Background: The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination. Results: We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome. Conclusions: In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.

Studies of symbioses have traditionally focused on explaining one-to-one interactions between organisms. In reality, symbioses are often much more dynamic. They can involve many interacting members, and change depending on context. In studies of the ambrosia symbiosis—the mutualism between wood borer beetles and fungi—two variables have introduced uncertainty when explaining interactions: imprecise symbiont identification, and disregard for anatomical complexity of the insects. The black twig borer, Xylosandrus compactus Eichhoff, is a globally invasive ambrosia beetle that infests >200 plant species. Despite many studies on this beetle, reports of its primary symbionts are conflicting. We sampled adult X. compactus and infested plant material in central Florida to characterize the fungal symbiont community using dilution series, beetle partitioning, and DNA-based identification. X. compactus was consistently associated with two fungal taxa, Fusarium spp. and Ambrosiella xylebori . Multivariate analyses revealed that A. xylebori was strongly associated with the beetle mycangium while Fusarium spp . were associated with the abdomen and external surfaces. The Fusarium spp. carried by X. compactus are not members of the Ambrosia Fusarium Clade, and are probably not mutualists. Fungal community composition of the mycangium was less variable than external body surfaces, thus providing a more consistent fungal inoculum. This is the first report of spatial partitioning as a mechanism for maintenance of a multimember ambrosia fungus community. Our results provide an explanation for discrepancies among previous reports, and suggest that conflicting results are not due to differences in symbiont communities, but due to inconsistent and incomplete sampling.

A variety of commercial instruments are available for sampling and quantifying microscopic airborne organisms from the environment. Although most samplers are highly sensitive, they are also expensive, costing thousands of dollars per unit, a price that is out of reach for many researchers, especially those looking to design experiments with replication. While looking at options to monitor pine stands for the presence of Fusarium circinatum, the causal agent of pitch canker disease, on multiple sites with several units per site, we developed a simple, low-cost spore trap that allows surveying spore abundance in outdoor environments. The trap consists of a rotating motor that holds a metal rod and two petroleum jelly-coated microscope slides. As the motor rotates, the slides collect airborne particles. To test whether the traps allowed detection of F. circinatum spores, we placed six traps on three sites: an actively-managed slash pine commercial stand located in Lake Butler, FL, a semi-managed loblolly and slash pine stand near Gainesville, FL, and a site with little perturbance at Goethe State Forest, FL, consisting of mainly slash pine trees. The slides were replaced weekly, and F. circinatum was detected by quantitative PCR using species-specific primers. Results show detection of low levels ( $\overline{\mathrm{X}}$ = 1.7–77.1 picograms ± SE = 0.3–39.7) of the pathogen spores with high reproducibility. These traps offer a low-cost solution to spore, pollen, or small insect trapping experiments for initial or general assessment of a pathogen or species population. Their low cost has the added benefit that multiple traps can be deployed per experiment, thus increasing statistical power by using multiple replications.

With future global temperatures predicted to increase, the relationship between a host, pathogen, and environment, becomes less predictable and epidemics may pose a greater risk to forests worldwide. Resistance breeding is an important disease management tool, but because tree species require long breeding times, it is necessary to develop techniques for testing current pathogen isolates against their hosts. Pitch canker disease of pines, caused by the pathogen Fusarium circinatum, is no exception and represents a threat to pine forests and commercial plantations worldwide, as it thrives at warm temperatures and high humidity. We tested growth of 15 F. circinatum isolates in culture at three temperatures: 25, 27, and 31 °C. We also evaluated the sporulation and pathogenicity of eight of the isolates on two susceptible Pinus elliotti (slash pine) open-pollinated families and one tolerant open-pollinated Pinus taeda (loblolly pine) family. Our results showed significant differences among isolates in the temperature and pathogenicity tests. All isolates showed a significant decrease in growth at 31 °C, although some showed similar growth at 25 and 27 °C. Several of the new isolates tested were more pathogenic than the isolates that the USDA Forest Service Resistance Screening Center (RSC) had been using. The new isolates have now been incorporated into their operational screening program.

Fusarium circinatum causes pine pitch canker (PPC) disease and associated symptoms such as resinous lesions, mechanical weakness, and crown dieback that may lead to mortality in Pinus and Pseudotsuga spp. There are no ameliorative techniques available for the disease, and the genetic resistance among populations to support commercial plantation deployment has not been well characterized. In this study, we characterize the genetic control of PPC disease tolerance (and/or resistance) and predict the tolerance of families in existing breeding populations: open-pollinated (OP) half-sib and control-pollinated full-sib (FS) slash pine (Pinus elliottii var. elliottii Engelm.), OP loblolly pine (Pinus taeda L.), and advanced-generation OP hybrid slash × P. caribaea (Pinus elliottii var. elliottii Engelm. × Pinus caribaea var. bahamensis, caribaea, and hondurensis) using F. circinatum isolates obtained from three locations in Georgia and FL, USA. We describe a new experimental design that improves the accuracy of breeding value predictions, provides more precise genetic parameter estimates, and facilitates comparisons within and among taxa as well as comparisons among isolates. We found strong evidence for genetic control of the ratio of stem damage by F. circinatum, especially in slash pine and slash × P. caribaea hybrids. Loblolly and slash × P. caribaea hybrids exhibited less damage than slash pine. We observed a spectrum of virulence among F. circinatum isolate sources, which were not equally virulent in different pine taxa.