Title: The role of Ophiostomatoid fungi and their insect vectors in decline of longleaf pine (Pinus palustris).

Project summary Efforts to restore longleaf pine (Pinus palustris) in the southeastern United States (US) have been hampered by a decline which prevents mature trees from attaining their biological longevity. While hypotheses have been advanced to explain the causes of the decline, no single causal agent or process has yet been demonstrated to be uniformly associated with declining trees. Fungi are the most common forest pathogens, and members of the genus Leptographium and other fungi of the Ophiostomataceae have been found in association with roots of declining pines in many regions of the US. In this proposal we will elucidate the role of Ophiostomatoid fungi in longleaf pine decline, as these fungi have been found to play an important role in the decline of other pine species including loblolly pine (P. taeda) and shortleaf pine (P. echinata) in the southeastern US. The role of these fungi will be considered in the context of host biology and environmental factors, as declines are often attenuated by a suite of interrelated agents. The data collected in this study will be compared with a model designed to predict loblolly pine decline in order to identify parameters of importance in longleaf decline, thus informing managers who want to restore longleaf pine and avoid premature decline and mortality.

Objectives The southeastern US was once dominated by longleaf pine (Pinus palustris Mill.), with a range of over 37 million hectares (Frost 2006). The species was found predominantly in the Atlantic and Gulf coastal plains, the inland sandhills and lower elevations of the piedmont (Frost 2006). Longleaf pine grows well on deep sandy, infertile, nutrient poor soils (Carey 1992), and is known to be a fire-loving species. Prescribed burning is a common tool in the restoration of longleaf pine (Frost 2006). While fire historically has been an essential component of the longleaf ecosystem, prescribed burning may exacerbate decline on some sites (Otrosina et al 2002). Other site factors have also been shown to be related to decline in loblolly (Eckhardt 2003), and these too should be considered in the case of longleaf. Longleaf pine is considered to be relatively immune to disease and pest problems common to other southeastern pines (Frost 2006). Of the major pests and pathogens common in the area, including southern pine beetle, fusiform rust, pitch canker, annosus root disease, and littleleaf disease, longleaf is typically considered the least susceptible of the pines. A notable exception is brown spot needle blight, which typically affects only grass stage (early juvenile) longleaf. Forest declines, in contrast with other types of forest diseases such as blights, wilts, and root rots, are not typically caused by single pathogenic agents. Rather they are complexes of biotic agents and abiotic factors (Manion and Lachance 1992). Thus a broad-scale, interdisciplinary approach is warranted to identify the suite of factors that contribute to the decline. In this study we propose to investigate longleaf pine decline considering conditions relating to the tree hosts, the environmental factors associated with declining trees, and pests and pathogens that may also play an important exacerbating role in the decline. Previous authors have found evidence to suggest that fungi of the genus Leptographium (Ascomycota: Ophiostomataceae) are involved in the decline of loblolly pine (Eckhardt 2003) and longleaf pine (Otrosina et al 1999) in the southeastern US. Ophiostomatoid fungi reproduce and disperse via sexually- and asexually- derived spores that are insect-vectored, so an investigation into the role of the beetles that vector them is also necessary in an investigation of the biology of these fungi. Of the Ophiostomatoid fungi previous found to be associated with southern pines, Leptographium serpens appears to be the most virulent. It is therefore reasonable to consider that L. serpens may be an introduced species that may accelerate decline of longleaf pine, whereas apparently native fungi such as L. procerum and L. terebrantis are less aggressive, causing lesions smaller in size but still affecting root physiology (Eckhardt et al 2004). If L. serpens is an exotic organism, the introduction may explain the recent increase in incidence of this disease of loblolly and longleaf pine, and further support the agency of these fungi in the decline. Also important in any pathosystem is the response of the host. Physiological changes that distinguish healthy trees from diseased trees are more difficult to diagnose in the case of decline, where the first symptoms to develop may be growth reduction and changes in the condition of the crown, which are imperceptible to most casual observers. Thus an assessment of growth rate and crown condition is important to identify sites and individuals likely to succumb. In addition to aspects of pathogen, vector, and host biology, abiotic and environmental influences on all of these are important in determining the extent of decline. If the host and the pathogen are both present but the environment is not conducive to disease development, the stand will continue to be asymptomatic. Slope, aspect, position and other microsite characteristics are commonly considered important factors in areas where the topography is highly varied. In the southeastern US, many authors dismiss the importance of microsite variation as negligible, although a decline risk model for loblolly pine suggests that slope and aspect are highly correlated with disease incidence (Eckhardt 2003). Therefore it is prudent to consider these site factors in the proposed study, to determine the degree to which these factors may overlap between loblolly and longleaf pine. As the decline is accompanied by a disruption of root function, consideration of soil parameters will also be relevant and germane.

Objective 1: Characterization of longleaf pine decline symptomatology at Fort Benning, GA.

In general, symptoms of declines are slow to develop, and may include reduction in height and radial growth, foliar chlorosis and increase in crown transparency (Manion and Lachance 1992). In order to assess the health of the tree, a number of measurements can be applied. Height and diameter at breast height (DBH, 1.37 m) are typical static measures which can be repeated over time to assess the rate of growth. Another estimate of growth rates is obtained from increment cores, which in the case of longleaf only give a lower boundary to the age due to the length of time spent in the grass stage. Increment cores can be used be used to measure growth increment by examination of the distal rings and comparing annual growth rates over the past 5 and 10 years. The condition of the crown is an indicator of overall tree health, and can be measured using the crown condition protocols of the USDA Forest Service to assess live crown ratio, crown light, position, transparency, and dieback (USDA-FS 2005b). Resin is considered to be one of the main defenses against pests and pathogens of longleaf, being exuded by the host to physically evict the invader, as well as being a chemical defense due to the presence of noxious secondary compounds. The amount of resin exuded after injury thus represents the defensive ability of the tree. We will assess the resin production ability of trees within our plots and determine the mass to measure the ability of trees to resist attack by biotic agents.

Objective 2: Characterization of site factors associated with longleaf pine decline at Fort Benning, GA, testing of loblolly risk model and development of longleaf risk model.

Consideration of habitat features associated with declining trees and asymptomatic trees is valuable in determining which sites may be at greatest risk for development of decline symptoms. In a previous study, microclimate factors including slope and aspect were determined to be important in predicting incidence of decline of loblolly pine (Eckhardt 2003). Loblolly pine growing on sites having slope greater than 5%, and a southwestern aspect were more likely to exhibit decline symptomatology than flatter sites with other aspects. To test how this model applies to longleaf, we will establish 32 plots, divided into 16 sites predicted to be expressing decline, and 16 sites predicted to remain healthy, i.e. be comparatively reduced in crown symptoms and have lower root incidence of Ophiostomatoid species. Other site factor data will also be collected relative to slope position, the concavity of a site, elevation, total basal area, and pine basal area. Edaphic factors are often important in determining whether a pathogen may exist on a site, or if symptom expression will develop. To this end, we intend to collaborate with Dr. Emily Carter (USDA-Forest Service, Auburn, AL) who will collect and examine soil samples from all of the plots to assay soil series, bulk density, total porosity, and other potentially important physical and chemical properties. Another important abiotic factor in longleaf pine ecosystems is fire. Historically, longleaf pine forests were maintained by frequent fires, and prescribed fire is used regularly at Fort Benning in their longleaf restoration efforts. In this study, we will include timing of the most recent prescribed fire as a predictor variable in our model of decline incidence. A product of the loblolly decline study was a risk model to predict the incidence of decline. An important deliverable of this study is a test of the model’s efficacy relative to longleaf, and adjustment of the model to better fit the latter host. It is anticipated that the loblolly model will provide a framework for predicting a site’s likelihood to develop longleaf pine decline, and that the loblolly model will be attenuated by differences in longleaf biology for an improved, better fitting longleaf model.

Objective 3: Assessment of vector population and incidence of surface infestation with Ophiostomatoid fungi.

Ophiostomatoid fungi are vectored by beetles, most typically members of the sub- family Scolytinae (bark and ambrosia beetles) and other pinophagous Curculionidae (weevils). Crawling beetles known to infest roots will be sampled from each of the subplots by the use of pitfall traps baited with 95% ethanol and turpentine. Insects will be collected weekly and identified and rolled on selective media to screen for the presence of Ophiostomatoid fungal propagules on their carapaces. Isolates derived will be transferred to axeny and identified to species to determine which fungi are present and likely to be found on roots in each plot, and relative inoculum load.

Objective 4: Determine the incidence of Ophiostomatoid fungi on roots of longleaf pine in situ.

To investigate the incidence of infection with Ophiostomatoid fungi, it is necessary to examine roots directly. The two lateral root method will be employed on the central sub plot of each macroplot (see procedures, below) on three longleaf pines of comparable size (height and DBH). Roots will be excavated, assessed for damage from fire, insect, mechanical or other agents, and processed in the laboratory to determine the presence of Ophiostomatoid fungi. The average depth for the length of the excavated root will also be estimated, and adjacent soil collected approximately every 0.3 m for attempts to recover fungi of interest from the soil. To assess the ability of Ophiostomatoid species to colonize fine roots, we will collect and fix fine roots on excavated lateral roots for histological analysis in collaboration with Dr. Charles Walkinshaw (USDA-FS Emeritus) who will look for fungal structures in fine root tissue.

Objective 5: Determine the nativity of Leptographium serpens in the Southeastern United States

Many important questions about the basic biology of Leptographium serpens merit research. This species, unlike L. terebrantis and L. procerum, has only recently been positively identified in North America (Harrington 1988, Eckhardt 2003). Greenhouse pathogenicity tests on loblolly pine have shown that L. serpens is more aggressive than L. terebrantis and L. procerum (Eckhardt et al 2004). Given that the fungus has a cryptic history in the US, is more aggressive than more frequently collected fungi with a different etiology and symptomatology, and has an apparently restricted range not overlapping with the hosts in the southeastern US, it is logical to hypothesize that L. serpens is an exotic species, recently introduced into the southeastern US. Furthermore, this species is thought to have been introduced into plantation forests in South Africa, as the vector there is known to have been introduced. Definitive testing of the nativity of fungi can be achieved by measuring gene flow with molecular tools such as microsatellite analysis (microsatellites) or amplified fragment length polymorphisms (AFLPs). This part of the proposed project will be performed in collaboration with and financed by Dr. Michael Wingfield, of the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria in South Africa. Dr. Wingfield’s laboratory houses the world’s largest collection of Leptographium spp., including many isolates from South Africa and Europe where L. serpens has been collected frequently (MJ Wingfield, personal communication).

Procedures Study site and plot selection The field sites for the proposed study will be installed at Fort Benning Military Reservation (FB), near Columbus, Georgia. Fort Benning is managed by the United States Army, and contains many nesting and foraging sites of the red-cockaded woodpecker (RCW) which are managed to enhance habitat for this Federally-listed Endangered Species. Red cockaded woodpecker requires live, mature longleaf pine for its nesting cavities, thus Fort Benning is thus actively increasing longleaf pine acreage for RCW habitat. Plots will be 0.067 ha as described by the USDA Forest Service’s Forest Inventory and Analysis (USDA-FS 2005a), and comprised of four circular subplots 7.32 m in radius, as diagrammed in Figure 1 (after USDA-FS 2005a). All destructive sampling (root digging, increment coring) and crown rating will be conducted on the central subplot to reduce overall impact on the plot. Crown rating and insect collection will be conducted on the outlying subplots. Potential plot locations will be identified using a risk model developed by Dr. Lori G. Eckhardt (2003) to predict incidence of loblolly pine decline. The most important site factors shown to predict loblolly pine decline were slope and aspect. Sites on slopes of 5% or greater, and with south to southwesterly aspects were at greatest risk for loblolly pine decline symptom expression. Thus, to test the efficacy of this model with longleaf, four plots in four age classes (<10 years old, 10-20 years old, 20-40 years old, and >40 years old) in two decline categories (Healthy and Decline) will be installed throughout forested stands containing solely or predominantly longleaf pine at Fort Benning, for a total of 32 plots. It is surmised that incidence of Leptographium infection will be higher on sites predicted to harbor decline in the loblolly risk model.

Figure 1. Plot layout (after USDA-FS 2005a)

Assessment of host symptomology

In each year of this three-year study, all trees (>12.7 cm DBH) in the subplots will have their diameter tallied with a diameter tape (Forestry Suppliers, Inc), and height measured using an electronic clinometer (Häglof). On each of the 32 plots, trees in all subplots will have their crowns rated according to the protocol of the USDA Forest Service Forest Inventory and Analysis (USDA-FS 2005b). Crown rating is used to assess tree health by determining live crown ratio, foliar density, crown transparency, crown position, and crown dieback, and is assessed by two observers examining a single tree from orthogonal vantage points. Visible damage will also be assessed including but not limited to cankers, galls, parasitic plants, large wounds (>30% of diameter), broken tops and loss of apical dominance. All of the above listed protocols will be repeated annually for three years. Increment cores will be collected in the second year to estimate tree ages, and determine 5 and 10 year growth increment. We anticipate older stands on predicted decline sites to have greater crown transparency and reduced 5 year growth increment relative to the younger stands on predicted healthy sites.

Resin sampling to assess host defense response

A subset of trees (30 trees/age class/decline risk category) from the central subplots in each plot will be tapped to determine resin production during the first year. Sampled trees will have the bark shaved down on the south face, before tapping the cambium with a circular punch (1 cm diameter), and a tared 15mL conical tube attached with a spout to the tree to collect resin at 1.37 m. After 24 (± 1.5) hours, the tubes will be collected, capped, and kept at 4 °C until mass determination. Decline sites would be expected to have reduced standing resin reserves relative to healthy sites.

Infection of roots by Leptographium spp.

Roots of three longleaf trees in the central subplot will be sampled by excavating two lateral roots, approximately opposite each other, outward until the drip line in a protocol modified from that of Otrosina et al. (1997). Excavated roots will be sampled approximately every 0.3 m from the base of the tree to drip line, and the sampled segments kept at 4 °C until processing at Auburn University School of Forestry and Wildlife Sciences (AUSFWS) within 1 week. The root segments will be washed in tap water to dislodge soil prior to cutting the roots into pieces approximately 6 mm × 6 mm and surface sterilization in a solution of 80% ethanol, 10% bleach (active sodium hypochlorite 0.3%), and 10% distilled water (v:v:v), prior to plating on malt-extract agar (MEA) and MEA amended with the antibiotics cycloheximide and streptomycin (CSMA) to allow for the growth of ophiostomatoid fungi. Leptographium spp. and closely related taxa are cycloheximide-tolerant (Harrington 1981), though unamended MEA may allow a rare intolerant species of interest to be observed and isolated. Four root segments will be plated on 20 petri plates of CSMA and MEA, for a total of 160 segments per tree. Ophiostomatoid fungi growing on the media or segments will be transferred to CSMA or MEA amended with streptomycin only (SMEA) and serially transferred until axenic cultures are obtained, at which point the isolates will be transferred to MEA for species identification and archiving on slants. One half (16) of the plots will be excavated summer 2006, and the remainder summer 2007.

Incidence of pinophagous beetle activity and infestation with Leptographium spp.

Beetles known to infest roots will be sampled from each of the three distal subplots by the use of pitfall traps placed at the subplot center. The traps (adapted from Klepzig et al 1991) are constructed of sections of PVC tubing 10 cm diameter and 20 cm long and capped at one end. Eight radial holes are drilled in to allow insects to enter trap and fall into a cup which sits inside the trap. An unfixed cap contains two vials filled with 95% ethanol and steam distilled southern pine turpentine (Hercules) as attractant to pinophagous insects. Three sections of longleaf pine twig, approximately 5 cm long and 2 cm in diameter are placed in the cup as substrate for the captured insects and as additional bait. Each trap is visited weekly and the contents transferred to a clean specimen cup and stored at 4 °C for analysis at AUSFWS. The trap cup is cleaned with 70% ethanol and swabbed with liquid Teflon (Northern Products, Woonsocket RI) to prevent insects from crawling out of the trap. Fresh twigs are placed in the trap cup, and the ethanol and turpentine evacuated and replaced. At AUSFWS, insects are sorted from the specimen cups and identified. The identified vector insects are then “rolled” on CSMA and MEA to dislodge fungal propagules, and Ophiostomatoid fungi serially transferred to CSMA until axenic cultures are obtained, at which point the fungi are transferred to MEA for archiving and species determination.

Comparison of loblolly pine decline model and longleaf pine decline observations

The data will be analyzed using logistic regression models to account for the number of categorical variables being considered, and the nature of the response variable (likelihood of decline incidence expressed as a proportion). All factors will be incorporated into a stepwise regression procedure (PROC REG STEPWISE) in SAS (2001) to determine which variables are significant in the model.

Population genetic analysis of Leptographium serpens

Isolates of L. serpens collected by Dr L.G. Eckhardt in the southeastern United States will be compared with isolates from South Africa (SA) and other provenances in the laboratory of Dr. M.J. Wingfield at FABI. The isolates were selected to represent the breadth of the known range of L. serpens in the southeastern US, and to maximize the scale of inquiry, from several plots in an area, with several trees from each plot, and several isolates from each tree. The isolates will be grown out in South Africa at FABI, and the DNA isolated and purified to prepare for microsatellite analysis. Genomic DNA from the isolates will be screened using primers developed for Ophiostoma ips (Zhou et al 2002), which until recently was congeneric with L. serpens (Zipfel et al. 2006). If these primers do not amplify the L. serpens isolates, either new primers will be developed, or amplified fragment length polymorphism (AFLP) analysis may be attempted. As the pathogen is known to be introduced into SA, it is expected that the measured degree of gene-flow in SA isolates will be comparable with the southeastern US if L. serpens is indeed an introduced organism in the US.

Justification The problem of longleaf pine decline is relevant to all areas where longleaf restoration is ongoing, viz. from North Carolina south to Florida and east to Texas. Enhanced knowledge about Ophiostomatoid fungi would be useful throughout the native and introduced range of their hosts, that is, throughout the world’s temperate forests, as international trade moves trees and fungi. An imminent start on this project is necessary to enhance the decision making ability of managers trying to preserve mature longleaf pine. Many species of conservation concern, including RCW and the gopher tortoise, are reliant on this species for habitat. The timely gathering of data on forest pathogens and relevant site factors is necessary due to the potential longevity of the hosts, which means any improvement in knowledge now will reap benefits decades later. The Auburn University School of Forestry and Wildlife Sciences enjoys modern laboratory facilities and equipment thanks largely to a new building completed and occupied in 2005. Auburn University is within 50 miles of Fort Benning, allowing for quick and frequent access to study sites and prompt processing of materials collected, and is the nearest land-grant university. The USDA Forest Service also maintains a research unit in Auburn, where the soil analysis will be performed in collaboration with Dr. Emily Carter. Auburn University is also the headquarters of the Longleaf Alliance, an organization of landowners, managers, growers and academicians dedicated to restoring longleaf pine in the southeastern US. The principals of the Longleaf Alliance, experts on longleaf biology, ecology and restoration, are housed at the School of Forestry and Wildlife Sciences at Auburn University. Another perceived benefit of this study is the interdisciplinary nature of the PhD research assistantship. The graduate training required will prepare the incumbent via a program of study in forest entomology and pathology, disciplines which have traditionally been distinct. Study of both disciplines constitute a more holistic forest health training, and include ecological interactions between trees, insects and fungi.

Literature Review Longleaf pine restoration Longleaf pine (Pinus palustris Mill.) once dominated the forested landscape of the southeastern United States, ranging from North Carolina to Florida to Texas (Jose et al 2006). This range has been reduced dramatically from over 37 million hectares pre- European settlement to approximately 1 million hectares in 2000, a reduction of approximately 97% (Frost 2006). This reduction has been effected by several factors; exploitation for naval stores (e.g. turpentine and rosin) and timber, human development, suppression of fire, and replacement with other species. This has occurred to the detriment of many plant and animal taxa co-adapted with and dependent upon longleaf pine, including species of conservation concern such as the red-cockaded woodpecker (RCW, Picoides borealis) (Conner et al 2001) and the gopher tortoise (Gopherus polyphemus) (Means 2006). Red-cockaded woodpecker is unique among Piciformes in that it makes its nesting cavity in live trees, preferring mature longleaf pine infected with red heart (Phellinus pini), which decays the heartwood and facilitates excavation (Conner et al 2001). Ecosystem-level restoration of the longleaf forest has advanced in areas throughout the southeastern US, thanks in large part to the Longleaf Alliance (Anonymous 2006), which encourages the planting of longleaf pine and return of prescribed burning to enhance habitat value.

Southern pine decline and longleaf decline

Observation of decline of southern pines may have begun with littleleaf disease (Campbell and Copeland 1954). In littleleaf disease, shortleaf pine (Pinus echinata) and less often loblolly pine (P. taeda) were observed to be stunted, producing yellowed and shortened needles and more transparent crowns. Littleleaf symptoms occur most frequently on clayey, poorly-drained soils. In the original study, Phytophthora cinnamomi Rand (Oomycota: Pythiaceae) was isolated and implicated as a putative causal agent. Subsequent study by Hess et al (1999) and Eckhardt (2003) suggested that a distinctly different decline-type disease was affecting loblolly pine predominantly, and Leptographium spp. regularly isolated from symptomatic trees. Longleaf pine also has exhibited similar symptoms, with trees failing to attain historically-known potential longevity on some sites due to decline in growth starting at about age 40 (Otrosina et al 1999). An investigation of longleaf pine decline at the Savannah River Site in South Carolina yielded two most likely candidate fungal agents: species of Leptographium isolated from roots of younger trees; and Heterobasidion annosum isolated from the roots of older trees (Otrosina et al 1999). While prescribed fire is common in longleaf forests and the species is known to be fire adapted, on some sites mortality was observed over periods of years after fire, suggesting that some agent other than direct burning of the trees was operating to precipitate mortality (Otrosina et al 2002). Again, Leptographium spp. were recovered.

Leptographium spp. as pathogens

Leptographium spp. (teleomorph= Grosmannia sensu Zipfel et al 2006, Ascomycota: Ophiostomataceae) have been investigated for their role as pathogens of conifers and symbionts of pine-infesting beetles (Harrington and Cobb 1988, Jacobs and Wingfield 2001). The most damaging species is probably Leptographium wageneri, causal agent of black stain root disease, which affects pines and other conifers in the western United States (Wagener and Mielke 1961). This fungus infects the outer sapwood, causing girdling tyloses which appears black. There is strong evidence to support the role of Leptographium wageneri as a primary pathogen (Harrington and Cobb 1983). Other species of Leptographium have also been linked to diseases of conifers in the US, although the manifestation of disease is more often wedge-shaped occlusion of xylem accompanied by a blue stain indicative of the hyphal growth of these pigmented fungi. Several species have been implicated in diseases of conifers, including Leptographium procerum (e.g. Lackner and Alexander 1983, Klepzig et al 1991, Eckhardt et al. 2004) and L. terebrantis (Highley and Tattar 1985, Rane and Tattar 1987, Klepzig et al. 1991, Otrosina et al 1999, Eckhardt et al. 2004). In these cases, the degree to which these fungi act as primary pathogens is not as clear as in L. wageneri (Harrington and Cobb 1983, Wingfield 1986). Pathogenicity tests of Leptographium procerum on eastern white pine (P. strobus L.) have been inconclusive, with some authors finding marked decline in health (Lackner and Alexander 1983), and others little or no disruption of the trees physiology (Wingfield 1986). Most authors agree that L. terebrantis is more aggressive than L. procerum on most of the pine species tested (Wingfield 1986, Rane and Tattar 1987, Eckhardt et al 2004), but not as aggressive as L. wageneri (Harrington and Cobb 1983) Leptographium serpens was associated with decline of Pinus pinea in Italy (Lorenzini and Gambogi 1976) and has been reported in the United States, but many of these first reports from North America are unauthenicated and doubtful (Harrington 1988). Early reports of L. serpens in South Africa indicate that it may be pathogenic on Monterey pine (P. radiata) (Wingfield and Knox-Davies 1980) though subsequent inoculation experiments contradict the earlier reports (Zhou et al 2002). When compared with other Leptographium spp. under greenhouse conditions, L. serpens caused more damage to loblolly pine seedlings than L. terebrantis or L. procerum (Eckhardt et al 2004). In this latter study, isolates were compared with type material to confirm the identity of the fungus. While the three Leptographium spp. found most often on loblolly are likely to be important in longleaf, other Ophiostomatoid fungi are likely to be found also, including some new to science (Jacobs et al 2006).

Accomplishments to date

As shown in the attached timeline, the 32 plots have been established at Fort Benning, and insect trapping commenced in March 2006 and continued through the 5 May 2006. Insects collected over that time of species of interest (Hylastes salebrosus, H. tenuis, Dendroctonus terebrans, Hylobius pales, Pachylobius picivorus) have been rolled and Ophiostomatoid species transferred to axeny and are currently awaiting transfer to slants for archiving and identification. Insect traps were resituated in late August 2006 to determine whether insect populations exhibit a second peak in the fall. Henceforth, insect trapping will continue year-round, as insect numbers appear to remain significantly high even after anticipated drops. Insect trapping will be coordinated by George Matusick during my tenure at FABI (15 January-22 July 2007). Roots have been excavated on ½ (n=16) of the plots (June 2006), these roots have been processed, Ophiostomatoid fungi isolated and transferred to axeny and are also awaiting transfer to slants for archiving and identification. The remaining 16 plots will be excavated in July or August 2007, upon my return from South Africa. Crown rating was performed during June-August 2006, and will be repeated in 2007 and 2008. Resin sampling was performed in August 2006.

20 20 20 06 07 08 Protoc J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D ol establis h plots X X insect collection X X X X X X X X X X X X X X X X X X X X X X X X X X root dig X X soil collection X X crown rating X X X X X X X X X microsatellite analysis X X X X X X X data analysis X X X X X X X X X X X X X X