Fungi associated with the southern pine beetle: avoidance of induced defense response in loblolly pine

Summary Loblolly pines (Pinus taeda) are rapidly killed by colonizing southern pine beetle (Dendroctonus frontalis). The female beetles carry two species of fungi (Ceratocystis minor var. barrasii and an unnamed basidiomycete) within a mycangium. The insects are also frequently associated with a blue-staining form of C. minor. These fungi are inoculated into the tree during colonization. The tree has an induced defensive response that involves resin soaking and necrosis of affected tissue isolating the invading organlsms. The blue-staining fungus stimulates formation of this response in the tree, but the two mycangial fungi do not. These results suggest that the beetles are closely associated with two highly pathogenic fungi that do not stimulate one of the critical components of tree defense.     

Induction of somatic embryogenesis in loblolly pine (Pinus taeda L.)

Summary Several factors that may affect induction of somatic embryogenesis in loblolly pine (Pinus taeda L.) were investigated in 1994 and 1995. Megagametophytes containing immature zygotic embryos were excised from seeds as explants. Potassium chloride, silver nitrate, myo-inositol, coconut water, or polyamine was added to the control media (U.S. patent no. 5,036,007) to determine the effects of each single ingredient or their combinations on the initiation of embryogenic tissue. Supplements of myo-inositol at 22.2 mM resulted in increases in frequencies of cell mass extrusion and proliferation compared with the control media in consecutive years. Addition of silver nitrate showed the potential to promote initiation of embryogenic culture. The combination of 10 mM potassium with 29.4 μM silver nitrate achieved the highest frequencies in both extrusion and proliferation of embryogenic tissue. The combination of silver nitrate at 29.4 μM with addition of myo-inositol at 11.1 or 22.2 mM achieved a higher conversion rate from extrusion to proliferation. Polyamine did not significantly affect the induction of somatic embryogenesis, but coconut water was inhibitory. Published with approval of the Director of Arkansas Agricultural Experimental Station.     

Water status and growth of loblolly pine (Pinus taeda L.) callus

Water status of Pinus taeda L. callus supported on Murashige and Skoog (MS) liquid medium was characterized over an 8 week period using thermocouple psychrometry. Medium with 30 gl⁻¹ sucrose was used to produce a high water potential (Ψ_w) of −0.4 MPa (H), and the same medium was used to create a moderate Ψ_w of −0.7 MPa (M) by the addition of 10% polyethylene glycol (PEG, w/v, MW=8000). Calli were produced from cotyledon explants on H medium for 2 weeks and then transferred to either M or H medium. Callus absorption of PEG accounted for 40% of the callus dry weight and less than 7% of the callus fresh weight. Callus dry weight (without the PEG fraction) on M medium was 40% of that observed on H medium. Fresh weight on M medium was only 15% of that observed on H medium. The Ψ_w of both H and M media remained constant throughout the culture period. On H medium, callus Ψ_w and osmotic potential (Ψ_s) both increased 0.05 MPa/week with the callus Ψ_w approaching that of the external medium. On M medium, callus Ψ_w and Ψ_s both decreased more than 0.1 MPa/week with the callus Ψ_w decreasing greatly below that of the external medium. The latter was attributed to a rapidly produced osmotic shock induced upon callus transfer and/or PEG which caused less callus hydration and resulted in reduced growth. Callus turgor potential (Ψ_p) was estimated to be +0.02 to +0.09 MPa and turgor was maintained as callus Ψ_w increased or decreased. After 8 weeks, cell volumes from callus on M medium were 50 to 60% less than on H medium, suggesting that reduced cell volumes were related to turgor maintenance.     

Partial age-specific life tables for Monochamus titillator in Dendroctonus frontalis infested loblolly pines

Partial age-specific life tables were constructed for Monochamus titillator(Fabricius) (Coleoptera: Cerambycidae) in Dendroctonus frontalisZimmermann (Coleoptera: Scolytidae) infested loblolly pine (Pinus taedaL.) trees. Successive life stages including egg, early larvae, mid-stage larvae, late larvae, and adult emergence were sampled within six sample trees. Generation mortality ranged from 60.94% to 98.61% in sample trees. Highest mortality typically occurred to eggs and mid-stage larvae. Possible mortality factors included resinosis, predaceous beetles, parasitoids, and woodpeckers. Dendroctonus frontalisbrood stages were determined for consecutive M. titillatorsampling. Monochamus titillatorand D. frontaliscoexisted and likely interacted in the phloem of host trees for at least 20 days.     

High-throughput genotyping and mapping of single nucleotide polymorphisms in loblolly pine (Pinus taeda L.)

The development and application of genomic tools to loblolly pine (Pinus taeda L.) offer promising insights into the organization and structure of conifer genomes. The application of a high-throughput genotyping assay across diverse forest tree species, however, is currently limited taxonomically. This is despite the ongoing development of genome-scale projects aiming at the construction of expressed sequence tag (EST) libraries and the resequencing of EST-derived unigenes for a diverse array of forest tree species. In this paper, we report on the application of Illumina’s high-throughput GoldenGate™ SNP genotyping assay to a loblolly pine mapping population. Single nucleotide polymorphisms (SNPs) were identified through resequencing of previously identified wood quality, drought tolerance, and disease resistance candidate genes prior to genotyping. From that effort, a 384 multiplexed SNP assay was developed for high-throughput genotyping. Approximately 67% of the 384 SNPs queried converted into high-quality genotypes for the 48 progeny samples. Of those 257 successfully genotyped SNPs, 70 were segregating within the mapping population. A total of 27 candidate genes were subsequently mapped onto the existing loblolly pine consensus map, which consists of 12 linkage groups spanning a total map distance of 1,227.6 cM. The ability of SNPs to be mapped to the same position as fragment-based markers previously developed within the same candidate genes, as well as the pivotal role that SNPs currently play in the dissection of complex phenotypic traits, illustrate the usefulness of high-throughput SNP genotyping technologies to the continued development of pine genomics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effects of ultraviolet-B radiation on loblolly pine

Summary Depletion of stratospheric ozone and the resulting increase in ultraviolet-B (UV-B) radiation may negatively impact the productivity of terrestrial ecosystems. This concern has led to a number of studies that report the influence of supplementing UV-B radiation on plant growth and development. However, only two of these field studies have included tree species and both were singleseason experiments. In this study, loblolly pine (Pinus taeda L.) from seven seed sources was grown under natural and supplemental levels of UV-B radiation. Irradiation treatments were continued for three seasons on plants from four of the seven groups and for 1 year only for three groups. The supplemental irradiances simulated those that would be anticipated with stratospheric ozone reductions of 16% and 25% over Beltsville, Md. The effects of UV-B radiation during the 1st year on plant growth varied among the seed sources. The growth of plants from two of the seven seed sources tested showed significant reductions following a single irradiation season and plants from one group tended to be larger under increased UV-B radiation. However, after 3 years of supplemental irradiation, plant biomass was reduced in all four groups by 12–20% at the highest simulated ozone depletion. These results suggest that the effects of UV-B radiation may accumulate in trees and that increased UV-B radiation could significantly reduce the growth of loblolly pine over its lifetime. However, they also point to a need for multiple season research in any analysis of potential consequences of global change on the long-term growth of trees.     

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.     

Nutrient proportions in foliage of semi-mature loblolly pine

Summary Nitrogen and phosphorus limitations to growth are common in many loblolly pine (Pinus taeda) stands. Interactions of these nutrients may complicate interpretation of foliar nutrient analysis for predicting response to forest fertilization. Proportions of foliar nutrient concentrations (and the changes in these proportions following fertilization) were examined in 36 semi-mature loblolly pine plantations in the southeastern United States. Mean proportions of nutrient concentrations (NPKCaMg) for non-fertilized stands were 1009.336.517.29.2. Potassium and phosphorus were higher. Nitrogen fertilization generally decreased the PN ratio and enhanced growth, indicating a nitrogen deficiency in most stands under study. Additions of nitrogen and phosphorus together yielded a significant increase in the PN ratio. Effects of fertilization effects on other nutrient concentration ratios were also examined.Paper No. 9401 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695.     

Winter nitrogen fertilization of loblolly pine seedlings

Loblolly pine (Pinus taeda L.) seedlings were nitrogen fertilized during winter in a bare root forest tree nursery located in the coastal plain of the southeastern United States. Total application rates were 0, 50, 100, and 200 kg/N/ha applied in split applications 4 weeks apart in January and February. Seedlings were lifted and outplanted in March, 4 weeks after the second fertilization and measured at 3 and 6 months after outplanting. No seedling morphological differences were encountered at the time of lifting and outplanting although seedling shoot nitrogen content was 28% greater in the highest fertilization treatment compared to the check. Shoot nitrogen concentrations fell after outplanting regardless of treatment, decreasing from an average of 1.51% across all treatments at the time of planting to 0.64% at 6 months after planting. When measured at 6 months after outplanting, seedling dry weight and height growth after planting was shown to increase by 12% and 24%, respectively, for the high nitrogen treatment. This and other studies across a variety of sites have found positive post-outplanting seedling growth response after nutrient loading in the nursery.     

Ultrastructure of the mycangium of the southern pine beetle,Dendroctonus frontalis (Coleoptera: Curculionidae,Scolytinae): complex morphology for complex interactions

Yuceer, C, Hsu, C.‐Y., Erbilgin, N and Klepzig, K.D. 2011. Ultrastructure of the mycangium of the southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae, Scolytinae): complex morphology for complex interactions. —Acta Zoologica (Stockholm) 92 : 216–224. The southern pine beetle (SPB) (Dendroctonus frontalis Zimmermann) is the most economically important pest of southern pine forests. Beetles carry fungal cells within specialised cuticular structures, called mycangia. Little is known about the mycangia ultrastructure or function. We used cryo‐fracturing and scanning electron microscopy to examine the ultrastructural features of SPB mycangia and surrounding tissues. Mycangia, one on each side of anterior portion of the prothorax, are terminated on the dorsal side at a ‘mycangial bridge’. This sclerotised mycangial bridge does not appear to provide a passage between the two mycangia, suggesting that each mycangium functions independently. Mycangia are surrounded by abundant tracheoles connecting the structures to the outside via openings within the prothorax. Previously unknown pits overlying the mycangial gland cells were also observed in both the inner wall and anterior fold of prothorax. We hypothesise that these openings and pits may play roles in determining which fungi enter, and grow within, the mycangium.     

Effect of shade stress on growth,morphology, and carbon dynamics of loblolly pine branches

Summary Shade treatments were applied to the terminal portions of branches in loblolly pine trees to test whether portions of branches were autonomous with respect to carbohydrates. The shade treatments reduced light by 50% and 72% compared with full sun conditions resulting in significant reductions in net photosynthesis. Branch growth (branch diameter and length, branch and needle biomass) decreased significantly within the shaded portion of the branch. Branch and needle morphology were also affected by shading. Lateral branches subtending terminal shoots were labelled with ¹⁴C 2 times during the growing season. No movement of ¹⁴C into the terminal portions of the branch was evident during the first growth flush. However, during the second flush of growth small but statistically significant amounts of ¹⁴C were imported into the terminal portion of the shaded branches from subtending laterals. It was concluded that loblolly pine shoots were usually autonomous with respect to carbohydrate supply, but that carbohydrate movement into the terminal shoot from subtending foliage could occur under conditions of very high stress.     

cDNA isolation,functional expression,and characterization of (+)-alpha-pinene synthase and (-)-alpha-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis

The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens. Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine. The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions. Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes. Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases. The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms. The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed.     

Somatic embryogenesis in loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> L.): improving culture initiation with abscisic acid and silver nitrate

Loblolly pine (Pinus taeda L.) culture initiation was improved by the addition of abscisic acid (ABA) (3.7 µM), silver nitrate (20 µM), and guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt (10 µM) to the medium and by raising cytokinin levels in the presence of 50 mg/l activated carbon (AC). Basal medium contained modified 1/2-P6 salts, 50 mg/l AC, Cu and Zn added to compensate for adsorption by AC, 1.5% maltose, 2% myo-inositol, 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l -naphthaleneacetic acid (NAA), 0.55 mg/l 6-benzylaminopurine (BA), 0.53 mg/l kinetin, and 2 g/l Gelrite. Across 32 open-pollinated families initiation ranged from 0 to 53.4%, with an average of 17.9%. Further optimization of cytokinins to 0.63 mg/l BA and 0.61 mg/l kinetin along with the removal of ABA maintained initiation at 18.2% across 19 families. Survival of 2001 new initiations was tracked for 4–6 months. Survival averaged 28.8%. A test of 68 new initiations tracked closely for 4 months demonstrated that at least 80% of the cultures lost did not grow after transfer to the multiplication media, suggesting that many new initiations abort during the initiation process.Abbreviations ABA Abscisic acid - AC Activated carbon - BA 6-Benzylaminopurine - 8-Br-cGMP Guanosine 3,5-cyclic monophosphate, 8-bromo-, sodium salt - NAA -Naphthaleneacetic acid Communicated by G.C. Phillips     

The effects of annual fertilization and complete competition control on fascicle morphology of different aged loblolly pine stands

The goal of this study was to determine how increased nutrient availability affects foliage morphology of loblolly pine (Pinus taeda L.) without introducing the confounding influence of light availability. Morphology of fascicles from the terminal leader (radius, length, specific needle area, density, and needles per fascicle) and terminal leader traits (length, foliated length, total leaf area, and total fascicles) were measured for different aged (ranged from 5-year-old to 12-year-old) loblolly pine stands growing on the Piedmont and lower Coastal Plain of Georgia, USA. A factorial combination of annual fertilization and complete interspecific competition control was applied as stand level treatments. Competition control and stand age generally did not affect fascicle morphology. In contrast, annual fertilization significantly increased fascicle length (5%), needles per fascicle (4%), and total leaf area of the terminal (18%), while decreasing specific needle area (4%). Fertilization also increased terminal leader growth (5%) and total number of fascicles (6%). Therefore, loblolly pine foliage morphology does have plasticity to respond to increased nutrient availability. However, the magnitude of these morphological changes is small compared to changes in total canopy leaf area.     

The suppression of Dendroctonus frontalis and subsequent wildfire have an impact on forest stand dynamics

Question: Interacting disturbance effects from Dendroctonus frontalis outbreaks and wildfire are thought to maintain Pinus spp. composition in the southeastern U. S. Our objective was to assess forest composition, structure, and succession following the interaction of two frequently occurring disturbance events in southern Pinus spp. forests: cut‐and‐leave suppression, a commonly used means for managing D. frontalis outbreaks, and wildfire. Location: Western Gulf Coastal Plain, Louisiana, USA. Method: Pinus taeda stands with cut‐and‐leave suppression and subsequent wildfire were compared to stands undisturbed by D. frontalis but with the same wildfire events twenty years after Pinus spp. mortality. The woody plant community was assessed in three different size classes and used to predict future forest types with the Forest Vegetation Simulator (50 years). Results: P. taeda is the most abundant (> 50%) species of saw‐ and poletimber‐sizes following cut‐and‐leave suppression with wildfire and in stands only with fire. Using canonical correspondence analysis, vegetation assemblages were primarily explained by slope position and elevation (7.6% variation explained). Fire intensity and stand age also accounted for variance in the ordination (4.4% and 3.1%, respectively). Dominant and co‐dominant P. taeda forest types were predicted by the model to be the most abundant forest types in each disturbance regime. In addition, new regeneration represents high hazard for future mortality from D. frontalis. Conclusion: Our study demonstrates that cut‐and‐leave suppression with additional wildfire disturbance maintains P. taeda composition, and does not alter forest composition differently from stands receiving only wildfire. As a result, predicted Pinus spp. basal area under both disturbances is great enough to facilitate future bark beetle disturbance.     

Isolation and characterization of 16 microsatellite loci in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae: Scolytinae)

We isolated 16 polymorphic microsatellite loci in the mountain pine beetle (Dendroctonus ponderosae Hopkins) and developed conditions for amplifying these markers in four multiplex reactions. Three to 14 alleles were detected per locus across two sampled populations. Observed and expected heterozygosities ranged from 0.000 to 0.902 and from 0.100 to 0.830, respectively. Three loci deviated from Hardy-Weinberg equilibrium in one sampled population. One of these loci may be sex linked. These markers will be useful in the study of population structure in this important pest species.     

Lipid transfer protein genes of loblolly pine are members of a complex gene family

A loblolly pine (Pinus taeda L.) cDNA with properties of a nonspecific lipid transfer protein (nsltp) is reported. In contrast to simple family structures reported for a variety of angiosperm nsltp genes, the putative pine nsltp gene is a member of a complex family.     

Peroxidase activity of desiccation-tolerant loblolly pine somatic embryos

Summary Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 μM abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG₆₀₀₀). Scanning electron microscopy of desiccated somatic embryos showed that the size and external morphology of the desiccation-tolerant somatic embryos recovered to the pre-desiccation state within 24–36 h, whereas the non-desiccation-tolerant somatic embryos did not recover and remained shriveled, after rehydration. Peroxidase activity of desiccated somatic embryos increased sharply after 1 d of desiccation treatment at 87% relative humidity (RH), and desiccation-tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation-tolerant somatic embryos may have allowed them to catalyze the reduction of H₂O₂ produced by drought stress, and protected them from oxidative damage.     

Root regeneration as an indicator of aluminium toxicity in loblolly pine

Loblolly pine (Pinus taeda L.) seedlings were grown in sand culture and exposed to solution Al concentrations of 0, 5, 10, 20, 40, and 80 mg.l⁻¹ in two separate studies. Root regeneration potential (RRP) of loblolly pine was found to be very sensitive to Al. Both the total number and total length of new white roots produced during a 24-day period declined with as little as 5 mg Al.l⁻¹ in solution.