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Laminated Root Rot caused by the fungal pathogen Coniferiporia sulphurascens is a damaging disease within many Douglas-fir forests of the Pacific Northwest of North America. Management of these forests in a changing climate and fire regime will require changes to silvicultural practices. A long-term study (ca. 30 years) in Oregon, USA provides an opportunity to investigate the effects of thinning on disease dynamics in an area of historically high laminated root rot incidence. The effects of three thinning prescriptions on tree mortality caused by C. sulphurascens were compared on ca. 160 ha within the Siuslaw National Forest. Observations were compared with predictions from the Forest Vegetation Simulator and its Western Root Disease Model extension. No significant effect of thinning treatment on mortality (p = 0.981), or annual basal area increment (p = 0.372) was observed. In contrast to observations, the Forest Vegetation Simulator over estimated growth, while the Western Root Disease Model extension was consistent with field measurements. Thinning treatments appear to have minimal impacts on laminated root rot induced mortality but also do not result in the expected increase in growth rate typically associated with a thinning treatment. • Long-term study reveals no impact of thinning on pathogen induced mortality. • Thinning of root disease affected stands has no impact on growth rate of crop trees. • The Forest Vegetation Simulator overestimates growth in diseased stands. • The Western Root Disease Model accurately predicts growth in diseased stands. [ABSTRACT FROM AUTHOR]

The hemibiotroph Colletotrichum gloeosporioides and the necrotroph Cytospora chrysosperma cause poplar foliage and stem disease, respectively, resulting in substantial economic losses. In this study, Populus trichocarpa ptc-miR472a was down-regulated in leaves treated with salicylic acid, jasmonic acid (JA) or bacterial flagellin (flg22). Here, ptc-miR472a and a short tandem target mimic (STTM) of miR472a were overexpressed in P. alba × P. glandulosa, and overexpression lines of miR472a and silenced lines of STTM472a were generated. Compared with the STTM472a and wild type lines, lower reactive oxygen species accumulation was detected in miR472a overexpressing plants treated with flg22, C. gloeosporioides or C. chrysosperma. In addition, the miR472a overexpressing lines exhibited the highest susceptibility to the hemibiotroph, C. gloeosporioides, but the highest effective defence response to the necrotroph, C. chrysosperma. The JA/ethylene marker gene ERF1 was rapidly up-regulated in miR472a overexpressing plants. Furthermore, five phased, secondary, small interfering RNAs (phasiRNAs) were confirmed in the miR472a overexpressing and STTM472a lines, triggering phasiRNAs predicted to enhance NBS-LRR silencing. Taken together, our results revealed that ptc-miR472a exerts a key role in plant immunity to C. gloeosporioides and C. chrysosperma by targeting NBS-LRR transcripts. This study provides a new strategy and method in plant breeding to improve plant disease resistance. [ABSTRACT FROM AUTHOR]

Abstract: Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of Coniferiporia sulphurascens and Coniferiporia weirii, two well‐defined and distinguishable fungal species of the family Hymenochaetaceae. The pathogens are regulated in Council Directive 2000/29/EC (Annex IAI, under the previous name Inonotus weirii for both species) as a harmful organism whose introduction into the EU is banned. The two pathogens are native to North America, where C. sulphurascens causes laminated root rot primarily in Douglas fir (Pseudotsuga menziesii) and grand fir (Abies grandis), while C. weirii causes cedar laminated root and butt rot mainly in cedars (Thuja plicata and Cupressus nootkatensis). C. weirii has been reported from Japan and China, and C. sulphurascens from China, Russia and Turkey. Neither species has been reported from the EU. C. sulphurascens may infect all conifers, while C. weirii is reported to mainly cause disease in tree species of Thuja spp. and Cupressus spp. The two pathogens could enter the EU mainly via wood with bark, isolated bark and plants for planting (including artificially dwarfed plants) of Pinaceae and Cupressaceae. Both fungi could establish in the EU, as hosts are present and climatic conditions are favourable. The two pathogens would be able to spread following establishment by the pathways mentioned for entry and also by dissemination of basidiospores and root contact with infected root/wood. Should the pathogen be introduced in the EU, impacts can be expected on coniferous woodlands, plantations and ornamental trees, thus leading to reduced tree growth and ecosystem service provision. The key uncertainties concern (i) the distribution of the two pathogens in Asia, (ii) the level of susceptibility of conifers native to Europe and (iii) the role of plants for planting as a pathway of entry and spread. For both pathogens, the criteria assessed by the Panel for consideration as a potential quarantine pest are met. As the two pests are not present in the EU, not all the criteria for consideration as regulated non‐quarantine pests are met. [ABSTRACT FROM AUTHOR]

Viruses are serious threat to chilli crop production worldwide. Resistance screening against several viruses resulted in identifying a multiple virus resistant genotype 'IHR 2451'. Degenerate primers based on the conserved regions between P-Loop and GLPL of Resistance genes (R-genes) were used to amplify nucleotide binding sites (NBS)-encoding regions from genotype 'IHR 2451'. Alignment of deduced amino acid sequences and phylogenetic analyses of isolated sequences distinguished into two groups representing toll interleukin-1 receptor (TIR) and non-TIR, and different families within the group confirming the hypotheses that dicots have both the types of NBS-LRR genes. The alignment of deduced amino acid sequences revealed conservation of subdomains P-loop, RNBS-A, kinase2, RNBS-B, and GLPL. The distinctive five RGAs showing specific conserved motifs were subjected to BLASTp and indicated high homology at deduced amino acid level with R genes identified such as Pvr9 gene for potyvirus resistance, putative late blight resistance protein homolog R1B-23 and other disease resistance genes suggesting high correlation with resistance to different pathogens. These pepper RGAs could be regarded as candidate sequences of resistant genes for marker development. [ABSTRACT FROM AUTHOR]

Phellinus sulphurascens (previously the Douglas-fir form of Phellinus weirii ) is an important native pathogen causing laminated root rot in forests of western North America. Visual crown symptoms, or attacks by bark or ambrosia beetles appear only during advanced stages of the disease with extensive infection in the lower bole. Ethanol synthesis is one of many physiological responses in tree tissues stressed by pathogens. Ethanol, acetone and other volatiles from root tissues of healthy and diseased trees were analyzed using headspace gas chromatography. Xylem and phloem from 20 diseased trees at two western Oregon sites contained higher concentrations of ethanol, acetone, or other headspace volatiles than 20 healthy trees on one or more dates in September, November, or the following May. Root cross-sections from eight diseased trees were sampled along perpendicular transects and found to contain extremely variable ethanol concentrations, with highest xylem quantities in a 0–2 cm zone outside the infection boundary and lowest amounts inside the infection. Acetone concentrations were the opposite. Logistic regression models were built and tested to determine which volatiles could predict diseased trees. A model using xylem ethanol concentrations as the only parameter was selected and validated with measurements from 80 trees on the edges of P. sulphurascens infection centers at two different western Oregon sites. This model successfully predicted trees with laminated root rot (78% overall correct classification and 68% for known diseased trees), but worked best for those with infections observed in both root cores and the root collar (100% correct). Early detection of P. sulphurascens infected trees remains a challenge. Our ethanol analysis method is useful for research, but provides limited benefits for identifying individual P. sulphurascens hazard trees, or for extensive ground surveys in the forest. Whether ethanol is released to the atmosphere in sufficient quantities to confirm infection before the late appearance of crown symptoms, or bark beetles remains unknown. If it is, then development of sensors capable of tree side detection requiring minimal tissue sampling would be useful in managing this disease. We also propose a mechanism for how ethanol with host monoterpenes could play a central role in pioneering bark beetle primary host selection of trees infected with this pathogen. [ABSTRACT FROM AUTHOR]

Abstract: In order to study the defense response to turnip mosaic virus (TuMV) infection in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), we cloned the LRR II subfamily genes which comprises six members. They were high homologous to the function-known LRR II genes of Arabidopsis. We investigated their expression through quantitative real-time PCR analysis. TuMV infection induced the expression of these genes locally and systematically, and regulated the endogenous accumulation of salicylic acid (SA). Exogenous SA spraying was able to induce resistance to the susceptibility of the TuMV-infected plants, which might function via inhibiting the viral duplication. Though TuMV-induced SA accumulation was not the determinant in regulating gene expression, it mediated the reaction oxygen species (ROS) burst as a channel of defense. [Copyright &y& Elsevier]

To better understand the molecular basis of interactions between the laminated root rot pathogen Phellinus sulphurascens Pilát and its principal host, Douglas-fir [DF; Pseudotsuga menziesii (Mirb.) Franco.], we constructed a cDNA library from roots of P. sulphurascens-infected DF seedlings. From a total of 3600 random cDNA clones, we identified 56 clones that matched with barley wound induced (barwin)-type pathogenesis-related 4 (PR4) genes that have been reported in planta. At least three PR4 genes comprising 426- to 444-bp full-length open reading frames, Pm PR4a1, Pm PR4a2, and Pm PR4b1, were identified from DF seedlings. Deduced amino acid sequences for the proteins encoded by these genes confirmed that two of them are acidic (Mr, 12.8-13.0 kDa; p I, 4.12-4.53) and one is a basic (Mr, 13.0 kDa; p I, 8.53) PR4 protein. Quantitative reverse transcriptase-PCR analyses of these transcripts showed significantly higher transcription in P. sulphurascens-infected vs. control DF tissues. Upregulation of the transcripts in roots of a P. sulphurascens-resistant DF family was significant. Our results suggest that DF PR4 genes play an important role in the defence mechanism of DF against P. sulphurascens infection and thus could be useful in DF breeding. [ABSTRACT FROM AUTHOR]

Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 ( S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato. [ABSTRACT FROM AUTHOR]

Abstract: Interactions between roots of Douglas-fir (DF; Pseudotsuga menziesii) seedlings and the laminated root rot fungus Phellinus sulphurascens were investigated using scanning and transmission electron microscopy and immunogold labelling techniques. Scanning electron micrographs revealed that P. sulphurascens hyphae colonize root surfaces and initiate the penetration of root epidermal tissues by developing appressoria within 2d postinoculation (dpi). During early colonization, intra- and intercellular fungal hyphae were detected. They efficiently disintegrate cellular components of the host including cell walls and membranes. P. sulphurascens hyphae penetrate host cell walls by forming narrow hyphal tips and a variety of haustoria-like structures which may play important roles in pathogenic interactions. Ovomucoid–WGA (wheat germ agglutinin) conjugated gold particles (10nm) confirmed the occurrence and location of P. sulphurascens hyphae, while four specific host pathogenesis-related (PR) protein antibodies conjugated with protein A–gold complex (20nm) showed the localization and abundance of these PR proteins in infected root tissues. A thaumatin-like protein and an endochitinase-like protein were both strongly evident and localized in host cell membranes. A DF-PR10 protein was localized in the cell walls and cytoplasm of host cells while an antimicrobial peptide occurred in host cell walls. A close association of some PR proteins with P. sulphurascens hyphae suggests their potential antifungal activities in DF roots. [Copyright &y& Elsevier]

Abstract: We carried out a comparative proteomic study to explore the molecular mechanisms that underlie the defense response of Douglas-fir (DF, Pseudotsuga menziesii) to laminated root rot, a disease caused by Phellinus sulphurascens. 2-DE was conducted on proteins extracted from roots of laboratory-grown, young DF seedlings inoculated with P. sulphurascens. A total of 1303 proteins was detected in 7 dpi infected and uninfected root samples. Among these 1303 proteins, 277 showed differential expression that was statistically significant (p <0.05). Of these 277 proteins, 74 upregulated and 85 downregulated proteins showed at least a two-fold change from controls. Forty seven upregulated and 23 downregulated proteins were selected to be excised and analyzed using LC–MS/MS followed by peptide matching. Our results indicate that the major proteins differentially expressed in P. sulphurascens-infected DF seedlings include those in the following functional groups: disease/defense (27%), metabolism (16%), transcription factors (11%), signal transduction (10%), secondary metabolism (7%) and energy (4%). A number of additional proteins involved in cell structure (3%) and protein synthesis (3%) were also identified. By providing an initial database of candidate pathogenesis-related proteins for the DF–Phellinus sulphurascens pathosystem the results of this study will enable future detailed investigation of gene expression and function. [Copyright &y& Elsevier]

Abstract: Removal of stumps and fertilization after harvesting have been proposed to reduce root diseases in succeeding stands. Potential impacts such as compaction and loss of soil C or N could limit the use of this technique. This research examined forest soils in five Pacific Northwest stands 22–29 years after stump removal and fertilization with N. Results show that small long-term soil bulk density increases caused by stump removal are not likely to either limit tree growth or discourage the use of stump removal to reduce root rot. However, an extended decrease was noted in mineral soil total N and C and forest floor depth caused by stump removal. The stumped areas show a mineral soil nitrogen concentration that is 20% lower than the non-stumped areas. Mineral soil carbon concentrations were 24% lower and the forest floor depth was 24% lower. A non-significant trend of lower foliar N was also observed with stump removal. These results were consistent in all five soil types. This reduction in the organic component of the soil may be a concern for nutrient cycling and long-term productivity on poor sites. [Copyright &y& Elsevier]

Several aspects of the host-pathogen interaction between Douglas-fir (Pseudotsuga menziesii) and the fungal pathogen Phellinus sulphurascens were investigated in an in vitro inoculation system using young seedlings and fungal mycelia. Light microscopy confirmed that P. sulphurascens mycelia can successfully penetrate host epidermal cells within 3 days postinoculation (dpi). Extensive fungal colonization and cortical cell decay occurred within 14 dpi. Western immunoblot studies showed significant upregulationi (five to sixfold) of four specific pathogenesis-related (PR) proteins in infected roots. These proteins were a Douglas-fir thaumatin-like protein (PmTLP), an endochitinase protein (ECP), a Douglas-fir PR10 (DF-PR10) protein (Pseinl), and a 10.6-kDa antimicrobial peptide (PmAMP1). The highest accumulation of PmTLP and PmAMPI occurred at 12 dpi, whereas accumulations of the ECP and DF-PRIO proteins peaked at 7 dpi. For both inoculated and control Douglas-fir seedlings, only one of the four PR proteins, PmAMPI, was clearly detectable in needles. Immunolocalization experiments using fluorescein isothio-cyanate-conjugated secondary antibodies confirmed accumulation of all four PR proteins mainly in and around cell walls of root cortical tissues. Overall, the highest immunofluorescence was observed in infected roots at 12 dpi, whereas labeling in control roots was negligible at all sample times. The ECP produced the highest fluorescence; the DF-PR10 the lowest. Upregulation and localization of these PR proteins in cortical tissues of inoculated roots suggest that they play a defensive role in response to infection by P suiphurascens. This in vitro inoculation system will facilitate further proteomic and genomic studies of this important pathosystem. [ABSTRACT FROM AUTHOR]

Abstract: Phellinus weirii (Murr.) Gilb. causes laminated root rot (LRR), a major disease affecting growth and survival of Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) and other commercially important conifer species throughout the Pacific Northwest. This disease is known to spread to a replacement stand by root contact between replacement trees and residual infected stumps and roots from the harvested stand. One strategy to manage LRR is to reduce the residual inoculum on a site through application of a fungicidal chemical to infested stumps. The first two studies in this series established that chloropicrin (trichloronitromethane), applied to infested stumps, largely eliminates P. weirii from most of the belowground biomass and determined the effective dosage to apply. This third study approximated an operational application and based treatment success on reduction of LRR in the replacement stand. The study area was an 8-ha, 65-year-old naturally regenerated second-growth stand that was predominantly Douglas-fir. The stand was surveyed preharvest and postharvest (clearcut). Each P. weirii-infected entity (standing dead or down trees and stumps) was marked, and the location of its center was mapped. Circular plots (0.04ha) were located so as to include concentrations of infested stumps. Plots were stratified based on their estimated biomass of inoculum into eight replicate blocks of four plots each. Three chloropicrin treatments and an untreated control were assigned randomly to four plots in each of the eight blocks: (a) 100% labeled dosage to all stumps, (b) 20% labeled dosage to all stumps, (c) 100% labeled dosage to only visibly P. weirii-infected (stump-top stain or advanced decay) stumps, and (d) control (nothing done to the stumps). Holes were drilled into stump tops, a dose of chloropicrin poured in, and the holes plugged. The labeled dosage was 3.3ml of chloropicrin per kilogram of estimated stump and root biomass. Douglas-fir seedlings were planted in the winter following treatment application. When the stand was considered established, each plot was thinned to an inter-tree spacing of 2.4m and the trees tagged. Diameter at breast height, total height, and mortality of trees were recorded every 2–5 years. A total of 1041 tagged trees were observed for 16 growing seasons following treatment. Application of chloropicrin to stumps in the harvested stand did not influence the rate of LRR-caused mortality or growth of Douglas-fir seedlings in the replacement stand. [Copyright &y& Elsevier]

Abstract: Phellinus weirii causes laminated root rot (LRR), a major disease affecting growth and survival of Pseudotsuga menziesii (Douglas-fir) and other commercially important conifer species throughout the Pacific Northwest. Increasing tree vigor and resistance to pathogens through application of K fertilizer is a suggested disease management strategy and one we wished to test. Plots were established with Douglas-fir seedlings planted densely (12,000seedlingsha−1) around stumps of LRR-killed trees on a harvested site west of the Cascade Range crest near Morton, Washington. Treatments, randomly assigned to plots, were: 224kgKha−1, 448kgKha−1, 224kgK+224kgNha−1, and a no-fertilizer control, with 11 replicates. Roots of seedlings receiving the K+N treatment showed the only increase in phenol/sugar (P/S) ratios after one growing season, owing to a reduction in sugar concentrations. Although increases in P/S ratios are negatively correlated with fungal infection rates, the LRR mortality for all fertilizer treatments, including the K+N, was similar to the mortality on the no-fertilizer plots 7 years post-treatment. For this study site we concluded that the single application of K or K+N to Douglas-fir at the rates tested were not an effective management strategy for LRR. [Copyright &y& Elsevier]

Abstract: Phellinus weirii (Murr.) Gilb. causes laminated root rot (LRR), a major disease affecting growth and survival of Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) and other commercially important conifer species throughout the Pacific Northwest. Stump removal or nitrogen (N) fertilization, or both, have been suggested as management strategies for some root diseases but have not been tested previously for LRR in a replicated field study. Independent studies, using the same experimental design, were established in five Douglas-fir stands in Oregon and Washington. The stands were surveyed preharvest and postharvest (clearcut). Each P. weirii-infected entity (standing dead or down trees and stumps) was marked, and the location of its center was mapped. In each clearcut, a 2×4 set of factorial treatments of stump removal in combination with N fertilization was applied to 0.04ha circular plots and replicated five to seven times. Treatments, applied in the fall, included stump removal with a bulldozer (either all stumps removed or the plot left undisturbed) and broadcast fertilization with ammonium nitrate (0, 336, 672, or 1345kgNha−1). Douglas-fir seedlings were planted in the winter following treatment application. When the stand was considered established, each plot was thinned to a spacing of about 2.4m inter-tree spacing and the trees tagged. Diameter at breast height, total height, and mortality of trees were recorded every 2–5 years. A total of 7827 tagged trees on 239 plots were observed for up to 27 growing seasons following treatment. Stumping caused a significant reduction in seedling mortality due to LRR at all five study areas. The mean LRR-caused mortality on stumped and nonstumped plots was 2.4 and 9.1%, respectively. Fertilization did not cause a significant change in LRR-caused mortality of seedlings at any of the study areas. Nitrogen fertilization before planting increased growth of Douglas-fir seedlings but only in some stands. Nitrogen fertilizer can have a positive residual effect on growth of Douglas-fir for at least 23 years. Effects on soil of heavy equipment used in stumping operations did not reduce subsequent Douglas-fir survival or growth. There was a mean reduction in wood volume of 24.8% in areas where LRR was observed compared to areas where it was not found. [Copyright &y& Elsevier]

The population structure and ecological roles of the indigenous pathogen Phellinus weirii, cause of laminated root rot in conifer forests of western North America, are examined. This pathogen kills trees in slowly expanding mortality centers, creating gaps in the forest canopy. It is widespread, locally abundant, and very long-lived. It is among the most important disturbance agents in the long intervals between stand-replacing events such as wildfire or harvest in these ecosystems and shapes the structure and composition of both wild and managed forests. Trees are infected and killed regardless of individual vigor. Management of public lands is changing dramatically, with renewed emphasis on natural forest structures and processes but pathogens, especially root rot fungi, remain a significant challenge to "ecosystem management."