Deciphering common and specific transcriptional immune responses in pea towards the oomycete pathogens Aphanomyces euteiches and Phytophthora pisi

Primers used in this study for RT-qPCR analysis. (DOCX 20 kb

Propagation through seed or somatic embryogenesis: comparing the effects of two methods and methyl jasmonate treatment on Norway spruce resistance to Heterobasidion infection

The clonal propagation method somatic embryogenesis (SE) has been shown to affect plant resistance to an insect pest. In a field trial, Norway spruce SE-plants (emblings) experienced less damage by the bark-chewing pine weevil compared to seedlings propagated through seeds from the same mother tree. Moreover, if emblings were treated with the defense-inducing hormone methyl jasmonate (MeJA), their resistance became much greater compared to the effects of SE and MeJA alone. Thus, we evaluated in a full factorial design if propagation method (seed or SE) and MeJA treatment (treated or not) can affect Norway spruce resistance to infection by the fungal pathogen Heterobasidion parviporum. Emblings and seedlings were half-sibs originating from four different Norway spruce families. We found that emblings and seedlings exhibited similar fungal lesion lengths (LL) and sapwood growth (SWG) in a constitutive state (no MeJA). Once treated, seedlings exhibited a 26% and 28% reduction in LL and SWG growth respectively, in line with previous studies. For emblings, it was the opposite. MeJA increased LL and SWG by 41% and 16% respectively. This is unexpected given the genetic relatedness of the material, and the previously documented effects of SE. It is possible that SE is causing changes in Norway spruce resistance that are effective against insects, but not pathogens. Also, MeJA treatment may be affecting embling terpene composition in ways that benefit fungal growth, as suggested from separate experiments. Further studies are needed to uncover the mechanisms behind the increased susceptibility of Norway spruce emblings to fungal infection

Fungal Pathogens Associated with Tomicus Species in European Forests: Regional Variations and Impacts on Forest Health

Pinus species are extensively abundant in Europe and, as pioneer trees, prominently influence local ecology. However, pine forests in Lithuania, Montenegro, and Ukraine have been significantly damaged by pine bark beetles (Tomicus sp.), which are closely associated with ophiostomatoid and other pathogenic fungi. This study aimed to identify the diversity of ophiostomatoid and other fungi associated with Tomicus sp. in these three countries. Fungi were isolated from beetles and identified. High-throughput sequencing of ITS2 rDNA yielded 285,828 reads, of which 91,141 high-quality reads were retained, representing 561 fungal operational taxonomic units (OTUs). The most important groups of fungi included ophiostomatoids, yeasts, and plant pathogens. While the fungal communities associated with Tomicus spp. were influenced more by environmental factors than by beetle species, the presence of known pathogens such as Ophiostoma spp. indicates that Tomicus spp. could play a significant role in dispersing harmful fungi. Although the virulence of these fungi may vary, their association with potentially pathogenic species suggests that Tomicus spp. may contribute to forest health decline, especially if environmental conditions or host susceptibility change

Development of a molecular genetics and cell biology toolbox for the filamentous fungus Diplodia sapinea

Diplodia sapinea (Fr.) Fuckel is a widespread fungal pathogen affecting conifers worldwide. Infections can lead to severe symptoms, such as shoot blight, canker, tree death, or blue stain in harvested wood, especially in Pinus species. Its impact on forest health is currently intensified, likely due to climate change, posing an increasing threat to global ecosystems and forestry. Despite extensive and successful research on this pathogen system, fundamental questions about its biology and plant-associated lifestyle remain unanswered. Addressing these questions will necessitate the development of additional experimental tools, including protocols for molecular genetics and cell biology approaches. In this study, we continue to address this need by establishing an Agrobacterium-mediated genetic transformation protocol for D. sapinea, enabling targeted mutagenesis and heterologous gene expression. We utilized this methodology to localize the histone H2B by tagging it with the fluorescent protein mCherry. Additionally, we established a time- and space-efficient laboratory-scale infection assay using two-week-old Pinus sylvestris seedlings. Integrating these tools in a proof-of-concept study enabled the visualization of D. sapinea in planta growth through the fluorescently labeled reporter strain

Diplodia sapinea as a contributing factor in the crown dieback of Scots pine (Pinus sylvestris) after a severe drought

The frequency and impact of drought on global ecosystems have increased within the last century, while drought has affected tree health in many regions. Diplodia sapinea is a widespread, opportunistic pathogen infecting most conifers, causing Diplodia tip blight, thriving on hosts impacted by stress such as drought, heat, or mechanical wounding. In summer of 2018, a large-scale drought was recorded all over Europe. In late summer, pine trees all over Gotland showed crown dieback, where necrotic twigs and needles were found, especially in the upper part of the crowns. Symptoms were consistent with a potential outbreak of D. sapinea. Effects of the combination of drought and Diplodia tip blight on mortality or recovery of Scots pine in Nordic conditions are unknown. This study confirmed the presence and potential contribution of D. sapinea in the observed damages of Scots pine. Shoot blight and drought led to crown defoliation which was observed one year post-drought, while trees showed a clear recovery of newly grown shoots within the second year. Severely affected pines (>70% of the upper third of the crown with shoot blight) showed increased mortality. Recovery of the surviving trees was independent of previous dieback levels. Diplodia sapinea was most abundant in twigs with shoot blight of the symptomatic trees compared to healthy-looking twigs from the same trees and asymptomatic trees in affected and healthy pine stands. Sampling on affected and healthy sites showed possible endophytic infections with low abundance within healthy-looking twigs. Spore deposition of D. sapinea was monitored on healthy and affected sites for two consecutive years after crown damages occurred to confirm the presence of the opportunistic pathogen in the affected region. Spore deposition was observed during all seasons and correlated with high precipitation during sampling. Our observations provide insights into the emergence of Diplodia tip blight in the Northern countries and underline the potential impact of D. sapinea on tree health in the course of a changing climate

Forest Damage

Heterobasidion root and butt rot pose a greater risk in continuous cover forestry (CCF) than in rotation forestry (RF) in conifer-dominated forests, regardless of whether selective, gap or shelterwood cutting is used.Damage from wind, snow, spruce bark beetle, and large pine weevil are likely to be less severe in CCF than in RF. However, the conversion of RF to CCF may briefly expose stands to windthrow.Browsing by large herbivores on saplings may limit regeneration of tree species other than spruce in continuous cover forestry and reduce tree species diversity, but alternative silvicultural practices may also increase forage availability in the field and shrub layer. Browsing damage outcomes for saplings in CCF are difficult to predict.For many types of damage in CCF, substantial knowledge gaps complicate the assessment of damage risk

Transcriptomic analysis identifies candidate genes for Aphanomyces root rot disease resistance in pea

BackgroundAphanomyces euteiches is a soil-borne oomycete that causes root rot in pea and other legume species. Symptoms of Aphanomyces root rot (ARR) include root discoloration and wilting, leading to significant yield losses in pea production. Resistance to ARR is known to be polygenic but the roles of single genes in the pea immune response are still poorly understood. This study uses transcriptomics to elucidate the immune response of two pea genotypes varying in their levels of resistance to A. euteiches.ResultsIn this study, we inoculated roots of the pea (P. sativum L.) genotypes 'Linnea' (susceptible) and 'PI180693' (resistant) with two different A. euteiches strains varying in levels of virulence. The roots were harvested at 6 h post-inoculation (hpi), 20 hpi and 48 hpi, followed by differential gene expression analysis. Our results showed a time- and genotype-dependent immune response towards A. euteiches infection, involving several WRKY and MYB-like transcription factors, along with genes associated with jasmonic acid (JA) and abscisic acid (ABA) signaling. By cross-referencing with genes segregating with partial resistance to ARR, we identified 39 candidate disease resistance genes at the later stage of infection. Among the genes solely upregulated in the resistant genotype 'PI180693', Psat7g091800.1 was polymorphic between the pea genotypes and encoded a Leucine-rich repeat receptor-like kinase reminiscent of the Arabidopsis thaliana FLAGELLIN-SENSITIVE 2 receptor.ConclusionsThis study provides new insights into the gene expression dynamics controlling the immune response of resistant and susceptible pea genotypes to A. euteiches infection. We present a set of 39 candidate disease resistance genes for ARR in pea, including the putative immune receptor Psat7g091800.1, for future functional validation

Fungal Communities Vectored by Ips sexdentatus in Declining Pinus sylvestris in Ukraine: Focus on Occurrence and Pathogenicity of Ophiostomatoid Species

Drought-induced stress and attacks by bark beetle Ips sexdentatus currently result in a massive dieback of Pinus sylvestris in eastern Ukraine. Limited and fragmented knowledge is available on fungi vectored by the beetle and their roles in tree dieback. The aim was to investigate the fungal community vectored by I. sexdentatus and to test the pathogenicity of potentially aggressive species to P. sylvestris. Analysis of the fungal community was accomplished by combining different methods using insect, plant, and fungal material. The material consisted of 576 beetles and 96 infested wood samples collected from six sample plots within a 300 km radius in eastern Ukraine and subjected to fungal isolations and (beetles only) direct sequencing of ITS rDNA. Pathogenicity tests were undertaken by artificially inoculating three-to-four-year-old pine saplings with fungi. For the vector test, pine logs were exposed to pre-inoculated beetles. In all, 56 fungal taxa were detected, 8 exclusively by isolation, and 13 exclusively by direct sequencing. Those included nine ophiostomatoids, five of which are newly reported as I. sexdentatus associates. Two ophiostomatoid fungi, which exhibited the highest pathogenicity, causing 100% dieback and mortality, represented genera Graphium and Leptographium. Exposure of logs to beetles resulted in ophiostomatoid infections. In conclusion, the study revealed numerous I. sexdentatus-vectored fungi, several of which include aggressive tree pathogens

Scots Pines With Tolerance to Melampsora pinitorqua and Diplodia sapinea Show Distinct Metabolic Profiles

Diplodia sapinea causes Diplodia tip blight (DTB) and is recognised as an opportunistic necrotrophic pathogen affecting conifers. While DTB is associated with abiotic stress, the impact of biotic stress in the host on D. sapinea's lifestyle shift is unknown. Observed co-occurrences of D. sapinea and Melampsora pinitorqua, causing pine twisting rust on Scots pine (Pinus sylvestris), instigated an investigation into their interaction with and influence on the defence mechanisms of the host. We hypothesised that M. pinitorqua infections predispose the trees to D. sapinea by stressing the host and altering the shoot metabolites. Pines in a plantation were sampled over time to study pathogen biomass and host metabolites. Symptoms of both pathogens were consistent over years, and the preceding season's symptoms affected the metabolic profiles pre-infection and M. pinitorqua's proliferation. Symptoms of M. pinitorqua altered shoot metabolites more than fungal biomass, with co-symptomatic trees exhibiting elevated M. pinitorqua biomass. Specific phenolic compounds had a strong positive association with the shoot symptom x D. sapinea interaction. D. sapinea's biomass presymptoms was independent of previous disease symptoms and infection by M. pinitorqua. Some trees showed disease tolerance, with delayed rust infections and minimal DTB symptoms. Further investigations on this trait are needed

Evaluation of pea genotype PI180693 partial resistance towards aphanomyces root rot in commercial pea breeding

The cultivation of vining pea (Pisum sativum) faces a major constraint with root rot diseases, caused by a complex of soil-borne pathogens including the oomycetes Aphanomyces euteiches and Phytophtora pisi. Disease resistant commercial varieties are lacking but the landrace PI180693 is used as a source of partial resistance in ongoing pea breeding programs. In this study, the level of resistance and their interaction with A. euteiches virulence levels of six new back-crossed pea breeding lines, deriving from the cross between the susceptible commercial cultivar Linnea and PI180693, were evaluated for their resistance towards aphanomyces root rot in growth chamber and green house tests. Resistance towards mixed infections by A. euteiches and P. pisi and commercial production traits were evaluated in field trials. In growth chamber trials, pathogen virulence levels had a significant effect on plant resistance, as resistance was more consistent against A. euteiches strains exhibiting high or intermediate virulence compared with lowly virulent strains. In fact, line Z1701-1 showed to be significantly more resistant than both parents when inoculated with a lowly virulent strain. In two separate field trials in 2020, all six breeding lines performed equally well as the resistant parent PI180693 at sites only containing A. euteiches, as there were no differences in disease index. In mixed infections, PI180693 exhibited significantly lower disease index scores than Linnea. However, breeding lines displayed higher disease index scores compared with PI180693, indicating higher susceptibility towards P. pisi. Data on seedling emergence from the same field trials suggested that PI180693 was particularly sensitive towards seed decay/damping off disease caused by P. pisi. Furthermore, the breeding lines performed equally well as Linnea in traits important for green pea production, again emphasizing the commercial potential. In summary, we show that the resistance from PI180693 interacts with virulence levels of the pathogen A. euteiches and is less effective towards root rot caused by P. pisi. Our results show the potential use of combining PI180693 partial resistance against aphanomyces root rot with commercially favorable breeding traits in commercial breeding programs