D7k: Pine pitch canker - strategies for management of Fusarium circinatum in greenhouses and forests (PINESTRENGTH)

Fusarium circinatum (teleomorph: Gibberella circinata)  was first detected in North America, since when the pathogen has spread into Central and South America, South Africa, Asia and, more recently, Europe. F. circinatum is now considered the most important pathogen affecting Pinus seedlings and mature trees in many countries globally; asymptomatic seedlings may be planted out, resulting in very serious losses in forests. The main aim of PINESTRENGTH is to establish a European-focused network to increase knowledge of the biology, ecology and pathways of spread of F. circinatum, to examine the potential for the development of effective and environmentally-friendly prevention and mitigation strategies and to deliver these outcomes to stakeholders and policy makers. To that end, a multidisciplinary approach is being taken, including researchers, forest managers and policy makers from 35 countries to date. Furthermore, any interested party is encouraged to join this COST Action, participating in some of the six Working Groups (WG1 The pathogen-diagnosis, WG2 Interactions with other forest pests and pathogens, WG3 Pathway of disease spread, WG4 Pest risk analyses, WG5 Management of the disease in forest and nurseries and WG6 Coordination, identifying research gaps and dissemination). For further information, please check http://www.pinestrength.eu/.

Fusarium circinatum, the causal agent of Pine Pitch Canker is one of the most aggressive pathogens for pine. The fungus, considered to be native in America, was detected in several countries in America, Asia and also in South Africa. In Europe, the fungus was first reported in Spain, and then in Italy, France and Portugal. After the first detection in Portugal in 2007, an action plan was implemented by the Portuguese forest authority (ICNF), following EU regulation (Commission Decision 2007/433/EC).  In the scope of that plan, an intensive survey was implemented every year (in nurseries, forests and seed production Centre) and hundreds of samples were analysed in three national laboratories.  During consecutive years, positive results were only found in nurseries and none in forest samples. In 2016, a positive result was recorded by INIAV Mycology laboratory in an allotment of P. radiata seeds. Tracing the track of those seeds, a deep survey was done on the P. radiata provenance forest site. Despite few symptomatic trees were observed in that area, F. circinatum was detected for the first time in forest samples. Also in 2016, the fungus was detected in another site of young P. radiata trees in the center of Portugal. After these two records in field, nine years after the first detection in Portugal, ICNF forced the eradication of these two foci and intensified the management in field using a combination of tools and procedures to control and prevent the disease´s spread in forest. 

The pitch canker fungus (PCF), Fusarium circinatum, that has become distributed around the globe, is well known as the cause of resinous stem cankers of pine.  In South Africa, PCF is also the cause of nursery and post-planting disease mortality.  PCF was first identified in a forestry nursery in South Africa in 1990 and within a decade was found in all forest nurseries in the country.  Production of the two main pine species planted, Pinus patula and P. radiata, was severely impacted.  Following the nursery infections, post-planting mortality, up to two years after planting, was observed associated with PCF.  Genetic diversity studies on PCF indicated that subsequent outbreaks of pine pitch canker disease in established plantations in South Africa were associated with nursery incidence of PCF.  This presentation reviews the industry and research response to this serious problem.  Post-planting mortality was shown to result from asymptomatic, infected planting stock despatched from nurseries rather than tree-to-tree infection processes in-field.   Two interventions were developed to combat the disease.  Firstly, hygiene measures were identified to reduce nursery inoculum to levels that thus diminishing not only nursery disease losses but also asymptomatic infection incidence and transplant mortality in field.  Secondly, planting stock, genetically tolerant to PCF was developed for certain planting sites in the form of P. patula x P. tecunumanii hybrids.  Development of a genetically diverse, asexual PCF population presents challenges for continued control of this disease.  The presentation reflects on the importance of industry and academic collaboration and opportunities for further research.

Pinus radiata is one of the most economically important pine species in the world due to its fast growth rate. Nevertheless, in turn, this conifer specie is the most susceptible host to Pine Pitch Canker disease caused by the fungal pathogen Fusarium circinatum. The fact that there are no effective means of controlling this devastating disease has led the search for new measures such as virocontrol. The infection of F. circinatum by putative mycoviruses apparently reduces the percentage of spore germination and mycelial growth, although it does not seem to affect the size of the lesions caused in P. radiata seedlings. In this investigation, mRNA sequencing (RNA-Seq) is used to identify differentially expressed genes during the interaction of P. radiata with isogenic strain of F. circinatum infected by mitovirus FcMV2-1 and free of mycoviruses including uninfected plants. The essay was carried out at two time points (4 and 11 days post inoculation) in order to obtain a more comprehensive view of the fungal invasion. Transcriptome analysis showed altered gene expression during the different interactions in the pathosystem formed by virus-fungus-plant.

Pine pitch canker, caused by Fusarium circinatum, represents a considerable threat to conifer forests worldwide and is currently associated to significant economic losses. Although essential for the development of disease mitigation strategies, little research focused on host susceptibility/tolerance mechanisms has been conducted. Therefore, we aim to explore the response of Pinus spp. with contrasting responses to F. circinatum infection using a multidisciplinary approach. A highly susceptible and a relatively tolerant pine species (Pinus radiata and Pinus pinea, respectively) were artificially infected with the pathogen and several analyses were carried out for both species when infected P. radiata seedlings exhibited disease symptoms. Morpho-physiological, hormonal and oxidative stress-related changes were assessed, as well as targeted gene expression, proteomic and metabolomic profiles, and epigenetic variation. Several species-specific responses were found by applying this holistic methodology. These are likely to be correlated with the outcome of F. circinatum infection for each species. Moreover, the integration of the data obtained and the construction of networks provide a comprehensive overview of pine defence mechanisms upon F. circinatum attack, defining new interaction, signalling pathways and related targets according to species susceptibility. These studies contribute to fulfil knowledge gaps on tree stress responses and allow developing markers for the implementation of environmentally sound integrated pest management solutions to control pine pitch canker.

The endophytic community is mainly composed by fungi and bacteria that live inside the trees without causing any visible symptom and together with the epiphytes conform the microbiome of a plant. The microbiome is currently considered as an integral determinant of a plant phenotype, and influencing ecologically important characteristics of the host, such as stress or disease tolerance. It has been shown that the structure and diversity of endophyte communities depend on the host, the geographical location and abiotic and biotic factors. However, there is a lack of studies investigating the vertical stratification of endophyte communities. In this study we analyze the composition of the endophyte communities in different heights of several Pinus radiatatrees previously inoculated with the forest pathogen Fusarium circinatum, responsible for the Pine Pitch Canker disease. The total DNA was extracted and subjected to a NGS analysis with the Illumina platform. A total of 536 taxa of fungi were obtained in the 20 samples analyzed. We observed that the communities of endophytes were similar in the heights of 15, 65 and 115 cm but different from the species found at 165 cm and from the samples collected in the branches. The results of this study can provide further knowledge of the ecology of fungal endophytes and their paper as biological antagonists. 

Fusarium circinatum is present in Pinus radiata nurseries in Chile since 2001 when was first reported by Wingfield et al (2001). The fungus is present in most of the P. radiata nurseries and is well-established despite the permanent effort made to reduce its negative impact in plant production and plantations establishment. This pathogen is still confined to nurseries and there is no evidence of the pitch canker disease in plantations in Chile or in other country in South America where is present. Monitoring is focused to have early detection in plantations and for that a national effort is made by forestry companies and the government, with special attention to insects that could be associated with F. circinatum, like the small banded pine weevil Pissodes castaneus, among others. Fusarium circinatum in Chile has very low genetic diversity and only one mating type, probably related with a low number of introductions into the country. Some initiatives have been made to select genetic material of P. radiata tolerant to F. circinatum and also on the definition of risk areas where P. radiata could have high stress, allowing the fungus its expression. Good practices at the nursery are crucially important to reduce the amount of inoculum, and these practices should include a rigorous elimination of symptomatic plants, sanitization of machines, tools, hands and shoes of the workers. A combination of tools whit chemical and biological fungicides, bioestimulants, sanitization and quality plant improvement, show that is possible to reduce mortality during the plantations establishment.

Pathogenicity tests with Polish and Lithuanian Scots pine provenances were carried out revealing tolerance of some of them to Fusarium circinatum infections performed via wounds and via soil. While 3 month-old seedlings were not successfully infected (no symptoms and positive results of pathogen DNA tests in the tissue of roots and shoots) in contrast to the older plants (1 year-old and more), which showed external symptoms of disease (and positive DNA tests). Interaction between soil borne fungi Fusarium genus and Phytophthora species revealed different reaction of F. circinatum and other species of fusaria, sometime stimulating growth of its mycelium another time inhibiting it. SEM photographs show the interaction zones. Biological control with the use of bacteria Bacillus subtilis and hyper parasite Trichoderma asperellum was quite efficient in dual organism tests in vitro. Detection and identification of pathogens with an electronic nose is a promising tool for quarantine organisations. Chromatograms from GCMS reveal specific substances differencing fungi from oomycets.

Fusarium circinatum is a fungus and is the causal agent of the Pine Pitch Canker. It affects different Pinus spp. and is characterized by resinous lesions and branch dieback, causing reduced growth and extensive tree death. Fusarium circinatum may also be present in Pinus spp. seeds without showing symptoms and its detection in nurseries results in the inhibition of sale for a period of two years. Thus, it is essential to determine the most efficient methods in the treatment/elimination of F. circinatum in Pinus spp. seeds. 

Here, we used seeds from P. pinaster, P. radiata and P. pinea, inoculated with F. circinatum to analyze the effects of different concentrations of hydrogen peroxide (H₂O₂), sodium hypochlorite (NaClO), thiophanate-methyl and difenoconazole. In order to prove the efficiency of the treatment, the seeds were plated in specific medium and incubated at 22°C during 7 days; after that, the Fusarium spp. colonies were transferred to PDA and SNA medium, for morphological identification; it was also extracted DNA from the PDA colonies and it was performed a PCR reaction with F. circinatum specific primers. 

The treatment with H₂O₂ (20% and 30%) was the one with the best outcome in all Pinus spp. seeds tested. With this treatment, we were able to eliminate the presence of Fusarium circinatum in all the tested seeds, being a reproducible result. Furthermore, it was possible to observe germinating seeds in the DCPA medium in 7 days of growth, which means that the H₂O₂ treatment might not affect the germination capacity.

Virus infections generally are ubiquitous in major fungal families including plant-pathogenic fungi. The effects of these mycoviruses range from asymptomatic to detrimental conferring hypovirulence to their host. Three different mycoviruses of the genus Mitovirus has been identified infecting Spanish isolates of the conifer pathogen Fusarium circinatum (FcMV1, FcMV2-1 and FcMV2-2). Mitoviruses has been associated with the hypovirulence phenomenon in many phytopathogenic fungi, motivating the study of F. circinatum mycoviruses in an attempt to limit the effects of the devastating Pine Pitch Canker disease. Although the presence of mitoviruses does not seem to affect the size of the lesions caused by F. circinatum, it does so reducing the percentage of spore germination and mycelial growth. Thus, metabolic activity reduction could be related to the presence of mitoviruses but it is necessary the use of more accurate methods to determine their effect beyond the visible symptoms. The development of new tools based on host genome studies helps to address the intellectual challenge of understanding the fungal–virus interactions. For the purpose to identify genes linked to the presence of the mitovirus FcMV1, standard differential expression analysis was carried out using RNA-Seq. Total RNA from virus-infected and cured isogenic F. circinatum strains was isolated and sequenced. CLC Genomics Workbench V7.5 was used to perform the bioinformatic analysis. As a result, 14 genes were differentially expressed in response to mitovirus FcMV1 infection. Our analysis provides further detailed insights into mitovirus-F. circinatum interaction.

Fusarium species, as well as other endophytic or pathogenic fungi forming fungal communities, have been reported to be phoretically associated with bark beetles vectors worldwide. This is the case of Fusarium circinatum, the causal agent of pine pitch canker, which threats Pinus radiata plantations in northern Spain. The main objective of this study was to study the fungal communities present on bark beetles and their galleries in pine pitch canker diseased stands with special attention to Fusarium species.

Ten funnel traps and 12 piles of logs were displayed in a P. radiata plot affected by F. circinatum. Traps were baited with different attractive compounds, four with pityol and six with ethanol and α-pinene. In addition, fresh green shoots with Tomicus piniperda’s feeding galleries were collected from the ground in 25 P. radiata affected plots. Collected insects and gallery tissues were processed and cultured with the aim of isolating and identifying associated fungi. A total of 24 different fungal species were obtained from the bark beetles galleries from logs and shoots while 18 were obtained from the insects’ exoskeleton.. Ten different Fusarium species were isolated from tissues and insects. Fusarium circinatum was isolated from bark beetles exoskeletons (1.05 % of the P. pubescens) and from their galleries (3.5 % of T. piniperda’s feeding gallery). These results are an approach to the fungal communities associated with bark beetles in P. radiata PPC diseased stands in northern Spain, suggesting an association between bark beetles and Fusarium species, which is highlighted by the presence of F. circinatum in T. piniperda’s galleries.

Pine Pitch Canker disease, caused by the pathogenic fungus Fusarium circinatum, affects conifer species worldwide. However, the virulence of the pathogen may be affected by the presence of mycoviruses. The aim of this laboratory-based study was to investigate the probability and rate of transmission of F. circinatummitoviruses FcMV1 and FcMV2-2 via microconidia. Ten isolates of mitovirus-infected F. circinatumwere subcultured to produce a total of 100 single-spore colonies (ten replicates per isolate). The total RNA and cDNA obtained from each spore isolate (monosporic culture) were amplified by PCR with specific primers for detection of F. circinatummitoviruses FcMV1 and FcMV2-2. The mitoviruses were detected in a high percentage of the individual spore isolates (between 60% and 100% depending on the fungal isolate). However, the probability of transmission was not statistically significantly associated with either the F. circinatumisolate or the viral strain. A high proportion of transmission via microconidia is critical for development of a biological control program against Pine Pitch Canker (PPC) disease in forests. However, further studies are needed to establish the effect of these mitoviruses on the virulence of F. circinatum.

Nowadays, European forests are exposed to a high number of invasive alien pathogens. Among them, Fusarium circinatum is considered as one of the most important pathogens of conifers globally. Little is known regarding the infection and defense mechanisms involved in this pathosystem, even though this information is crucial to develop management strategies. Interspecific variation in pine susceptibility against F. circinatum is the key to identify the genes involved in these mechanisms. In the present study three pine species (P. radiata, P. pinaster and P. pinea) were selected according to a negative gradient of susceptibility. RT-qPCR was used to identify the differential expression of 8 target genes (5 from the pathogen and 3 from the host) according to the host species at two different sampling points (12 and 20 days after inoculation). Differential expression of F. circinatum pathogenicity genes was dependent on the host species and sampling point. In particular, the lowest and highest expression level of F. circinatum genes were found in P. radiata and P. pinea, respectively. This concurs with the susceptibility of these pine species. An opposite pattern was found between both pine species according to disease progression (12 vs. 20 dpi), which seems to point out that seedlings could not avoid infestation but the resistant species (P. pinea) could develop any resistance mechanism to prevent its progress. Pathogenesis-related genes of the host showed significant differential expression according to the species, but no effect of the sampling point was found.

The microbiome of a plant is composed of endophytic (internal) and epiphytic (external) microbes, mainly bacteria and fungi. The endophytic community (i.e. organisms that live inside the plant without causing any visible symptom) is increasing in importance because their interaction with the host and with other organisms such as pathogens. It is known that the degree of tolerance of a host to a certain pathogen is a combination of genetic resistance, the environment and the microbes that live inside the plant. In the present study we focus on the diversity of fungal endophytic communities in different pine species (Pinus radiata, P. pinaster, P. sylvestris, P. nigra andP. uncinata) previously inoculated with the forest pathogen Fusarium circinatum and grown in the same environment. The study of fungal species was carried out with NGS (Next Generation Sequencing) technologies. The results of our study showed that existed a common core microbiome among all the samples that was composed of several endophytic fungi: Alternaria alternata, Pleospora herbarum, Cryptococcus terreus andCladosporium shpaerospermum. We also found differences between the endophytic communities of each pine species being the community of Pinus uncinatathe one that presented more dissimilarities with the others. The results of our study could provide valuable information about species composition and the role of the fungal species as biological antagonists of forests pathogens.