Using direct amplification and next-generation sequencing technology to explore foliar endophyte communities in experimentally inoculated western white pines

Fungal endophytes can influence survivability and disease severity of trees. Here we characterized the endophyte community in Pinus monticola (western white pine), an important species in the northwest USA, largely decimated by pathogenic fungi. We also assessed the ability to successfully inoculate seedlings with desirable endophytes, with the long-term goal of providing a protective microbiome and added defense from pathogens. P. monticola seedlings were inoculated in the field with potential pathogen antagonists and fungi isolated from healthy mature trees. Following inoculations direct amplification and next generation sequencing were used to characterize fungal endophyte communities, and explore interspecific competition, diversity, and co-occurrence patterns in needle tissues. Negative co-occurrence patterns between inoculated fungi and potential pathogens, as well as many other species, suggest early competitive interactions. Our study explores early endophyte community assemblage and shows that fungal inoculations may influence tree growth.     

Fungal endophytes reduce necrosis length produced by Gremmeniella abietina in Pinus halepensis seedlings

Gremmeniella abietina (Lagerberg) Morelet is a pathogenic fungus that causes severe damage in coniferous forests, causing the death of the trees, in Central and Northern Europe, North America and Japan. Biological control (i.e. the use of biologically antagonistic organisms) is being considered as an alternative and an eco-friendly method to deal with plant diseases. Among such organisms several fungal endophytes have been successfully used to reduce or inhibit the growth of pathogens. Thus, the aim of this study was to evaluate the ability of several fungal endophytes to reduce the G. abietina spreading on pine seedlings, with the goal of exploring the mechanisms involved in that reduction. The experiment was carried out on two-year old Pinus halepensis seedlings under greenhouse conditions. Five fungal endophytes (Trichoderma spp., Aureobasidium pullulans, Aureobasidium spp., endophyte 20.1 and Leotiomycete spp.) obtained from healthy P. halepensis trees were used to evaluate their effect on six G. abietina isolates. The pathogen and the endophyte were both inoculated in every seedling. At the end of the experiment seedlings were cut and brought to the laboratory where the necrosis length and total phenol content of the plant were measured. The pathogen presence was determined by a nested PCR with specific primers of G. abietina. The presence of all endophytes significantly reduced the necrosis length caused by G. abietina in most of the cases. However, the phenolic content of the plant, which is an indicator of the activation of the plant’s defence mechanisms, had not increased with the endophyte inoculation. Therefore, the reduction in the necrosis observed could be attributed to a direct effect of the endophyte on the Gremmeniella spreading. These results suggest that the use of fungal endophytes could be an effective way to protect against G. abietina infections.     

Novel forest decline triggered by multiple interactions among climate,an introduced pathogen and bark beetles

Novel forest decline is increasing due to global environmental change, yet the causal factors and their interactions remain poorly understood. Using tree ring analyses, we show how climate and multiple biotic factors caused the decline of whitebark pine (Pinus albicaulis) in 16 stands in the southern Canadian Rockies. In our study area, 72% of whitebark pines were dead and 18% had partially dead crowns. Tree mortality peaked in the 1970s; however, the annual basal area increment of disturbed trees began to decline significantly in the late 1940s. Growth decline persisted up to 30 years before trees died from mountain pine beetle (Dendroctonus ponderosae), Ips spp. bark beetles or non‐native blister rust pathogen (Cronartium ribicola). Climate–growth relations varied over time and differed among the healthy and disturbed subpopulations of whitebark pine. Prior to the 1940s, cool temperatures limited the growth of all subpopulations. Growth of live, healthy trees became limited by drought during the cool phase (1947 –1976) of the Pacific Decadal Oscillation (PDO) and then reverted to positive correlations with temperature during the subsequent warm PDO phase. In the 1940s, the climate–growth relations of the disturbed subpopulations diverged from the live, healthy trees with trees ultimately killed by mountain pine beetle diverging the most. We propose that multiple factors interacted over several decades to cause unprecedented rates of whitebark pine mortality. Climatic variation during the cool PDO phase caused drought stress that may have predisposed trees to blister rust. Subsequent decline in snowpack and warming temperatures likely incited further climatic stress and with blister rust reduced tree resistance to bark beetles. Ultimately, bark beetles and blister rust contributed to tree death. Our findings suggest the complexity of whitebark pine decline and the importance of considering multiway drought–disease–insect interactions over various timescales when interpreting forest decline.     

Detection of foliar endophytes and their metabolites in Picea and Pinus seedling needles

The needles of Picea glauca (white spruce) and Pinus strobus (white pine) trees infected with toxigenic fungal endophytes contain varying concentrations of their secondary metabolites that are toxic to either insect pests or needle pathogens. In the present study, liquid chromatography-mass spectrometric methods to determine needle concentrations of metabolites of four endophyte species were developed. The endophytes considered were a Phialocephala sp. (vermiculine) and Phialocephala scopiformis (rugulosin) from white spruce, as well as a Xylaria sp. (griseofulvin) and Lophodermium nitens (pyrenophorol) from white pine needles. To ensure that needles were infected with the associated fungal endophyte, suitable qPCR-based methods were also developed. There was a high degree of concordance between the qPCR analysis of the fungal mycelium and the LC-MS/MS quantification of the associated metabolites. Concentrations of the antifungal compounds griseofulvin and pyrenophorol were present in amounts that affect conifer needle diseases including white pine blister rust caused by Cronartium ribicola. Similarly, concentrations of the antiinsectan compounds vermiculine and rugulosin were in the range known to reduce the growth of Choristoneura fumiferana and mitigate foliage damage.     

Influences of the biophysical environment on blister rust and mountain pine beetle,and their interactions,in whitebark pine forests

Aim To understand how the biophysical environment influences patterns of infection by non‐native blister rust (caused by Cronartium ribicola) and mortality caused by native mountain pine beetles (Dendroctonus ponderosae) in whitebark pine (Pinus albicaulis) communities, to determine how these disturbances interact, and to gain insight into how climate change may influence these patterns in the future. Location High‐elevation forests in south‐west Montana, central Idaho, eastern and western Oregon, USA. Methods Stand inventory and dendroecological methods were used to assess stand structure and composition and to reconstruct forest history at sixty 0.1‐ha plots. Patterns of blister rust infection and mountain pine beetle‐caused mortality in whitebark pine trees were examined using nonparametric Kruskal–Wallis ANOVA, Mann–Whitney U‐tests, and Kolmogorov–Smirnov two‐sample tests. Stepwise regression was used to build models of blister rust infection and mountain pine beetle‐related mortality rates based on a suite of biophysical site variables. Results Occurrence of blister rust infections was significantly different among the mountain ranges, with a general gradient of decreasing blister rust occurrence from east to west. Evidence of mountain pine beetle‐caused mortality was identified on 83% of all dead whitebark pine trees and was relatively homogenous across the study area. Blister rust infected trees of all ages and sizes uniformly, while mountain pine beetles infested older, larger trees at all sites. Stepwise regressions explained 64% and 58% of the variance in blister rust infection and beetle‐caused mortality, respectively, indicating that these processes are strongly influenced by the biophysical environment. More open stand structures produced by beetle outbreaks may increase the exposure of surviving whitebark pine trees to blister rust infection. Main conclusions Variability in the patterns of blister rust infection and mountain pine beetle‐caused mortality elucidated the fundamental dynamics of these disturbance agents and suggests that the effects of climate change will be complex in whitebark pine communities and vary across the species’ range. Interactions between blister rust and beetle outbreaks may accelerate declines or facilitate the rise of rust resistance in whitebark pine depending on forest conditions at the time of the outbreak.     

Foliar nutrients shape fungal endophyte communities in Western white pine (Pinus monticola) with implications for white-tailed deer herbivory

Asymptomatic fungal endophytes colonize tissues of woody plants worldwide, with largely unknown ecological effects. Using culture-based methods and ITS1-5.8S-ITS2 rDNA sequence analysis, we investigated differences between foliar endophyte communities in disease-resistant hybrid and wild-type Pinus monticola (Western white pine) trees with observed variation in tree growth, vigor, and browsing damage by white-tailed deer (Odocoileus virginianus). We isolated 69 phylotypes of endophytic fungi in at least 39 genera, including 26 that have not previously been reported in P. monticola. Principal components analysis revealed that endophyte communities differed between browsed seedlings, unbrowsed seedlings, and unbrowsed adult trees. Sulfur, nitrate and calcium concentrations correlated with endophyte community differences among tree groups based on a distance-based redundancy analysis. Our results indicate that foliar nutrient variation influences endophyte community assembly and deer herbivory in P. monticola on a small landscape scale (80 hectares).     

庞泉沟自然保护区常见禾本科植物内生真菌分布及其影响因素

以庞泉沟自然保护区4种不同群落类型林地和灌丛下的优势禾草为研究对象,调查不同禾草内生真菌感染率,研究内生真菌感染的禾草种群与原生生境的土壤理化性质、土壤酶活性及土壤微生物群落结构的相互关系。结果发现,影响紫羊茅、中亚薹草和野燕麦染菌率的主要因子是土壤水分含量和碳氮比,且土壤C、N、S以及土壤水分含量与紫羊茅的内生真菌染菌率成正相关,土壤蔗糖酶、脲酶和酸性磷酸酶与紫羊茅的感染率显著正相关,但中亚薹草与紫羊茅的结果相反。丝盖伞科是华北落叶松林和油松林土壤微生物群落的优势真菌,但不同染菌植被群落类型下的优势细菌微生物群落组成不同。总体看来,4种森林群落类型下禾草内生真菌染菌率与土壤理化性质相关性不同,且受到的生态因子影响大小也有差异,内生真菌感染导致各植被群落类型的土壤微生物群落结构具有差异,其优势土壤真菌和土壤细菌组成不同,这有助于进一步认识原生生境下不同感染率的禾草种群对土壤微生物群落结构的影响及其在生态系统中的各异生态学功能。     

Location,but not defensive genotype,determines ectomycorrhizal community composition in Scots pine (Pinus sylvestris L.) seedlings

1. For successful colonization of host roots, ectomycorrhizal (EM) fungi must overcome host defense systems, and defensive phenotypes have previously been shown to affect the community composition of EM fungi associated with hosts. Secondary metabolites, such as terpenes, form a core part of these defense systems, but it is not yet understood whether variation in these constitutive defenses can result in variation in the colonization of hosts by specific fungal species.
2. We planted seedlings from twelve maternal families of Scots pine (Pinus sylvestris) of known terpene genotype reciprocally in the field in each of six sites. After 3 months, we characterized the mycorrhizal fungal community of each seedling using a combination of morphological categorization and molecular barcoding, and assessed the terpene chemodiversity for a subset of the seedlings. We examined whether parental genotype or terpene chemodiversity affected the diversity or composition of a seedling''s mycorrhizal community.
3. While we found that terpene chemodiversity was highly heritable, we found no evidence that parental defensive genotype or a seedling''s terpene chemodiversity affected associations with EM fungi. Instead, we found that the location of seedlings, both within and among sites, was the only determinant of the diversity and makeup of EM communities.
4. These results show that while EM community composition varies within Scotland at both large and small scales, variation in constitutive defensive compounds does not determine the EM communities of closely cohabiting pine seedlings. Patchy distributions of EM fungi at small scales may render any genetic variation in associations with different species unrealizable in field conditions. The case for selection on traits mediating associations with specific fungal species may thus be overstated, at least in seedlings.

     

Colonization History,Host Distribution,Anthropogenic Influence and Landscape Features Shape Populations of White Pine Blister Rust,an Invasive Alien Tree Pathogen

White pine blister rust is caused by the fungal pathogen Cronartium ribicola J.C. Fisch (Basidiomycota, Pucciniales). This invasive alien pathogen was introduced into North America at the beginning of the 20th century on pine seedlings imported from Europe and has caused serious economic and ecological impacts. In this study, we applied a population and landscape genetics approach to understand the patterns of introduction and colonization as well as population structure and migration of C. ribicola. We characterized 1,292 samples of C. ribicola from 66 geographic locations in North America using single nucleotide polymorphisms (SNPs) and evaluated the effect of landscape features, host distribution, and colonization history on the structure of these pathogen populations. We identified eastern and western genetic populations in North America that are strongly differentiated. Genetic diversity is two to five times higher in eastern populations than in western ones, which can be explained by the repeated accidental introductions of the pathogen into northeastern North America compared with a single documented introduction into western North America. These distinct genetic populations are maintained by a barrier to gene flow that corresponds to a region where host connectivity is interrupted. Furthermore, additional cryptic spatial differentiation was identified in western populations. This differentiation corresponds to landscape features, such as mountain ranges, and also to host connectivity. We also detected genetic differentiation between the pathogen populations in natural stands and plantations, an indication that anthropogenic movement of this pathogen still takes place. These results highlight the importance of monitoring this invasive alien tree pathogen to prevent admixture of eastern and western populations where different pathogen races occur.     

Endophyte inoculation enhances Ulmus minor resistance to Dutch elm disease

Certain fungal endophytes improve plant resistance to biotic stresses in forest trees. In this study, three stem fungal endophytes belonging to classes Cystobasidiomycetes, Eurotiomycetes and Dothideomycetes were selected from 210 isolates for their potential as enhancers of Ulmus minor resistance to Ophiostoma novo-ulmi. We evaluated phenotypic traits of these endophytes that could be beneficial for inhibiting O. novo-ulmi in the host plant. Under in vitro conditions, the Dothideomycetous isolate YCB36 strongly inhibited O. novo-ulmi growth, released antipathogenic VOCs, chitinases and siderophores, and overlapped with the pathogen in nutrient utilization patterns. These functional traits could explain the 40% reduction in leaf wilting due to O. novo-ulmi in elm trees pre-inoculated with this endophyte. Ulmus minor trees inoculated with this endophyte showed increased leaf stomatal conductance and higher concentrations of flavonoids and total phenolic compounds in xylem tissues, suggesting induction of defence metabolism.