Search Results (38)

Pitch canker caused by the fungus Fusarium circinatum is a destructive disease that affects pines in Europe, South Africa, and North America, particularly along the southeastern and western coasts of the United States. This study systematically elucidated the function of the Leucine-rich repeat (LRR) protein FcLRR1 in the pine pitch canker pathogen Fusarium circinatum. A total of 13 LRR proteins were identified via bioinformatic analysis. Using a gene knockout system, we demonstrated that deletion of FcLRR1 significantly impaired vegetative growth, conidiation, and conidium germination; led to a complete loss of macroconidia production; and drastically reduced abiotic stress tolerance and virulence. Transcriptome profiling revealed 612 downregulated genes, which were significantly enriched in pathways such as starch and sucrose metabolism, indicating that FcLRR1 modulated energy supply and pathogenicity through carbon source utilization. Through genome-wide protein structure modeling and yeast two-hybrid assays, we identified and validated the interaction between FcLRR1 and ALG-11, among other candidate proteins, further supporting its involvement in carbon metabolism, cell wall integrity, and pathogenesis. This study represents the first functional characterization of an LRR-containing protein in a forest pathogenic fungus and provides a foundational basis for developing targeted disease control strategies. Full article

Fusarium circinatum Nirenberg & O’Donnell (the causal agent of Pine Pitch Canker, PPC), one of the most devastating threats to pine forests and nurseries worldwide, induces canker disease on a wide range of pine species. However, its status as a quarantined pathogen and the scarcity of reliable genetic manipulation tools have long impeded in-depth genomic research on this fungus, and the infection mechanisms of F. circinatum remain an urgent area for investigation. A key approach to expounding its pathogenicity is to perform gene editing on candidate genes, which requires an efficient transformation system. Protoplast-mediated transformation is a critical means for investigating plant-pathogen interactions. During the course of this study, we constructed a PEG/CaCl₂-mediated protoplast transformation method for F. circinatum. Following systematic optimization of transformation conditions, strain A015-1 was selected as the model organism. The optimal enzymolysis system consisted of 5 mg/mL Lysing enzymes, 12.5 mg/mL Driselase, and 7.5 mg/mL Snailase, with incubation at 30 °C for 3 h under shaking at 80 rpm. All positive transformants exhibited strong green fluorescent signals. A total of 31 transformants were obtained after hygromycin B (HPH) selection, and PCR verification confirmed successful amplification of the gfp and hph gene fragments in 30 transformants, corresponding to a positive rate of 96%. Transformation efficiency was calculated as the number of PCR-positive transformants per microgram of plasmid DNA, yielding an efficiency of 1 transformant/μg plasmid DNA under optimal conditions. Furthermore, no significant differences were observed in vegetative growth, development, or pathogenicity between the transformants and the wild-type (WT) strain. In addition, Green fluorescent protein (GFP) was efficiently transformed into F. circinatum protoplasts and functionally expressed. Collectively, this study successfully established a stable transformation system for F. circinatum, providing a foundational platform for analyzing virulence-related functional genes involved in host infection and deciphering the molecular mechanisms underlying the pathogen’s colonization of pine hosts. Full article

Mycoviruses are viruses that infect fungi, including plant pathogens. The infection of these mycoviruses is sometimes associated with impaired phenotypes of their fungal hosts, a phenomenon known as hypovirulence. Thus, using mycoviruses as biological control agents has emerged as a promising tool to combat forest diseases. The invasive ascomycete fungus Fusarium circinatum, which causes pine pitch canker (PPC) disease in Pinus tree species and other coniferous trees, is infected by the mycovirus Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1, and FcMV2-2. However, its impact on pathogen fitness remains unclear. The most accurate method used to identify the effect of a mycovirus on its host is the generation of isogenic lines with and without the mycovirus. The present study aimed to cure F. circinatum isolates infected by FcMV1 using different approaches. For this purpose, three replicates of each isolate were exposed to thermal treatment (38 °C) and antibiotic treatment (ribavirin, cycloheximide, kanamycin, and rifampicin mixed with cAMP)(cyclic adenosine monophosphate) for five successive passages. The viral titer of FcMV1 was then assessed using qPCR (quantitative polymerase chain reaction) after the first week and after the fifth week of the treatment. The results revealed differences in treatment efficacy among F. circinatum isolates, with some showing very low virus titers at the end of the experiment. Both thermal and antibiotic treatment effectively reduced the viral load in all isolates. In addition, the antibiotic cycloheximide and rifampicin +cAMP reduced the viral titer more than ribavirin and kanamycin. The isolate Fc179 was found to be more prone to antibiotic treatment than the other two isolates (001 and Va221). This study demonstrated the possibility of using some isolates of F. circinatum for fine-tuning cures for mitovirus, in order to create virus-free strains for biological control in the future. Full article

Fusarium circinatum, the causative agent of pine pitch canker disease, is a pathogenic fungus that poses a significant threat to pine forests globally. It infects various Pinus species, causing resinous cankers, needle discoloration, and tree death. The disease severely impacts forest ecosystems, necessitating cost-effective and environmentally friendly management strategies. Contaminated pine seeds and seedlings are the main pathways for introducing this fungus to disease-free areas. To mitigate this disease and prevent its spread, it is crucial to implement new processes in forest plant production systems that align with the existing conditions of forest nurseries, ensuring effective and sustainable management. With this in mind, a national collaborative study involving 14 Portuguese partners was initiated to develop new prevention and mitigation strategies. In this work, four different treatments—MennoFlorades, Captan, ethanol, and hot water—were tested for their ability to eliminate F. circinatum from contaminated Pinus seeds in vitro. The most effective treatments were selected for further in vitro assays and real-context nursery germination trials to assess their impacts on seed germination, plant production, and certification. MennoFlorades, Captan, and hot water were tested in the nursery, with hot water showing the most promising results due to its negligible impact on seedlings, eco-friendly nature, ease of implementation, and cost-effectiveness. These findings offer promising prospects for preventing pine pitch canker outbreaks in nurseries and, consequently, in forests. Full article

In ascomycetous fungi, sexual mate recognition requires interaction of the Ste2 receptor protein produced by one partner with the α-factor peptide pheromone produced by the other partner. In some fungi, Ste2 is further needed for chemotropism towards plant roots to allow for subsequent infection and colonization. Here, we investigated whether this is also true for the pine pitch canker fungus, Fusarium circinatum, which is a devastating pathogen of pine globally. Ste2 knockout mutants were generated for two opposite mating-type isolates, after which all strains were subjected to chemotropism assays involving exudates from pine seedling roots and synthetic α-factor pheromone, as well as a range of other compounds for comparison. Our data show that Ste2 is not required for chemotropism towards any of these other compounds, but, in both wild-type strains, Ste2 deletion resulted in the loss of chemotropism towards pine root exudate. Also, irrespective of mating type, both wild-type strains displayed positive chemotropism towards α-factor pheromone, which was substantially reduced in the deletion mutants and not the complementation mutants. Taken together, these findings suggest that Ste2 likely has a key role during the infection of pine roots in production nurseries. Our study also provides a strong foundation for exploring the role of self-produced and mate-produced α-factor pheromone in the growth and overall biology of the pitch canker pathogen. Full article

Radiata pine is one of the most commonly planted tree species in Chile due to its fast growth and desirable wood and pulp properties. However, its productivity is hampered by several diseases. Pitch canker disease (PCC) caused by Fusarium circinatum, is considered the most damaging disease to the pine forest industry. Several control measures have been established, with biological control emerging as an environmentally friendly and effective way for F. circinatum control. Previous studies support the value of Clonostachys rosea in reducing PCC damage, with evidence suggesting a potential induced systemic resistance (ISR) triggered in radiata pines by this agent. Ten-month-old radiata pine plants were pre-treated with C. rosea on a substrate at 8 and 1 days before inoculation with F. circinatum on the stem tip, and expression levels were determined for DXS1, LOX, PAL, and PR3 genes 24 h later. Lesion length was 45% lower on plants pre-treated with C. rosea and infected with F. circinatum compared to non-pre-treated and infected plants. Additionally, LOX and PR3 were induced 23 and 62 times more, respectively, in comparison to untreated plants. Our results indicate that C. rosea causes an ISR response in pre-treated plants, significantly increasing the expression of resistance genes and reducing lesion length. Full article

Pine pitch canker, caused by the ascomycete Fusarium circinatum, poses a substantial threat to pine trees and Douglas firs (Pseudotsuga menziesii), and has been identified as a pervasive issue in forests and nurseries worldwide, particularly in regions where susceptible conifers are cultivated. Given its prevalence in the Iberian Peninsula, assessments of the susceptibility of diverse European provenances of Scots pine (Pinus sylvestris)—specifically those from Poland, Lithuania, and Ukraine—have been conducted. Preliminary evaluations of Polish provenances have raised concerns about the potential threat to Scots pine stands in Poland posed by pitch canker. Under controlled conditions, we examined the impact of F. circinatum inoculation on the survival of seeds and seedlings from ten provenances of Scots pine. In response, the initial assessment of F. circinatum pathogenicity was undertaken in a controlled greenhouse environment. This evaluation uncovered a heightened susceptibility of pine seedlings to pitch canker among the tested provenances. Notably, one Lithuanian provenance demonstrated superior resistance to pitch canker, while two Polish provenances exhibited a higher prevalence of symptomless seedlings. These findings underscore the need for further exploration and identification of resilient individuals within these provenances, offering valuable insights for developing strategies to mitigate the impact of pitch canker on Scots pine in Europe. Full article

The pine pitch canker pathogen, Fusarium circinatum, is globally regarded as one of the most important threats to commercial pine-based forestry. Although genome sequences of this fungus are available, these remain highly fragmented or structurally ill-defined. Our overall goal was to provide high-quality assemblies for two notable strains of F. circinatum, and to characterize these in terms of coding content, repetitiveness and the position of telomeres and centromeres. For this purpose, we used Oxford Nanopore Technologies MinION long-read sequences, as well as Illumina short sequence reads. By leveraging the genomic synteny inherent to F. circinatum and its close relatives, these sequence reads were assembled to chromosome level, where contiguous sequences mostly spanned from telomere to telomere. Comparative analyses unveiled remarkable variability in the twelfth and smallest chromosome, which is known to be dispensable. It presented a striking length polymorphism, with one strain lacking substantial portions from the chromosome’s distal and proximal regions. These regions, characterized by a lower gene density, G+C content and an increased prevalence of repetitive elements, contrast starkly with the syntenic segments of the chromosome, as well as with the core chromosomes. We propose that these unusual regions might have arisen or expanded due to the presence of transposable elements. A comparison of the overall chromosome structure revealed that centromeric elements often underpin intrachromosomal differences between F. circinatum strains, especially at chromosomal breakpoints. This suggests a potential role for centromeres in shaping the chromosomal architecture of F. circinatum and its relatives. The publicly available genome data generated here, together with the detailed metadata provided, represent essential resources for future studies of this important plant pathogen. Full article

Fusarium circinatum causes pine pitch canker (PPC) disease and associated symptoms such as resinous lesions, mechanical weakness, and crown dieback that may lead to mortality in Pinus and Pseudotsuga spp. There are no ameliorative techniques available for the disease, and the genetic resistance among populations to support commercial plantation deployment has not been well characterized. In this study, we characterize the genetic control of PPC disease tolerance (and/or resistance) and predict the tolerance of families in existing breeding populations: open-pollinated (OP) half-sib and control-pollinated full-sib (FS) slash pine (Pinus elliottii var. elliottii Engelm.), OP loblolly pine (Pinus taeda L.), and advanced-generation OP hybrid slash × P. caribaea (Pinus elliottii var. elliottii Engelm. × Pinus caribaea var. bahamensis, caribaea, and hondurensis) using F. circinatum isolates obtained from three locations in Georgia and FL, USA. We describe a new experimental design that improves the accuracy of breeding value predictions, provides more precise genetic parameter estimates, and facilitates comparisons within and among taxa as well as comparisons among isolates. We found strong evidence for genetic control of the ratio of stem damage by F. circinatum, especially in slash pine and slash × P. caribaea hybrids. Loblolly and slash × P. caribaea hybrids exhibited less damage than slash pine. We observed a spectrum of virulence among F. circinatum isolate sources, which were not equally virulent in different pine taxa. Full article

Fusarium circinatum is one of the many threats to forests and Pinus nurseries all over the world, being classified as a quarantine organism by several organizations and governing bodies, such as the European and Mediterranean Plant Protection Organization (EPPO) and the European Union (EU), with associated phytosanitary measures in place to prevent its spread through the various means of dispersal. One such means of dispersal is the water used for irrigation in nurseries, which can contain fungal propagules. Three different treatments, namely, Desogerme, Intra Hydrocare and sodium hypochlorite (NaClO), were tested for their efficacy in eliminating F. circinatum spores in water at several concentrations. Those that showed 100% disinfection rates were selected for further assays regarding seed germination and water quality impact. From these studies, Desogerme 1% and Intra Hydrocare 4% were then selected for large-scale seed germination and plant certification assays in nurseries, where they showed promising results in regard to the prevention of infections in nurseries, and in this way, contribute to the efforts of mitigating this disease. Full article

Fusarium circinatum is an introduced fungal pathogen extended to the northern regions of Spain that causes Pine Pitch Canker (PPC) disease. In this work, we analyzed the pathogen’s genetic diversity to study changes over time and space since the first outbreak occurred in Spain. Using six polymorphic SSR markers, 15 MLGs were identified in 66 isolates, and only three haplotypes were found with frequencies higher than one. In general, genotypic diversity was low and decreased shortly over time in the northwestern regions while maintained at País Vasco, where only one haplotype (MLG32) was detected 10 years. This population also included isolates of a single mating type (MAT-2) and VCGs identified in only two groups, while isolates from NW regions were of both mating types and VCGs represented in 11 groups. The existence of haplotype MLG32 maintained on time and widely distributed suggests its good adaptation to the environment and the host. Results showed that the pathogen in País Vasco remains clearly differentiated from other northwestern populations. This fact was supported with no evidence of migration among regions. Results are explained by the asexual reproduction, but also selfing at least to a lesser extent that leads to identification of two new haplotypes. Full article

This study investigated the complex phenotypic and genetic response of Monterey pine (Pinus radiata) seedlings to co-infections by F. circinatum, the causal agent of pine pitch canker disease, and the oomycetes Phytophthora xcambivora and P. parvispora. Monterey pine seedlings were wound-inoculated with each single pathogen and with the combinations F. circinatum/P. xcambivora and F. circinatum/P. parvispora. Initially, seedlings inoculated only with F. circinatum showed less severe symptoms than seedlings co-inoculated or inoculated only with P. xcambivora or P. parvispora. However, 30 days post-inoculation (dpi), all inoculated seedlings, including those inoculated only with F. circinatum, showed severe symptoms with no significant differences among treatments. The transcriptomic profiles of three genes encoding pathogenesis-related proteins, i.e., chitinase (PR3), thaumatin-like protein (PR5), phenylalanine ammonia-lyase (PAL), and the pyruvate decarboxylase (PDC)-encoding gene were analyzed at various time intervals after inoculation. In seedlings inoculated with single pathogens, F. circinatum stimulated the up-regulation of all genes, while between the two oomycetes, only P. xcambivora induced significant up-regulations. In seedlings co-inoculated with F. circinatum and P.xcambivora or P. parvispora none of the genes showed a significant over-expression 4 dpi. In contrast, at 11 dpi, significant up-regulation was observed for PR5 in the combination F. circinatum/P.xcambivora and PDC in the combination F. circinatum/P. parvispora, thus suggesting a possible synergism of multiple infections in triggering this plant defense mechanism. Full article

Fusarium circinatum causes one of the most important diseases of conifers worldwide, the pine pitch canker (PPC). However, no effective field intervention measures aiming to control or eradicate PPC are available. Due to the variation in host genetic resistance, the development of resistant varieties is postulated as a viable and promising strategy. By using an integrated approach, this study aimed to identify differences in the molecular responses and physiological traits of the highly susceptible Pinus radiata and the highly resistant Pinus pinea to F. circinatum at an early stage of infection. Dual RNA-Seq analysis also allowed to evaluate pathogen behavior when infecting each pine species. No significant changes in the physiological analysis were found upon pathogen infection, although transcriptional reprogramming was observed mainly in the resistant species. The transcriptome profiling of P. pinea revealed an early perception of the pathogen infection together with a strong and coordinated defense activation through the reinforcement and lignification of the cell wall, the antioxidant activity, the induction of PR genes, and the biosynthesis of defense hormones. On the contrary, P. radiata had a weaker response, possibly due to impaired perception of the fungal infection that led to a reduced downstream defense signaling. Fusarium circinatum showed a different transcriptomic profile depending on the pine species being infected. While in P. pinea, the pathogen focused on the degradation of plant cell walls, active uptake of the plant nutrients was showed in P. radiata. These findings present useful knowledge for the development of breeding programs to manage PPC. Full article

Mycoviruses are known to be difficult to cure in fungi but their spontaneous loss occurs commonly. The unexpected disappearance of mycoviruses can be explained by diverse reasons, from methodological procedures to biological events such as posttranscriptional silencing machinery. The long-term effects of a virus infection on the host organism have been well studied in the case of human viruses; however, the possible residual effect on a fungus after the degradation of a mycovirus is unknown. For that, this study analyses a possible residual effect on the transcriptome of the pathogenic fungus Fusarium circinatum after the loss of the mitovirus FcMV1. The mycovirus that previously infected the fungal isolate was not recovered after a 4-year storage period. Only 14 genes were determined as differentially expressed and were related to cell cycle regulation and amino acid metabolism. The results showed a slight acceleration in the metabolism of the host that had lost the mycovirus by the upregulation of the genes involved in essential functions for fungal development. The analysis also revealed a weak expression in the annotated genes of the RNA silencing machinery. To our knowledge, this is the first time that a potential residual effect on the host transcriptome caused by the past infection of a mycovirus is reported. Full article

Pine forests in the southern United States are a major contributor to the global economy. Through the last three decades, however, there have been concerns about the decline of pine forests attributed mostly to pests and pathogens. A combination of biotic agents and environmental factors and their interaction often influences outbreaks and the resultant damage in the forests. Southern pines experience periodic mortality from bark beetles and root rot fungi and losses from fusiform rust and pitch canker have long been important for management. In recent years, there is also growing evidence of increasing damage from foliar disease in southern pines. Early detection of diseases following changes in foliar characteristics and assessment of potential risks will help us better utilize our resources and manage these forests sustainably. In this study, we used Forest Inventory and Analysis (FIA) data to explore the intensity of foliar disease in three common pines: loblolly (Pinus taeda L.), longleaf (Pinus palustris Mill.), and slash (Pinus elliottii Engelm.) in spatial and temporal terms using tree-level and climatic variables. Results from a tree-level model suggests that crown ratio may be an important factor in pine foliar disease (p < 0.1). We applied the MaxEnt model, a presence-only species distribution model (SDM), to explore any association of foliar disease incidences with the climatic variables at a landscape level. Results indicate that mean dew point temperature, maximum vapor pressure deficit, and precipitation during cold months had more influence over disease incidences than other climatic variables. While the sample size is limited as this is an emerging disease in the region, our study provides a basis for further exploration of disease detection methods, disease etiology studies, and hazard mapping. Full article