Role of Phytotoxins in Pine Wilt Diseases

Characteristic rapid death of pines after infection by Bursaphelenchus xylophilus suggests the involvement of phytotoxins in the pine wilt disease syndrome. Crude extract from diseased pine is toxic to pine seedlings, whereas an extract from healthy pine is not. The response of seedlings to the crude toxin is more prominent in susceptible pine species than in resistant ones. Benzoic acid, catechol, dihydroconiferyl alcohol, 8-hydroxycarvotanacetone (carvone hydrate), and 10-hydroxyverbenone, which are toxic, low molecular weight metabolites, can be isolated from diseased pines. Other unidentified toxins are also found. The toxicity of some of these metabolites correlates positively to the susceptibility of pines to B. xylophilus. Some of these abnormal metabolites show synergistic toxicity when in combination. The D-isomer of 8-hydroxycarvotanacetone, dihydroconiferylalcohol, and 10-hydroxyverbenone inhibited the reproduction of B. xylophilus. Cellulase excreted by pinewood nematode also may be involved in rapid wilting.     

Charcoal and activated carbon as adsorbate of phytotoxic compounds – a comparative study

This study compares the potential of natural charcoal from Scots pine ( Pinus sylvestris L.) and activated carbon to improve germination under the hypothesis that natural charcoal adsorbs phytotoxins produced by dwarf-shrubs, but due to it's chemical properties to a lesser extent than activated carbon. Activated carbon has been used in many bioassays as an adsorbate to clean aqueous solutions.
We used aqueous extracts from young leaves of Calluna vulgaris (L.) Hull and Vaccinium myrtillus (L.) as phytotoxin sources in two different concentrations (10 and 14 gr. of dried leaves in 100 ml distilled water). Germination of pine seeds was prevented by the higher concentration of both species, while the lower ones did not show significantly reduced germination. Both ericaceous species showed a very similar potential to prevent germination of Scots pine seeds.
Supplemented carbon (activated carbon, powdered or granulated pine charcoal) restored germination in strong extracts. Adding activated carbon resulted in germination of almost 100%. With pine charcoals added, lower germination percentages were observed. The charcoal powder was more effective (60% for C. vulgaris ; 28% for V. myrtillus ) than the charcoal granulate (30% and 16%, respectively) in restoring germination.
Chemical and surface analysis of the three carbon supplements revealed that activated carbon had by far the biggest active surface area (641 m² g⁻¹), and thus many more cavities to bind phytotoxins than natural charcoal (total surface area of 142 m² g⁻¹).
We conclude, that charcoal produced by forest fires can have a positive effect on seed germination, but to a much lesser extent than activated carbon. Previous studies, which used activated carbon as an equivalent for charcoal, overestimated the effect of charcoal on germination.     

Responses of Pinus clausa, Pinus serotina and Pinus taeda seedlings to anaerobic solution culture. I. Changes in growth and root morphology

Seedlings of pond pine ( Pinus serotina Michx.), sand pine [ P. clausa (Engelm.) Sarg.] and two edaphic seed sources of loblolly pine ( P. taeda L., dry- and wet-site seed sources) were grown in non-circulating, continuously flowing solution culture under aerobic (250 μ M O₂) and anaerobic (≤ 23 μ M O₂) conditions. Survival was 100% for all seedlings at 11 weeks. Although shoot height, biomass and leaf emergence of loblolly and pond pine seedlings were not significantly affected by 15 or 30 days of anaerobic growth conditions, root biomass was significantly reduced. Sand pine suffered the largest reduction in biomass, showing extensive root dieback and shoot chlorosis with retarded leaf development. These anaerobically induced symptoms of flooding injury were less severe in dry-site loblolly pine, and absent in wet-site loblolly and pond pine seedlings. Adventitious or new, secondary roots, and stem or taproot lenticels were particularly abundant under the 30-day anaerobic treatment in wet-site loblolly and pond pine seedlings, present to a lesser degree in dry-site loblolly pine, and nearly absent in sand pine seedlings. These results indicate that much of the immediate damage from flooding is due to the anoxic condition of the root rather than to the build-up of phytotoxins or soil nutrient imbalances. On the basis of overall seedling vigor, root plasticity and growth, we suggest the following flooding-tolerance/intolerance species (seed source) gradient: pond pine ≥ wet-site loblolly pine > dry-site loblolly pine > sand pine.     

A review of fungal phytotoxins: from basic studies to practical use

Recent materials are summarized, pertaining to classification of fungal phytotoxins, methods of their isolation, and assays for biological activity. Producers of phytotoxic substances have been characterized, and the chemical nature of phytotoxins has been subjected to analysis. The review gives consideration to the mechanisms of action of phytotoxins on susceptible plants and the mechanisms of plant resistance to such agents. Other matters discussed include prospects of utilizing basic knowledge of the nature and mechanisms of action of phytotoxins for (1) developing means of plant protection against diseases and weeds and (2) identifying or classifying fungi (chemosystematics).     

Flow cytometry as a method for assaying the biological activity of phytotoxins

A flow cytometric method has been developed for the qualitative and quantitative evaluation of the biological activities of phytotoxins from plant pathogenic fungi. The method utilized fresh wheat (Triticum aestivum L.) leaf protoplast preparations treated with purified phytotoxins, triticone A-B and triticone D. Subsequently, protoplasts were exposed to fluorescein diacetate, and analyzed by flow cytometry. Information acquired included fluorescence owing to esterase activity on fluorescein diacetate, and chlorophyll autofluorescence. Results indicate that triticone A-B has a rapid dose-dependent toxic effect on wheat protoplasts but triticone D has no toxic effect. This method can also yield information on the mechanism of action of phytotoxins that are relatively unstable or available only in small quantities.     

Mountain Pine Beetles Use Volatile Cues to Locate Host Limber Pine and Avoid Non-Host Great Basin Bristlecone Pine

The tree-killing mountain pine beetle (Dendroctonus ponderosae Hopkins) is an important disturbance agent of western North American forests and recent outbreaks have affected tens of millions of hectares of trees. Most western North American pines (Pinus spp.) are hosts and are successfully attacked by mountain pine beetles whereas a handful of pine species are not suitable hosts and are rarely attacked. How pioneering females locate host trees is not well understood, with prevailing theory involving random landings and/or visual cues. Here we show that female mountain pine beetles orient toward volatile organic compounds (VOCs) from host limber pine (Pinus flexilis James) and away from VOCs of non-host Great Basin bristlecone pine (Pinus longaeva Bailey) in a Y-tube olfactometer. When presented with VOCs of both trees, females overwhelmingly choose limber pine over Great Basin bristlecone pine. Analysis of VOCs collected from co-occurring limber and Great Basin bristlecone pine trees revealed only a few quantitative differences. Noticeable differences included the monoterpenes 3-carene and D-limonene which were produced in greater amounts by host limber pine. We found no evidence that 3-carene is important for beetles when selecting trees, it was not attractive alone and its addition to Great Basin bristlecone pine VOCs did not alter female selection. However, addition of D-limonene to Great Basin bristlecone pine VOCs disrupted the ability of beetles to distinguish between tree species. When presented alone, D-limonene did not affect behavior, suggesting that the response is mediated by multiple compounds. A better understanding of host selection by mountain pine beetles could improve strategies for managing this important forest insect. Moreover, elucidating how Great Basin bristlecone pine escapes attack by mountain pine beetles could provide insight into mechanisms underlying the incredible longevity of this tree species.     

Differential selection of North American and Scandinavian conifer browse by northwestern moose (Alces alces andersoni) in winter

Scandinavian moose (Alces alces) eat Scots pine (Pinus sylvestris) in winter. Although North American moose are known to eat conifers such as true firs (Abies spp.) in winter, substantial consumption of pine by moose in North America has not been documented. Here, we document short-term winter preferences of human-habituated northwestern moose (Alces alces andersoni) for branches of mature North American and European conifer species as determined by a cafeteria-style feeding trial. Moose selected for species such as Douglas fir (Pseudotsuga menziesii; from which they took the smallest bite diameters) while avoiding species such as lodgepole pine (Pinus contorta; from which they took the largest bites) and hybrid white spruce (Picea glauca × engelmanii). The amount of species-specific biomass consumed by moose was negatively correlated with bite diameters taken from branches of those species and did not appear to be significantly influenced by differences in twig morphology between species. Our trial suggests that northwestern moose readily consume conifers in winter and, from the species we tested, prefer Douglas fir. While no clear preference existed between Scots pine and lodgepole pine, moose avoided lodgepole pine, but not Scots pine, relative to Douglas fir. Our trial suggests that northwestern moose are more likely to feed on the branches of Douglas fir than pine, which may be of interest to foresters managing conifers within the North American range of moose, particularly where Scots pine are being considered for planting.     

Fungal phytotoxins: from basic studies to practical use (a review)

Recent materials are summarized, pertaining to classification of fungal phytotoxins, methods of their isolation, and assays for biological activity. Producers of phytotoxic substances have been characterized, and the chemical nature of phytotoxins has been subjected to analysis. The review gives consideration to the mechanisms of action of phytotoxins on susceptible plants and the mechanisms of plant resistance to such agents. Other matters discussed include prospects of utilizing basic knowledge of the nature and mechanisms of action of phytotoxins for developing means of plant protection against diseases and weeds and identifying or classifying fungi (chemosystematics).     

Mixing Effects of Understory Plant Litter on Decomposition and Nutrient Release of Tree Litter in Two Plantations in Northeast China

Understory vegetation plays a crucial role in carbon and nutrient cycling in forest ecosystems; however, it is not clear how understory species affect tree litter decomposition and nutrient dynamics. In this study, we examined the impacts of understory litter on the decomposition and nutrient release of tree litter both in a pine (Pinus sylvestris var. mongolica) and a poplar (Populus × xiaozhuanica) plantation in Northeast China. Leaf litter of tree species, and senesced aboveground materials from two dominant understory species, Artemisia scoparia and Setaria viridis in the pine stand and Elymus villifer and A. sieversiana in the poplar stand, were collected. Mass loss and N and P fluxes of single-species litter and three-species mixtures in each of the two forests were quantified. Data from single-species litterbags were used to generate predicted mass loss and N and P fluxes for the mixed-species litterbags. In the mixture from the pine stand, the observed mass loss and N release did not differ from the predicted value, whereas the observed P release was greater than the predicted value. However, the presence of understory litter decelerated the mass loss and did not affect N and P releases from the pine litter. In the poplar stand, litter mixture presented a positive non-additive effect on litter mass loss and P release, but an addition effect on N release. The presence of understory species accelerated only N release of poplar litter. Moreover, the responses of mass loss and N and P releases of understory litter in the mixtures varied with species in both pine and poplar plantations. Our results suggest that the effects of understory species on tree litter decomposition vary with tree species, and also highlight the importance of understory species in litter decomposition and nutrient cycles in forest ecosystems.     

More chemistry of the thaxtomin phytotoxins

Chemical and biochemical studies indicated the possible involvement of N-acetyltryptophan and 4-nitrotryptophan as intermediates in biosynthesis of the thaxtomin phytotoxins. A search for other potential pathways indirectly resulted in the identification of three unusual thaxtomin analogues derived from the o-thaxtomin A isomer. Investigations to resolve the identity of a previously described thaxtomin A di-glucoside were not supportive of the proposed structure.     

Respiration of wood ant nest material affected by material and forest stand characteristics

We studied differences in respiration of materials from different parts of wood ant nest (top, bottom, and rim) and from the nest surroundings (humus layer and mineral soil). Samples were taken from 8 wood ant (Formica aquilonia) nests in each of the two types of forest (birch and pine) in eastern Finland. The differences were related to material and forest stand characteristics (i.e., moisture, pH, carbon content, and C:N ratio). As a result, the highest respiration per g DW was measured at the top of ant nests in the birch forest. However, respiration did not significantly differ between the parts of ant nests in the pine forest. Respiration of the humus layers in both forest stands was on average higher, whereas respiration of the mineral soils in both forest stands was lower in comparison with respiration of the nest materials. The respiration per g C did not show any significant differences between different parts of nests and surrounding soil. The most important factors influencing respiration of the materials appeared to be moisture, carbon content, and pH. In conclusion, respiration of wood ant nest material is affected by the specific material and forest stand characteristics.     

The genetics of shortleaf pine (<Emphasis Type="Italic">Pinus echinata</Emphasis> mill.) with implications for restoration and management

Shortleaf pine (Pinus echinata Mill.) is an important commercial timber resource and forest ecosystem component in the southeastern USA. The species occurs in mainly drier sites as an early- to mid-successional species, is fire-adapted, and it plays an important role in the fire ecology of the region. However, shortleaf pine genetics are not well-studied, especially in this era of molecular genetics and genomics. Most genetics research about the species has focused on provenance testing. Generally, shortleaf pine performs well in colder areas, when compared to loblolly pine (Pinus taeda L.), a close relative, which is faster growing and the most common plantation species in the region. Though not as advanced in genetic improvement as loblolly pine, tree breeders have improved shortleaf pine in one to two generations of selection, and diverse, genetically improved shortleaf pine materials are available to foresters and landowners throughout the southeastern USA. Researchers have also studied the genetic variation of shortleaf pine using various molecular markers and have found that shortleaf pine is generally a prolific outcrosser, a trait it shares with other non-isolated members of the family Pinaceae. In recent years, however, it has shared less genetic material across long ranges, probably because of habitat fragmentation. Various anthropogenic factors also affect shortleaf pine’s future, as recent studies show that shortleaf pine introgression with loblolly pine puts the species—and the resiliency of southeastern forests—at risk. Importantly, fire exclusion is a likely cause of the increase in introgression. Herein, we provide further details and up-to-date genetic information and resources for foresters and ecologists interested in the restoration and management of shortleaf pine.     

Root-Associated Bacteria Contribute to Mineral Weathering and to Mineral Nutrition in Trees: a Budgeting Analysis

The principal nutrient source for forest trees derives from the weathering of soil minerals which results from water circulation and from plant and microbial activity. The main objectives of this work were to quantify the respective effects of plant- and root-associated bacteria on mineral weathering and their consequences on tree seedling growth and nutrition. That is why we carried out two column experiments with a quartz-biotite substrate. The columns were planted with or without pine seedlings and inoculated or not with three ectomycorrhizosphere bacterial strains to quantify biotite weathering and pine growth and to determine how bacteria improve pine growth. We showed that the pine roots significantly increased biotite weathering by a factor of 1.3 for magnesium and 1.7 for potassium. We also demonstrated that the inoculation of Burkholderia glathei PML1(12) significantly increased biotite weathering by a factor of 1.4 for magnesium and 1.5 for potassium in comparison with the pine alone. In addition, we observed a significant positive effect of B. glathei PMB1(7) and PML1(12) on pine growth and on root morphology (number of lateral roots and root hairs). We demonstrated that PML1(12) improved pine growth when the seedlings were supplied with a nutrient solution which did not contain the nutrients present in the biotite. No improvement of pine growth was observed when the seedlings were supplied with all the nutrients necessary for pine growth. We therefore propose that the growth-promoting effect of B. glathei PML1(12) mainly resulted from the improved plant nutrition via increased mineral weathering.     

Characterization of phytotoxin and secreted proteins identifies of Lasiodiplodia theobromae, causes of peach gummosis

Lasiodiplodia theobromae, a phytopathogenic fungus that causes peach gummosis, is considered one of the major constraints to peach production worldwide. Here, we report the characteristics of toxic metabolites and the proteomics investigation of the secreted proteins of L. theobromae from its in vitro culture. The phytotoxins of L. theobromae from the culture filtrate of Richard’s liquid medium showed high toxicity on peach current year shoots with large lesion diameter and high gum weight. The toxicity measurement showed that 23.6 and 21.2 mg gum were induced from peach shoots by solvent fractions of ethyl acetate and dichloromethane, respectively, with significant differences from other organic solvents. A total of 23 proteins were identified by liquid chromatography–mass spectrometry from the in vitro secretome of L. theobromae. Sequence analysis indicated that 14 proteins were extracellular proteins based on signal peptides and localization. The expression profiles of the analyzed fungal genes were significantly upregulated from 1 day postinoculation (dpi) to 2 dpi, indicating that the early stage is an important stage for the infection of L. theobromae. The present study has provided insights into the extracellular phytotoxins and secreted proteins that are possibly associated with pathogenicity of the peach gummosis.     

Brown Pine Leaf Extract and Its Active Component Trans-Communic Acid Inhibit UVB-Induced MMP-1 Expression by Targeting PI3K

Japanese red pine (Pinus densiflora) is widely present in China, Japan, and Korea. Its green pine leaves have traditionally been used as a food as well as a coloring agent. After being shed, pine leaves change their color from green to brown within two years, and although the brown pine leaves are abundantly available, their value has not been closely assessed. In this study, we investigated the potential anti-photoaging properties of brown pine leaves for skin. Brown pine leaf extract (BPLE) inhibited UVB-induced matrix metalloproteinase-1 (MMP-1) expression to a greater extent than pine leaf extract (PLE) in human keratinocytes and a human skin equivalent model. HPLC analysis revealed that the quantity of trans-communic acid (TCA) and dehydroabietic acid (DAA) significantly increases when the pine leaf color changes from green to brown. BPLE and TCA elicited reductions in UVB-induced MMP-1 mRNA expression and activator protein-1 (AP-1) transactivation by reducing DNA binding activity of phospho-c-Jun, c-fos and Fra-1. BPLE and TCA also inhibited UVB-induced Akt phosphorylation, but not mitogen activated protein kinase (MAPK), known regulators of AP-1 transactivation. We additionally found that BPLE and TCA inhibited phosphoinositide 3-kinase (PI3K), the upstream kinase of Akt, in vitro. In summary, both BPLE and its active component TCA exhibit protective effects against UVB-induced skin aging. Taken together, these findings underline the potential for BPLE and TCA to be utilized as anti-wrinkling agents and cosmetic ingredients, as they suppress UVB-induced MMP-1 expression.     

Temperature effects on seed imbibition and leakage mediated by viscosity and membranes

The possible involvement of membranes and water viscosity in the temperature effects on imbibition and solute leakage of radish (Raphanus sativa var. Early Scarlet Globe) seeds and excised sugar pine (Pinus lambertiana Dougl.) embryos was evaluated. In these two seed materials, the temperature effect on initial rates of imbibition and solute leakage could be accounted for primarily by changes in water viscosity, the relationship being approximately linear. It appears that membranes are involved both in water uptake and solute leakage. Heat-killed radish seeds and sugar pine embryos exhibited significantly higher rates of imbibition and solute leakage than did viable ones. In addition, sugar pine embryos exhibited an abrupt change in rates of imbibition and solute leakage between 15 and 20°C, resulting in abnormally high water uptake and solute leakage above this temperature.     

Epidemiology of a Salmonella enterica subsp. enterica Serovar Typhimurium Strain Associated with a Songbird Outbreak

Salmonella enterica subsp. enterica serovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. This Salmonella serovar is also responsible for die-offs in songbird populations. In 2009, there was an S. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and human S. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This same S. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 total S. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature of S. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.     

Co-invading ectomycorrhizal fungal succession in pine-invaded mountain grasslands

Ectomycorrhizal (EM) fungal communities that associate with invading pines (Pinus spp.) are expected to be poor in species diversity. However, long-term successional trajectories and the persistence of dispersal limitations of EM fungi in the exotic range are not well understood. We sampled the roots and surrounding soil of Pinus elliottii and P. taeda trees invading mountain grasslands of Argentina. We also sampled the EM fungal spore bank in grassland soil near (∼150 m) and far (∼850 m) from the original pine plantations. We found 86 different co-invasive EM fungal OTUs. Differential dispersal capacities among EM fungi were detected in the spore bank of grassland soil, but not under mature pines. After thirty years of invasion, the age, but not the degree of spatial isolation of pine individuals affected the EM fungal composition. We showed how EM fungal succession occurs during pine invasions, which may have clear consequences for ecosystem functioning of co-invaded sites.     

Newly Discovered Transmission Pathway of Bursaphelenchus xylophilus from Males of the Beetle Monochamus alternatus to Pinus densiflora Trees via Oviposition Wounds

The transmission of Bursaphelenchus xylophilus from Monochamus alternatus males to Pinus densiflora trees via oviposition wounds has been determined. Nematode-infested males, with mandibles fixed experimentally to prevent feeding, were placed for 48 hours with pine bolts containing oviposition wounds that had been made by nematode-free females. After removal of the nematode-infested males, the pine bolts were held for 1 month and then examined for the presence of nematodes. Reproducing nematode populations were recovered from pine bolts that were exposed to male beetles carrying a high number of nematodes. No reproducing nematode population could be recovered from pine bolts exposed to beetles with a small number of nematodes. Nematode reproduction in the pine bolts was not related to the number of oviposition wounds per bolt. Fourth-stage dispersal B. xylophilus juveniles, collected from beetle body surfaces, were inoculated on pine bolt bark 0, 5, 10, and 15 cm away from a single artificial, small hole. These dauer juveniles successfully entered some bolts. The probability of successful nematode reproduction decreased with increased distance between inoculation point and artificial hole. The results indicated that B. xylophilus can move a significant distance to oviposition wounds along the bark surface and enter a tree via the wounds. The new transmission pathway is considered important for the nematode to persist in pine forests such as in North America where pine wilt disease does not occur.