles or with Sirex adult emergence holes. We then felled the trees and sealed the cut ends with Waxlor® or AnchorSeal®.
Inoculation Harvesting of nematodes from culture flasks is detailed in Calder and Bedding (2002). To summarize briefly, we washed the nematodes from flasks with tap water, counted 108 them, and placed them in breathable baggies (Kordon®) in aliquots of 1 million per 10 ml for transport to the release sites. At a release site, we mixed the contents of one baggie with 5 g of polyacrylamide gel (Alcosorb400® or AquaRocks®) and 500 ml of water, whisked until the water was absorbed, and decanted the mixture into plastic squeeze bottles. In the 2006 and 2007 releases, we used the nematode dosage recommended by Ecogrow: that is, 1 million per batch of inoculum to treat 10 trees. However, we seemed to be using less inoculum per tree than was Ecogrow. We compensated for this apparently lower density of nematodes in 2008 and 2009 by increasing the dosages (Table 1). To prepare the trees, we punched 50 inoculation holes at 15 cm spacing along the lower 7.5 m of the tree trunk using a South African punch hammer. We then injected gel inoculum into each hole using the squeeze bottle and pressed the gel into place with a fingertip. The trees were left on the ground exposed to ambient conditions during the winter. Because we were unaware of the presence of the North American nematode strain until 2008, we assumed that all nematode infections resulted from the Kamona inoculations and did not sample control trees. In 2008, we felled a few trees at each site that were infested but not inoculated and reared them to check for nematodes. In 2009, we created real controls by felling a third or a half of the trees at each site and injecting them with pure gel (i.e., not containing nematodes) (Table 1).
Sample collection We collected billet samples from inoculated trees during late winter to early spring (March – May). We cut three billets from each tree: one at the top and another at the bottom of the inoculation zone and a third at approximately the center of the zone. Remaining woody material, including the tree crown, was chipped to 2.5 cm dimension so as to kill S. noctilio larvae and thus prevent any escape of nematodes into the environment. We returned the billets to the CPHST laboratory in Syracuse, New York, where we sealed the cut ends with Waxlor® or AnchorSeal® and placed them in screened barrels for adult emergence. One to three months later, all emerging insects were collected and stored in a freezer before identification and dissection. All siricids as well as parasitic Hymenoptera, cerambycids, and bark beetles were dissected to assess infection by nematodes. Dissections of siricids, primarily S. noctilio, ranged widely, from < 300 to >3,000 per year (Table 1).
Analysis Population size of S. noctilio and the hymenopterous parasitoids was expressed as density per m3 of wood. Because we did not record diameters of each of the three billets from each tree, we used the dbh as an aggregate estimate of diameter for a sample. Thus, sample volume was estimated as,
volume = π· (dbh/2)2 · length
where length = 2.04 m, the total length of the three billets. Of course, this approximation will somewhat underestimate insect density, but we assume that it will generally be consistent across sites and tree species.
109
B. Controlled release 2006
Objectives
Test the Australian inoculation technique
Assess the establishment of Kamona nematodes in North American and European pine species
Assess the establishment of Kamona nematodes under winter conditions in North America
Results
All study sites in this initial release were in pine plantations in New York: four with Scots pine and one with red pine (Table 2). The tree infestation rate by Sirex was generally high, ranging from 67% to 97% of the trees infested at a site and suggesting that our ability to identify trees attacked by Sirex was good. Of the trees infested, the numbers containing nematode-infected Sirex were generally low, less than half in all but one case. Note that we do not report infection density and rates for the Chard and Green sites because not all Sirex at those sites were dissected (Table 2). In addition, those values for the remaining three sites should be taken cum grano salis because some samples had dried or became moldy so that nematode infection was difficult to assess. As mentioned above, we speculate that the Ecogrow nematodes that were used for inoculation probably did not establish. Thus, the low rates of infection — in the 2-3% range — may reflect the activity of the North American strain already present (Table 3).
At almost 36% females, the Sirex population in red pine had, by far, the highest sex ratio, although it was not significantly different from those at two of the Scots pine plantations. The parasitism rate by Ibalia was uniformly high, ranging from 18-23%, with no significant differences among sites (Table 4). By contrast, the parasitism rate by Rhyssa peaked at 6.3% in one Scots pine plantation but was less than 1% at the other four plantations.
C. Controlled release 2007
Objectives
Test the Australian inoculation technique
Assess the establishment of Kamona nematodes in North American and European pine species
Assess the establishment of Kamona nematodes under winter conditions in North America
Results
Releases were made in four New York sites, including one red pine and three Scots pine plantations (Table 5). The infestation rate at the sites varied from 70% to 94%, again attesting 110 our ability to identify Sirex signs. Sirex populations in 2007 exhibited the highest densities in the four years of releases, with the three sites on Scots pine averaging over 1,000 individuals per m3 of wood (Table 6). Note that Sirex density was quite low by comparison. We speculate that the woodwasp had only recently invaded this site in northern Pennsylvania. The infection rate of sampled trees ranged widely, with 23% to 78% containing B. siricidicola. The infection rate for Sirex individuals in a tree also varied considerably, from as low as 2.2% up to 40.8% (Table 7). These higher infection rates overall suggest that the Kamona strain nematodes were able to establish populations in the 2007/8 season, presumably because of the warmer weather earlier in the season when they were released. As in 2006, the Sirex sex ratio was highest in the red pine site, although not significantly different from the values in two Scots pine plantations (Table 7).
Parasitism rates by Ibalia were relatively high, ranging from about 16% to 26%, and with no significant differences among the study sites. Also as in 2006, percentage parasitism by Rhyssa was less than 1% with the exception of one site (Table 7).
Although over 3,000 S. noctilio adults emerged in our barrels in 2007, just five S. edwardsii and sixteen S. nigricornis did so (Table 8). Over half of the total number emerged from red pine samples collected at Highland State Forest. One of the individuals of S. edwardsii was infected, by a nematode that resembled B. siricidicola, whereas two individuals of S. nigricornis were infected by nematodes that were very unlike B. siricidicola.
A small nematode release was also made in Michigan in 2007, in Wolcott Mills Metropark, Macomb County, where S. noctilio was first trapped in the state. Five Scots pines with obvious resin beads were inoculated with Kamona strain nematodes on 22 October 2007. Just 15 S. noctilio adults emerged in the spring, none containing nematodes. The failure to establish in trees was probably a result of low temperatures on the late inoculation date.
D. Controlled release 2008
Objectives
Investigate the “nonsterilization problem”, in which juvenile nematodes do not enter Sirex eggs
Determine if it is an artifact of rearing overwintering Sirex at lab temperatures
Results
Releases were conducted at five sites, including four plantations of Scots pine in New York and one of red pine in Pennsylvania (Table 9). Sample sizes ranged from seven to 27 trees and the woodwasp infestation rates were generally high, ranging from 57% to 86% (Table 9) . The infection rates of sampled trees by S. noctilio containing nematodes were lower, varying from 36% to 55%. Sirex densities were down from the peak in 2007, but were still high and relatively 111 uniform across the sites. The Sirex sex ratio ranged widely, and as observed in the previous two seasons, it was highest in the red pine plantation (Hills Creek SP), although not significantly different from those in two Scots pine plantations (Table 10) .
The density of nematode-infected S. noctilio was remarkably similar across sites, not differing significantly at the 0.05 level (Table 10). Nonetheless, the infection rate varied considerably among the sites, from 6.1% to 40.1% (Table 11). The percentages of Sirex eggs that were sterilized (i.e., that had juvenile nematodes inside) were low at the four New York sites, ranging from 0% to 25%. By contrast, 93% of the Sirex females were sterilized at the Pennsylvania site (Table 11). Subsequent samples taken at that site did not find any Sirex containing nematodes. Thus, we speculate that all infection at Hills Creek was a result of the activity of the released Kamona strain nematode, which is inferred to be a sterilizing strain. Conversely, the low level of sterility at the New York sites suggests that we are observing primarily the activity of the North American strain. It should be noted in passing that, having become aware of the presence of the North American strain in 2008, we took samples of 1-2 trees at each site from trees with resin beads to provide controls for the infection rates. No nematode-infected S. noctilio emerged from those trees.
The parasitism rate of Ibalia was more variable in 2008 than in previous years, but was still consistently high (Table 11). As in other years, the rate was highest in the red pine plantation, and the parasitoid sex ratio was also highest there.
Our billet samples in the 2008 release were also used to address a possible cause of the nonsterilization problem. The failure of nematodes to enter the host egg arises because of a lack of synchrony between the development of juvenile nematodes and the hardening of the chorion of the host egg. Essentially, the juveniles are released from the mother nematode after the egg shell has begun to harden and are unable to penetrate it. Because many processes in invertebrates are affected by temperature, we hypothesized that the observed asynchrony may have been an artifact of the relative lengths of time at outdoor temperatures versus the heated rearing garage. To test this idea, we divided our samples from each site into groups of three. We moved the three groups into the garage at different dates: April 3, May 1, and May 29. Our temperature hypothesis was refuted; there was no difference in sterilization over the three dates.
E. Controlled release 2009
Objectives
Investigate the “nonsterilization problem”, in which juvenile nematodes do not enter Sirex eggs
Compare infection rates of the Kamona and New York strain nematodes in North American pines 112
Compare infection rates by the Kamona nematode strain when mass-reared on two different New York isolates of the fungus, Amylostereum areolatum.
Results
We continued releases for a second year at Hills Creek State Park, where the Sirex infestation in the red pine stands was the highest among sites at 80% (Table 12). Sirex densities dropped off considerably and were significantly higher at Hills Creek than elsewhere (Table 13). The woodwasp sex ratio was also highest at that site although not significantly so. Percentages of trees having nematode-infested Sirex ranged from 4% to 38% although it must be stressed that these included both inoculated trees and uninoculated controls (Table 12). The infection rates of Sirex ranged from 2% to 35%. Notably the rates at the two Richland sites were significantly different although they were just 500 m apart. As observed in previous years, parasitism rates by Ibalia did not differ significantly between sites.
Comparisons of Kamona inoculated and control trees were made at the Trenton Greenbelt (Table 15). Clearly, the 8% infection rate for the controls indicates the presence of the North American strain, and it is interesting that infection increased only slightly (to 13%) in trees inoculated with Kamonas. Table 16 compares five crown classes of Scots pines ― from green to dead ― at Trenton Greenbelt. Interestingly, Sirex density, sex ratio, and percent parasitism by Ibalia did not differ significantly among the classes.
Nematode strains were compared with a control in the red pine stands at Hills Creek (Table 17). As expected, the control group contained no infected Sirex. Although the difference was not significant, the infection rate by North American nematodes was over four times that by Kamonas. However, nematodes were outside the eggs in all cases. Only one infected female was found overall. Her body cavity was packed with nematodes, but she had no eggs.
Crown condition in the Hills Creek red pine plantations did not affect Sirex density or the Ibalia parasitism rate (Table 18). The only significant differences seen were in the Sirex sex ratio, which was highest in the healthy (green) crown class for no readily apparent reason.
Results comparing the nematode infection rate between strains at the first Richland Scots pine site were not as expected (Table 19). Although the Sirex density did not differ significantly among treatments and control, the only infected woodwasps were found in the control trees, which were not inoculated with nematodes. It seems very likely that these counterintuitive results were due to small sample sizes. Possible effects of the fungal isolate used for mass rearing of nematodes were investigated at the second Richland plantation (Table 20). Sirex population density and that of infected Sirex did not differ significantly among the isolates tested. However, the nematode infection rate was highest with the Hajek isolate and zero with the Otis isolate, with the control intermediate and unexpectedly positive. Again, small sample size may be to blame. 113
F. Controlled release 2010
Objectives
Investigate the effect of release timing on the infection rate of Kamona strain nematodes in red and Scots pines
Compare the infection rates of Kamona strain nematodes in red, Scots, and white pines
Compare the infection rates and sterilization of Sirex eggs by the Kamona and New York strains inside Scots pine
Results
The controlled releases in 2010 differed from previous releases (Tables 21). Three sites were located in Pennsylvania whereas only one was in New York and all were red pine plantations. Experiments at all four sites compared a Kamona treatment with an untreated control. Unfortunately, the rate of infestation of trees by woodwasps was generally low, ranging from 28% to 65%. The percentages of tree containing infected Sirex wer even lower, ranging from zero to 24%, despite the fact that about half of the trees at each site had been inoculated. At the Ole Bull and Bell Hill sites, infection rates conformed with expectations; rates were lower in the controls than in the treatments. However, at Hills Creek, the infection rate was twice as high in the controls as the treatments (Table 22). Nematodes were not found inside eggs at any of the sites, suggesting that either the Kamona strain did not establish or that it is not able to sterilize the North American strain of S. noctilio.
Percent parasitism by Ibalia varied widely among sites, ranging from 2.6% to 39.5% (Table 22). As observed previously, the parasitism rate by Rhyssa was generally low, ranging from 0% to 11.1%.
Native Sirex nigricornis were also found at two sites, with numbers exceeding those of S. noctilio in one case (Table 22).
G. Controlled release 2011
Objectives
Investigate nematode infection rate and egg sterilization in S. noctilio and North American Sirex species
Compare infection rates of the Kamona and New York strain nematodes in North American pines
Evaluate efficacy of herbicide girdled trap trees and the date of their inoculation on the nematode infection rate 114
Results
Trap trees for Sirex nematode release. The objectives of this project were to investigate the effect of the time of nematode inoculation on the rate of infection and sterilization and to evaluate trap trees for implementing biological control using nematode. All trees, which grew on four sites in central New York, were girdled chemically with Banvel® on 12 July 2011. Even numbers were inoculated with Kamona nematodes in gel or with gel alone (i.e., as controls) while standing (i.e., to prevent deterioration of the bark and wood over winter). Ten red pines were inoculated on 12 July, ten red pines and ten Scots pines on 24 August, and twenty red pines on 5 October. The trees were felled, and sample billets were cut in March and April of 2012. Billets were stored in screened barrels at the Syracuse Lab, and emerging Sirex adults were collected and kept in a freezer before dissection at the Otis Lab.
The results were disappointing. Part of the problem was that many of the trees did not have Sirex, in the worst case having no infested trees out of 10 (Table 23). The trends followed expectations. Percent infection was lowest at the earliest date, when the herbicide would not have much time to act, highest at the middle date, and lower again at the last date because lower temperatures hindered nematode establishment (Table 24). Again, the lack of significance can probably be attributed to the low sample sizes. The results for effect of treatment versus control on the nematode infection rate were likewise inconclusive (Table 25). One might have expected results like those at Hills Creek, with rate of the control at zero and that of the nematode inoculation at a high level (46%). However, the rates for the trap trees were not significantly different, and moreover, the infection rate for the controls was slightly higher than that for the nematodes. In these cases, the results undoubtedly were confounded by the presence of the North American nematode strain. 115
Scots pine
red pine
Fulton, NY First catch 116
Fig. 1. Nematode release locations in New York and Pennsylvania in 2007 (A), 2008 (B), 2009 (C), 2010 (D), and 2011 (E). Sirex noctilio was first trapped in Fulton, New York in 2004. The longest time series for an individual site was at Hills Creek State Park in Pennsylvania from 2008 to 2011.
Table 1. Summary of controlled release experiments with the Kamona strain of Beddingia siricidicola in New York and Pennsylvania during 2006-2012.
Year 2006/7 2007/8 2008/9 2009/10 2010/11 2011/12
Sites 5 NY 4 NY, 1 MI 4 NY, 1 PA 4 NY, 1 PA 1 NY, 3 PA 3 NY, 3 PA
Total trees 91 95 85 105 84 102 6-7 Nov 1-3 Oct 23-26 Sep 21-24 Sep 23-26 Sep Early Jul to Release date 2006 2007 2008 2009 2010 early Oct Uninoculated no no few ⅓ of total ½ of total ½ of total controls? Kamona & Nema strains Kamona Kamona Kamona North Kamona Kamona American Nema dosage 1× 1× 3× 2× 1× 2× NY wild NY wild NY wild NY wild NY wild type type Fungus strains Australian type type (OAA) type (OAA) (OAA, (OAA, (Hajek) Hajek) Hajek) Establishment? unlikely likely likely likely likely likely
Total Sirex 2,224 3,024 1,423 429 356 292
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Table 2. Characteristics of the five New York study sites used in the 2006 controlled release
Site Chard Excavation Green Lopitz Rodman
Town Cazenovia Palermo Palermo Parrish Pompey
County Madison Oswego Oswego Oswego Onondaga N 42.89780 N 43.37412 N 43.31987 N 43.41405 N 42.9635 Coordinates W 75.89126 W 76.30727 W 76.28429 W 76.03961 W 75.95603 Pine species red Scots Scots Scots Scots
Mean DBH (cm) 19.3 19.0 16.3 17.8 17.2
Mean sample vol (m3) 0.062 0.060 0.047 0.053 0.049
No. trees sampled 22 9 11 18 30
No. trees with Sirex 20 6 10 12 29
No. trees with nemas — 3 — 4 16
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Table 3. Mean numbers (and 0.05 confidence limits) of S. noctilio, nematode-infected S. noctilio, I. leucospoides, and Rhyssa species emerged per m3 wood in 2006 controlled release and mean Sirex sex ratios. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level.
Sirex sex ratio (% Ibalia density Rhyssa density Site Sirex density (per m3) females) (per m3) (per m3)
302.4 b 35.9 a 131.4 bc 4.9 a Chard (167.1,437.8) (20.9,52.4) (48.7,214.1) (-1.7,11.5)
182.2 b 8.8 b 88.1 c 44.0 a Excavation (–42.8,407.3) (0.4,38.4) (-21.3,197.5) (-20.3,108.2)
703.0 a 15.4 b 258.7 ab 28.9 a Green (198.2,1207.9) (5.3,29.5) (17.4,500.0) (-27.5,85.3)
290.6 b 18.5 ab 147.0 c 28.0 a Lopitz (87.1,494.0) (8.0,32.1) (-1.1,295.1) (-28.9,85.0)
913.5 a 19.5 ab 291.2 a 0.0 a Rodman (655.9,1171.2) (16.7,22.6) (215.9,366.6) (0.0,0.0)
119
Table 4. Mean percentages of S. noctilio infected by B. siricidicola and parasitized by I. leucospoides and Rhyssa species in the 2006 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back- transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level.
Site Percent parasitism by Ibalia Percent parasitism by Rhyssa
17.9 a 0.6 b Chard (9.4.28.50 (0.0,2.8)
19.6 a 6.3 a Excavation (4.4,41.9) (0.2,27.1)
23.2 a 0.3 b Green (12.2,36.4) (0.1,2.1)
22.0 a 0.2 b Lopitz (4.2,48.5) (0.1,1.2)
21.5 a 0.0 b Rodman (15.4,28.3) (-,-)
120
Table 5. Characteristics of four New York study sites used in the 2007 controlled release
Highland State Site Ventre Schoeller Spafford Forest
Town Fabius Manlius Richland Spafford
County Onondaga Onondaga Oswego Onondaga
N 42.82244 N 42.99006 N 43.55951 N 42.80559 Coordinates W 75.93418 W 75.00350 W 76.04728 W 76.25930
Pine species red Scots Scots Scots
Mean dbh (cm) 16.3 15.2 10.3 15.0
Mean sample volume (m3) 0.044 0.037 0.018 0.037
No. trees sampled 22 17 40 18
No. trees with Sirex 17 15 28 17
No. trees with nematodes 9 12 9 14
121
Table 6. Mean numbers (and 0.05 confidence limits) of S. noctilio, nematode-infected S. noctilio, I. leucospoides, and Rhyssa species emerged per m3 wood in 2007 controlled release and mean Sirex sex ratios. Data were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level.
Density of infected Sirex density Sirex sex ratio Ibalia density Rhyssa density Site Sirex (per m3) (% females) (per m3) (per m3) (per m3)
158.3 b 39.8 a 32.9 b 94.0 b 11.8 a Highland SF (74.7,241.9) (19.2,62.6) (-1.7,67.5) (20.8,167.2) (0.33,23.2)
1266.3 a 28.6 ab 486.5 a 514.0 a 14.2 a Ventre (825.6,1707.0) (21.9,35.8) (176.7,796.3) (304.1,723.9) (-12.5,5,40.8)
1093.0 a 33.3 a 138.5 b 458.8 a 21.0 a Schoeller (530.2,1655.7) (20.3,47.7) (15.9,261.2) (223.5,694.2) (-0.6,42.5)
1031.4 a 13.3 b 549.5 a 413.5 a 21.6 a Spafford (599.1,1463.7) (8.0,19.7) (265.9,833.1) (187.2,639.7) (-20.5,63.6)
122
Table 7. Mean percentages of S. noctilio infected by B. siricidicola and parasitized by I. leucospoides and Rhyssa species in the 2007 controlled release. Data were arcsine square root transformed for analysis, and means and confidence limits were then back- transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05
Percent infection by Percent parasitism by Ibalia sex ratio (% Percent parasitism by Site nematodes Ibalia females) Rhyssa
9.6 bc 25.5 a 65.5 a 2.6 a Highland SF (1.0,25.5) (9.8,45.6) (43.4,84.6) (0.0,11.6)
23.3 ab 24.1 a 41.2 a 0.1 a Ventre (9.0,41.8) (16.9,32.2) (22.9,60.7) (0.0,0.3)
2.2 c 22.9 a 62.4 a 0.4 a Schoeller (0.2,6.5) (12.9,34.8) (44.5,78.7) (0.0,2.1)
40.8 a 16.2 a 49.8 a 0.1 a Spafford (20.7,62.6) (8.3,26.3) (27.3,72.2) (0.0,0.5)
123
Table 8. Total numbers of nontarget native Sirex species per site in the 2007 controlled release
Site Sirex edwardsii numbers Sirex nigricornis numbers
Highland SF 4 7
Ventre 0 0
Schoeller 1 8
Spafford 0 1
124
Table 9. Characteristics of the New York and Pennsylvania study sites used in the 2008 controlled release
Hills Creek State Site Rodman Route 69 West Monroe Zink Park
Town Charleston Pompey Parish West Monroe LaFayette
County, Tioga, Oswego, Onondaga, NY Oswego, NY Onondaga , NY State PA NY
N 41.80393 N 42.96359 N 43.39664 N 43.34969 N 42.90666 Coordinates W 77.19140 W 75.95603 W 76.03198 W 76.08673 W 76.05862
Pine species red Scots Scots Scots Scots
Mean dbh (cm) 12.5 18.1 14.8 17.9 16.4
Mean sample volume (m3) 0.027 0.054 0.036 0.052 0.046
No. trees sampled 26 14 27 7 11
No. trees with Sirex 17 8 19 6 8
No. trees with nematode 13 5 10 3 6
125
Table 10. Mean numbers (and 0.05 confidence limits) of S. noctilio, nematode-infected S. noctilio, and I. leucospoides emerged per m3 wood in 2008 controlled release and mean Sirex sex ratios. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level.
Sirex sex ratio Density of infected Sirex Site Sirex density (per m3) Ibalia density (per m3) (% females) (per m3)
613.2 a 57.4 a 108.7 a 345.9 a Hills Creek (317.7, 908.7) (38.8, 75.0) (30.5, 186.8) (188.6, 503.3)
443.0 a 9.0 b 231.0 a 141.8 ab Rodman (53.6, 832.3) (1.9, 20.5) (-6.7, 468.6) 12.9, 270.7)
414.3 a 29.5 ab 105.2 a 168.6 ab Route 69 (191.3, 637.4) (11.7, 51.3) (12.4, 198.1) (54.8, 282.4)
491.8 a 41.7 a 116.6 a 257.9 ab West Monroe (-207.0, 1190.6) (2.0, 90.7) (-63.4, 296.5) (12.9, 503.0)
229.1 a 7.4 b 124.2 a 94.1 b Zink (38.1, 420.0) (0.5, 21.5) (-18.0, 266.4) (-0.7, 188.9)
126
Table 11. Mean percentages of S. noctilio infected by B. siricidicola, infected and sterilized eggs, emerging females of I. leucospoides, and parasitism by I. leucospoides in the 2008 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level.
Percent infection by Percent infected Sirex w/ Ibalia sex ratio (% Percent parasitism by Site nematodes nemas in eggs females) Ibalia
11.3 ab 92.7 a 57.2 a 40.6 a Hills Creek (3.8,22.2) (53.8,96.0) (40.6,73.1) (28.8,53.1)
24.1 ab 0.8 b 53.2 a 15.2 b Rodman (2.2,59.1) (3.8,13.6) (16.1,88.3) (2.7,35.2)
10.2 b 0.0 b 37.4 ab 24.3 ab Route 69 (2.3,22.9) (-,-) (15.8,62.0) (12.2,38.8)
6.3 b 10.4 b 22.6 ab 28.0 ab West Monroe (0.5,30.0) (14.0,74.5) (1.9,56.5) (3.4,64.3)
40.1 a 25.0 b 12.3 b 21.2 ab Zink (4.3,84.2) (97.5,12.8) (0.0,40.7) (3.4,48.7)
127
Table 12. Characteristics of the New York and Pennsylvania study sites used in the 2009 controlled releases
Hills Creek State Site Greenbelt Schoeller 1 Schoeller 2 Park
Town Charleston Trenton Richland Richland
County, State Tioga, PA Oneida, NY Oswego, NY Oswego, NY
N 41.80393 N 43.25833 N 43.55951 N 43.55951 Coordinates W 77.19140 W 75.19167 W 76.04728 W 76.04728
Pine species red Scots Scots Scots
Mean dbh (cm) 13.8 15.7 9.8 11.3
Mean sample volume (m3) 0.032 0.044 0.016 0.021
No. trees sampled 30 24 24 12
No. trees with Sirex 24 16 6 6
No. trees with nematode 5 9 1 4
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Table 13. Mean numbers of S. noctilio, nematode-infected S. noctilio, and I. leucospoides emerged per m3 wood in 2009 controlled release and mean Sirex sex ratios. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P <0.05) are given in parentheses.
Sirex density Sirex sex ratio (% Density infected Sirex Ibalia density Site (per m3) females) (per m3) (per m3)
292.8 a 30.1 a 28.1 ab 128.7 a Hills Creek (175.0, 410.6) (15.7, 46.7) (-4.8, 61.0) (44.9, 212.4)
22.9 b 6.7 a 6.6 b 10.2 b Richland 1 (0.8, 44.9) (16.0, 64.7 ) (-7.0, 20.1) (-0.6, 20.9)
144.7 b 8.2 a 62.5 a 55.8 ab Richland 2 (8.8, 280.6) (1.9, 43.7) (-0.6, 125.6) (-16.1, 127.7)
137.1 b 19.7 a 31.5 ab 36.3 ab Trenton (63.3, 210.8) (7.3, 36.2) (10.4, 52.7) (-10.4, 82.9)
129
Table 14. Mean percentages of S. noctilio infected by B. siricidicola and parasitized by I. leucospoides in 2009 controlled releases. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P<0.05) are given in parentheses.
Site Percent infection by nematodes Percent parasitism by Ibalia
2.0 b 14.1 a Hills Creek SP (0.0,7.3) (6.1, 24.8)
6.7 b 20.9 a Richland 1 (16.0,64.7) (0.2, 70.6)
35.2 a 11.3 a Richland 2 (0.9,85.0) (0.4, 46.9)
11.6 ab 4.5 a Trenton (2.4,26.6) (0.3, 13.1)
130
Table 15. Mean densities of Sirex and infected Sirex per m3 wood and infection rates for the Kamona strain of B. siricidicola at the Trenton Greenbelt site in the September 2009 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P<0.05) are given in parentheses.
Sirex density Density of infected Sirex Percent infection by Treatment (per m3) (per m3) Beddingia
119.3 a 34.4 a 13.4 a Kamona (57.6, 181.1) (4.7, 64.2) (1.1, 35.9)
172.6 a 25.8 a 8.2 a Control (-47.5, 392.7) (-6.7, 58.2) (0.6, 37.3)
131
Table 16. Mean densities of Sirex per m3 wood, Sirex sex ratios, and Ibalia parasitism rates for several crown conditions at the Trenton Greenbelt site in the September 2009 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P<0.0) are given in parentheses.
Sirex density Crown condition Sirex sex ratio Percent parasitism by Ibalia (per m3) 144.0 a 20.6 a 3.0 a Green (5.4, 282.5) (0.4, 59.5) (0.3, 15.1) 120.6 a 21.4 a 1.3 a Green-red (-53.8, 295.0) (28.8, 99.8) (13.4, 31.9) 178.5 a 11.0 a 9.7 a Red (-22.6, 379.5) (14.5, 76.7) (74.8, 98.9) 100.3 a 24.5 a 7.1 a Dead (12.5, 188.1) (1.2, 83.1) (6.7, 51.2)
132
Table 17. Mean densities of Sirex and infected Sirex per m3 wood and infection rates for two strains of B. siricidicola at the Hills Creek State Park site in the September 2009 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P< 0.05) are given in parentheses. N = 30
Sirex density Density of infected Sirex Percent infection by Treatment (per m3) (per m3) Beddingia 248.1 a 23.3 a 1.7 a Kamona (106.4, 389.7) (-17.2, 63.9) (1.0,12.4) 271.7 a 61.0 a 8.7 a New York (-0.0, 543.5) (-39.3, 161.3) (0.6, 39.2) 358.7 a 0.0 a 0.0 a Control (101.1, 616.3) (-,-) (-,-)
133
Table 18. Mean densities of Sirex per m3 wood, Sirex sex ratios, and Ibalia parasitism rates for several crown conditions at the Hills Creek State Park in the September 2009 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P< 0.05) are given in parentheses.
Sirex density Crown condition Sirex sex ratio Percent parasitism by Ibalia (per m3)
349.6 a 38.5 b 11.4 a Green (191.2, 507.9) (20.4, 58.5) (3.3, 23.7) 182.1 a 0.0 a 7.7 a Green-red (-451.3; 815.4) (-,-) (2.5, 43.8) 353.5 a 25.2 ab 27.8 a Red (20.8, 686.2) (0.5, 69.0) (2.3, 67.1) 0.0 a Dead — — (-,-)
134
Table 19. Mean densities of Sirex and infected Sirex per m3 wood and infection rates for two strains of B. siricidicola at the Schoeller 1 site in the September 2009 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P<0.05) are given in parentheses.
Sirex density Density of infected Sirex Percent infection by Treatment (per m3) (per m3) Beddingia
11.9 a 0.0 a 0.0 a Kamona (-7.0, 30.7) (-,-) (-,-)
25.4 a 0.0 a 0.0 a New York (-34.7, 85.4) (-,-) (-,-)
31.2 a 19.7 a 25.0 a Control (-14.8, 77.4) (-26.9, 66.3) (97.5, 12.8)
135
Table 20. Mean densities of Sirex and infected Sirex per m3 wood and infection rates for Kamona nematode treatments mass cultured on two fungal isolates at the Schoeller 2 site in the September 2009 controlled release. Percentages were arcsine square root transformed for analysis, and means and confidence limits were then back-transformed. Means in columns that are followed by the same letter were not significant using an lsd test at the 0.05 level. Confidence limits (P<0.05) are given in parentheses
Sirex density Density of infected Sirex Percent infection by Treatment (per m3) (per m3) Beddingia
159.0 a 119.2 a 75.0 a Hajek fungus isolate (-31.4, 349.3) (-23.5, 262.0) (-,-)
80.3 a 0.0 a 0.0 b Otis fungus isolate (-175.2, 335.7) (-,-) (-,-)
194.8 a 68.1 a 10.8 ab Control (-352.8, 742.5) (-148.7, 285.0) (48.9, 98.4)
136
Table 21. Characteristics of the New York and Pennsylvania study sites used in the 2010 controlled releases
Site Bell Hill Road Hills Creek SP Armenia Mtn Ole Bull SP
Town Guilford Charleston Ward Stewardson
County, State Otsego, NY Tioga, PA Tioga, PA Potter, PA
N42 37.824 N41 48.236 N41 43.659 N41 32.591 Coordinates W75 10.586 W77 11.484 W76 58.300 W77 42.673
Pine species red red red red
Mean DBH (cm) 15.3 17.3 15.7 15.2
Mean sample vol (m3) 0.026 0.036 0.031 0.029
No. trees sampled 17 33 18 15
No. trees with Sirex 11 21 5 5
No. trees with nematodes 4 2 0 2
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Table 22. Controlled releases in New York and Pennsylvania sites in 2010. Experiments at all five sites compared a Kamona treatment with an untreated control. The trials at Hills Creek SP and Bell Hill Road also contrasted nematode infection rates for two release dates, in late August and late September
Site Hills Creek SP Armenia Mtn Ole Bull SP L Harrison SP Bell Hill Rd Total S. noctilio 280 5 6 4 61
Total S. nigricornis 0 0 28 0 20 % paras by Ibalia 39.5 33.3 2.6 65 6.9 % paras by Rhyssa 2.4 11.1 7.9 17 0.0
% S. noctilio infect 5.4 0.0 66.7 0 22.2 with Kamonas
% S. noctilio infect 11.2 0.0 0.0 0 11.6 with controls
% nemas in eggs 0.0 — 0.0 — 0.0
Combined percent parasitism of S. noctilio and S. nigricornis (where S. nigricornis includes S. edwardsii)
138
Table 23. Characteristics of the New York and Pennsylvania study sites used in the 2011 controlled releases
Site Rose Carbone Narbonne Honeywell Hills Creek L. Harrison F. Slocum
Town Rose Forestport Forestport Tully Charleston Delmar Kingston
Onondaga, County, State Wayne. NY Oneida, NY Oneida, NY Tioga, PA Tioga, PA Luzerne, PA NY N41 48.236 N41 41.791 N41 20.863 Coordinates ― ― ― ― W77 11.484 W77 27.306 W75 52.843
Pine species red red red Scots red red red
Mean DBH (cm) 15.9 19.1 14.9 17.9 14.6 13.5 13.8
Mean sample vol (m3) 0.038 0.051 0.043 0.047 0.036 0.032 0.026
No. trees sampled 20 10 10 10 24 12 16
No. trees with Sirex 6 8 3 9 13 2 8
No. trees with nemas 1 5 1 6 5 0 1
139
Table 24. Timing of trap tree inoculation 2011
Inoculation time of trap Sample size Percent infection Sirex density trees (omitting zeros) (insects/m3) Early (July) 7 7.1 a 206.3 a
Middle (August) 12 29.2 a 120.9 a
Late (October) 5 12.2 a 243.5 a
Table 25. Infection rate as affected by nematode treatment 2011
Sites Percent infection Sirex density (insects/m3)
Nematodes (N) Control (N) Nematodes (N) Control (N)
All trap trees 17.8 (14) a 21.3 (10) a 204.3 (14) a 125.2 (10) a
Hills Creek SP 45.7 (6) a 0.0 (5) b 277.4 (6) a 133.8 (5) a
140
H. Sirex noctilio population trends in New York and Pennsylvania
Figure 2 shows the dynamics of S. noctilio during the six years of the outbreak. Sampling sites during this period ranged from Lake Ontario in central New York through the northern tier counties in central Pennsylvania. Means for individual years for “all sites” are not entirely comparable because we generally did not use the same sites from year to year. Nevertheless, a trend is clear. Populations peaked in 2008 and tapered off to low levels by 2012. We sampled Hills Creek State Park in Pennsylvania for the past four years, and dynamics at that “permanent” site echo the overall dynamics. However, they appear to lag behind the general trend by a year or so, possibly reflecting the distance from the S. noctilio introduction point in Fulton, New York to northern Pennsylvania. The general explanation for these dynamics is probably that the region was ideal Sirex habitat, having largely unmanaged stands stocked with susceptible host species. Sirex populations simply exploited the most vulnerable trees and then dropped to low levels when they disappeared. It would be interesting to know if this outbreak pattern is like a wave at the leading edge, presumably somewhere south and west of Pennsylvania.
Fig. 2. Sirex populations at all sites and Hills Creek SP, PA 2500
2000 )
3 All sites m Hills Creek SP 1500 (per
density 1000 Sirex 500
0 2007 2008 2009 2010 2011 2012 Year
I. Density independent parasitism by Ibalia leucospoides
Although the species has been transported around the world in classical biological control programs for Sirex and is the most effective of the hymenopterous parasitoids, I. leucospoides has rarely achieved rates of parasitism beyond 20-25% in our sites. It has been suggested in the 141 literature that the parasitoid generally acts in a density independent manner, attacking hosts at about the same rate regardless of their density. Thus, the parasitoid, while providing a valuable modicum of control, will not be able to respond effectively to Sirex outbreaks. We explore the question of density independence in Table 3, regressing percent parasitism on Sirex density. In four of the five years, these regressions had negligible correlations, with slopes not significantly different from zero. Only one regression (2010) exhibited a significant slope and positive correlation, suggesting possible direct density dependence.
What is the reason for this appearance of density independence? Possibly, I. leucospoides is limited by the length of its ovipositor. We investigated this by looking at the relationship between parasitism and average bole diameter per tree (Fig. 3). Unfortunately, we have data on sample billet diameters only for 2011. Although there was a lot of variation, the relationship was significant and inverse, suggesting that Sirex larvae in narrower boles may suffer relatively higher parasitism, but those in wider boles suffer progressively lower rates, possibly leveling out asymptotically at about 20-25%.
Table 26. Regression of percent parasitism by I. leucospoides on Sirex density Year Sample size Slope r2 P(>F) 2007 78 0.0020 0.016 ns 2008 83 0.0008 0.013 ns 2009 60 0.0009 0.003 ns 2010 54 0.0157* 0.193* 0.001 2011 58 0.0035 0.004 ns
142
Laboratory experiments during 2006 – 2012
A. Mass rearing (2006-2010) 1. Effect of temperature on nematode population increase (Fig. 4) 2. Effect of inoculum size and growth time to harvest on nematode population increase (grow for 8, 10, 12, 14, and 16 weeks) (Table 27)
B. Investigation of sterilization behavior (invasion of eggs) in North American B. siricidicola (2008)
We obtained uninfested 5 ft long Scots pine logs from Falmouth, MA, and placed them in closed SonoTube® cages. We exposed them to Sirex females reared from infested, untreated billets from Richland, NY, and allowed oviposition. After the oviposition period, we dissected females to determine possible presence of North American nematodes. We held logs for 2-3 months to allow woodwasp development to large larval stage. We then inoculated infested logs with Sirex nematodes and held them for Sirex emergence. We dissected emerging wasps and determined location of any nematode juveniles contained (that is, in eggs, testes, body cavity). Note that this experiment failed to produce expected results. We ultimately reared only five tiny but healthy Sirex males. The quarantine setup was apparently not conducive to oviposition by the woodwasp females. In addition, the logs dried out quickly, probably resulting in a failure to establish by the nematodes.
C. Hybridization of the Kamona and New York nematode strains (2009-2012)
Carrie developed hybrid colonies of Kamona and New York nematodes in 2010-2011. The process was laborious and time consuming. She required hundreds of plates to get two successful hybrids. Often the nematode mating pairs could not be found after they were placed on plates. Carrie and Dyanna tried various approaches to confine the pairs, but to no avail.
D. Determination whether the Kamona strain is able to live and reproduce on the North American fungus, Amylostereum chailletii.
I addressed this question experimentally in 2008. The Kamona strain cannot develop and reproduce on the American fungus species.
143
E. Collection of native Sirex species and develop cultures of their associated fungi and nematodes. (2008-2012)
I have isolated fungi alone or fungi with nematodes from Sirex californicus, S. cyaneus, S. nigricornis, and Urocerus cressoni.
F. Investigation of the linear growth of A. areolatum fungus in artificially infected pine logs in the lab. (2008-2009)
This experiment was not successful. Fungus samples became contaminated and the logs dried out quickly
144
Figure 4. Response of nematode population increase to temperature in Kamona strains of Beddingia siricidicola grown on two isolates of Amylostereum areolatum. All flasks were harvested after 10-12 weeks. A. Maximum numbers, 903,000 nemas, at 16.4˚ C; B. maximum numbers, 1,040,000 nemas, at 16.3˚; C. maximum numbers, 2,370,000, at 15.6˚; D. maximum numbers, 5,300,000 nemas, at 10.0˚..
1,600,000 2,000,000 E7 Kamonas on Otis Aa E6 Kamonas on Otis Aa A 1,400,000 B
flask 1,200,000 1,500,000 flask
1,000,000 per per
1,000,000 800,000 600,000 500,000 400,000 y = ‐10757x2 + 353418x ‐ 2E+06 2
Nematodes y = ‐19053x + 619973x ‐ 4E+06 Nematodes 200,000 R² = 0.1858 0 R² = 0.3128 0 5 10152025 Temperature (oC) 5 10152025 Temperature (oC) 145
Figure 4. Continued C D
6,000,000 10,000,000 E8 Kamonas on Otis Aa E8 Kamonas on Hajek Aa 5,000,000 y = 77381x2 ‐ 3E+06x + 2E+07 y = ‐10092x2 + 315352x + 96869 8,000,000 flask
flask R² = 0.2955
4,000,000 R² = 0.0085
per 6,000,000 per
3,000,000 4,000,000 2,000,000 2,000,000 1,000,000 Nematodes Nematodes
0 0 5 10152025 5 10152025 o Temperature ( C) Temperature (oC) 146 Table 27. Nematode harvests in 2008 ̶ 2010. Beddingia siricidicola in 500 ml flasks on Areolatum spp. Setup Total No. per No. No. Sample Nema vol Study date Strain Temp Inoc nemas flask flasks weeks date (ml) Fungus 1/23/2008 E7 full 10,850,000 986,364 11 12 4/16/2008 Otis 2/1/2008 E6 full 10,260,000 932,727 12 12 4/25/2008 Otis 2/11/2008 E7 18 full 14,352,000 1,304,727 12 16 6/3/2008 18 Otis 2/22/2008 E6 18 full 5,016,000 418,000 12 16 6/16/2008 13 Otis 2/26/2008 E6 full 10,200,000 850,000 12 12 5/20/2008 Otis 3/6/2008 E7 room full 5,456,000 454,667 12 13 5/29/2008 Otis 3/19/2008 E7 full 9,050,000 754,167 12 12 6/11/2008 Otis 3/21/2008 E6 room full 19,810,000 1,650,833 12 12.5 6/13/2008 26 Otis Growth 3/26/2008 E6 13 full 16,796,000 1,399,667 12 12 6/19/2008 34 Otis Growth 3/26/2008 E6 18 full 11,400,000 950,000 12 12 6/19/2008 20 Otis Growth 3/26/2008 E6 room full 8,100,000 736,364 12 12 6/19/2008 18 Otis Growth 3/26/2008 E6 23 full 3,300,000 275,000 12 12 6/19/2008 12 Otis 4/1/2008 E7 18 full 8,006,667 667,222 12 12 6/26/2008 14 4/11/2008 E7 18 full 6,513,000 542,778 12 12 7/3/2008 14 Cryo 5/14/2008 E6 full 17,000,000 1,700,000 10 13 8/6/2008 Otis Cryo 5/16/2008 E7 full 7,013,333 584,444 12 13 8/8/2008 Otis Viability 6/5/2008 E6 full 15,310,000 1,275,833 12 12 8/28/2008 30 Otis 6/9/2008 E7 full 5,306,667 482,424 12 12 9/1/2008 12 Otis Release 7/1/2008 E6 room full 86,900,000 2,172,500 40 12 9/20/2008 70 Otis Release 7/1/2008 E6 room full 18,850,000 1,570,833 12 17 10/31/2008 32 Otis 7/17/2008 E6 full 9,813,462 892,133 12 13 10/9/2008 16 Otis 7/24/2008 E7 full 9,200,000 766,667 12 13 10/16/2008 14 Otis Growth 8/7/2008 E6 13 full 9,936,000 828,000 12 12 10/31/2008 16 Otis Growth 8/8/2008 E7 13 full 9,008,000 750,667 12 12 10/31/2008 14 Otis 10/2/2008 E6 room full 6,891,429 574,286 12 12 12/23/2008 Otis Inoc size 10/24/2008 E6 room full 7,600,000 950,000 8 12 1/20/2009 14 Otis Inoc size 10/24/2008 E6 room half 4,880,000 610,000 8 12 1/20/2009 8 Otis Inoc size 10/24/2008 E6 room quarter 5,546,667 693,333 8 12 1/20/2009 9 Otis 10/28/2008 E7 room full 7,582,667 689,333 12 12 1/22/2009 14
Growth 11/14/2008 E6 room full 6,336,000 582,000 12 12 2/5/2009 14 Otis 147
Table 27 (cont'd). Nematode harvests in 2008 ̶ 2010. Beddingia siricidicola in 500 ml flasks on Areolatum spp. Total No. per No. No. Sample Nema vol Study Setup date Strain TempInoc nemas flask flasks weeks date (ml) Fungus Growth 11/14/2008 E6 24 full 6,644,000 553,667 12 12 2/5/2009 9 Otis 11/28/2008 NY room full 6,286,667 523,889 12 12 2/26/2009 7 Inoc size 1/23/2009 E7 room full 4,878,000 813,000 6 12 4/17/2009 8 Otis Inoc size 1/23/2009 E7 room double 4,602,000 767,000 6 12 4/17/2009 7 3/11/2009 E7 room full 7,113,333 592,778 12 12 6/3/2009 10 3/27/2009 E6 room full 13,581,333 1,131,778 12 12 6/29/2009 17 Growth 4/17/2009 E6 15 full 7,544,872 1,257,479 6 12 7/8/2009 12 Growth 4/17/2009 E6 room full 5,260,256 876,709 6 12 7/8/2009 9 Growth 4/20/2009 E7 15 full 8,723,077 1,453,846 6 12 7/15/2009 9 Growth 4/20/2009 E7 room full 3,439,423 573,237 6 12 7/15/2009 5 Growth 5/13/2009 E7 room full 3,948,718 658,120 6 12 8/4/2009 6 Growth 5/13/2009 E7 15 full 8,024,359 1,337,393 6 12 8/4/2009 10 5/18/2009 E8 room full 15,990,000 2,665,000 6 12 8/10/2009 20 Cryo 5/19/2009 E8 room full 18,540,000 3,090,000 6 12 8/17/2009 Growth 6/4/2009 E7 room full 1,893,333 315,556 6 12 9/2/2009 5 Growth 6/4/2009 E7 15 full 2,653,333 442,222 6 12 9/2/2009 5 6/11/2009 E8 room full 28,320,000 2,832,000 10 12 9/10/2009 32 Release 6/17 & 6/29 E8 room full 16,092,000 315,529 51 13 & 12 9/19/2009 18 Otis Release 7/17/2009 E8 room full 22,524,000 3,754,000 6 9 9/19/2009 20 Hajek Release 7/15 & 8/6 NY room full 5,168,000 215,333 24 9 & 6 9/19/2009 8 6/18/2009 E6 room full 9,064,000 755,333 12 16 10/9/2009 10 Otis 6/26/2009 E7 room full 6,866,667 572,222 12 15 10/9/2009 6 Otis Growth 7/21/2009 E7 room full 4,746,667 791,111 6 12 10/16/2009 6 Otis Growth 7/21/2009 E7 15 full 6,820,000 1,136,667 6 12 10/16/2009 10 Otis Growth 8/7/2009 E6 10 full 1,614,000 403,500 4 12 10/29/2009 4 Otis Growth 8/7/2009 E6 15 full 5,896,000 1,474,000 4 12 10/29/2009 11 Otis Growth 8/7/2009 E6 room full 5,320,000 1,330,000 4 12 10/29/2009 9 Otis Growth 8/11/2009 E7 room full 2,723,000 453,833 6 12 11/4/2009 6 Otis Growth 8/11/2009 E7 15 full 3,066,667 511,111 6 12 11/4/2009 8 Otis 8/13/2009 E8(8) 10 full 3,836,000 639,333 6 12 11/5/2009 10
148
Table 27 (cont'd). Nematode harvests in 2008 ̶ 2010. Beddingia siricidicola in 500 ml flasks on Areolatum spp. Total No. per No. No. Sample Nema vol Study Setup date Strain Temp Inoc nemas flask flasks weeks date (ml) Fungus Growth 8/14/2009 E8 room full 4,573,333 762,222 6 13 11/9/2009 6 Otis Growth 8/14/2009 E8(15) 15 full 9,240,000 1,540,000 6 13 11/9/2009 11 Otis Growth 8/14/2009 E8(10) room full 8,294,000 1,382,333 6 13 11/9/2009 10 Otis Full 9/4/2009 E6 room full 3,733,333 622,222 6 12 11/30/2009 8 Otis Reg 9/4/2009 E6 room reg 3,406,667 567,778 6 12 11/30/2009 6 Otis 10/22/2009 E8(11) room full 28,853,333 4,808,889 6 7 12/8/2009 20 10/22/2009 E8(11) 10 full 1,023,333 170,556 6 7 12/8/2009 >1 10/22/2009 E8(11) 15 full 68,060,000 11,343,333 6 7 12/8/2009 48 9/17/2009 E8(9) room full 17,160,000 3,432,000 6 12 12/9/2009 25 9/17/2009 E8(9) 10 full 19,215,000 3,843,000 6 12 12/9/2009 21 9/17/2009 E8(9) 15 full 19,506,667 3,901,333 6 12 12/9/2009 20 Growth 10/28/2009 E8 room full 2,975,000 495,833 6 8 12/21/2009 3 Otis Growth 10/28/2009 E8 15 full 2,265,000 337,500 6 10 1/6/2010 2 Otis 10/30/2009 NY room full 1,336,333 222,722 6 8 12/22/2009 4 10/30/2009 NY 15 full 732,000 122,000 6 8 12/22/2009 2 11/20/2009 E8 room full 19,493,333 3,248,889 6 8 1/20/2010 20 Hajek 11/20/2009 E8 10 full 35,820,000 5,970,000 6 8 1/20/2010 34 Hajek 11/20/2009 E8 15 full 8,756,667 1,459,444 6 8 1/20/2010 10 Hajek Growth 11/20/2009 E8 room full 18,456,667 3,076,111 6 10 2/1/2010 Hajek Growth 11/20/2009 E8 room full 10,530,000 1,755,000 6 12 Hajek 10/30/2009 NY room full 2,400,000 400,000 6 12 1/25/2010 5 10/30/2009 NY 15 full 1,306,667 217,778 6 12 1/25/2010 2 Host 10/30/2009 WA room full 1,980,000 330,000 6 12 1/25/2010 5 cyan Host 10/30/2009 WA room full 5,168,000 861,333 6 12 1/25/2010 12 cali 10/30/2009 NY room full 1,422,000 245,333 6 14 2/4/2010 2 10/30/2009 NY 15 full 1,306,667 217,778 6 14 2/4/2010 2 Growth 11/20/2009 E8(15) 15 full 15,722,000 2,620,333 6 10 2/1/2010 Hajek Growth 11/20/2009 E8 15 full 15,877,333 2,646,222 6 12 Hajek Growth 11/20/2009 E8(10) 10 full 9,920,000 1,653,333 6 10 2/1/2010 Hajek Growth 11/20/2009 E8 10 full 42,331,667 7,055,278 6 12 Hajek 149
Table 27 (cont'd). Nematode harvests in 2008 ̶ 2010. Beddingia siricidicola in 500 ml flasks on Areolatum spp. No. per No. No. Sample Nema vol Study Setup date Strain Temp Inoc Total nemas flask flasks weeks date (ml) Fungus Growth 3/23/2010 E8 room full 27,489,333 4,581,333 6 10 Hajek Growth 3/23/2010 E8 room full 20,825,000 3,470,833 6 12 Hajek Growth 3/23/2010 E8 15 full 8,645,667 1,440,944 6 8 Hajek Growth 3/23/2010 E8 15 full 14,824,667 2,470,778 6 10 Hajek Growth 3/23/2010 E8 15 full 6,190,000 1,031,667 6 12 Hajek Growth 3/23/2010 E8 10 full 5,860,000 976,667 6 8 Hajek Growth 3/23/2010 E8 10 full 54,535,000 9,089,167 6 10 Hajek Growth 3/23/2010 E8 10 full 19,121,667 3,186,944 6 12 Hajek Growth 9/17/2009 E8 room full 17,160,000 3,432,000 6 12 Growth 10/22/2009 E8 room full 28,853,333 4,808,889 6 7 Growth 12/17/2009 E8(9) room full 38,362,500 6,393,750 6 6 1/29/2010 Growth 12/17/2009 E8(14) room full 27,772,000 4,628,667 6 8 2/16/2010 Growth 12/17/2009 E8(14) room full 39,600,000 3,300,000 12 12 3/17/2010 Growth 12/29/2009 E8(15) room full 22,958,000 3,826,333 6 8 2/25/2010 Growth 9/17/2009 E8 15 full 19,506,667 3,901,333 6 12 Growth 10/22/2009 E8 15 full 68,060,000 11,343,333 6 7 Growth 10/22/2009 E8 10 full 1,023,333 170,556 6 7 Growth 12/29/2009 E8(15) 15 full 103,080,000 17,180,000 6 8 2/25/2010 Growth 3/19/2010 E8 room full 8,512,000 1,418,667 6 12 PA fungus Growth 3/19/2010 E8 15 full 9,263,333 1,543,889 6 8 PA fungus Growth 3/19/2010 E8 15 full 16,970,667 2,828,444 6 10 PA fungus Growth 3/19/2010 E8 15 full 9,650,000 1,608,333 6 12 PA fungus Growth 10/28/2009 E8 15 full 3,043,333 507,222 6 1/21/2010 Otis Growth 10/29/2009 E8 room full 4,304,000 717,333 6 1/21/2010 Otis Growth 5/7/2008 E6 18 full 12,642,333 1,053,528 12 8/5/2008 Otis Growth 5/5/2008 E7 18 full 13,867,333 1,155,611 12 8/5/2008 Otis 150
G. Investigation of the effects of low temperature on growth of fungus and nematode cultures (2010-2011)
Carrie monitored growth and survival of nematodes and fungus on plates held at 5˚ C. Plates can be stored at that temperature without noticeable adverse effects for 3-6 months.
H. Rearing of S. noctilio artificially in lab using eggs from recently dissected females placed in rearing flasks with fresh wood chips of Scots pine growing A. areolatum. (2010)
Our objective was to grow Sirex males “in vitro”. We had no success.
I. Dissection of Rhyssa spp. from field collections in attempt to isolate B. wilsoni. (2006- 2012)
We have dissected hundreds of ichneumonids over the years without finding nematodes.
J. Investigation of the viability of B. siricidicola in breathable baggies over time (2009)
Carrie monitored the survival of nematodes left over from the 2009 release. She held samples under “travel conditions”, 5-10˚ C, subjecting some to continuous chill (Table 28) and others to chill followed by room temperature (Table 29). She also conducted an experiment monitoring survival in a Percival at 4˚ C up to nine days (Table 30). In general, Carrie concluded that nematodes can survive well for 3-4 weeks in breathable baggies at temperatures around 5-10˚ C.
Table 28. Survival of nematodes in leftover breathable baggies used in 2009 release. Temperature range 5‐10˚, N = 6 Average Days at Sample nematode Average percent alive temperature baggie count
1 206 92 9 2 301 93
1 233 91 16 2 440 92
1 299 93 25 2 199 90 151
Table 29. Survival of nematodes in breathable baggies inside Percival. Temperature range 3.7‐4.6˚, N = 6 Average Average Days at temperature Sample baggie nematode count percent alive
1 277 93 1 2 302 93
1 265 92 2 2 322 90
1 243 93 3 2 224 94
1 280 96 4 2 257 96
1 283 90 5 2 360 89
1 359 89 8 2 442 90
1 367 91 9 2 303 90
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Table 30. Survival of nematodes in leftover breathable baggies used in 2009 release. Chilled at 5‐10˚ for 31 days and then at room temperature for days indicated, N = 6 Days at room Average Average Sample baggie temperature nematode count percent alive
1 292 92 1 2 278 91
1 273 92 2 2 208 92
1 335 91
7 2 319 89
1 324 72
13
2 ― ―
153
L. Lab inoculation of Kamona nematodes into billets infested with S. noctilio
Objective To shorten the time needed to determine infection rates of the Kamona and New York nematodes. Billets were collected from three trees with obvious resin beads in two state parks in Pennsylvania in October 2010. On return to the Otis quarantine, they were inoculated with either a nematode strain (E8 Kamona or New York) or a gel-only control and held for emergence in screened barrels. Unfortunately, none of the adult Sirex, which emerged in January-March, was infected with nematodes, probably because the billets desiccated rapidly in the quarantine lab. Combined parasitism by Ibalia and Rhyssa was high, 35-58%. Note: this experiment was repeated in 2012 with similar negative results.
Table 31. Emergence from billets collected in PA and inoculated with two strains of Kamona nematodes.
No. No. S. noctilio % parasitism % parasitism Wood collection site % females trees emerged by Ibalia by Rhyssa Frances Slocum SP 3 33 9 32 26 Leonard Harrison SP 3 15 27 9 26
154
Table 32. Quarantine study: emergence of insects from red pine billets from L. Harrison SP and F. Slocum SP on 10/21/2010. Resin Sirex Exit S. noc S. noc Barrel Treatmnt Park First Date Last Date Rhyssa Ibalia Ichn beads larvae holes ♂♂ ♀♀ 1 E8 LH 12/10/2010 2/7/2011 0 32 4 3 0 2 2 1 2 E8 LH 12/10/2010 1/27/2011 2 5 4 0 0 9 1 0 3 E8 FS 12/8/2010 3/31/2011 13 39 13 11 1 3 8 0 4 E8 FS 12/10/2010 3/25/2011 6 2 2 0 0 1 0 2 7 E6 LH 12/10/2010 3/21/2011 5 27 8 8 3 1 1 3 8 E6 LH 12/10/2010 4/11/2011 4 8 13 2 0 24 6 0 9 E6 FS 12/10/2010 5/27/2011 1 32 9 7 4 7 3 0 10 E6 FS 12/10/2010 1/11/2010 7 2 6 0 0 0 0 0 11 Control LH 12/10/2010 3/18/2011 7 32 9 7 2 1 0 0 12 Control LH 12/8/2010 8/12/2011 5 18 8 3 0 12 1 0 13 Control FS 12/10/2010 10/5/2011 7 31 17 15 0 8 9 0 14 Control FS 12/10/2010 12/10/2011 2 1 4 0 0 0 0 0
155
L. Lab inoculation of Kamona nematodes into billets infested with native Sirex species
Objective To investigate possible susceptibility of S. edwardsii and S. nigricornis to the Kamona strain. Experimental material was provided by Forest Service cooperators in Louisiana. Stacked billets from three pine trees (two P. taeda and one P. echinata) were exposed to Sirex oviposition and subsequently shipped to Otis. Billets were inoculated with either Kamona strain nematodes (E6 and E8) or a gel-only control. Sirex adults emerged only from the P. echinata billets, and none of those adults was infected with nematodes. As with the previous experiment, nematodes may have failed to establish because the billets desiccated. Alternatively, they may not have established because they cannot survive or reproduce on A. chailletii. Although the P. echinata billets were exposed only to S. nigricornis females, emerging adults included 82 S. nigricornis and 81 S. edwardsii, suggesting that the two “species” are actually only one (i.e., S. nigricornis). Note: this experiment was repeated in 2012 with similar negative results.
Table 33. Pine billets shipped from the Forest Service in Louisiana in late January 2011 Pine First S. edw S. edw S. nig S. nig Bark Barrel sp. Treatmnt Date Last Date ♂♂ ♀♀ ♂♂ ♀♀ Weevils btls 1 taeda E8 4/6/2011 4/21/2011 0 0 0 0 11 0 2 echinata E8 4/5/2011 11/2/2011 39 10 31 14 18 1 3 taeda E8 4/5/2011 4/21/2011 0 0 0 0 10 0 4 taeda E6 4/6/2011 4/21/2011 0 0 0 0 31 19 5 echinata E6 4/21/2011 11/2/2011 8 15 14 13 0 0 6 taeda E6 4/6/2011 9/7/2011 0 0 0 0 3 0 7 taeda Control 4/21/2011 4/21/2011 0 0 0 0 5 0 8 echinata Control 4/21/2011 10/27/2011 9 0 11 0 1 0 9 taeda Control 4/21/2011 4/21/2011 0 0 0 0 1 0
156
Nematode cultures at Otis Beddingia lab – January 2013
E8 on Amylostereum areolatum (Aussie) New Kamona strain. Arrived 4/27/2008 (10 plates) first subculture 5/2/2009
E8 on Amylostereum areolatum (Otis)-Placed E8 on OAa (5/18/2009)
E8-PA-placed on PA 6/09
E8 on Amylostereum areolatum (Hajek strain) 6/4/2009
E6OAa-shipment 6- 11-28-2006 shipment was lost at JFK for a day by DHL. Placed on OAA 12-18-2006.
C-E6/PAAa- shipment 6- 11-28-2006 shipment was lost at JFK for a day by DHL- cryogenically frozen 2008. Thawed 11/2010.
C-E6/Aussie- shipment 6- 11-28-2006 shipment was lost at JFK for a day by DHL- cryogenically frozen 2008. Thawed 11/2010.
E7/Aussie-shipment 7 (E7) 1-29-07 Arrived-10 plates. Labeled “Sirex plates, 3rd generation ½ strength PDA Jan 11, 2007. Sent 10 more plates 1-22-2007.
E7OAa- shipment 7. Arrived 1-29-2007. 10 plates. Placed on OAA 2-18-2007.
WA Nemas-Extracted from Sirex californicus and placed on A. chailletii. Isolated 8/20/2009
WA Nemas-Extracted from Sirex cyaneus and placed on A. chailletii. Isolated 8/20/2009
MCNY/Otis 2010-set up from male Sirex, 2-23-2010 from a male Sirex from Central NY (Miriam Cooperband’s barrel)
MCNY/PA-2010 set up from male Sirex, 2-23-2010 from a male Sirex from Central NY (Miriam Cooperband’s barrel)
MCNY/Hajek 2010 -set up from male Sirex, 2-23-2010 from a male Sirex from Central NY (Miriam Cooperband’s barrel)
157
MCNY/Otis-2011 set up from male 3-7-2011 from a male Sirex from Central NY (Miriam Cooperband’s barrel).
MCNY/PA 2011- set up from male Sirex, 3-7-2011 from a male Sirex from Central NY (Miriam Cooperband’s barrel).
MCNY/Hajek 2011-set up from male Sirex, 3-7-2011 from a male Sirex from Central.
E8/NY Hybrids/Hajek (2010)- 2 juvenile from both strains were set up on nearly 100 Hajek fungus plates. Only one plate was successful. Not sure which strain was the female, so we are not able to use this colony for mitochondrial DNA testing.
E8/NY Hybrids/Aussie (2011)-April 2011. E8 female and NY male.
E8/NY Hybrids/PAAa (2011)-April 2011. E8 female and NY male.
NY/E8 Hybrids/Hajek (2012)-March 2012. NY female and E8 male.
MCNY 2012/Aussie (MC2e)-March 2012. Adirondacks, NY ?
MCNY 2012/Hajek (MC1C)-March 2012. Adirondacks, NY ?
MCNY 2012/MC1E fungus (MC3A)-4/24/2012. Adirondacks, NY ?
BHNY/Aussie-9/9/2011. Tree N16.
BHNY/PAAa-9/9/2011. Tree N16.
BHNY/PAAa-9/9/2011. Tree N9 (treated tree-male Sirex)
NY (N1)/Otis. 7/6/2012. Forestport, NY (treated tree-male Sirex)
NY (C9)/PAAa-5/29/2012. Forestport, NY (treated tree-male Sirex)
PA (H21)/Otis-7/6/2012. Hills Creek State Park, PA (treated tree-male Sirex).
Old NY nemas- isolated from Sirex female, central NY 6/18/2008 (no longer have this colony). 158
Fungal cultures at Otis Beddingia lab – January 2013
A.chai- isolated from S. nigricornis, Spafford, NY 10/7/2007
Frances Slocum A. chailletii. (from Urocerus sp.) Female collected from Frances Slocum State Park, Pennsylvania. Isolated fungus at Otis 9/2011.
A.chai (MC) from Sirex from Miriam Cooperband’s barrels. Isolated 2-2009. Central NY
A. chailletti (from Urocerus sp.) Female collected from Bear Mountain, New York 8/26/2009. Isolated fungus at Otis 9/3/2009.
Otis fungus- set up 2.3.2006 from frozen Sirex females. Only 1 out of 4 yielded a successful culture.
(PAAa)-Amylostereum areolatum extracted from a female Sirex noctilio from Leonard Harrison State Park, PA. Collected 8/24/2009 and isolated at Otis 9/3/2009.
Hajek fungus-(GR94-1) isolated 2/19/2008.
WA Fungus-Washington fungus extracted from Sirex californicus 8/4/2009.
Aussie fungus- isolated from original nematode shipment 11/24/2006.
MC2A fungus- islolated 3-12-2012. Adirondacks, NY?
MC1B Fungus- islolated 3-12-2012. Adirondacks, NY?
MC1E Fungus- islolated 3-12-2012. Adirondacks, NY?
159
Amylostereum, Deladenus siricidicola and Sirex noctilio in North America
Progress: 2008-2009
Ann E. Hajek
The European woodwasp Sirex noctilio has been present in North America since at least September 2004; however, based on the distribution detected for this species on the southeastern shore of Lake Ontario in 2005, S. noctilio had certainly already been present for at least a few years before it was first detected. This woodwasp vectors a fungal symbiont, Amylostereum areolatum, which is instrumental in helping the woodwasp to kill trees. The woodwasp/fungus association has caused extensive damage to pine plantations throughout many countries in the Southern Hemisphere (Hurley et al. 2007). The nematode Deladenus siricidicola has been used extensively for biological control of S. noctilio in the Southern Hemisphere. D. siricidicola has two forms: a parasitic form that sterilizes female S. noctilio and a mycophagous form that eats A. areolatum. At least 4-5 species of woodwasps in the genus Sirex are native to eastern North America. The areas S. noctilio has invaded previously, in the Southern Hemisphere, were all locations where pines (that tree species that Sirex noctilio attacks) and Sirex species are not native. Thus, in these areas, S. noctilio is the only Sirex present in plantations of introduced Pinus species. The invasion of S. noctilio in North America marks the first time that S. noctilio has invaded an area where trees that it might attack are native and where there are also native species of Sirex. Relatively little has been known about the Amylostereum species and strains present in North America and their associations with Sirex species. A. areolatum has been studied in the Southern Hemisphere (e.g., Slippers et al. 2001, 2002, 2003) and in Europe (e.g., Thomsen & Koch 1999, Vasiliauskas et al. 1998; Vasiliauskas and Stenlid1999). Several experts in this field of study have assumed that A. areolatum was not native to North America and therefore this species would now only be present in North America only due to its introduction S. noctilio (I. Thomsen, personal communication; Smith and Schiff 2002). However, with support from USDA APHIS, we have made some surprising findings.
Some of our progress to date includes:
Strains of Amylostereum in North America We’ve found at least three strains of A. areolatum in the U.S. Two of these have been isolated from S. noctilio and one of the ones from S. noctilio was also isolated from the native S. edwardsii. The third strain of A. areolatum was isolated from a native species, Sirex nitidus (this 160 name is not yet published but is based on identification by Dr. Henri Goulet; this species was known as S. cyaneus in Schiff et al. (2006)). S. nitidus and its associated fungus were collected in an area of central Maine from which S. noctilio is not yet known and therefore this strain could not have originated from the new S. noctilio infestation. A paper reporting these results has now been published in Mycological Research. (Nielsen et al. 2009).
Nielsen, C., Williams, D. W., Hajek, A.E. 2009. Putative source of the invasive Sirex noctilio fungal symbiont, Amylostereum areolatum, in the eastern United States and its association with native siricid woodwasps. Mycol. Res. (in press). These different strains of A. areolatum differ in vegetative compatibility grouping (VCG) and in the sequences of the nuclear ribosomal DNA intergenic spacer region. VCGs are used among mycologists to test the relationship between different cultures of a fungal species (incompatibility reactions occur between different clones whereas identical clones intermingle freely when confronting each other while growing). Thus, for A. areolatum introduced with Sirex noctilio to North America at least two vegetative compatibility groups are present (and these VCG groupings agree with DNA sequences). This information is also presented in the paper cited above (Nielsen et al. in press).
The Amylostereum areolatum strains that are present in the United States in S. noctilio (IGS: D and BD) are not the same as the strain that is causing so many problems in the Southern Hemisphere (IGS: AB). One strain the US is the same as an isolate from Lautenthal, Germany from Picea abies in 1967 (information about this isolate (IGS: D according to Slippers et al. 2002 = CBS isolate 305.82) was recorded incorrectly in the 2002 publication by Slippers et al. but is correct here; B. Slippers pers. comm.). The second strain we’ve found in North America is BD; this strain is a mixture of the D strain and the B strain (one hypothesis is that there are different nuclei within the fungal cells, which each carry two nuclei). The D strain is the same as above (618 base pairs). The B part of this strain (638 base pairs) has been isolated previously from Sirex juvencus in Lithuania and Denmark and from Sirex noctilio in Denmark and the Southern Hemisphere (Slippers et al. 2002).
Amylostereum chailletii in S. nigricornis and A. areolatum in S. noctilio and S. edwardsii were isolated from these woodwasps emerging from the same tree--even the same section of the same tree.
Two North American Sirex that Henri Goulet thinks could possibly be the same species (S. nigricornis and S. edwardsii) could differ in the fungal species they usually carry. This would suggest that they really are different species. However, we need more samples to be certain about 161 which fungal species these natives usually carry. Based on 2007 samples, we've only isolated A. areolatum from S. edwardsii and A. chailletii from S. nigricornis (however, see below).
We presently have approximately 117 isolates of Amylostereum, all of which have been isolated from siricid mycangia. We estimate that approximately 83 of them are A. areolatum and 34 of them are A. chailletii. These 2008 and 2009 collections include 41 isolates from 5 species of Sirex and 7 isolates from 2 species of Uroceros, with collections coming from 12 sites in 6 states. We are not aware of a collection of cultures similar to this worldwide!! Due to the value of this collection, these cultures have been deposited in a Forest Service collection of wood rot fungi (USDA, Forest Service, Center for Forest Mycology Research, Madison, WI; contact person: Dan Lindner) so that there is a back-up of our cultures. While molecular techniques and VCGs have been used to evaluate a number of the fungal isolates collected in 2007 (as reported in Nielsen et al. in revision), we still need to evaluate the remainder, which include the isolates made in 2008 and 2009.
Parasitoids associated with Sirex noctilio in North America In 2009, we published a note in The Canadian Entomologist, documenting species of hymenopteran parasitoids emerging from pines in New York State that were predominantly infested by S. noctilio. Interestingly, the most common parasitoid species, Ibalia leucospoides, was more abundant in smaller diameter wood, while we found this was the part of the tree with more males S. noctilio. Therefore, we hypothesize that this parasitoid would parasitize more male than female S. noctilio, which would lessen its control of this woodwasp (Long et al. 2009).
Long, S.J., D.W. Williams, A.E. Hajek. 2009. Sirex species (Hymenoptera: Siricidae) and their parasitoids in Pinus sylvestris in eastern North America. Can. Entomol. 141: 153-157.
Following, the results specifically from our studies over the past year are presented.
Objective 1 Optimizing growth of Amylostereum areolatum for enhanced nematode production Alexandra Jimenez, a Cornell undergraduate student, spent the 2008-2009 school year, as well as most of the following summer, conducting studies along these lines. She wrote an honor’s thesis, which I’ve sent to Dave Williams (our collaborator at USDA, APHIS). Following is the abstract from her senior honors thesis:
In 2004 the invasive woodwasp Sirex noctilio was found in the northeastern United States. Female wasps carry a symbiotic fungus Amylostereum areolatum and inject the fungus, toxic mucus and eggs into pine trees. Inside the tree, the larvae feed on the fungal-infested wood until they exit at maturity. In Australia, pine plantations were devastated by this wasp, but the nematode Deladenus siricidicola provided an effective means of biological control; this nematode is either parasitic on S. noctilio or feeds on A. areolatum. This nematode is mass 162 produced on fungal cultures of a strain of A. areolatum called Aussie for use as a biological control agent. To combat S. noctilio in North America, the nematode must be able to grow and reproduce successfully on North American strains of A. areolatum. This study aimed to first optimize the growth rates of three North American strains of A. areolatum (ScyMe, Gr94-1, and Sed DF) and Aussie by using four different media (Green bean agar [GBA]; Hagem agar; ½ strength PDA with 2x agar [½ PDA-h]; and PDA with thiamine hydrochloride [PDA-th]). Fungal growth rates were calculated by measuring colony area changes over the test period. GBA increased the growth rates for nearly all A. areolatum strains tested; Hagem agar slowed down the growth rates of all the strains. The North American fungal strain Gr94-1 on GBA had a growth rate comparable to the Aussie strain on Hagem agar. These two strain-media combinations were used for the second part of this study, a nematode growth assay. The successful propagation of D. siricidicola on the North American strain of fungus Gr94-1 was compared with the growth of the nematode on the Aussie strain. The initial concentration of nematodes needed to maximally increase the population of nematodes was tested. Nematode assays were conducted by inoculating fungal plates (approximately 3 cm in diameter) with five concentrations of nematodes. Due to contamination the Gr94-1 plates were not viable. The Aussie test plates inoculated with nematodes indicated that inoculating plates with concentrations of 680 nematodes/ml or higher achieve a maximum yield after 14 days.
I would add the following: Green bean agar significantly increased growth of all fungal strains but we have found that it is very easily contaminated with bacteria probably because it is very rich in nutrients. Hagem agar produced very slow growth of all strains tested. Therefore, for further studies we have been using ½ PDA-hard, the media used for nematode mass production in Australia, which is also easier for working with the nematodes because it is a little harder in consistency.
The final study from Alexandra’s senior thesis was a trial comparing grown of Kamona strain Deladenus siricidicola on different Amylostereum strains. She spent a lot of time and effort setting this up twice but both times, the study did not yield useable results. We feel the problem is that we could not standardize the density of nematodes being added to treatments so this is presently (11/09) a focus of our efforts. We are working toward being able to inoculate plates with only one age of nematode (e.g., eggs). We will try using a Swinnex filter to separate eggs from nematodes, but to do this we needed the dimensions of the eggs. The eggs of the Kamona strain of Deladenus siricidicola are 50.0 + 1.9 microns long and 31.0 + 1.0 microns wide (n = 20).
Objective 2 Isolate the North American nematodes parasitizing Sirex spp. and investigate their biology We have repeatedly detected nematodes that we assume to be native in Sirex in samples from both traps and emerging from wood (Tables 1, 2). We have introduced them to plates of 163
Amylostereum but have thus far been unable to get the native nematodes in culture, perhaps because we are offering the wrong strains of fungus or perhaps because the initial densities of nematodes are too low (our hypotheses at present). Below, are tables showing the numbers of native Sirex that we have found parasitized by nematodes. As you will see, in general levels of parasitism by nematodes in the native Sirex are often quite low (Table 1, 2).
In almost all of the 2009 female Sirex in which nematodes were found, nematodes- sometimes alive-were in the ovaries and not the mycangia. In only a few cases (2) nematodes were found in the mycangia. Thus, presence of nematodes in females is virtually always from finding them in Sirex tissues (i.e. the ovaries) and not the mycangia.
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Table 1. Native nematodes detected in female siricids.
Female Sirex year species # # females w/ coll females nematodes dissected 2007 S. noctilio 142 1 female from AH2 section DH emerged on 8/22 S. edwardsii 3 0 S. 31 0 nigricornis S. nitidus 2 0 U. cressoni 1 0 2008 S. noctilio 27 0 S. edwardsii 13 1 from Mt Morris, PA trap caught 10/14 S. 22 2 1 from Gerrard Fort PA trap on nigricornis 10/15, 1 from LA trap on 11/20 S. nitidus 12 0 U. cressoni 12 0 S. cyaneus 2 0 U. 2 0 albicornis 2009 S. noctilio 3 0 S. cyaneus 26 11 all from FP3, all had nematodes in ovaries but not in mycangia S. 1 1 from Rice creek WRA Oswego, nigricornis NY trap on 9/3. Had nematodes in ovaries and mycangia U. 7 0 albicornis U. cressoni 2 0
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Table 2. Native nematodes detected in male siricids
Male Sirex year species # males # males w/ coll dissected nematodes 2008 S. noctilio 13 1 possible Deladenus from R5T3 (FLNF) section QV on 8/4 S. nitidus 23 2 one from NF2 section C (Huntington Forest) had lots of nematodes, the other from NF2 section B had few. Both emerged on 9/2. S. cyaneus 8 0
2009 S. cyaneus 69 25 only 6 with live nematodes/nemas in other specimens were dead 166
Objective 3 Investigate growth of the S. noctilio-parasitic nematode Deladenus siricidicola in different pine species.
Methods We conducted studies comparing effects of adding red pine, white pine or Scotch pine to agar. First, each pine species was harvested and ground using a Wiley mill (Thomas Scientific, Swedesboro, NJ) with a 20 mesh sieve. The ground pine was sent to Penn State where it was irradiated at 2.5 Megarads (Radiation Science and Engineering Center, Pennsylvania State University, University Park, PA); this was necessary so that when we added this to media for growing fungus and nematodes, there would be no contamination but we would not have altered the basic chemistry of the wood. We inoculated three 60 mm petri dishes of five types of media (1.5% agar containing different amounts of pine) with the Otis strain of Amylostereum areolatum: 400 g/L red pine 400 g/L white pine 10 g/L red pine 10 g/L white pine 10 g/L Scotch pine
Nematode source for study. ½ PDA-h (half strength potato dextrose agar plates with 2.5% instead of 1.5% agar) were inoculated with nematodes growing on Otis Aa A. areolatum on 2 July. These plates were flooded with 10 mL of sterile 50g/L gentocin water for 30 minutes. The water was pipetted off and transferred into 15 mL tubes. To quantify the nematode concentration, the initial wash was diluted ten-fold with the gentocin water. The number of live nematodes and eggs in ten 20uL drops were counted (at 20X) and averaged to determine the amount of inoculum needed to make a 1250 nematode/mL solution.
Experiment. Two plates were used for the nematode growth experiment and one plate was used to quantify fungal growth. To inoculate plates with fungus, 3 mm diameter plugs were transferred to pine plates on Jul 24 at 23 C in continuous darkness. For the nematode growth study, four days later, fungal plates were each inoculated with 250 nematodes in 200 microliters of sterile 50g/L gentocin water on Jul 28. Nematodes were pipetted around the edge of the fungal colony.
Fourteen days later, nematode growth plates were flooded with 3-5 mL of gentocin water for 30 minutes. Liquid from each plate was pipetted into a 15 mL tube. The volume of each tube was recorded. Five 20 uL samples were taken from each tube and placed on a petri dish. The number of live nematodes and eggs in each drop were counted and averaged. This average was used to determine the total number of nematodes harvested from each plate.
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Results The media with 40% ground wood was extremely thick and difficult to work with and, at least for white pine, the nematodes did not seem to grow as well. When harvesting nematodes from the 40% media, after only 30 minutes of flooding plates, many nematodes were still within the media and the fungus was growing within the media too. To harvest these plates, plates were flooded for 30 minutes and then the media was slurried and nematodes were also extracted from the slurry. In addition, the 40% Scots pine media became contaminated and these samples were therefore excluded.
Curiously, the nematodes on the 1% wood media seemed be more successful than those on the 40% wood media. Numbers of juveniles and adults as well as numbers of eggs were greater on the 1% media. At this point, the differences among tree species have not been completely determined but we can say that on the 1% media, nematode growth and egg production was not inhibited by any of the tree species.
The studies are ongoing. More replication is necessary and, as can be seen, the error bars from these studies were far too large. At this time, we feel that once more we are not certain we have adequately inoculated plates with the same concentrations of the same stages of nematodes. Therefore, before continuing this line of research, we feel that our top priority must be to develop methods for making sure that the same numbers of nematodes (and the same stages) are introduced to different treatments. In addition, we need to add more replicates in the future. Future studies will certainly use the 1% treatment instead of the 40% and, at this point, we’ve learned some important methods (i.e., using the Wiley mill for grinding wood and where to have wood irradiated) to use in the future.
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Figure 1. Numbers of A. adult and juvenile D. siricidicola (per 100 mm plates) and B. D. siricidola eggs on media incorporating 1% versus 40% of wood from different species of trees. The 40% from Scots pine became contaminated and was excluded from the study. A Adults & juveniles from different media 5000
1% 4000 40% inoculation
3000 post
d
14 2000
1000 collected
0
Number Red pine White pine Scots pine
B 400 Nematode egg counts from different media
300 1% inoculation
40% post
d 200 14
collected
100
Number 0 Red pine White pine Scots pine
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Objective 4 Continue investigations of the Sirex/Amylostereum communities present in eastern North America
We have continued to isolate Amylostereum from mycangia of siricids (Table 3). One of our goals was to get more isolates from the native siricid that is usually in fir trees: S. cyaneus and we were successful with this endeavor. However, we also tried to obtain specimens of S. edwardsii and S. nigricornis from areas where S. noctilio has been established the longest (e.g., near Fulton and Syracuse). While we obtained some specimens for this purpose, we were unable to get isolates from them.
In fact, we have found through our studies that we cannot isolate fungus from every female because every female is not carrying fungal cells (at least not fungal cells that will start growing on media) (Table 3). We have been surprised to find that in some samples a significant number of Sirex females did not contain Amylostereum in their mycangia. If we add the number where a fungus that was not Amylostereum grew from mycangia to the number with no fungal growth, the number of females not carrying Amylostereum is even higher. We are not aware of other studies like this, that have reported that some siricid females (besides Xeris) do not carry the fungus in their mycangia. 170
Table 3. Number of fungal isolates from different species of siricids.
Year species # # # fungal contaminating # no Amylostereum coll females dissected isolates fungus not fungal but lost to Amylostereum growth contamination 2007 S. noctilio 283 142 63 10 38 26 S. 3 3 2 0 1 0 edwardsii S. 42 31 8 2 16 5 nigricornis S. nitidus 2 2 2 0 0 0 U. cressoni 1 1 0 0 1 0 2008 S. noctilio 27 27 15 0 12 0 S. 13 13 7 0 0 6 edwardsii S. 22 22 11 0 2 9 nigricornis S. nitidus 12 12 7 0 1 4 U. cressoni 12 12 7 1 5 0 S. cyaneus 2 2 1 1 0 0 U. 2 2 2 0 0 1 albicornis 2009 S. noctilio 3 3 3 0 0 0 S. cyaneus 26 26 6 0 19 1 S. 1 1 1 0 0 0 nigricornis U. 7 7 2 0 4 1 albicornis U. cressoni 2 2 1 0 1 0 171
Progress with Sirex and Deladenus: 2010
Objective 1 Conduct studies on native species of Sirex and their associated fungi and nematodes
Objective 2 Conduct studies on the interactions between the commercial Deladenus (Beddingia) siricidicola nematode and A. areolatum strains already present in North America. Note that information gained from these studies is important for future biological control of S. noctilio in North America using D. siricidicola.
Specific studies
Optimizing growth of Amylostereum areolatum and Deladenus siricidicola to enhance nematode production.
We tried D. siricidicola on A. chailletii strains from northeastern North America, and this fungus was not acceptable for growth of the Ecogrow strain. The A. chailletii strains we tested were from S. nigricornis and S. cyaneus and were compared with two strains of A. areolatum on which these nematodes grew well. One problem with our previous studies was that we did not have enough precision regarding how to consistently add a specific number of nematodes to fungal plates. Thus, we decided that we should add only eggs to the fungal plates. Studies were then conducted to evaluate how long after fungus was added to plates nematodes should be inoculated onto plates. Our results are still being analyzed but they suggest that it is very important to know when to inoculate with the fungus and with nematodes when mass producing the nematodes. In addition, different strains of A. areolatum differ significantly in growth rate.
Investigating growth of the S. noctilio-parasitic nematode D. siricidicola in different pine species. We hypothesized that the species of pine tree could influence the growth of D. siricidicola; although the nematodes eat fungus, they are living within pine trees when they are not parasitic on Sirex. Red, white and Scots pine were passed through a mill, the pine chips were sterilized using gamma radiation, and the sterile chips were added to water agar at two concentrations. We inoculated wood chip plates with a strain of A. areolatum from Oswego Co, NY and added Ecogrow nematodes (a diversity of different ages). Results from this study were extremely variable and did not indicate any trends in nematode growth associated with pine species. Due to the lack of a suggestion of any trend by pine species, we did not repeat these studies.
Continue investigations of the Sirex/Amylostereum communities present in eastern North America. Amylostereum isolates were evaluated from 6 species of Sirex from North America, and we worked on the IGS and ITS regions. At this time, we have only gotten identifications for fungal 172 species. At present, we have 125 isolates of A. areolatum and A. chailletii and we plan to continue collecting more isolates during the 2010 field season.
Work with a molecular probe to differentiate between the North American and European strains of the nematode D. siricidicola. We have made progress on molecular work with the nematode D. siricidicola using the Ecogrow strain (mass-produced in Australia) and a strain of D. siricidicola isolated from S. noctilio from Pompey, NY. Using the CO1 gene, we developed a method using restriction enzymes that differentially cut the two nematode strains so that we could distinguish between them. However, then we decided that because we do not understand the diversity of nematodes that we might find within Sirex individuals in the US, this method for differentiating between the two strains that are in culture is not the proper approach. We have samples from 19 Sirex adults from North America. Throughout the 2010 field season we will continue taking samples, when possible, and these will be included in this work. Our goal is to identify the samples to species and, for D. siricidicola, to find a region of the genome that varies among the different samples so that we can differentiate among potential strains of D. siricidicola.
Investigation of non-target impacts of D. siricidicola (not included in 2009 objectives). We dissected and evaluated non-target insects collected by David Williams as part of his research on parasitism by D. siricidicola in the field. Specimens had been reared from wood that had been inoculated with this nematode in the field in fall 2008. None of the total of 200 Ibalia leucospoides ensiger that were dissected contained nematodes.
Progress with Sirex and Deladenus: 2011
Results will be presented by study objective: Continue molecular work with Amylostereum from North American native Sirex species and any parasitic nematodes from native siricids (predominantly Sirex) and Sirex noctilio in the northeastern United States. We have been making excellent progress with the molecular studies of both the fungus and the nematode in northeastern U. S. samples:
Amylostereum
Samples: Dr. Ryan Kepler joined the lab in mid-January 2011 and he reorganized all of our fungal samples. Then, during 2011, we isolated some more fungal samples from a variety of locations. At present, we are concentrating on a paper about the fungi carried by native Sirex. For this paper, we have a total of 177 isolates of Amylostereum from native eastern North American Sirex: 173
108 S. nigricornis (predominantly pine) 44 S. edwardsii (pine; Dr. Goulet is synonymizing this species with nigricornis) 10 S. cyaneus (predominantly fir) 15 S. nitidus (predominantly spruce) Samples come from New York, Maine, Louisiana, Georgia and Pennsylvania and have been collected from 2007 to 2011. None of the samples included in the Nielsen et al. (2009) publication are being included in the group of samples for this upcoming paper.
Molecular analysis: Previously (Nielsen et al. 2009) we have conducted molecular work with this fungus using primers for the intergenic spacer (IGS) region of the nuclear ribosomal DNA because Dr. Bernard Slippers’ et al. (2002) had found the most variability in this region and then we could compare our results with previous results. Dr. Kepler wanted to develop other regions because there are multiple copies of the IGS in each strain of Amylostereum areolatum, which complicates analysis. He also felt that it would be preferable to find markers that varied among fungal strains by nucleotides. The IGS strains often vary largely by indels (gaps in the DNA); although indels are very normally seen as the variability among very closely related strains, standard programs do not deal with them very well. It is also preferable to use several markers and while we previously had used both ITS and IGS, the ITS demonstrated very little variability within species. Therefore, Dr. Kepler spent some time trying to develop more and other markers besides IGS that would not be in multiple copies and would differ among strains by nucleotides but he was not successful. Then, a paper on the variability among strains of A. areolatum associated with S.noctilio (but isolated only from wood) from Canada was published this fall (Bergeron et al. 2011). In this paper, the authors had used 5 different sets of mitochondrial and nuclear primers (multi-locus genotype analysis) that each occurred as a single copy in a strain of A. areolatum. While they included IGS in a minor way, they principally used 5 different primer pairs. Bergeron et al. (2011) also did not include any native Sirex in their study (in fact, they did not include any fungal samples that had been isolated from Sirex species as all of their samples were isolated from infested wood). With the publication of this paper, Dr. Kepler and I thought that he should try working with the 5 primer pairs that were so successfully used for Amylostereum areolatum in the Bergeron et al. (2011). However, we quickly learned another difference between our approaches. Dr. Bergeron’s study was primarily conducted with cultures, so they had unlimited amounts of their samples. Many of our samples come directly from mycangia and we have a very limited amount of fungal DNA. This meant that we often did not have enough DNA in our samples to visualize the bands from single copy loci. Therefore, we went back to the drawing board (= IGS) to conduct a study investigating the use of A. chailletii versus different strains of A. areolatum by native eastern Sirex species. We still had a problem with how to tell apart the strains of A. areolatum because there are multiple copies. The cloning approach used by Nielsen et al. (2009) is very costly and time-consuming so Dr. Kepler is now conducting fragment analysis. 174
Publication: We are working hard toward completing a paper (first draft by December 15) reporting our results regarding the species and strains of Amylostereum being carried by native Sirex in eastern North America. This will include an empirical evaluation, based on wasps emerging from all of our wood samples and caught in traps. Then, we also have a subset of native Sirex emerging from the same barrels as S. noctilio and we will evaluate the fungi carried in these instances. We are aiming this paper toward submission to Biology Letters
Deladenus
Samples: Erin Morris, a doctoral student in my laboratory has been using molecular techniques with samples of Deladenus dissected from adult Sirex collected from our reared material as well as traps. At present she is working with the following Sirex samples (out of the total number of samples that we have). The goal with this study is to investigate the genetic relatedness of the Deladenus parasitizing different Sirex species but especially the native Sirex versus S. noctilio.
13 of 18 Sirex nigricornis 2 of 2 Sirex edwardsii 21 of 38 Sirex cyaneus 2 of 4 Sirex nitidus 1 of 1 S. californicus
In her studies, she is including the non-sterilizing strain of D. siricidicola (of which she is including at least 10-14 and we have more samples than this) as well as the Kamona strain. Also included in her studies have been some nematodes from Sirex juvencus and S. noctilio from Hungary.
Molecular methods: Erin has been using 4 different loci to investigate the different nematode strains and species present in her samples: LSU 28S, SSU 18S, ITS and CO1. She is sequencing the loci from each sample.
Publication and Poster: Erin is presently working hard toward completing her analyses for a molecular review of the nematodes being carried by native eastern North American Sirex. The results will include a phylogenetic tree showing relationships among native Deladenus in eastern North American Sirex and the nematodes from Sirex noctilio. Although it’s a little premature (and confidential at this moment), Erin has been finding that S. nigricornis and edwardsii parasitized by Deladenus are almost always parasitized by a nematode that is very distantly related to D. siricidicola; we think that this could be what Robin Bedding described as D. proximus (type specimens were from S. nigricornis from South Carolina; Bedding 1974). We think that the nematodes from Sirex cyaneus are most probably what Robin Bedding described as 175
D. canii. The Deladenus parasitizing Sirex nitidus is very similar to D. siricidicola and perhaps this is D. wilsoni. This publication will include no morphological work so we will not be able to say for sure whether these species names are correct. Erin plans to complete a poster for the USDA Interagency meeting in Annapolis in January 2012 and she plans to write up the study as a publication shortly thereafter, in 2012. However, since these results are part of Erin’s PhD thesis, please treat this information confidentially at present.
Erin’s work will provide information about whether native Sirex are carrying the nematodes that parasitize S. noctilio (e.g., the non-sterilizing strain of D. siricidicola). In fact, of the 13 nematodes that she has sequenced from S. nigrocornis, only one was D. siricidicola, non- sterilizing strain. She has 5 more S. nigricornis with Deladenus so we will evaluate those to see what nematodes they are carrying.
Fungal associations of native Deladenus. We have collected the data about fungal species and strains associated with the different Deladenus carried by native Sirex but have not working on this aspect of the data set but we have not focused on analysis of nematode association with fungi as of yet since our focus thus far has been host associations (but this is certainly to come).
2. Isolate living strains of nematodes from siricids reared from infested wood and investigate their growth on cultures of A. areolatum and A. chailletii isolated from different siricids. We isolated several strains of nematodes from Sirex noctilio. We are very pleased with this because our laboratory has not been successful with this procedure previously. At present, only one of the strains of nematodes is still alive and we will now work on freezing it (a goal for the first week of December 2011). It came from red pine from Pack Experimental Forest in the southern Adirondacks and is the non-sterilizing strain of D. siricidicola and feeds on A. areolatum.
3. Continue studies optimizing growth of the commercial strain of D. siricidicola to identify an optimal balance of the relative concentrations of nematodes and amount of fungal growth for different strains of A. areolatum. Erin Morris completed data analysis and submitted a manuscript along the lines of this objective. This paper has been submitted to the journal BioControl:
Morris, E.E., Jimenez, A., Long, S.J., Williams, D.W., Hajek, A.E. Variability in growth of Deladenus siricidicola on strains of the white rot fungus Amylostereum areolatum. BioControl (in review).
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Erin conducted extensive studies of the growth of Kamona strain D. siricidicola on different fungal strains, with emphasis on how long it takes for nematodes to develop to adults when feeding on different fungal strains. She plans to conduct electron microscopy to demonstrate when the fungus attacks nematode eggs. We think that the reason D. siricidicola does not grow as well on the Ecogrow strain of A. areolatum is that this fungal strain grows so quickly and the nematodes can’t control it (by harvesting the edges) so it then overgrows the nematode colony and is able to eat the nematode eggs. Once Erin’s poster and publication on the Deladenus phylogeny are complete, then she will begin to analyze these data.
4. Will the commercial strain of D. siricidicola hybridize with the strain of D. siricidicola present in the field in northeastern North America? Objective 1 required immense efforts over the past year and we were unable to conduct these studies with the personnel and time available.
5. Are bacteria associated D. siricidicola? Our inspiration for conducting this study, Dr. Patricia Stock of the University of Arizona, Tucson, AZ was unavailable due to health challenges. In addition, we do not have any good evidence of significant interactions of D. siricidicola with bacteria and so, without her guidance (and with the overload of other studies and accomplishments) we made no progress with this objective.
6. Evaluation of non-target insects from trees inoculated with commercially available D. siricidicola. Non-target samples from trees inoculated with Kamona in 2009, with emergence in 2010. We had an exceptionally busy year, filled with studies with living siricids, nematodes and fungus so, unfortunately, we have not evaluated these samples yet. We also did not get to this because the main person in the lab who is very well-versed in this work (Stefan Long) was out for a month due to appendicitis. In the near future, we will dissect every individual sent to us and will search for nematodes within them. In particular, we will search in hemocoels and reproductive structures under a microscope. Nematodes that we find will be evaluated using molecular primers to determine whether they are the same as the nematodes that were initially injected into the wood.
Included in these efforts was an evaluation of the occurrence of the D. siricidicola non-sterilizing strain in S. noctilio and native Sirex.
We evaluated nematodes in samples of Sirex reared from wood into which Dave Williams’ group injected Kamona nematodes in 2007 in Highland State Forest in Onondaga County, NY. All of the siricids that emerged from the wood were Sirex noctilio except for 3 native Sirex. Erin Morris used cytochrome oxidase I and the ribosomal large subunit (28S) to 177 evaluate some of the nematodes in the Sirex that were sent. For others she PCRed with CO1 followed by restriction enzymes.
Dave and Carrie had hypothesized that the one Sirex edwardsii contained ‘Beddingia-like’ nematodes and the two Sirex nigricornis samples contained nematodes that were more ‘sausage- like’ and that these were likely to be ‘native’ nematodes. The nematodes in the one Sirex edwardsii were neither Kamona nor the D. siricidicola non-sterilizing strain. The nematodes in the two Sirex nigricornis were not Kamona and not the D. siricidicola non-sterilizing strain. The nematodes in these two native Sirex species were all the same and were also the same as nematodes found in our samples of S. nigricornis and edwardsii in New York, Pennsylvania and Louisiana.
We have a total of 116 samples of S. noctilio from this study and DNA has been extracted from all of these. A subsample has been sequenced thus far: Site # sequenced out of Rt 69 4 30 West Monroe 3 17 MC 0 6 Zink's 0 14 Hill's Creek 4 18 Rodman 5 30
Results to date are below. Dave provided us with data about whether nematodes were in S. noctilio eggs or not and very time that nematodes were not found in the eggs our sequence data showed that the nematode was the non-sterilizing strain. Conversely, when nematodes were found within S. noctilio eggs by Dave, the sequence data showed that the nematode was Kamona.
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Site Erin's number Route 69 Females treatment in eggs? CO1 RE type 146 S17‐3 E6/OAa no NS strain Males 148 S1‐5 E6/OAa no NS strain 149 S1‐1 E6/OAa no NS strain 152 S1‐9 E6/OAa no NS strain West Monroe Males 79 W5‐1 E6/OAa no NS strain 76 W5‐7 E6/OAa no NS strain 80 W6‐6 E6/OAa no NS strain Hills Creek Males 101 H15‐1 E6/OAa yes Kamona 96 H24‐1 E6/OAa yes Kamona 99 H3‐1 E6/OAa yes Kamona 106 H24‐3 E6/OAa yes Kamona Rodman Females 115 R6‐1 E6/OAa no NS strain Males 124 R4‐1 E6/OAa no NS strain 116 R6‐2 E6/OAa no NS strain 120 R6‐5 E6/OAa no NS strain 119 R6‐11 E6/OAa no NS strain
Shortly, we will evaluate the remaining samples using CO1 and 3 restriction enzymes that will differentiate between Kamona, NS strain (non-sterilizing D. siricidicola) and what we are calling D. proximus (the nematode we find in S. nigricornis and edwardsii which might be in the same trees as S. noctilio). 179
Table 1. Fungal cultures stored by Dr. Iben Thomsen, University of Copenhagen that Dr. Kepler attempted to revive in July 2011.
Iben R. Cultur Status Fungal Date(d/m/ Site Wasp species Substrate Sourc Coll. By Thoms Vasiliauska e Species y) e(frb en ID# s ID# starte =fruit d body) DK2 No A. chailletii 22/10/92 Rude Skov, Holte, L. decidua frb JK Zealand, DK DK3a Yes Growi A. chailletii 22/10/92 Rude Skov, Holte, Picea abies frb IMT ng Zealand, DK DK3b Yes Growi A. chailletii 22/10/92 Rude Skov, Holte, Picea abies frb IMT ng Zealand, DK DK4 Yes Growi A. chailletii 22/10/92 Rude Skov, Holte, Picea abies frb JK ng Zealand, DK DK5 Yes Failed A. chailletii 22/10/92 Rude Skov, Holte, Picea abies frb JK Zealand, DK DK6 Yes Failed A. chailletii 22/10/92 Rude Skov, Holte, Picea abies frb IMT Zealand, DK DK7 No A. chailletii 22/10/92 Rude Skov, Holte, Picea abies frb IMT Zealand, DK DK8 Yes Failed A. chailletii 20/10/92 Frederikslund Skov, Picea abies frb JK Holte, DK DK9 Yes Failed A. 20/10/92 Frederikslund Skov, Picea abies frb JK areolatum Holte, DK DK10 Yes Growi A. chailletii 20/10/92 Rude Skov, Holte, Abies grandis frb IMT ng Zealand, DK DK12 Yes Growi A. 20/10/92 Rude Skov, Holte, Picea abies frb JK ng areolatum Zealand, DK 180
DK17 Yes Growi A. 20/10/92 Rude Skov, Holte, Picea abies frb IMT ng areolatum Zealand, DK DK19 No A. chailletii 20/10/92 Rude Skov, Holte, Picea abies frb IMT Zealand, DK DK20 Yes Growi A. 20/10/92 Rude Skov, Holte, Picea abies frb IMT ng areolatum Zealand, DK DK21 Yes Growi A. chailletii 22/10/92 Rude Skov, Holte, Abies grandis frb IMT ng Zealand, DK DK22 No A. chailletii 20/10/92 Stubberup Skov, Picea abies frb IMT/JK Vemmetofte, Zealand, DK DK23 Yes Growi A. chailletii 30/03/92 Stubberup Skov, Picea abies frb IMT/JK ng Vemmetofte, Zealand, DK DK25 Yes Growi A. chailletii 30/03/92 Stubberup Skov, Picea abies frb IMT/JK ng Vemmetofte, Zealand, DK DK26 Yes Growi A. chailletii 30/03/92 Stubberup Skov, Picea abies frb IMT/JK ng Vemmetofte, Zealand, DK DK27 No A. chailletii 30/03/92 Stubberup Skov, Picea abies frb IMT/JK Vemmetofte, Zealand, DK DK28 Yes Failed A. chailletii 30/03/92 Stubberup Skov, A. frb IMT/JK Vemmetofte, Zealand, nordmanniana DK DK29 No A. chailletii 30/03/92 Stubberup Skov, A. frb IMT/JK Vemmetofte, Zealand, nordmanniana DK 181
DK31 Yes Growi A. chailletii 30/03/92 Stubberup Skov, A. frb IMT/JK ng Vemmetofte, Zealand, nordmanniana DK DK32 Yes Growi A. chailletii 30/03/92 Stubberup Skov, A. frb IMT/JK ng Vemmetofte, Zealand, nordmanniana DK DK37 Yes Growi A. 03/10/93 Sorø Sønderskov, Picea abies frb IMT ng areolatum Zealand DK38 Yes Failed A. chailletii 03/10/93 Sorø Sønderskov, Picea abies frb IMT Zealand DK40 Yes Failed A. chailletii 03/10/93 Sorø Sønderskov, Picea abies frb IMT Zealand DK42 No A. chailletii 03/10/93 St. Bøgeskov, Sorø Picea abies frb IMT DK44 No A. chailletii 21/10/93 St. Bøgeskov, Sorø Picea abies frb IMT DK45 Yes Failed A. chailletii 21/10/93 Svinkløv Pl. Thy Picea abies frb JVH DK48 Yes Failed A. chailletii 03/11/93 Geel Skov, Holte, Picea abies frb BKA Zealand, DK DK51 Yes Failed DK52 Yes Growi A. chailletii 11/93 Skojoldenaesholm Abies alba wood IMT ng DK53 Yes Growi A. chailletii 11/93 Skojoldenaesholm Picea abies wood IMT ng DK54 Yes Growi A. chailletii 11/93 Palsgaard, Jutland, DK P. menziessi wood IMT ng DK55 Yes Growi A. 15/12/93 Randbøl Picea abies frb IMT ng areolatum DK56 No A. 01/94 Geel Skov, Holte, Picea abies ? frb BKA areolatum Zealand, DK DK58 No A. 12/03/94 Frijsenborg Picea abies wood IMT 182
areolatum DK59 Yes Growi A. 12/03/94 Kalvebod Faelled Picea abies ? frb BTO ng areolatum DK60 Yes Failed A. chailletii 12/03/94 Høstermark Sk, Jutland, Abies alba frb JVH DK DK61 Yes Failed A. chailletii 03/94 Høstermark Sk, Jutland, Abies alba frb JVH DK DK62 Yes Growi A. 25/04/94 Rude Skov, Holte, conifer stump frb JK ng areolatum Zealand, DK DK63 No A. chailletii 25/04/94 Store Dyrehave Picea abies frb IMT/JK DK66a No A. chailletii 25/04/94 Store Dyrehave P. menziessi frb IMT DK66b Yes Growi A. chailletii 25/04/94 Store Dyrehave P. menziessi wood IMT ng DK76 Yes Failed A. 24/10/94 Rude Skov, Holte, Picea abies frb IMT areolatum Zealand, DK (=62) DK77 Yes Failed A. chailletii 19/11/94 Rude Skov, Holte, P. menziessi wood IMT Zealand, DK DK78 No A. chailletii 19/11/94 Ulborg, Jutland, DK Abies grandis wood IMT DK79 No A. chailletii 19/11/94 Gråsten Picea abies wood IMT DK80 No A. chailletii 29/11/94 Bregentved P. menziessi wood IMT DK78 No A. chailletii 29/11/94 Ulborg, Jutland, DK Abies grandis wood IMT DK90a No A. chailletii 18/04/96 Uggerløse Skov Picea abies frb IMT DK90b No A. chailletii 18/04/96 Uggerløse Skov Picea abies wood IMT S201 I‐1 Yes Growi A. 10/94 Sweden, Tierp (Uppsala ng areolatum L202 V‐02 Yes Growi A. 02/96 Lithuania, sample plot V ng areolatum L203 RA‐3 Yes Growi A. 04/94 Lithuania, sample plot R ng areolatum 183
L204 7 A‐14 Yes Growi A. 04/94 Lithuania, sample plot 7 ng areolatum L205 11 A‐5 Yes Growi A. 04/94 Lithuania, sample plot 11 ng areolatum L206 V‐60 Yes Growi A. chailletii 03/95 Lithuania, sample plot V ng L209 LL A‐9 Yes Growi A. 04/94 Lithuania, sample plot 11 ng areolatum L210 V‐01 Yes Growi A. chailletii 02/96 Lithuania, sample plot V ng S212 I‐12 Yes Growi A. 10/94 Sweden, Tierp (Uppsala ng areolatum L213 V‐113 Yes Growi A. chailletii 03/95 Lithuania, sample plot V ng L214 11 A‐214 Yes Growi A. 04/94 Lithuania, sample plot 11 ng areolatum L215 SA‐5 Yes Growi A. 04/94 Lithuania, sample plot S ng areolatum L216 11 A‐116 Yes Growi A. chailletii 04/94 Lithuania, sample plot 11 ng L218 V‐0118 Yes Growi A. chailletii 02/96 Lithuania, sample plot V ng L220B V‐22B Yes Growi A. chailletii 03/95 Lithuania, sample plot V ng L220C V‐22C Yes Growi A. 03/95 Lithuania, sample plot V ng areolatum L221 11 A‐221 No A. 04/94 Lithuania, sample plot 11 areolatum S222 I‐22 Yes Growi A. chailletii 10/94 Sweden, Tierp (Uppsala 184
ng L223 V‐0123 Yes Growi A. chailletii 02/96 Lithuania, sample plot V ng S225 III‐25 Yes Growi A. 10/94 Sweden, Bysala (Köping) ng areolatum S226 III‐16 Yes Growi A. 10/94 Sweden, Bysala (Köping) ng areolatum S227 III‐27 Yes Growi A. 10/94 Sweden, Bysala (Köping) ng areolatum S228 III‐28 Yes Growi A. 10/94 Sweden, Bysala (Köping) ng areolatum L229 RA‐229 No A. 04/94 Lithuania, sample plot R areolatum L233 V‐3 Yes Growi A. chailletii 02/96 Lithuania, sample plot V ng L234 V‐34 Yes Growi A. chailletii 03/95 Lithuania, sample plot V ng L236 V‐36 Yes Growi A. 03/95 Lithuania, sample plot V ng areolatum S240 III‐4 No A. 10/94 Sweden, Bysala (Köping) areolatum L241 V‐14 Yes Growi A. chailletii 02/96 Lithuania, sample plot V ng L254 V‐154 Yes Growi A. 03/95 Lithuania, sample plot V ng areolatum L255 V‐155 Yes Growi A. chailletii 03/95 Lithuania, sample plot V ng L257 V‐0157 Yes Growi A. 02/96 Lithuania, sample plot V ng areolatum 185
L260 7 A‐160 Yes Growi A. 04/94 Lithuania, sample plot 7 ng areolatum L261 V‐0166 Yes Growi A. 02/96 Lithuania, sample plot V ng areolatum L262 v‐26 Yes Growi A. 03/95 Lithuania, sample plot V ng areolatum S266 III‐60 Yes Growi A. 10/94 Sweden, Bysala (Köping) ng areolatum L274 V‐74 Yes Growi A. 03/95 Lithuania, sample plot V ng areolatum L279 V‐079 Yes Growi A. 02/96 Lithuania, sample plot V ng areolatum L280 V‐0180 Yes Growi A. 02/96 Lithuania, sample plot V ng areolatum S282 II‐82 Yes Growi A. 10/94 Sweden, Tierp (Uppsala ng areolatum L296 KA‐396 Yes Growi A. chailletii 04/94 Lithuania, sample plot K ng L297 2 A‐97 Yes Growi A. chailletii 04/94 Lithuania, sample plot 21 ng L299 2 A‐99 Yes Growi A. chailletii 04/94 Lithuania, sample plot 21 ng L230 Yes Growi A. ng areolatum L274 Yes Failed A. areolatum L279 Yes Failed A. areolatum L269 Yes Growi A. 186
ng areolatum L280 Yes Failed A. areolatum L219 Yes Growi A. chailletii ng L293 Yes Growi A. chailletii ng L281 Yes Growi A. chailletii ng L264 Yes Growi A. chailletii ng L244 Yes Growi A. chailletii ng DK531 Yes Failed A. 07/07/94 Frederikshåb Pl. Randbøl, Sirex juvencus IMT areolatum Jutland, DK DK532 Yes Growi A. 07/07/94 Frederikshåb Pl. Randbøl, Sirex juvencus IMT ng areolatum Jutland, DK DK539 Yes Growi A. 18/07/94 Frederikshåb Pl. Randbøl, Sirex juvencus IMT ng areolatum Jutland, DK DK540 Yes Growi A. 18/07/94 Frederikshåb Pl. Randbøl, Sirex juvencus IMT ng areolatum Jutland, DK DK541 Yes Growi A. 19/07/94 Frederikshåb Pl. Randbøl, Sirex juvencus IMT ng areolatum Jutland, DK DK544 Yes Growi A. 25/07/94 Frederikshåb Pl. Randbøl, Sirex juvencus IMT ng areolatum Jutland, DK DK545 Yes Failed A. 28/07/94 Rude Skov, Holte, Sirex juvencus IMT areolatum Zealand, DK DK547 Yes Growi A. 01/08/94 Rude Skov, Holte, Sirex juvencus IMT ng areolatum Zealand, DK 187
DK550 Yes Failed A. 08/94 Rude Skov, Holte, Sirex juvencus IMT areolatum Zealand, DK DK557 Yes Failed A. 01/08/95 Rude Skov, Holte, Sirex juvencus IMT areolatum Zealand, DK DK566 Yes Failed A. 01/08/95 Virum Sirex juvencus AHA areolatum DK530 Yes Failed A. chailletii 05/94 Gilleleje, Helsingor, Urocerus Picea abies IMT Zealand, DK gigas DK534 Yes Failed A. chailletii 11/07/94 Rude Skov, Holte, Urocerus Picea abies IMT Zealand, DK gigas DK535 No A. chailletii 11/07/94 Rude Skov, Holte, Urocerus Picea abies IMT Zealand, DK gigas DK537 Yes Growi A. chailletii 18/07/94 Rude Skov, Holte, Urocerus Picea abies IMT ng Zealand, DK gigas DK538 Yes Growi A. chailletii 18/07/94 Rude Skov, Holte, Urocerus Picea abies IMT ng Zealand, DK gigas DK546 Yes Growi A. chailletii 29/07/94 Rude Skov, Holte, Urocerus Picea abies IMT ng Zealand, DK gigas DK554 Yes Failed A. chailletii 20/07/95 Grib Skov, Hillerod, Urocerus Larix SHA Zealand, DK gigas DK558 No A. chailletii 27/07/95 Rude Skov, Holte, Urocerus Picea abies IMT Zealand, DK gigas DK559 Yes Growi A. chailletii 27/07/95 Rude Skov, Holte, Urocerus Picea abies IMT ng Zealand, DK gigas CP678 No Growi A. 10/83 Rold Skov, Jutland, DK Picea abies JK ng areolatum CP679 Yes Growi A. 10/83 Rold Skov, Jutland, DK Picea abies JK ng areolatum CP680 No A. 10/83 Rold Skov, Jutland, DK Picea abies JK 188
areolatum CP681 No A. 10/83 Rold Skov, Jutland, DK Picea abies JK areolatum CP692 No A. 11/83 Rold Skov, Jutland, DK Picea abies JK areolatum CP782 Yes Growi A. 03/87 Teglstrup Hegn, Picea abies JK ng areolatum Helsingor, Zealand, DK CP784 Yes Growi A. 03/87 Teglstrup Hegn, Picea abies JK ng areolatum Helsingor, Zealand, DK CP832 No A. 01/07/88 Grib Skov, Hillerod, Picea abies JK areolatum Zealand, DK CP886 Yes Growi A. 02/90 Farum Sk.dstr. Picea abies JK ng areolatum CP955 Yes Growi A. 02/10/92 Teglstrup Hegn, Picea abies JK ng areolatum Helsingor, Zealand, DK CP724 No A. chailletii 02/85 Farum Sk.dstr., Farum, Picea abies JK Zealand, DK CP940 No A. chailletii 03/92 Willestrup Picea sitchensis IMT CP942 Yes Failed A. chailletii 04/92 St. Dyrehave Picea abies JK CP953 Yes Growi A. chailletii 02/10/92 Teglstrup Hegn, Picea abies JK ng Helsingor, Zealand CP954 Yes Growi A. chailletii 02/10/92 Teglstrup Hegn, Picea abies JK ng Helsingor, Zealand CO962 Yes Growi A. chailletii 07/11/92 Naesseskoven, Holte Picea abies JK ng CP966 Yes Growi A. chailletii 22/11/92 Geel Skov, Holte, Picea abies JK ng Zealand, DK CP983 Yes Failed A. chailletii 01/01/93 Geel Skov, Holte, Picea abies JK Zealand, DK 189
CP986 Yes Failed A. chailletii 12/01/93 Løvenholm, Jutland, DK Picea abies JK CP012 Yes Growi A. chailletii 17/06/93 Grib Skov, Hillerod, Picea abies JK ng Zealand, DK CP017 Yes Growi A. chailletii 07/07/93 Klosterheden, Jutland, Picea abies JK ng DK CP022 Yes Growi A. chailletii 25/10/93 Feldborg, afd 273, Picea abies JK ng Jutland, DK CP023 Yes Failed A. chailletii 26/10/93 Gludsted, afd 139, Abies alba JK Jutland, DK CP031 Yes Growi A. chailletii 04/01/94 Løvenholm, Jutland, DK Picea abies JK ng 192 Yes Failed A. chailletii
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Progress: 2012
Emphasis over the past few months has been on convincing scientists in Europe to work with us collecting siricid specimens. This is not so simple because it’s not so easy to collect siricids. However, we have 3 collaborators who are very actively working on collecting specimens.
European collaborators During fall and winter, we established contacts with forest entomologists in several European countries to find collaborators who would assist us with collecting siricids from conifers in Europe. We have sent extensive information to all of them. The major contacts and their involvement include Belgium, Spain and Italy:
1. Belgium: Dr. Jean-Claude Grégoire [email protected] Biological Control and Spatial Ecology Lab, Université Libre de Bruxelles, 50 av FD Roosevelt - CP 160/12, 1050 Bruxelles, Belgium Dr. Gregoire’s graduate student, Quentin, will be collecting siricids and putting them into buffer. We hope to go and help (especially to establish fungal cultures) but timing might be difficult to figure out because they have not done this before. At this time (early July), we have sent information about how to collect and about the buffer we prefer.
2. Spain: Dr. Juan Alberto Pajares Alonso [email protected] Universidad Valladolid, Sustainable Forestry Management Dr. Pajares has a lot of experience and a big project at this time working with Monochamus. He has wood caged from which he expects Monochamus to emerge and he also hopes that siricids will emerge, at which point they will be collected. He also is trapping with intercept panel traps, with advice from Dr. Lombardero from Galicia, Spain (Dr. Matt Ayres did work with Dr. Lombardero looking for Sirex in Galician pines and this was not easy so she suggested trapping). Dr. Pajares has experience with traps and pheromones and with isolating fungal associates of wood borers and, once again, since he is unsure about the timing for siricid emergence in Spain, he is not interested in having us visit his lab to help.
3. Italy: Dr. Sergio Angeli
Dr. Angeli will be trapping/collecting siricids in the Dolomite region of Italy. We sent 8 intercept panel traps (with netting collection bags; our design) and CTAB buffer to Dr. Angeli, along with extensive instructions. Dr. Angeli will also go to lumber yards to collect siricids.
We also have collaborations at different stages of development with the following scientists: 191
Switzerland: Dr. Beat Wermelinger ETH, Zurich
Dr. Wermelinger did not have space to collaborate this year (I asked him last year too) but he is herbicided trees in the Valais region this summer and will rear out of them next summer.
Czech Republic: Dr. Petr Srutka Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6, Czech Republic
Dr. Srutka worked with and published on fungal associates of Xiphydria woodwasps so he is our associate with the greatest and most appropriate experience. Dr. Srutka is going to try to rear from infested wood this year (he also tried last year).
Turkey: Dr. Suleyman Akbulut
192
References
Bedding RA (1968) Deladenus wilsoni n. sp. and D. siricidicola n. sp. (Neotylenchidae), entomophagous-mycetophagous nematodes parasitic in siricid woodwasps. Nematologica 14: 515-525.
Bedding R. A. 1974. Five new species of Deladenus (Neotylenchidae), entomophagous- mycetophagous nematodes parasitic in siricid woodwasps. Nematologica 20:204-225.
Bedding RA, Akhurst RJ (1978) Geographical distribution and host preferences of Deladenus species (Nematoda: Neotylenchidae) parasitic in siricid woodwasps and associated hymenopterous parasitoids. Nematologica 24:286-294.
Bergeron M.-J., I. Leal, B. Foord, G. Ross, C. Davis, B. Slippers, P. de Groot, & R. C. Hamelin. 2011. Putative origin of clonal lineages of Amylostereum areolatum, the fungal symbiont associated with Sirex noctilio, retrieved from Pinus sylvestris, in eastern Canada. Fungal Biology 115:750-758.
Hurley, B.P., Slippers, B., Wingfield, M.J. 2007. A comparison of control results for the alien invasive woodwasp, Sirex noctilio, in the southern hemisphere. Agric. For. Entomol. 9: 159–171.
Nielsen, C., Williams, D.W., Hajek, A.E. 2009. Putative source of the invasive Sirex noctilio fungal symbiont, Amylostereum areolatum, in the eastern United States and its association with native siricid woodwasps. Mycological Research 113: 1242-1253.
Slippers, B., Wingfield, M.J., Coutinho, T.A., Wingfield, B.D. 2001. Population structure and possible origin of Amylostereum areolatum in South Africa. Plant Pathol. 50: 206–210.
Slippers, B., Wingfield, B.D., Coutinho, T.A., Wingfield, M.J. 2002. DNA sequence and RFLP data reflect geographical spread and relationships of Amylostereum areolatum and its insect vectors. Molec. Ecol. 11: 1845–1854.
Slippers, B., Coutinho, T.A., Wingfield, B.D., Wingfield, M.J. 2003. The genus Amylostereum and its association with woodwasps: a contempary review. So. Afr. J. Sci. 99: 70–74.
Smith, D.R., Schiff, N.M. 2002. A review of the siricid woodwasps and their ibaliid parasitoids (Hymenoptera: Siricidae, Ibaliidae) in the eastern United States, with emphasis on the mid- Atlantic region. Proc. Entomol. Soc. Wash. 104: 174-194.
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Thomsen IM, (1996) Amylostereum areolatum and Amylostereum chailletii, symbiotic fungi of woodwasps (Sirex sp. and Urocerus sp.). PhD thesis, Danish Forest and Landscape Research Institute, Hørsholm, Denmark.
Thomsen IM, Harding S (2011) Fungal symbionts of siricid woodwasps: Isolation techniques and identification. Forest Pathology 41(4): 325–333.
Thomsen, I.M., Koch, J. 1999. Somatic compatibility in Amylostereum areolatum and A. chailletii as a consequence of symbiosis with siricid woodwasps. Mycol. Res. 103: 817-823.
Vasiliauskas, R., Stenlid, J. 1999. Vegetative compatibility groups of Amylostereum areolatum and A. chailetii from Sweden and Lithuania. Mycol. Res. 103: 824-829.
Vasiliauskas, R., Stenlid, J., Thomsen, I.M. 1998. Clonality and genetic variation in Amylostereum areolatum and A. chailletii from northern Europe. New Phytol. 139: 751-758.
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Endemic nematode survey of New York 2006-2012
Kelley Zylstra
In 2006 a survey was initiated in order to document the presence or absence of nematodes associated with the recent S. noctilio infestation discovered near Fulton NY. Until this point it was not known whether there were already other nematode species present with the invasive woodwasp population. In 2006, Sirex-infested host material taken from SUNY Oswego was placed in a field tent to rear S. noctilio. Individuals from that host material were dissected and were found infected with nematodes (Fig.1). This was the first confirmation of an endemic nematode associated with the invasive population of S. noctilio in the U.S. Starting in 2007 and continuing through 2012, a survey for the endemic nematode was conducted from all of the trap- caught S. noctilio from detection traps deployed across NY state. In early 2010, Evan Braswell identified the nematodes as a strain of D. siricidicola from samples I had sent. He said the nematodes collected in NY showed differences from that of the Australian (Kamona) strain being proposed for release.
Figure 1. Juvenile nematodes found inside adult S. noctilio in 2006 from SUNY Oswego campus
Annual Frequency of Nematode Occurrence of Trap-Caught Siricids Each year (2006-2012) survey traps were deployed in New York. Insect detection traps placed at each field site were checked bi-weekly, if not weekly, for each of the survey years. All siricids captured were retained in 70 % ethanol with accompanying locality information. Siricids were dissected to check for the presence of nematodes. In the beginning (2006-2007) data was recorded for presence/absence of nematodes associated with individuals. Starting in 2008, attention was made to determine if juvenile nematodes were making it inside of individual eggs. Typically, half a dozen or fewer adult nematodes are found in the abdominal cavity of an infected S. noctilio. Figure 2 demonstrates the percentage of the surveyed S. noctilio population that was found infected with nematodes each year, which ranges from 7.7-33.6%. The 195 percentage of S. noctilio that were found with nematodes inside of their eggs is shown in Table 1.
To examine the eggs for nematodes, a cluster of around 14 eggs were plated on the slide under the compound microscope. In 2011, the number of eggs per plated slide containing nematodes was noted. When nematodes were found in the eggs they were not found in every egg. Around 2 eggs per plated slide had nematodes inside.
% Occurance of Nematodes Found in S. noctilio Population Surveyed Annually
2012 10 out of 54 sampled 2011 50 out of 259 38 out of 113 2010 sampled sampled Year 76 out 387 2009 sampled 60 out 427
Survey 2008 sampled 47 out of 541 2007 sampled 7 out 91 2006 sampled
0 5 10 15 20 25 30 35 40 Percentage
Figure 2. The percent of the surveyed population of S. noctilio found infected with D. siricidicola nematodes. The data includes nematodes solely in the abdomen too, not just those that made it into the eggs. Table 1. The percentage of S. noctilio surveyed that were found with infected with D. siricidicola juvenile nematodes and the nematodes were inside of their eggs. Individuals Total Nematodes w/ Nemas Year Dissected Present In Eggs Percent 2012 54 11 5 45.5 2011 113 38 6 15.8 2010 259 47 16 34 2009 387 76 5 6.6 2008 427 60 16 26.7
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Native siricids were captured in detection traps and dissected for nematodes each year as well (Table 2). However, no molecular work has been done to determine the identification of the nematodes found.
Table 2. Dissection of native Siricidae.
2007 2008 Total Infected By % Total Infected By % Species Dissected Nematodes Parasitism Dissected Nematodes Parasitism
Urocerus spp. 26 0 0 13 2 15.4 T. columba 28 0 0 39 0 0 S. nigricornis 18 0 0 4 0 0 S. edwardsii 4 0 0 0 0 0 Xiphydria spp. 0 0 0 1 0 0
2009 2010 Total Infected By % Total Infected By % Dissected Nematodes Parasitism Dissected Nematodes Parasitism Urocerus spp. 30 4 13.3 10 0 0 T. columba 16 1 6.3 48 1 2.1 S. nigricornis 11 0 0 52 7 13.5 S. edwardsii 2 0 0 3 0 0 Xiphydria spp. 0 0 0 19 0 0
2011 2012 Total Infected By % Total Infected By % Dissected Nematodes Parasitism Dissected Nematodes Parasitism Urocerus spp. 3 0 0 9 1 11.1 T. columba 16 0 0 1 0 0 S. nigricornis 0 0 0 0 0 0 S. edwardsii 0 0 0 0 0 0 Xiphydria spp. 0 0 0 0 0 0
Future work needs to include identification of the strains to explore any differences that may be related to egg infectivity among individuals, and parasitism between S. noctilio and the native 197
siricid complex.
Microscope Pictures From Dissection
Figure 4. Juvenile nematodes spilling out of a S. noctilio egg that has been broken in half.
Figure 5. Juvenile nematodes inside of a S. noctilio egg.
Figure 6. Juvenile nematodes inside the ovarioles and eggs of S. noctilio 198
Figure 7. Juvenile nematode from S. noctilio
Figure 8. The end of a S. noctilio egg broken off to expose juvenile nematodes inside. 199
IV. COMMUNITY ECOLOGY
Sirex noctilio in New York State: reproductive success, interactions with native species and the forensic dendropathology of infested trees 2007- 2010
Matthew P. Ayres
The report includes the following components. Note that the full report is in Appendix B.
Executive Summary
Chapter 1. Spatial Patterns and Reproductive Success of an Invasive Woodwasp in New York State.
Chapter 2. Competitive interactions between Amylostereum areolatum and fungi associated with the southern pine beetle
Chapter 3. On-ground surveys for Sirex spp in Vermont, New York, and Pennsylvania.
Chapter 4. Diagnosing the presence of Sirex noctilio from examination of dying and recently dead pine trees.
Chapter 5. Retrospective identification of feeding galleries of Sirex noctilio from genetic identification of the fungal associate, Amylostereum areolatum.
Chapter 6. Comparison of the occurrence of Sirex noctilio within host trees in New York State vs. its native range in Spain.
Chapter 7. The nutritional and ecological role of Amylostereum areolatum fungus in relation to an invasive woodwasp Sirex noctilio.
Chapter 8. Evaluation of the fungal community in logs for dating the time since death of pine trees.
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Executive Summary
Dartmouth College and the USDA-APHIS-PPQ Center for Plant Health Science and Technology engaged in a three year cooperative study of certain aspects of the biology, ecology and spatial dynamics of Sirex noctilio (European woodwasp). This wood-boring insect is considered one of the most serious forest insect pest invaders worldwide. All pine species are believed to be at risk. Overall goals of the work to be undertaken by Dartmouth College were to (1) provide an improved basis for judging how long S. noctilio has been in a region based on examination of dead trees; (2) evaluate patterns of reproductive success of S. noctilio; and (3) identify and characterize ecological factors that will influence whether S. noctilio becomes a notable forest pest in North America. Having conducted studies addressing these questions, Dartmouth College herewith provides USDA-APHIS-PPQ-CPHST with the resulting information, protocols, methods, databases, models, and interpretations.
1. Detection We developed and evaluated a protocol for detecting Sirex noctilio by genetic identification of Amylostereum areolatum in logs after departure of Sirex (see Chapter 5). This was possible via culturing of fungi followed by PCR and sequencing of DNA barcode regions. Unfortunately, the technique proved to have very low efficiency because the fungus has limited persistence within trees or logs following departure of the woodwasps. Also, it now seems that A. areolatum can be associated with native Sirex, which reduces the certainty of diagnoses based on the fungal DNA.
However, we have developed an alternative protocol, that is cheap, easily transferable, and reasonably efficient, based on the size and patterning of persistent emergence holes and resin drips within and among stands in a region (Chapters 3-4). Because the host trees are generally within even-aged stands, counting annual rings within live and dead trees in a stand permits inferences regarding the duration of occupancy by relatively high numbers of woodwasps.
2. Population ecology and tendencies in population dynamics. Key factors in the population ecology of S.noctilio include: (1) variable fecundity, (2) skewed sex ratios, (3) host suitability, and (4) community interactions with competitors and antagonists (Chapters 1, 2, 6 - 8).
Amylostereum areolatum is crucial to larval development, especially for digestion of cellulose. Based on the nutritional stoichiometry of S. noctilio, we hypothesize that the community of symbionts also includes some N-fixing bacteria which are crucial for meeting the protein requirements of growing larvae and which are supported by microbially aided digestion of cellulose (Chapter 7).
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Native Ophiostomatoid (“bluestain”) fungi, propagated by native bark beetles, appear to be antagonistic to Sirex noctilio larvae (probably during the first 1-2 instars when mutualistic fungus needs to become established near cambium) (Chapter 1). A. areolatum has very slow growth and competes poorly with native Ophiostomatoid fungi (Chapter 2). Thus, native bark beetles and their phoretic mites, which propagate bluestain fungi are likely to provide resistance in American forests to populations of S. noctilio.
Fecundity and sex ratio are highly variable but unrelated to local abundance or host suitability. Therefore no destabilizing positive feedback in population dynamics from these sources (and lower risk of outbreaks than with positive feedback). (Chapter 1)
Parasitism rates by Ibalia can be very high (good for limiting outbreak risk) but were negatively related to local abundance, implying predator swamping, and a potential for outbreaks from predator release if populations become moderately abundant for any reason. (Chapter 1) red pine appear to be of comparable nutritional suitability for S. noctilio but produce fewer Sirex progeny, perhaps because they are less attractive to ovipositing adults (Chapter 1).
3. Ecology and population controls in native system To aid in projecting the potential impacts of Sirex noctilio in North America, we compared patterns of host tree attack in the recently invaded Finger Lakes region of New York state, with those in its native forests of northwestern Spain (Chapter 6). In the forests of both Spain and New York, S. noctilio were largely restricted to suppressed trees (smaller than average diameter) of the same size class that were also dying for other reasons. In both areas, Scots pine (native to Europe) was the species most likely to have attacks in non-suppressed trees and neither population appeared to be limited by suitable host trees. At least so far in the northeastern United States, the ecology of S. noctilio seems to be more like the situation in Europe (limited pestilence) than in the Southern Hemisphere (high pestilence).
Chapter 1. Spatial patterns and reproductive success of an invasive woodwasp in New York state
Jenna M. Sullivan, Jeffrey R. Garnas, and Matthew P. Ayres Biological Sciences, Dartmouth College, Hanover, NH 03755
ABSTRACT The ecological effects of introduced non-indigenous species are often minimal but sometimes profound. Predicting which introductions will be consequential is a challenge for ecologists and natural resource managers. We applied ecological theory to evaluate the trajectory of the recent invasion of North American forests by the European pine woodwasp, Sirex noctilio. S. noctilio has caused massive tree mortality following introductions to the southern hemisphere. In New York State, we found evidence of some natural controls on S. noctilio populations from interactions with indigenous species. There was a high incidence of 202 parasitism by the native wasp Ibalia leucospoides and of female infection by parasitic nematodes. Furthermore, there was a negative association between reproductive success by S. noctilio and the abundance of Ophiostoma ips, an indigenous bluestain fungus associated with indigenous bark beetles that is a putative competitor of the fungal mutualist of S. noctilio. However, there were also signals of destabilizing positive feedback in the population dynamics of S. noctilio: parasitism by I. leucospoides was least prevalent when S. noctilio abundance was low, which suggests predator swamping, and S. noctilio emergence was highly aggregated by tree, which is consistent with the hypothesis that increased numbers of woodwasp have increased success in overwhelming host tree defenses. Such positive feedbacks imply the potential for increasing abundance in coming years. However, at the scale of ~3200 km2, there was no spatial autocorrelation in the occurrence of S. noctilio, as would be expected if landscape pestilence arises from positive demographic feedbacks. The impacts of S. noctilio in North America will be modest if it mainly reproduces within stands of European Scots pine, which is a natural host species for S. noctilio that has been introduced into North America. Indeed, parental females were more common in Scots pine stands than red pine stands, but there were equal numbers of progeny emerging from the two species. Traditional forestry practices (e.g., thinning) could be used to limit pestilence from S. noctilio if the woodwasps preferentially attack suppressed trees within overstocked stands. Consistent with this, there was a higher incidence of S. noctilio in red pine stands with higher basal area. The expected future for S. noctilio in North America remains unclear; there would be value in continued studies of the relations between invasion ecology, insect entomology, and forest management in this system and elsewhere.
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Two projects on Sirex noctilio: progress in 2012
Kamal J.K. Gandhi
Project 1 Development of host preference-risk assessment maps for Eurasian woodwasp (Sirex noctilio) on southern conifer species
Objectives We will develop spatially-referenced risk models of S. noctilio that incorporate host preference information on southern conifer tree species. The two objectives of this project are as follows: 1) to assess the susceptibility of major conifer species known to be present in the southern forests to colonization by S. noctilio using feeding bioassays; and 2) based on this information, to provide a host risk assessment map for S. noctilio in southern conifer stands using geospatial techniques.
Brief description of tasks accomplished each year
Year 2009: We assessed the oviposition and colonization preferences of S. noctilio on conifer species that are present in varying degrees in southern forests. Two species of commercially important southern pines (loblolly and Virginia pine) were used for the bioassay study in 2009. Seven trees were felled in Georgia in late May. All trees were cut into 1m long logs in two diameter classes: 10-15 and 20-25 cm. Logs were transported to Syracuse, New York within five days of cutting, where three Scots pines (control species) were also cut into logs of two diameter classes. We conducted two experiments on S. noctilio as follows: 1. Host Choice: For the host choice experiment, four logs each of the three pine species (loblolly, Virginia, and Scots) in two diameter classes (small and large) were placed in random locations within a 4m2 arena. Forty one each of males and females of S. noctilio were released within the arena. Observations of the activity of adult S. noctilio on logs (e.g., ovipositing, sitting or walking) were taken every hour (three times) for three days. Logs were then individually enclosed in mesh sleeves, and stored at the APHIS lab. 2. Host No-choice: For the host no-choice study, two large diameter class logs of each of the three pine species were individually enclosed in mesh sleeves. Two male and female S. noctilio were introduced into each sleeve. Observations on the activity of adult S. noctilio on logs were taken once a day for three days. Logs were stored at the APHIS lab.
The adults of S. noctilio started emerging around August-September 2009, indicating rapid emergence of S. noctilio from our logs. We travelled to Syracuse in early December 2009 to collect the adults, and dissect the logs completely to collect all immature stages and count the number of exit holes to determine reproductive success of S. noctilio on southern pines. Adults 204 of the parental and progeny generations were also measured including attributes such as diameter of the pronotum, total length,length of the abdomen, and length of the ovipositor.
Our results from the 2009 experiment suggest that larger (18-26 cm diameter) bolts were more effective than smaller (11-17.8 cm diameter) bolts, as almost 23 times more S. noctilio were found walking/sitting on larger bolts. Sirex noctilio were most attracted to and developed the best on Virginia pine. Almost four times more S. noctilio were found walking/sitting and drilling on Virginia than Scots pine, with extremely low numbers on loblolly pine. Similarly, there were 10 times more exit holes of progeny on Virginia as compared to Scots pine with no progeny reared from loblolly pine. The strong activity of S. noctilio on Virginia pine suggests this species may be affected in the event of a S. noctilio range expansion to the southeastern U.S. Larger and more biologically applicable S. noctilio host choice studies needs to be conducted to elucidate the responses of S. noctilio to southeastern pines, and this study developed the necessary methodology to attempt such experiments under a laboratory setting in the future.
Year 2010: Bioassays of S. noctilio: In 2010, we continued our studies on the oviposition and colonization preferences of S. noctilio on southern conifer pines. Six species of commercially important southeastern pines including loblolly, shortleaf, slash, longleaf, white, and Virginia pine were used for the bioassay studies in 2010. Thirty six individual trees were felled in Georgia in early May. All trees were cut into 0.75 m long logs in diameter class of 20-30 cm. The large diameter class was chosen because it was preferred by S. noctilio over the smaller diameter class in the 2009 experiments. Logs were transported to Syracuse, New York within five days of cutting. Six Scots pines (control species) were also cut into 0.75 m logs in the same diameter class in Syracuse. Similar to 2009, we conducted two experiments on S. noctilio as follows: 1. Host Choice: For the host choice experiment, three logs each of the seven pine species (loblolly, shortleaf, slash, longleaf, white, Scots, and Virginia pine) were placed in random locations within a 4 m2 arena. 141 adults of S. noctilio were released within the arena. Observations of the activity of adult S. noctilio on logs (e.g., ovipositing, sitting or walking) were taken every hour (three times) for three days. Initial colonization observations (four observations in the first hour after release) were also conducted. Logs were then individually enclosed in mesh sleeves, and stored outside the APHIS lab under tarps for further processing. Four temperature and moisture loggers (HOBOs) were also installed under the tarps to gauge environmental conditions around the logs. 2. Host No-choice: For the host no-choice experiment, three logs each of the seven pine species (loblolly, shortleaf, slash, longleaf, white, Scots, and Virginia pine) were enclosed individually in mesh screens. Two adults of S. noctilio were released into each mesh screen on day 1 and 2 more adults in day 2. Observations of the activity of adult S. noctilio on logs (e.g., ovipositing, sitting or walking) were taken every hour (three times) for three days. Logs were then stored outside the APHIS lab for further processing 205
similar to that of the host choice experiment.
The adults of S. noctilio started emerging around August 2010, again indicating rapid emergence of S. noctilio from our logs. We travelled to Syracuse in November 2010 to move logs to an indoor emergence facility at SUNY, Syracuse to allow complete emergence of adults. In March 2011, we went back to Syracuse to collect the adults, and dissect the logs completely to collect all immature stages and count the number of exit holes to determine reproductive success of S. noctilio on southern pines. Adults of the parental and progeny generations were also measured such as diameter of the pronotum, total length, length of the abdomen, and length of the ovipositor.
Attributes of the southern tree species were determined to provide mechanisms for colonization preferences of S. noctilio. From each of the tested trees, we cut a 1-inch thick cookie towards each end of the logs (two cookies per tree); these wood cookies were frozen. For physical wood properties we assessed differences in surface area, specific gravity of the first ten rings of each tree, radial strip specific gravity, and density and area of resin canals among pine species. We also collected resin from each tree species at DBH height (two collections per tree) in late summer to determine differences in amounts and types of monoterpenes using GCMS among the southern pine species.
Our results from 2010 suggest that in the southeastern region, white and Virginia pine may experience the greatest colonization by S. noctilio, as it was attracted to and developed the best on these species. Sirex noctilio was found associated all seven species of pines tested in the choice experiments, but there was a clear preference for white, Virginia, and Scots pine. A possible mechanism of attraction of S. noctilio on pines may include lower specific gravity of wood. The resin of white pine seemed to have lower and/or higher percentages of many monoterpenes that distinguished this species from the other southeastern pines. The α- to ß- pinene ratio of white and Virginia pine was very similar to the 2.3:1 α- to ß-pinene ratio used in current S. noctilio lures.
GIS Mapping of S. noctilio: Utilizing data generated during the first phase of this project as weighted criteria and combining them with current versions of FHTET host tree layers, we have completed several iterations of the hazard map in ESRI Arc-GIS Desktop. A preliminary hazard model was bulit that included providing the highest weight to white pine followed by Scots, Virginia, longleaf, equal weight to loblolly and shortleaf, and slash pines. A slightly higher weight was given to the dryness index and tree density that are known to affect colonization dynamics. We are currently vetting our processing steps and optimizing our weighted criteria to create the final hazard map for S. noctilio.
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Project 2 An assessment of the distribution and biology of native Siricidae and associated Hymenopteran parasitoid species in Southeastern U.S.A.
Objectives Our project goals are to evaluate the biological control potential of native siricid species and their associated hymenopteran parasitoids on the exotic Sirex noctilio in the southeastern North America, should S. noctilio continue to spread and become established in this region. As related to the project goals, our supporting objectives are to determine the species complex and effective trapping methods for native siricids and their hymenopteran parasitoids in the southeastern U.S.A.
Brief description of tasks accomplished each year
Year 2009: During September- October 2009, we established sampling plots in three states in the southeastern region: Georgia (Piedmont region), Louisiana (Coastal region), and Virginia (Appalachian region). These sampling plots had experienced recent disturbance such as thinning and/or clear-cutting activities that may attract more native siricids and their parasitoids to this area. The timing of sampling varied across the regions to allow us to trap during the maximum activity of siricid wasps during the year. To assess the species complex of native woodwasps and their parasitoids in each state, we established 30 intercept panel traps that are widely used for monitoring S. noctilio. Each intercept trap was either: 1) unbaited, 2) baited with commercially available Sirex lure (alpha- and beta-pinenes), or 3) baited with Sirex lure and high release ethanol. We assessed the viability of these lures in catching siricids across southeastern U.S. In Georgia, we further tested whether funnel or intercept traps are more efficient in catching native siricids. Another 30 funnel traps with identical baits were placed in the same locations in Georgia. Hymenopteran parasitoids of siricids were further sampled using 10 Sante traps (similar to Malaise traps) placed high in the canopy. Hence, a total of 130 traps were used in 2009. Traps were emptied every 14 days until the catches of siricid wasps dropped to almost none in December 2009.
To assess differences in species complex of native woodwasps and their parasitoids colonizing different pine species, we created trap logs in each state. We left these trap trees in the field to allow them to be colonized both by siricids and their parasitoids. In late August 2009, we cut down three loblolly pine trees in Virginia. In late September 2009, we cut down two shortleaf and one longleaf pine in Georgia. In Louisiana, we also tested the effects of different methods of inducing tree decline and mortality, and at different times of the year on the colonization activity of woodwasps. In treatment 1, we girdled and applied herbicide (dicamba) to four loblolly pine trees between 1 September and 1 October, 2009. In treatment 2, we cut down four loblolly pine trees during the week of 2 November, 2009. In treatment 3, we cut down four loblolly pine trees during the week of 16 November, 2009. A panel intercept trap was attached to each of the trees 207
in Louisiana, and the traps were emptied every 14 days. The stumps and roots of trees from which boles were cut were also sampled by placing a small emergence cage on each of them. All emergence cages were checked every few days.
Results from the trapping experiments indicates that we have caught a total of 79 woodwasps with 34 individuals in Virginia, 23 in Louisiana, and 22 in Georgia. Five species of woodwasps have been identified; all these species are known to be present in the southeastern region. Sirex nigricornis was the most abundant woodwasp species followed by Sirex edwardsii (these two species will put together taxonomically in an upcoming publication by siricid systematists), Tremex columba, Eriotremex formosanus (exotic), and Urocerus cressoni. About equal number of woodwasps were caught in traps baited with Sirex lure, and Sirex lure with ethanol indicating that either of these lures could be used to monitor native woodwasps in these areas. Twice as many woodwasps were caught in the funnel than in the panel intercept traps in Georgia, although the overall numbers caught were fairly low to draw final conclusions. Overall, we did not trap any parasitoid species in any of the traps (intercept, funnel, Sante), suggesting that either these traps are not optimal for catching parasitoids, or that our timing for sampling was not conducive.
Year 2010: Brittany Barnes joined the project as a M.S. student in January 2010. We decided to focus on Georgia and Louisiana because it became clear that we need to conduct in-depth testing of different trap-types and lures to optimize our wasp collections, before spreading our sampling across the region. To find optimal sites to sample, in 2010, we installed five each of the intercept panel and funnel traps (ten total per site) adjacent to wood-piles in four sawmills in Georgia. These sawmills are considered hot-spots for siricid and their parasitoid diversity, due to the high volume of coarse-woody debris present in the area. We also tested four baits: 1) Sirex lure alone; 2) Sirex lure + ethanol; 3) ethanol alone; 4) Ips lure (racemic Ipsenol and Ipsdienol) along with Sirex lure and ethanol; and 5) an unbaited trap as control. In Louisiana, we further tested whether fresh pine volatiles are better attractants than commercially produced baits, since these baits caught fairly low numbers of siricids in previous year. We installed 30 intercept panel traps with 10 traps with each of the following baits: 1) Sirex lure alone; 2) lure with a bag of pine chips and foliage to emit volatiles; and 3) unbaited control. All of these traps were deployed in September and sampling finished in late-December 2010.
During the fall of 2010, all trap trees created in Virginia, Georgia, and Louisiana were retrieved, and cut up in sections. All the bole sections were placed in emergence cages to rear out woodwasps and their parasitoid species. To further enhance our collection of hymenopteran parasitoid species and woodwasps in 2011, we cut down and stacked 10 more loblolly pine trees in Georgia. The cut tops of the trees were placed on top of the logs to allow maximum attraction for wasps. Similar numbers of trees were cut in Louisiana as well. These trap-trees were left in the field for a year before retrieval and emergence of wasps in fall 2011.
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Results indicate that in Georgia, 52 native siricids were collected near wood-piles during the trapping period with 13 S. edwardsii and 39 S. nigricornis. There were no differences in siricid catches between different lures and trap-types. In Louisiana, 310 native siricids were collected during the trapping period with 113 S. edwardsii, 193 S. nigricornis, and four E. formosanus. The fresh pine lure captured a significantly greater number of siricids compared to the Sirex lure alone and the unbaited trap. This suggests that the currently used baits are not as effective as pine volatiles in catching siricids. Similar to 2009, we did not catch any siricid parasitoid species in our traps.
A total of 80 S. nigricornis emerged from three loblolly pine logs in Virginia and two wasps emerged from trees in Georgia. Siricid species emerged from late September to very early December, with male species peaking mid-October and females emerging fairly consistently from September through early November. To get a better estimate of siricid density, nine logs were split open from each of the three trees from Virginia for a total of 18 logs. A total of 15 siricid larvae were found in logs, with all of the larvae being found from one tree.
A total of 1,228 siricids and 486 siricid parasitoids (Ibalia leucopoides ensiger) emerged from 12 trap trees in 2010 in Louisiana. Both of the cut, buck, and stacked (early and late) trees had more siricid emergences than those trees that were sprayed with Dicamba. Cutting the trees early resulted in greater emergence of siricids then cutting later in November. Parasitoid emergences were significantly greater when the trees were cut, buck, and stacked early as compared to using dicamba. No woodwasps emerged from stumps and roots of the trees in Louisiana. Overall, we caught much greater numbers of siricids and parasitoids from trap trees than traps per se indicating that trap trees may be better in terms of obtaining larger number of siricid and parasitoid species.
Year 2011: Another 10 loblolly pine trees were cut-down and stacked in Georgia and Louisiana to rear out both siricids and parasitoids. These trees will be processed for emergence in fall 2012.
Trees that were cut down in fall 2010 in Georgia were retrieved from the forest and cut-into logs. These logs are currently placed in tents for emergence of woodwasps and their parasitoids, and we collected adults every few days. We collected 299 siricids and 7 parasitoids emerged from the logs with 131 S. edwardsii and 168 S. nigricornis in Georgia. Further, we finished and submitted an invited review of the general ecology and natural history of hymenopteran parasitoids of native siricids.
Year 2012: Specimens collected in previous years will be sent to systematists for final species verification, and will be deposited in the Georgia Museum of Natural History, Athens. We are currently considering which direction trapping for siricids and parasitoids should occur in the fall 209 of 2012. Also, we are currently in the process of writing up experiments conducted in these three years, and have started the process of submitting peer-reviewed papers.
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V. MODELING
Phenology and flight periodicity of Sirex noctilio in Eastern North America
Scott W. Myers
Field and laboratory studies were performed to determine phenology of flight activity and the thermal requirements for adult emergence of Sirex noctilio. To measure time from egg to adult under laboratory conditions adults were mated in 2 L containers, then females were introduced into cages with bolts of Pinus syvelstris for oviposition. Bolts were held at 22°C and adult emergence was recorded. Similarly, bolts of P. sylvestris cut from trees infested in the field by wild populations of S. noctilio were brought indoors for rearing at constant temperature. Adult emergence from laboratory infested bolts was almost entirely males, while field collected material produced a 2.7:1 male biased sex ratio. Mean degree-days to emergence was 1477.0 ± 13.4 (males) in laboratory infested bolts; and 1455.2 ± 11.2 (males) and 1577.8 ± 19.5 (females) 1n field collected material. Given the similar emergence times and the fact that winter collected material typically contained mid to late instar larvae rather than eggs, these data suggest a lag in development in field collected material that is likely the result of time required to break diapause. Field trapping studies were conducted concurrently over four years in central New York to compare flight activity with rearing data. Trap captures showed first flight activity and peak catch occurred at 709 and 1145 degree-days, respectively. The resulting degree-day model predicts early flight activity in early to mid-April for pine stands in southeastern United States, early to mid-May in the Mid Atlantic region, and late June to early July in the Northeast. 211
An assessment of the distribution and biology of native Siricidae and associated Hymenopteran parasitoid species in the Southeastern U.S.A.
Kamal J.K. Gandhi
Objectives Our project goals is to evaluate the biological control potential of native siricid species and their associated hymenopteran parasitoids on the exotic Sirex noctilio in the southeastern North America, should S. noctilio continue to spread and become established in this region. As related to the project goals, our supporting objectives are to determine the species complex and effective trapping methods for native siricids and their hymenopteran parasitoids in the southeastern U.S.A.
Brief description of tasks accomplished each year
Year 2009: During September- October 2009, we established sampling plots in three states in the southeastern region: Georgia (Piedmont region), Louisiana (Coastal region), and Virginia (Appalachian region). These sampling plots had experienced recent disturbance such as thinning and/or clear-cutting activities that may attract more native siricids and their parasitoids to this area. The timing of sampling varied across the regions to allow us to trap during the maximum activity of siricid wasps during the year. To assess the species complex of native woodwasps and their parasitoids in each state, we established 30 intercept panel traps that are widely used for monitoring S. noctilio. Each intercept trap was either: 1) unbaited, 2) baited with commercially available Sirex lure (alpha- and beta-pinenes), or 3) baited with Sirex lure and high release ethanol. We assessed the viability of these lures in catching siricids across southeastern U.S. In Georgia, we further tested whether funnel or intercept traps are more efficient in catching native siricids. Another 30 funnel traps with identical baits were placed in the same locations in Georgia. Hymenopteran parasitoids of siricids were further sampled using 10 Sante traps (similar to Malaise traps) placed high in the canopy. Hence, a total of 130 traps were used in 2009. Traps were emptied every 14 days until the catches of siricid wasps dropped to almost none in December 2009.
To assess differences in species complex of native woodwasps and their parasitoids colonizing different pine species, we created trap logs in each state. We left these trap trees in the field to allow them to be colonized both by siricids and their parasitoids. In late August 2009, we cut down three loblolly pine trees in Virginia. In late September 2009, we cut down two shortleaf and one longleaf pine in Georgia. In Louisiana, we also tested the effects of different methods of inducing tree decline and mortality, and at different times of the year on the colonization activity of woodwasps. In treatment 1, we girdled and applied herbicide (dicamba) to four loblolly pine 212 trees between 1 September and 1 October, 2009. In treatment 2, we cut down four loblolly pine trees during the week of 2 November, 2009. In treatment 3, we cut down four loblolly pine trees during the week of 16 November, 2009. A panel intercept trap was attached to each of the trees in Louisiana, and the traps were emptied every 14 days. The stumps and roots of trees from which boles were cut were also sampled by placing a small emergence cage on each of them. All emergence cages were checked every few days.
Results from the trapping experiments indicates that we have caught a total of 79 woodwasps with 34 individuals in Virginia, 23 in Louisiana, and 22 in Georgia. Five species of woodwasps have been identified; all these species are known to be present in the southeastern region. Sirex nigricornis was the most abundant woodwasp species followed by Sirex edwardsii (these two species will put together taxonomically in an upcoming publication by siricid systematists), Tremex columba, Eriotremex formosanus (exotic), and Urocerus cressoni. About equal number of woodwasps were caught in traps baited with Sirex lure, and Sirex lure with ethanol indicating that either of these lures could be used to monitor native woodwasps in these areas. Twice as many woodwasps were caught in the funnel than in the panel intercept traps in Georgia, although the overall numbers caught were fairly low to draw final conclusions. Overall, we did not trap any parasitoid species in any of the traps (intercept, funnel, Sante), suggesting that either these traps are not optimal for catching parasitoids, or that our timing for sampling was not conducive.
Year 2010: Ms. Brittany Barnes joined the project as a M.S. student in January 2010. We decided to focus on Georgia and Louisiana because it became clear that we need to conduct in- depth testing of different trap-types and lures to optimize our wasp collections, before spreading our sampling across the region. To find optimal sites to sample, in 2010, we installed five each of the intercept panel and funnel traps (ten in toto per site) adjacent to wood-piles in four sawmills in Georgia. These sawmills are considered hot-spots for siricid and their parasitoid diversity, due to the high volume of coarse-woody debris present in the area. We also tested four baits: 1) Sirex lure alone; 2) Sirex lure + ethanol; 3) ethanol alone; 4) Ips lure (racemic Ipsenol and Ipsdienol) along with Sirex lure and ethanol; and 5) an unbaited trap as control. In Louisiana, we further tested whether fresh pine volatiles are better attractants than commercially produced baits, since these baits caught fairly low numbers of siricids in previous year. We installed 30 intercept panel traps with 10 traps with each of the following baits: 1) Sirex lure alone; 2) lure with a bag of pine chips and foliage to emit volatiles; and 3) unbaited control. All of these traps were deployed in September and sampling finished in late-December 2010.
During the fall of 2010, all trap trees created in Virginia, Georgia, and Louisiana were retrieved, and cut up in sections. All the bole sections were placed in emergence cages to rear out woodwasps and their parasitoid species. To further enhance our collection of hymenopteran parasitoid species and woodwasps in 2011, we cut down and stacked 10 more loblolly pine trees in Georgia. The cut tops of the trees were placed on top of the logs to allow maximum attraction 213
for wasps. Similar numbers of trees were cut in Louisiana as well. These trap-trees were left in the field for a year before retrieval and emergence of wasps in fall 2011.
Results indicate that in Georgia, 52 native siricids were collected near wood-piles during the trapping period with 13 S. edwardsii and 39 S. nigricornis. There were no differences in siricid catches between different lures and trap-types. In Louisiana, 310 native siricids were collected during the trapping period with 113 S. edwardsii, 193 S. nigricornis, and four E. formosanus. The fresh pine lure captured a significantly greater number of siricids compared to the Sirex lure alone and the unbaited trap. This suggests that the currently used baits are not as effective as pine volatiles in catching siricids. Similar to 2009, we did not catch any siricid parasitoid species in our traps.
A total of 80 S. nigricornis emerged from three loblolly pine logs in Virginia and two wasps emerged from trees in Georgia. Siricid species emerged from late September to very early December, with male species peaking mid-October and females emerging fairly consistently from September through early November. To get a better estimate of siricid density, nine logs were split open from each of the three trees from Virginia for a total of 18 logs. A total of 15 siricid larvae were found in logs, with all of the larvae being found from one tree.
A total of 1,228 siricids and 486 siricid parasitoids (Ibalia leucopoides ensiger) emerged from 12 trap trees in 2010 in Louisiana. Both of the cut, buck, and stacked (early and late) trees had more siricid emergences than those trees that were sprayed with Dicamba. Cutting the trees early resulted in greater emergence of siricids then cutting later in November. Parasitoid emergences were significantly greater when the trees were cut, buck, and stacked early as compared to using dicamba. No woodwasps emerged from stumps and roots of the trees in Louisiana. Overall, we caught much greater numbers of siricids and parasitoids from trap trees than traps per se indicating that trap trees may be better in terms of obtaining larger number of siricid and parasitoid species.
Year 2011: Another 10 loblolly pine trees were cut-down and stacked in Georgia and Louisiana to rear out both siricids and parasitoids. These trees will be processed for emergence in fall 2012.
Trees that were cut down in fall 2010 in Georgia were retrieved from the forest and cut-into logs. These logs are currently placed in tents for emergence of woodwasps and their parasitoids, and we collected adults every few days. We collected 299 siricids and 7 parasitoids emerged from the logs with 131 S. edwardsii and 168 S. nigricornis in Georgia. Further, we finished and submitted an invited review of the general ecology and natural history of hymenopteran parasitoids of native siricids.
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Year 2012: Specimens collected in previous years will be sent to systematists for final species verification, and will be deposited in the Georgia Museum of Natural History, Athens. We are currently considering which direction trapping for siricids and parasitoids should occur in the fall of 2012. Also, we are currently in the process of writing up experiments conducted in these three years, and have started the process of submitting peer-reviewed papers.
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VI. MOLECULAR GENETICS
Molecular genetic Assessment of Beddingia (Deladenus) siricidicola Populations
Evan Braswell
Executive Summary CPHST Mission Lab documented genetic divergence between a biocontrol strain of Beddingia siricidicola and populations present in New York State. Furthermore, we provide genetic markers useful in measuring genetic exchange between these two strains.
Introduction Beddingia siricidicola Bedding is a nematode with dual life cycles. Under one life cycle, these nematodes are mycetophagus and feed specifically on Amylostereum areolatum, the white rot fungus. The alternative life cycle is of interest as it is insect parasitic, and the switch between these life cycles is controlled by the presence of insect host larvae. Specifically, B. siricidicola that develop near Sirex noctilio woodwasp larvae develop the parasitic phenotype. After infection, intra-host reproduction results in sterility for parasitized female S. noctilio woodwasps as their eggs become filled with juvenile nematodes.
This parasitic life cycle makes B. siricidicola a highly effective bio-control agent against the pine pest S. noctilio. In fact, a strain of B. siricidicola collected in 1990 from Kamona, Tasmania is widely used in bio-control. However, a number of species within the genus Beddingia exhibit similar morphologies making identification difficult.
CPHST Mission Lab was requested to assess whether the Kamona strain of B. siricidicola was genetically distinct from S. noctilio-attacking nematodes currently present in New York State. At the time, there was little information on the taxonomy of the New York nematodes. The New York populations have been morphologically identified as B. siricidicola, however concerns were raised whether the New York populations were genetically distinct from the Australian Kamona strain. Furthermore, as the Kamona strain was under development for release as a biocontrol agent, questions were raised regarding the ability to genetically identify hybrids and to track the Kamona strain following introduction using genetic tools.
Samples We acquired juvenile nematodes from David Williams’ (CPHST Otis) laboratory colonies of B. siricidicola. Samples included nematodes originating from a New York population and nematodes originating from the Kamona strain. We also acquired adult nematodes from dissected S. noctilio. Kelley Zylstra’s (PPQ) collections and subsequent dissections provided adult nematodes from numerous populations within New York State, 216 including Albion, Constantia, Fulton, Georgetown, Hannibal, Homer, Lafayette, Lewis Pt. Road, Monroe, Otisco, Palermo, Parish, Pompey, Richland, Solan, Spafford, Sullivan, Tully, and Volney.
DNA Extraction The small size and tough cuticle of juvenile nematodes caused difficulty with standard DNA extraction methodologies. After exploring several methodologies, including Qiagen’s QiaQuick kit, a boiling method, and a nematode specific Sodium Hydroxide method, we found that the ANDE method (Castalanelli et al. 2010) provided the most reliable results. While other methods certainly provided sufficient DNA for further analysis, they lacked the consistency when working with solitary juvenile nematodes that we found with the ANDE method. ANDE, or A Non-destructive DNA Extraction method, was designed for DNA extraction without disrupting the physical condition of insect exoskeletons. Unlike insects, nematode bodies were not retained while using this methology. Despite the difficulty of working with juvenile nematodes, DNA was reliably extracted from adult nematodes using both the ANDE method and the Qiagen DNeasy method.
Molecular discrimination We used polymerase chain reaction (PCR) to amplify a portion of the Cytochrome Oxidase I (COI) gene from the mitochondrial genome. The mitochondrial genome was chosen as it evolves rapidly and provides a higher level of variation. Oligonucleotide PCR primers were developed from an alignment of Tylenchida nematodes, with the following nucleotide sequences NCOIf1: 5’-CCTACTATGATTGGTGGTTTTGGTAATTG- 3’ and NCOIr2: 5’-GTAGCAGCAGTAAAATAAGCAC-3’, to easily and specifically amplify a variable region of the COI gene. PCR with these oligonucleotide primers was performed in reactions consisting of 39.75 µL of water, 5 µL of 10X buffer, 1 µL of dNTP mix (2.5 mM each), 1 µL each of the primers at 10 µM, 0.25 µL of ExTaq Hot Start polymerase (5 units/ µL) and 1 µL of template DNA. PCR was carried out with an initial denaturation step at 94°C for 60 seconds followed by 35 cycles of denaturation at 94°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 60 seconds before ending with a final extension step at 72°C for 5 minutes. Agarose electophoresis of successfully amplified PCR products resulted in a single band migrating the equivalent of a 900 base pair fragment.
Successfully amplified PCR products were exposed to Exonuclease I and Shrimp Alkaline Phosphatase (Work Instruction M-T-01-048) to remove unincorporated nucleotides and single stranded oligonucleotide primers. Cleaned PCR products were submitted for DNA sequencing and the resulting sequence chromatographs were edited using Sequencher.
We sequenced five individuals from each of the 19 locations within New York and 20 individuals from the Kamona strain. Two sites within the DNA sequenced region of the COI gene showed fixed differences between the New York populations and the Kamona strain. Specifically, at base 264, 5’- TTTTATATG”Y”ACAGTAAAG-3’, all sampled New York 217 populations carried the nucleotide Thiamine (T) whereas all Kamona strain individuals exhibited the nucleotide Cytosine (C). Similarly, at base 461, 5’-TAGTTTTGG”Y”GGTAACCCT-3’, all sampled New York populations carried the nucleotide Cytosine (C) whereas all Kamona strain individuals exhibited the nucleotide Thiamine (T).
Molecular methods for tracking genetic exchange To identify nuclear genetic markers with the potential to identify genetic exchange between the New York populations and the Kamona strain, we tested a number of oligonucleotide primer sets for amplification of informative Randomly Amplified Polymorphic DNAs (RAPDs). RAPDs are anonymous stretches of DNA amplified by random oligonucleotide primers. The presence, absence and migration distance of these amplified products during electrophoresis provide a window into variation in the nuclear genome. The nuclear genome, being composed of contributions from both parents, represents the ideal location to search for evidence of hybridization and genetic exchange.
We tested twenty RAPD primers, alone and in combination, under a number of PCR conditions. The most reliable reactions that exhibited unique banding patterns between the New York populations and the Kamona strain were using each of the following five oligonucleotide primers as the sole primer in the PCR reaction: OPA-04: 5’-AATCGGGCTG-3’, OPA-09: 5’- GGGTAACGCC-3’, OPA-12: 5’-TCGGCGATAG-3’, OPA-17: 5’-GACCGCTTGT-3’, and OPA-18: 5’-AGGTGACCGT-3’.
RAPD-PCR was performed, using a single oligonucleotide primer (above) in reactions consisting of 35.75 µL of water, 5 µL of 10X buffer, 4 µL of dNTP mix, 4 µL of the individual primer at 10 µM, 0.25 µL of ExTaq Hot Start polymerase and 1 µL of template DNA. PCR was carried out with an initial denaturation step at 94°C for 60 seconds followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 38°C for 30 seconds, and extension at 72°C for 60 seconds before ending with a final extension step at 72°C for 5 minutes. Agarose electophoresis of successfully amplified PCR products resulted in multiple bands migrating through the gel.
We analyzed five individuals for each of the 19 New York populations and 20 individuals from the Kamona strain. Discrimination of New York populations from the Kamona strain was determined by the migration patterns of these bands. PCR using the OPA-04 primer resulted in diagnostic electrophoretic bands at 900 base pairs for New York nematodes and 1,200 base pairs for Kamona strain nematodes. PCR using the OPA-09 primer resulted in diagnostic electrophoretic bands at 1,100 base pairs for New York nematodes and 400 base pairs for Kamona strain nematodes. PCR using the OPA-12 primer resulted in diagnostic electrophoretic bands at 750 base pairs for New York nematodes and 1,300 base pairs for Kamona strain nematodes. PCR using the OPA-17 primer resulted in diagnostic electrophoretic bands at 2,200 base pairs for Kamona strain nematodes and lacked the 2,200 base pair band in the New York 218 nematodes. Finally, PCR using the OPA-18 primer resulted in diagnostic electrophoretic bands at 1,700 base pairs for New York nematodes and 750 base pairs for Kamona strain nematodes.
Reference cited Castalanelli, M.A., D.L. Severtson, C.J. Brumley, A. Szito, R.G. Foottit, M. Grimm, K. Munyard, and D.M. Groth. 2010. A rapid non-destructive DNA extraction method for insects and other arthropods. Journal of Asia-Pacific Entomology 13(3):243-248.
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Discriminating the Kamona strain of Deladenus siricidicola, imported as a biological control agent of Sirex noctilio, from strains occurring naturally in North America
Ann E. Hajek
Background Sirex noctilio, a pine-killing woodwasp, was first detected outside its native range in Eurasia in New Zealand around the turn of the 20th Century. In the past century it has invaded pine plantations throughout the Southern Hemisphere, being discovered successively in Tasmania, Australia, South America, and South Africa. With its appearance in North America in 2004, Sirex woodwasp became a truly cosmopolitan forest pest.
Realizing the potential for very damaging outbreaks, the Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO) undertook a worldwide biological control campaign in the 1970s to seek out Sirex’s natural enemies. Of the many possible natural enemies that were identified, including a guild of parasitic Hymenoptera, the most promising agent was a parasitic nematode from Sopron Hungary, Deladenus siricidicola. The nematode has a curious and convenient biology; it is able to develop into two morphologically different forms dependent upon its propinquity to a host larva. If a host is distant or absent from a tree, a juvenile nematode develops into a fungus feeding form, eating the Amylostereum fungus that a female woodwasp injects into a pine tree to kill and rot the tree, which allows for development of her offspring. Alternatively, if the juvenile nematode develops near a host larva, parasitic forms are produced that enter the larva, mature to adults and reproduce. The resulting juveniles do not kill the host, but instead invade the host eggs, sterilizing them. The emerging woodwasp is unaware of the change and flies and oviposits as usual, with the result that she disperses the juvenile nematodes and decreases her own reproductive potential.
CSIRO scientists screened nematodes of several Deladenus species and strains from hundreds of sites in Northern Europe and the Mediterranean region. They ultimately chose a D. siricidicola population near Sopron, Hungary, for its high infection rate and relatively minor effect on the woodwasp host. Subsequently, this strain was known as “Kamona”. Australian scientists developed an entire production and delivery system around the Kamona strain, producing lab rearing protocols and tree injection devices. To date, their biocontrol system has worked remarkably well when coupled with a program of diligent monitoring as well as management of pine stands. Because of the spectacular success of the CSIRO program, we chose to evaluate it for possible use in the U.S. with suitable adaptations for the different climatic conditions. After S. noctilio was first found to be established in North America, USDA APHIS began to evaluate whether D. siricidicola Kamona should be introduced in North America for biological 220 control. In 2006, Dr. David Williams began testing whether D. siricidicola Kamona can survive the northeastern winter and whether it can parasitize the strain of S. noctilio that is present in North America (it is uncertain where this came from initially but different strains of this host can had different types of relationships with these parasitic nematodes). Soon after Dr. Williams began field evaluations of the Kamona strain, it became apparent that there was another nematode in the New York pines, a conspecific strain of D. siricidicola that does not sterilize S. noctilio females (= non-sterilizing strain, NS).
It is critical that these strains be distinguished in order to evaluate the survival and infectivity of the Kamona strain during field trials. It is obviously critical that the strains be identifiable to understand the association with these two components of parasitism. The facts that these are strains of the same species and that this species has limited morphological characters, rule out the use of morphological characters to differentiate them. Fortunately, the strains exhibit minor differences in some gene sequences and can be identified using molecular techniques.
Goals Identify the strains of Deladenus siricidicola found in controlled release studies in New York and Pennsylvania using molecular techniques and associate the strains with the physiological traits of infectivity and sterilization.
Methods Dr. Williams had purchased Kamona cultures from Ecogrow, the exclusive licensed producer in Australia, and mass reared them according to CSIRO Standard Operating Procedures. In the fall seasons from 2006 through 2012, Dr. Williams made controlled releases of the nematodes following CSIRO protocols, which included felling S. noctilio-infested trees, punching holes through the bark with a special hammer, and filling each hole with an inoculum of nematodes in a gel carrier. In the following spring, bolt samples were cut from the boles for lab rearing and the remainder of the tree was chipped to prevent escape of nematodes into the environment, hence the term “controlled release”; these controlled releases were conducted under USDA APHIS permits. Kamonas were not released outright in North America for S. noctilio control because they are known to be very virulent (in Australia) and there was concern that they might attack North American siricid species.
First, Dr. Williams had saved all of the nematode-infested S. noctilio emerging from wood injected with nematodes from 2008-2011. Dr. Williams had also made a release in 2012, but he did not have a location to rear out the wood (the USDA APHIS lab in Mattydale was not operating). We reared the wood at Cornell, with wood from Kamona-injected being reared in a quarantine (SARL) and the control wood being reared at a nearby barn. All siricids that emerged from rearing bolts in the spring of 2013 were dissected to search for Deladenus. Molecular probes (COI) that we had developed were used to confirm the presence of the sterilizing Kamona strain versus the ‘North American’ or ‘non-sterilizing’ strain of D. 221 siricidicola,that is already established in North America. The probes were also used to identify nematode strains from the controlled releases made in 2008, 2009, 2010, and 2011.
Accomplishments
Rearing We reared S. noctilio and associates from wood collected from 32 S. noctilio Kamona-injected trees and 30 control trees from 4 sites in Tioga County, Pennsylvania. These data were provided to Dr. David Williams in an excel file by e-mail and are summarized below.
Sirex noctilio Sirex nigricornis Males emerging Females Males emerging Females (nematodes) emerging (nematodes) emerging (nematodes) (nematodes) Kamona-injected 123 (5) 59 (27) 2 (0) 3 (3) Control 86 (35) 79 (3) 46 (1) 28 (1)
Molecular Analysis We extended the time for completion of this project because we were having trouble with extracting DNA from the nematodes. In discussions with Dr. Williams, it seems that similar problems had occurred at his labs when this evaluation was attempted.
We provided a description of the methods that were used for our molecular analyses to Dr. Williams .
Eventually, we successfully identified virtually all of the nematodes from the samples sent to us by Dr. Williams (from 2008-2009, 2009-2010, 2010-2011 and 2011-2012) and from the samples from our rearings (from the 2012-2013 samples) at Cornell. The data file containing these identifications (as Kamona or NS = nonsterilizing) was sent to Dr. Williams.
As a brief summary, for the 2008-2012 samples, we identified: NS 417 Kamona 50 unsuccessful 49
For the 2012-2013 samples: Kamona Controls NS 10 11 Kam 7 0