Characterization and distribution of fungi associated with needle defoliation of eastern white pine (Pinus strobus)

Over the past seven years eastern white pines (Pinus strobus) across the northeastern United States have been prematurely defoliating due to a disease complex known as White Pine Needle Damage (WPND). Previous research on WPND indicates that this outbreak, which started in 2010, is primarily attributed to the causal agent of brown spot needle blight, Lecanosticta acicola along with two other known P. strobus needle cast pathogens, Bifusella linearis and Lophophacidium dooksii. The current outbreak is thought to have arisen from several consecutive years of increased temperature and precipitation in the region. Current research, however, is lacking in both sampling distribution, the basic epidemiology of the primary causal agents, and the specific climatic factors that affect the development and spread of this outbreak. Furthermore, recent re-sampling of diseased P. strobus needles within this region has revealed a high isolation frequency of a putative new species closely related to Septorioides pini-thunbergii, a species associated with needle cast of Pinus thunbergii in Japan, which was initially not known to be associated with WPND. This thesis intends to fill this lack of information, first by examining the putative new species of Septorioides through phylogenetic analysis of six gene loci (SSU, LSU, ITS, β-tubulin, EF1, and RPB2) to describe and classify its association with WPND. Secondly, by expanding field based sampling of WPND within Vermont, New Hampshire, Maine, and their neighboring states to construct a more detailed distribution map. Thirdly, by utilizing long-term WPND monitoring plots and data collected from land-based weather stations to create a climatic model to predict the severity of defoliation events in the next year. Lastly, by establishing a field-based spore trapping experiment to determine how climatic factors affect the development and dispersal of L. acicola spores. Results from this thesis demonstrate the widespread occurrence and establishment of a new family (Septorioideaceae), and a new species Septorioides strobus, as well as its association with the current WPND outbreak. Continued sampling provided further support for the disease complex theory that L. acicoa, B. linearis, L. dooksii, and S. strobus were ubiquitous across the region and neither an individual species nor a specific combination of species had a dominating presence in particular states or regions, but were generally found in forest stands that compromised \u3e50% eastern white pine by basal area. Additionally, regional weather data confirmed the trend of increasing temperature and precipitation observed in this region with the previous year’s May, June, and July rainfall being the best predictor of the following years defoliation event, while the current year’s relative humidity was responsible for spore development and increased inoculum loads

Extent and Severity of Caliciopsis Canker in New England, USA: An Emerging Disease of Eastern White Pine (Pinus strobus L.)

Caliciopsis canker is an emerging problem in Pinus growing regions of Eastern North America. The fungal disease caused by Caliciopsis pinea is associated with overstocked stands and poor sites, but few quantitative data are available. The objective of this study, therefore, was to assess the extent and severity of Caliciopsis canker and to explore environmental variables associated with disease to identify areas at risk of damage. During 2014, 58 sites across New England with \u3e75% P. strobus basal area in public lands were surveyed. Most sites (72%) had Caliciopsis canker signs or symptoms. Caliciopsis pinea was successfully identified with molecular techniques. In sites with Caliciopsis canker, 36% of the mature pines were symptomatic. Pole sized and suppressed trees were more likely to be damaged than larger trees with dominant crown positions (p \u3c 0.05). Pinus strobus density for sites with Caliciopsis canker was 311 trees/ha (mean P. strobus stand diameter = 40 cm) compared to 220 trees/ha (mean white pine stand diameter = 43 cm) for sites without Caliciopsis canker (p = 0.1). Caliciopsis canker symptoms tended to appear more frequently in stands with excessively drained, coarse textured soils derived from glacial outwash (86%) or stands with poorly drained soils and low fertility (78%) than in stands with well drained, more fertile soils (59%) (p = 0.1). The severity of symptoms varied among soil groups and was greater for excessively drained, nutrient poor soils than for well-drained, more fertile soils (p = 0.027)

Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems

[EN] Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9-SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type.This research was funded by INIA, grant number: RTA 2017-00063-C04-03, LIFE programme, grant number: LIFE14 ENV/ES/000179 and by the Basque Government, grant number FUNGITRAP 19-00031. Red pine cone collection in New England and pathogen isolation was funded by USDA Forest Service.Aragonés, A.; Manzanos, T.; Stanosz, G.; Munck, IA.; Raposo, R.; Elvira-Recuenco, M.; Berbegal Martinez, M.... (2021). Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems. Microorganisms. 9(12):1-17. https://doi.org/10.3390/microorganisms9122565S11791

Caliciopsis moriondi, a new species for a fungus long confused with the pine pathogen C. pinea

Figure S1. One of the most parsimonious trees from EF1-α sequence datasets. Data type: Alignment of genomic sequences. Explanation note: One of the most parsimonious trees from EF1-α gene sequence datasets is shown (length = 66, CI = 0.9999, RI = 0.9998, RC = 0.9988, HI = 0.9888). The MP and Bayesian posterior probability are indicated next to the branches. C. pseudotsugae and C. orientalis are used as outgroup. This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/).Figure S2. One of the most parsimonious trees from Bt1 sequence datasets. Data type: Alignment of genomic sequences. Explanation note: One of the most parsimonious trees from Bt1 sequence datasets is shown (CI = 0.9268, RI = 0.9840, RC = 0.936428, HI = 0.912039). The MP and Bayesian posterior probability are indicated next to the branches. C. pseudotsugae and C. orientalis are used as outgroup. This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/).The genus Caliciopsis (Eurotiomycetes, Coryneliales) includes saprobic and plant pathogenic species. Caliciopsis canker is caused by Caliciopsis pinea Peck, a species first reported in the 19th century in North America. In recent years, increasing numbers of outbreaks of Caliciopsis canker have been reported on different Pinus spp. in the eastern USA. In Europe, the disease has only occasionally been reported causing cankers, mostly on Pinus radiata in stressed plantations. The aim of this study was to clarify the taxonomy of Caliciopsis specimens collected from infected Pinus spp. in Europe and North America using an integrative approach, combining morphology and phylogenetic analyses of three loci. The pathogenicity of the fungus was also considered. Two distinct groups were evident, based on morphology and multilocus phylogenetic analyses. These represent the known pathogen Caliciopsis pinea that occurs in North America and a morphologically similar, but phylogenetically distinct, species described here as Caliciopsis moriondi sp. nov., found in Europe and at least one location in eastern North America. Caliciopsis moriondi differs from C. pinea in various morphological features including the length of the ascomata, as well as their distribution on the stromata.Horizon 2020 Programme for Research & Innovationhttps://mycokeys.pensoft.netpm2020Forestry and Agricultural Biotechnology Institute (FABI

From Fields to Genomes: Towards a Comprehensive Understanding of the Lifestyle and Evolution of Claviceps purpurea the Ergot Fungus

Claviceps purpurea (ergot), an ascomycete and member of the family Clavicipitacea, is considered a pathogen of all grass species (family Poaecea) including economically important cereal crops which infects ovaries resulting in the development of a fungal sclerotium rather than a plant seed. Ergot infections poses significant impacts to agriculture and livestock due to various toxic alkaloids present in the sclerotia. Severe ergot poisoning in humans and livestock, ergotism, can cause corrosion/loss of extremities from gangrene, internal bleeding, diarrhea, and reduced pregnancy and abortion. Due to these serious health concerns, strict restrictions are placed on the amount of ergot contaminated grain that can be accepted for food and livestock feed. However, these toxic alkaloids are also heavily researched in the field of pharmacology and have been shown to provide some beneficial aspects in human medicine. Despite the abundance of pharmacological and agricultural research on C. purpurea researchers have been unsuccessful in identifying crop or wild grass varieties that have resistance to ergot infection, leading to critical challenges in the control of ergot disease outbreaks. Recent studies have also suggested that C. purpurea is more of a conditional defensive mutualist as opposed to a plant pathogen. Taken together, these factors demonstrate that there are still gaps of knowledge surrounding the epidemiology, lifestyle, evolution, and adaptability of this species. We implemented a comprehensive analysis into the life history of C. purpurea through a combination of field surveys, greenhouse inoculations, and deep genomic data mining to help elucidate these gaps. Field surveys were conducted to investigate the role wild grass populations surrounding cereal crop fields play in epidemiology of ergot outbreaks. Results revealed that unmanaged grasses along ditch banks, even in drought years, represent significant inoculum reservoirs of ergot, particularly when Bromus spp. are present, and should be a focal point in future research for better disease control. Greenhouse inoculations were conducted to elucidate the effects of C. purpurea infections on hosts through inoculations of a single isolate on two commercial cereal crops in a controlled setting. Our results show that the effect of C. purpurea infections can range from negative to positive, depending on infection rate, plant species, and plant tissue, but overall showed a general trend of neutral effects. However, we did observe a potential for increased root growth as infection rates increased, which could signify an interesting plant-microbe interaction that imparts a benefit, of infection, on highly rhizomatous grass hosts such as Bromus spp.. Lastly, through a collaborative effort we sequenced, assembled, and annotated 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture, plasticity, and evolution within the genera. We also conducted a detailed analysis of C. purpurea through construction of a pangenome and investigations of the recombination and positive selection landscape across the genome. Our genus-wide comparison revealed that despite having nearly identical life-strategies, these closely related species have substantially altered genomic architectures and plasticity that are likely driving genome adaptation. One key difference we observed was a shift from characteristic one-speed genomes in narrow host-range Claviceps species of sections Citrinae and Paspalorum to two-speed genomes in broader host-range lineages of sections Pusillae and Claviceps. Claviceps purpurea was observed to have a large accessory genome that is likely influenced by a large effective population size, high recombination rates, and transposable element (TE) mediated gene duplication. Due to a lack of repeat-point induced (RIP) mutation, prolific TE expansion is likely controlled by high recombination rates, which subsequently may be influencing the overall trend of purifying selection observed within the species. However, secondary metabolites genes were found to have the highest rates of positive selection on codons within genes, indicating that these genes are a primary factor affecting the diversification of the species into new ecological niches and to potentially help maintain its global distribution and broad host range

A large accessory genome and high recombination rates may influence global distribution and broad host range of the fungal plant pathogen Claviceps purpurea.

Pangenome analyses are increasingly being utilized to study the evolution of eukaryotic organisms. While pangenomes can provide insight into polymorphic gene content, inferences about the ecological and adaptive potential of such organisms also need to be accompanied by additional supportive genomic analyses. In this study we constructed a pangenome of Claviceps purpurea from 24 genomes and examined the positive selection and recombination landscape of an economically important fungal organism for pharmacology and agricultural research. Together, these analyses revealed that C. purpurea has a relatively large accessory genome (~ 38%), high recombination rates (ρ = 0.044), and transposon mediated gene duplication. However, due to observations of relatively low transposable element (TE) content (8.8%) and a lack of variability in genome sizes, prolific TE expansion may be controlled by frequent recombination. We additionally identified that within the ergoline biosynthetic cluster the lpsA1 and lpsA2 were the result of a recombination event. However, the high recombination rates observed in C. purpurea may be influencing an overall trend of purifying selection across the genome. These results showcase the use of selection and recombination landscapes to identify mechanisms contributing to pangenome structure and primary factors influencing the evolution of an organism

Characterization of Fungal Pathogens Associated with White Pine Needle Damage (WPND) in Northeastern North America

Eastern white pine is a crucial ecological and economic component of forests in the northern USA and eastern Canada, and is now facing an emerging problem in white pine needle damage (WPND). It is still unclear whether WPND results from one, or the combination of several fungal pathogens. Therefore, the first objective of this study was to characterize the fungi associated with WPND in the northeastern United States and document the damage being done to mature eastern white pine as a result of repeated defoliation. To date, 22 species of fungi, either cultured from diseased pine needles or formed fruiting bodies on pine needles were identified based on morphology and sequence data. Lecanosticta acicola and a putative new species of Septorioides were the species most frequently recovered from diseased needles, in addition to needle cast fungi Lophophacidium dooksii and Bifusella linearis, two obligate fungal pathogens that were frequently observed on pine needles in the northeast, but have not been known to cause excessive defoliation of eastern white pine. A second objective was to monitor yearly the health of 63 pairs of healthy and unhealthy trees in eight affected locations throughout New England. Since 2012, affected trees are increasingly and repeatedly chlorotic and defoliated every year. Trees that were initially healthy are now exhibiting symptoms. While L. acicola appears to be the primary pathogen causing WPND, several other common needle pathogens are being more frequently observed and the role of climate change may be important in the disease ecology of WPND. These defoliation events, while once a sporadic occurrence, have now become more frequent as observed in continued crown deterioration of eastern white pine in long-term monitoring plots followed during the course of this three-year study