Genetic d ion of fusiform rust and pitch canker disease traits in bblolly pine

Loblolly pine (Pinus taeda L.) exhibits genetic resistance to fusiforrn rust disease (incited by the biotrophic fungus, Cronartiurn quercuum f. sp. fusifom) and pitch canker disease (incited by the necrotrophic fungus, Fusarium circinatum). In this study, a total of 14,015 loblolly pine cuttings from 1,065 clones were screened in controlled greenhouse conditions to identify phenotypes of clones, families, and parents that guide a genetic dissection of disease traits associated with pitch canker and fusifom rust. A total of 23,373 phenotypic data points were collected for lesion length (pitch canker) and gall score, gall length, and gall width (fusifonn rust). We verified heritable fusifonn rust and pitch canker traits and calculated parental, clonal, and full-sib family rankings for both diseases. Genetic correlations revealed that traits associated with fusiform rust are genetically distinct from one another, and that the genetic mechanisms underlying pitch canker and fusifonn rust resistance are independent. The disease pheno typing described here is a critical step towards identifying specific loci and alleles associated with fusiform rust and pitch canker resistance. Communicated by D.B. Neaie G. C . Kayihan . D. A. Huber A. M. Morse . T. L. White J. M. Davis School of Forest Resources and Conservation, University of Florida, PO Box 1 104 10, Gainesville, FL 3261 1, USA J. M. Davis (E3) Program in Plant Molecular and Cellular Biology, University of Florida, PO Box 110410, Gainesviile, FL 3251 1, USA E-mail: [email protected] Tel.: + 1-352-8460879 Fax: + 1-352-8461277 Pinus species are both economically and ecologically important. Pines grown in the southeastern United States generate nearly half of the nation's pulpwood, with an annual harvest value of approximately $19 billion (McKeever and Howard 1996). Loblolly pine (Pinus taeda L.) is the most widely planted Pinus species in this region, averaging 74% of the annual seedling production (Carey and Kelley 1993). In addition to plantations, loblolly pine is the predominant species on 11.7 million ha of native forest (Baker and Langdon 1990), where it impacts the welfare of nearly 400 species of vertebrates (Schultz 1999). Loblolly pine is a host for two endemic pathogens, Cronartiurn quercuurn Berk. Miyable ex Shirai f. sp. fusiforme (Burdsall and Snow 1977), the inciting agent of fusiform rust disease, and Fusarium circinatum Nirenberg et O'Donnell (Nirenberg and O'Donnell 1998), the inciting agent of pitch canker disease. Fusiform rust is one of the most destructive fungal diseases in the southeastern United States, causing damage ranging from $25-$135 million per year (Cubbage et al. 2000). The major symptom of fusiform rust disease is the formation of stem galls that lead to decreases in survival, wood quality, and growth. Genetic variation in resistance at the family level has been demonstrated for fusiform rust (Kuhiman and Powers i988; McKeand et al. f 999). Based on controlled inoculation studies carried out on specific loblolly and slash pine Pinuf elliottii Engelm. var. elliottii) families, specific resistance-i.e., "gene-for-gene" interactions-has evolved (Powers 1 " S O ; Steker et al. 1997; Wilcox et al. 1996), as well as partial resistance in the form of short galls (Schmidt et al. 2000), which may be genetically distinct from specific resistance. Pitch canker is also a significant, although more episodic, disease problem (Dwinell et al. 1985). Symptoms of pitch canker disease include resinous lesions on stems and branches that cause seedling mortality, decreased growth rates, and crown dieback (Dwinell et al. 1985). A considerable amount of genetic variation for pitch canker resistance has detected in loblolly pine families Wuhlman et al. 1952) and clones w i n e l l and Barrows-Broaddus 1981); however, the genetic architecture of resistance is not well understood. Our goal in this work was to obtain precise estimates of pitch canker and fusifonn rust disease phenotypes expressed in loblolly pine. Precision was acquired by a combination of clonal propagation, which allows repeat observations of the same genotypes and is now feasible in loblolly pine prampton et al. 2000), testing of over one thousand pedigreed genotypes, and the use of a mixed linear model (GARENL) to adjust for environmental effects (Huber 1993). In this study, we identified traits, clones, farnilies, and parents that guide a genetic approach to dissecting disease traits in loblolly pine. We verified that pitch canker and fusifonn rust traits are heritable and identified the disease traits that are genetically distinct from one another. This work creates the baseline knowledge required for identifying genes that condition phenotypes of interest, either through genetic linkage analysis within defined pedigrees, or by association in populations of unrelated genotypes (FlintGarcia et al. 2003; Jannink et al. 2001). Genetic material, plant propagation, and experimental design The 63 loblolly pine families screened in this study were obtained from a circular mating design with some offdiagonal crossing (Fig. 1). Members of the Cooperative Fig. 1 A circular mating design was used to generate the plant material. Thirtytwo parents were crossed following a circular design, and the resulting progeny was used as the material screened for this study. The numbers in the cells above the diqgonnl are the number of clones used from a given cross, and the numbers below the diagonal are the family identification numbers Forest Genetics Research Program at the University of Florida and the North Carolina State UniversityIndustry Cooperative Tree Improvement Program provided the 32 parents and generated the full-sib families and clones screened in this study. Forty-nine seeds from each full-sib family were gernrinated and grown into hedges for clonal propagation. Maintenance of hedges and propagation of cuttings is reported in Baltunis et al. (2005). In brief, cuttings were set in July 200 1, assessed for rooting after 9 weeks, and clones with the highest rooting ability selected for this experiment. The number of clones within families and the number of ramets (i.e., rooted cuttings) for each clone was not equal, since families did not produce the same number of clones, and clones had different rates of rooting. Cuttings assigned to a greenhouse screen were chosen at random from the ramet pool of each available clone (Table I). The screens were grouped according to the disease (fusiform rust or pitch canker). The fusiform rust screens were conducted using two types of inoculm (a one-gall mix or a ten-gall mix), whereas both pitch canker screens used a single inoculum. The experimental design was a randomized complete block with single-tree plots arranged in an alpha lattice with an incomplete block size of 20. The clones were replicated with a maximum number of five rarnets per experiment. Ramets were pruned twice to stimulate synchronous elongation of multiple succulent shoots for inoculation. The initial pruning occurred in March 2002, 8 months after setting, by cutting back the shoots from 10-15 an to 3-4 cm each. The second pruning occurred 6 weeks prior to inoculations for both pitch canker and fusiform rust screens; shoots were succulent and 5-8 cm in length at the time of inoculation. After pruning, all trees were fertilized weekly with Miracle-Gro 15-30-1 5 until inoculation.