A novel tyrosinase from Armillaria ostoyae with comparable monophenolase and diphenolase activity suffers substrate inhibition

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyaestrain C18/9 (AoTyr) was characterized and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the two activities. In silico analysis and mutagenesis experiments showed that residue 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the substitution of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (kcat/Km) (1.63 folds) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. IMPORTANCE Tyrosinase is able to oxidize various phenolic compounds and its ability to convert monophenols into diphenols has caught great attention in the research field and industrial applications. However, the utilization of tyrosinase for the industrial synthesis of catechols has been limited due to the fact that the monophenolase activity of most of the known tyrosinases is much lower than diphenolase activity. In the present study, a novel tyrosinase with a comparable monophenolase/diphenolase activity ratio was characterized. The enzyme exhibits substrate inhibition in both monophenolase and diphenolase activities. In silico analysis followed by mutagenesis experiments confirmed the important roles of residue 262 and 266 in the substrate inhibition and activities modulation of the enzyme, and the variant D262N showed an enhanced monophenolase/diphenolase catalytic efficiency ratio as compared to the wild type enzyme.

Climate, radial growth, and mortality associated with conifer regeneration infected by root disease (Armillaria ostoyae)

Tree root disease caused by the pathogen Armillaria ostoyae (Romagn.) Herink has notable influence on bio-economic systems of southern British Columbia (BC) and the northwestern United States. Annual radial growth and mortality trends of regeneration associated with A. ostoyae during the first 21 years of a plantation were investigated. Our dendrochronological approach focused on a plantation established in 1991 with Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), western larch (Larix occidentalis Nutt.), and lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm. ex S. Wats.). We examined tree rings for reduced growth onset (RGO) to estimate infection periods. Among trees studied, we found that the number of RGO events peaked in 2006 followed by a steady decline in frequency. A similar trend was seen in annual mortality rates which reached maximum values in 2007 followed by an uninterrupted decrease. The average number of years from RGO to death based on host species ranged from 2.3 to 3.6 years. According to on logistic regression modeling, values associated with drought codes and duff moisture codes were significant in determining the year of death for infected trees. The predicted increase in frequency of summer droughts may lead to elevated mortality associated with A. ostoyae in young plantations.

Chromosomal assembly and analyses of genome-wide recombination rates in the forest pathogenic fungus Armillaria ostoyae

Recombination shapes the evolutionary trajectory of populations and plays an important role in the faithful transmission of chromosomes during meiosis. Levels of sexual reproduction and recombination are important properties of host-pathogen interactions because the speed of antagonistic co-evolution depends on the ability of hosts and pathogens to generate genetic variation. However, our understanding of the importance of recombination is limited because large taxonomic groups remain poorly investigated. Here, we analyze recombination rate variation in the basidiomycete fungus Armillaria ostoyae, which is an aggressive pathogen on a broad range of conifers and other trees. We constructed a dense genetic map using 198 single basidiospore progeny from a cross. Progeny were genotyped at a genome-wide set of single nucleotide polymorphism (SNP) markers using double digest restriction site associated DNA sequencing (ddRADseq). Based on a linkage map of on 11,700 SNPs spanning 1007.5 cM, we assembled genomic scaffolds into 11 putative chromosomes of a total genome size of 56.6 Mb. We identified 1984 crossover events among all progeny and found that recombination rates were highly variable along chromosomes. Recombination hotspots tended to be in regions close to the telomeres and were more gene-poor than the genomic background. Genes in proximity to recombination hotspots were encoding on average shorter proteins and were enriched for pectin degrading enzymes. Our analyses enable more powerful population and genome-scale studies of a major tree pathogen.

Genetic basis of susceptibility to Diplodia sapinea and Armillaria ostoyae in maritime pine

SummaryForest ecosystems are increasingly challenged by extreme events, e.g. pest and pathogen outbreaks, causing severe ecological and economical losses. Understanding the genetic basis of adaptive traits in tree species is of key importance to preserve forest ecosystemsAdaptive phenotypes, including susceptibility to two fungal pathogens (Diplodia sapinea and Armillaria ostoyae) and an insect pest (Thaumetopoea pityocampa), height and needle phenology were assessed in a range-wide common garden of maritime pine (Pinus pinaster Aiton), a widespread conifer in the western Mediterranean Basin and parts of the Atlantic coast.Broad-sense heritability was significant for height (0.497), needle phenology (0.231-0.468) and pathogen symptoms (0.413 for D. sapinea and 0.066 for A. ostoyae) measured after inoculation under controlled conditions, but not for pine processionary moth incidence assessed in the common garden. Genetic correlations between traits revealed contrasting trends for pathogen susceptibility to D. sapinea and A. ostoyae. Maritime pine populations from areas with high summer temperatures and frequent droughts were less susceptible to D. sapinea but more susceptible to A. ostoyae. An association study using 4,227 genome-wide SNPs revealed several loci significantly associated to each trait.This study provides important insights to develop genetic conservation and breeding strategies integrating tree responses to pathogens.

Soil disturbance and juvenile Douglas-fir growth following stump removal on moderately coarse textured soils in southwestern British Columbia: 10-year results

Mechanized treatment for root diseases such as Coniferiporia sulphurascens (Pilát) L. W. Zhou & Y. C. Dai (syn. Phellinus sulphurascens Pilát) and Armillaria ostoyae (Romagn.) Herink (syn. Armillaria solidipes Peck) is often avoided due to cost or the perception that removal of stumps creates detrimental soil disturbance or degradation that hinders site productivity. Our study tested five diseased stands that were treated by extracting stumps following harvesting and replanted with susceptible Douglas-fir. Soil disturbance surveys were conducted in treated and untreated plots, and individual planted spots were assessed and categorized for soil disturbance using existing and proposed new categories specific to disturbance caused by the stump removal. Tree measurements were taken at intervals over the first 10 years of stand development, and foliage was sampled for nutrient analysis. The percentage of total and counted disturbance was 20%–46% and 8%–11% greater, respectively, in treated versus untreated plots; however, mean tree growth in height and diameter was not statistically different between treatments and was more positive for treated plots at all sites but one. Tree nutrition and survival to age 10 was not negatively affected by stump removal. Total site productivity represented by basal area and tree volume differed widely between sites but was not significantly different between treatments.