Genome-wide identification and characterization of Fusarium circinatum-responsive lncRNAs in Pinus radiata.

BACKGROUND: One of the most promising strategies of Pine Pitch Canker (PPC) management is the use of reproductive plant material resistant to the disease. Understanding the complexity of plant transcriptome that underlies the defence to the causal agent Fusarium circinatum, would greatly facilitate the development of an accurate breeding program. Long non-coding RNAs (lncRNAs) are emerging as important transcriptional regulators under biotic stresses in plants. However, to date, characterization of lncRNAs in conifer trees has not been reported. In this study, transcriptomic identification of lncRNAs was carried out using strand-specific paired-end RNA sequencing, from Pinus radiata samples inoculated with F. circinatum at an early stage of infection. RESULTS: Overall, 13,312 lncRNAs were predicted through a bioinformatics approach, including long intergenic non-coding RNAs (92.3%), antisense lncRNAs (3.3%) and intronic lncRNAs (2.9%). Compared with protein-coding RNAs, pine lncRNAs are shorter, have lower expression, lower GC content and harbour fewer and shorter exons. A total of 164 differentially expressed (DE) lncRNAs were identified in response to F. circinatum infection in the inoculated versus mock-inoculated P. radiata seedlings. The predicted cis-regulated target genes of these pathogen-responsive lncRNAs were related to defence mechanisms such as kinase activity, phytohormone regulation, and cell wall reinforcement. Co-expression network analysis of DE lncRNAs, DE protein-coding RNAs and lncRNA target genes also indicated a potential network regulating pectinesterase activity and cell wall remodelling. CONCLUSIONS: This study presents the first comprehensive genome-wide analysis of P. radiata lncRNAs and provides the basis for future functional characterizations of lncRNAs in relation to pine defence responses against F. circinatum.

New Insights on the Integrated Management of Plant Diseases by RNA Strategies: Mycoviruses and RNA Interference.

RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease. Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.

Effect of High-Flow Oxygen Therapy vs Conventional Oxygen Therapy on Invasive Mechanical Ventilation and Clinical Recovery in Patients With Severe COVID-19: A Randomized Clinical Trial.

IMPORTANCE: The effect of high-flow oxygen therapy vs conventional oxygen therapy has not been established in the setting of severe COVID-19. OBJECTIVE: To determine the effect of high-flow oxygen therapy through a nasal cannula compared with conventional oxygen therapy on need for endotracheal intubation and clinical recovery in severe COVID-19. DESIGN, SETTING, AND PARTICIPANTS: Randomized, open-label clinical trial conducted in emergency and intensive care units in 3 hospitals in Colombia. A total of 220 adults with respiratory distress and a ratio of partial pressure of arterial oxygen to fraction of inspired oxygen of less than 200 due to COVID-19 were randomized from August 2020 to January 2021, with last follow-up on February 10, 2021. INTERVENTIONS: Patients were randomly assigned to receive high-flow oxygen through a nasal cannula (n = 109) or conventional oxygen therapy (n = 111). MAIN OUTCOMES AND MEASURES: The co-primary outcomes were need for intubation and time to clinical recovery until day 28 as assessed by a 7-category ordinal scale (range, 1-7, with higher scores indicating a worse condition). Effects of treatments were calculated with a Cox proportional hazards model adjusted for hypoxemia severity, age, and comorbidities. RESULTS: Among 220 randomized patients, 199 were included in the analysis (median age, 60 years; n = 65 women [32.7%]). Intubation occurred in 34 (34.3%) randomized to high-flow oxygen therapy and in 51 (51.0%) randomized to conventional oxygen therapy (hazard ratio, 0.62; 95% CI, 0.39-0.96; P = .03). The median time to clinical recovery within 28 days was 11 (IQR, 9-14) days in patients randomized to high-flow oxygen therapy vs 14 (IQR, 11-19) days in those randomized to conventional oxygen therapy (hazard ratio, 1.39; 95% CI, 1.00-1.92; P = .047). Suspected bacterial pneumonia occurred in 13 patients (13.1%) randomized to high-flow oxygen and in 17 (17.0%) of those randomized to conventional oxygen therapy, while bacteremia was detected in 7 (7.1%) vs 11 (11.0%), respectively. CONCLUSIONS AND RELEVANCE: Among patients with severe COVID-19, use of high-flow oxygen through a nasal cannula significantly decreased need for mechanical ventilation support and time to clinical recovery compared with conventional low-flow oxygen therapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04609462.

Dual RNA-Sequencing Analysis of Resistant (Pinus pinea) and Susceptible (Pinus radiata) Hosts during Fusarium circinatum Challenge.

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.

Fusarium circinatum isolates from northern Spain are commonly infected by three distinct mitoviruses.

Pitch canker is a serious disease of pines caused by the ascomycete fungus Gibberella circinata (anamorph = Fusarium circinatum). Three distinct mitovirus strains have been described in this fungus: Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1 and FcMV2-2. Here, we investigated the frequency and population variation of these viruses and closely related sequence variants in northern Spain using RT-PCR and sequencing. Each virus strain and similar sequence variants shared >95 % sequence identity and were collectively designated as virus types. All virus types were relatively common in Spain, with estimated prevalence of 18.5 %, 8.9 % and 16.3 % for FcMV1, FcMV2-1 and FcMV2-2, respectively.

Description and prevalence of a putative novel mycovirus within the conifer pathogen Gremmeniella abietina.

The European race of Gremmeniella abietina (Lagerberg) Morelet is the causal agent of stem canker and shoot blight on numerous conifers in Europe and North America. It comprises different species and biotypes in which the presence of mycoviruses has been determined. In this report, we describe the full-length sequence of the RNA-dependent RNA polymerase (RdRp) of a putative novel virus, Gremmeniella abietina RNA virus 6 (GaRV6), with 2165 nt and a GC content of 54.7 %. A BLASTp search using the deduced RdRp amino acid sequence confirmed GaRV6 to be related to members of a still unassigned virus taxon, which includes, e.g., Fusarium graminearum dsRNA mycovirus 4 (FgV-4) and the mutualistic Curvularia thermal tolerance virus (CThTV). The prevalence and genetic diversity of GaRV6 was also studied within the European race of G. abietina. We examined 162 isolates originating from Canada, the Czech Republic, Finland, Italy, Montenegro, Serbia, Spain, Switzerland, Turkey and the United States. According to direct specific reverse transcription (RT) PCR screening based on the RdRp sequence, the virus appears to be present only in Spain, where it is relatively abundant but genetically highly uniform.

Three mitovirus strains infecting a single isolate of Fusarium circinatum are the first putative members of the family Narnaviridae detected in a fungus of the genus Fusarium.

Fusarium circinatum Nirenberg & O'Donnell (teleomorph = Gibberella circinata) is the causal agent of pitch canker disease of pines. Since 2004 it has been present in Europe, particularly in northern Spain, affecting P. radiata and P. pinaster in plantations and nurseries. The disease has now also spread to other European countries, including France, Italy and Portugal. In this report, we describe three novel members of the genus Mitovirus from a Spanish isolate of F. circinatum: Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1 and FcMV2-2. Using a mitochondrial translation table, the complete 2419-bp genome of FcMV1 encodes an RNA-dependent RNA polymerase of 731 amino acids (GC-content ca 30 %). The partial genomes of FcMV2-1 and FcMV2-2 (2193 and 1973 bp, respectively) share ca 48 % RdRp sequence similarity at the aa level and might be regarded as conspecific, while FcMV1 is clearly distinct, showing 32-35 % polymerase similarity to the other strains. However, FcMV1 shared 46 % protein-level similarity with Thielaviopsis basicola mitovirus. This is the first study to report viruses in F. circinatum, as well as the first time that mitovirus genome sequences are described from Fusarium spp.

Ophiostomatoid fungi including two new fungal species associated with pine root-feeding beetles in northern Spain.

Many bark beetles live in a symbiosis with ophiostomatoid fungi but very little is known regarding these fungi in Spain. In this study, we considered the fungi associated with nine bark beetle species and one weevil infesting two native tree species (Pinus sylvestris and Pinus nigra) and one non-native (Pinus radiata) in Cantabria (Northern Spain). This included examination of 239 bark beetles or their galleries. Isolations yielded a total of 110 cultures that included 11 fungal species (five species of Leptographium sensu lato including Leptographium absconditum sp. nov., five species of Ophiostoma sensu lato including Ophiostoma cantabriense sp. nov, and one species of Graphilbum). The most commonly encountered fungal associates of the bark beetles were Grosmannia olivacea, Leptographium procerum, and Ophiostoma canum. The aggressiveness of the collected fungal species was evaluated using inoculations on two-year-old P. radiata seedlings. Leptographium wingfieldii, Leptographium guttulatum, and Ophiostoma ips were the only species capable of causing significant lesions.

Physiological, metabolic and hormonal responses of two Pinus spp. with contrasting susceptibility to brown-spot needle blight disease.

Needle blights are serious fungal diseases affecting European natural and planted pine forests. Brown-spot needle blight (BSNB) disease, caused by the fungus Lecanosticta acicola, causes canopy defoliation and severe productivity losses, with consequences depending on host susceptibility. To gain new insights into BSNB plant-pathogen interactions, constitutive and pathogen-induced traits were assessed in two host species with differential disease susceptibility. Six-month-old Pinus radiata D. Don (susceptible) and Pinus pinea L. (more resistant) seedlings were needle inoculated with L. acicola under controlled conditions. Eighty days after inoculation, healthy-looking needles from symptomatic plants were assessed for physiological parameters and sampled for biochemical analysis. Disease progression, plant growth, leaf gas-exchanges and biochemical parameters were complemented with hormonal and untargeted primary metabolism analysis and integrated for a holistic analysis. Constitutive differences between pine species were observed. Pinus pinea presented higher stomatal conductance and transpiration rate and higher amino and organic acids, abscisic acid as well as putrescine content than P. radiata. Symptoms from BSNB disease were observed in 54.54% of P. radiata and 45.45% of P. pinea seedlings, being more pronounced and generalized in P. radiata. For both species, plant height, sub-stomatal CO2 concentration and water-use efficiency were impacted by infection. In P. radiata, total soluble sugars, starch and total flavonoids content increased after infection. No differences in hormone content after infection were observed. However, secondary metabolism was induced in P. pinea visible through total phenolics, flavonoids and putrescine accumulation. Overall, the observed results suggest that P. pinea constitutive and induced traits may function as two layers of a defence strategy which contributed to an increased BSNB resistance in comparison with P. radiata. This is the first integrative study linking plant physiological and molecular traits in Pinus-Lecanosticta acicola pathosystem, contributing to a better understanding of the underlying resistance mechanisms to BSNB disease in pines.

The ADnet Bayesian belief network for alder decline: Integrating empirical data and expert knowledge.

The globalization in plant material trading has caused the emergence of invasive pests in many ecosystems, such as the alder pathogen Phytophthora ×alni in European riparian forests. Due to the ecological importance of alder to the functioning of rivers and the increasing incidence of P. ×alni-induced alder decline, effective and accessible decision tools are required to help managers and stakeholders control the disease. This study proposes a Bayesian belief network methodology to integrate diverse information on the factors affecting the survival and infection ability of P. ×alni in riparian habitats to help predict and manage disease incidence. The resulting Alder Decline Network (ADnet) management tool integrates information about alder decline from scientific literature, expert knowledge and empirical data. Expert knowledge was gathered through elicitation techniques that included 19 experts from 12 institutions and 8 countries. An original dataset was created covering 1189 European locations, from which P. ×alni occurrence was modeled based on bioclimatic variables. ADnet uncertainty was evaluated through its sensitivity to changes in states and three scenario analyses. The ADnet tool indicated that mild temperatures and high precipitation are key factors favoring pathogen survival. Flood timing, water velocity, and soil type have the strongest influence on disease incidence. ADnet can support ecosystem management decisions and knowledge transfer to address P. ×alni-induced alder decline at local or regional levels across Europe. Management actions such as avoiding the planting of potentially infected trees or removing man-made structures that increase the flooding period in disease-affected sites could decrease the incidence of alder disease in riparian forests and limit its spread. The coverage of the ADnet tool can be expanded by updating data on the pathogen's occurrence, particularly from its distributional limits. Research on the role of genetic variability in alder susceptibility and pathogen virulence may also help improve future ADnet versions.

Spanish ecological battleground: population structure of two invasive fungi, Cryphonectria parasitica and Fusarium circinatum.

Introduction: Invasive fungi distributed worldwide through globalization have caused devastating diseases in different forests, causing economic and ecologic disturbances. Two such invasive species are Cryphonectria parasitica and Fusarium circinatum, which were introduced to Europe from North America, separated temporally: C. parasitica was introduced about nine decades ago, whereas F. circinatum was introduced around two decades ago. As C. parasitica had a longer time to undergo genetic changes, we hypothesized that it has higher genetic diversity than the recently introduced F. circinatum in Spain. In addition, we studied the genetic characterization of both fungi present in similar ecological conditions in Northern Spain with the aim of providing data for biocontrol measures. Methods: Molecular genetic markers were used to test these hypotheses, including mating type and DNA sequencing of internal transcribed spacer (ITS) regions. In addition, we used vegetative compatibility (VC) type markers in C. parasitica as the information about VC type is essential to apply biocontrol against the fungus. Results and discussion: All the isolates of C. parasitica from the studied area belonged to only one VC type (EU-1) and one mating type (MAT-2). However, three distinct haplotypes of C. parasitica were identified through ITS sequencing, showing that multiple introductions might have happened to Cantabria. Among F. circinatum, no diversity was observed in ITS and MAT loci in the studied area but isolates from other Spanish regions showed the presence of both mating types. Overall, C. parasitica had higher genetic diversity than F. circinatum, despite both organisms appearing to reproduce clonally. This study helped understand the invasion patterns of C. parasitica and F. circinatum in northern Spain and will be useful in applying biocontrol measures against both pathogens.

Climate, host and geography shape insect and fungal communities of trees.

Non-native pests, climate change, and their interactions are likely to alter relationships between trees and tree-associated organisms with consequences for forest health. To understand and predict such changes, factors structuring tree-associated communities need to be determined. Here, we analysed the data consisting of records of insects and fungi collected from dormant twigs from 155 tree species at 51 botanical gardens or arboreta in 32 countries. Generalized dissimilarity models revealed similar relative importance of studied climatic, host-related and geographic factors on differences in tree-associated communities. Mean annual temperature, phylogenetic distance between hosts and geographic distance between locations were the major drivers of dissimilarities. The increasing importance of high temperatures on differences in studied communities indicate that climate change could affect tree-associated organisms directly and indirectly through host range shifts. Insect and fungal communities were more similar between closely related vs. distant hosts suggesting that host range shifts may facilitate the emergence of new pests. Moreover, dissimilarities among tree-associated communities increased with geographic distance indicating that human-mediated transport may serve as a pathway of the introductions of new pests. The results of this study highlight the need to limit the establishment of tree pests and increase the resilience of forest ecosystems to changes in climate.

Field and Laboratory Procedures for Fusarium circinatum Identification and Diagnosis.

Fusarium circinatum is a serious invasive pathogen affecting conifers and causes the disease commonly known as pine pitch canker. Due to the outbreak in European countries, regulations stipulate that Member States must conduct annual official surveys for the fungus on their territory and report the results to the European Commission. Here, we describe the field and laboratory protocols used for the identification and diagnostic of the pathogen.

Utilizing volatile organic compounds for early detection of Fusarium circinatum.

Fusarium circinatum, a fungal pathogen deadly to many Pinus species, can cause significant economic and ecological losses, especially if it were to become more widely established in Europe. Early detection tools with high-throughput capacity can increase our readiness to implement mitigation actions against new incursions. This study sought to develop a disease detection method based on volatile organic compound (VOC) emissions to detect F. circinatum on different Pinus species. The complete pipeline applied here, entailing gas chromatography-mass spectrometry of VOCs, automated data analysis and machine learning, distinguished diseased from healthy seedlings of Pinus sylvestris and Pinus radiata. In P. radiata, this distinction was possible even before the seedlings became visibly symptomatic, suggesting the possibility for this method to identify latently infected, yet healthy looking plants. Pinus pinea, which is known to be relatively resistant to F. circinatum, remained asymptomatic and showed no changes in VOCs over 28 days. In a separate analysis of in vitro VOCs collected from different species of Fusarium, we showed that even closely related Fusarium spp. can be readily distinguished based on their VOC profiles. The results further substantiate the potential for volatilomics to be used for early disease detection and diagnostic recognition.

Susceptibility of germinating seedlings of European and Eurasian populations of Pinus sylvestris to damping-off caused by Fusarium circinatum.

The effect of inoculation with Fusarium circinatum on survival of seed and seedlings of 19 populations of Pinus sylvestris was examined under environmentally controlled conditions, with four treatments (0, 50, 103, 106 spores ml-1). A single seed source of P. radiata was included as a positive control. Germination (emergence of the plumule above the compost) and health of seedlings was assessed daily, for 85 days. Spore density had a significant effect on germination: at 50 spores ml-1, only germination of a Northeast Scotland population was reduced. Treatment with 1000 spores ml-1, however, reduced germination of six populations of P. sylvestris and of P. radiata. Survival of emerged seedlings also varied with inoculum dose. Approximately 75% of seedlings survived 85 days after germination after inoculation with 50 spores ml-1. Seedlings of all populations were killed within 12-16 days of germination by the 103 and 106 spores ml-1 treatments. Emerged seedlings of the Austrian populations showed the highest susceptibility to F. circinatum following treatment with 50 spores ml-1, although 15% of seedlings of one Austrian population (AU3) survived to the end of the experiment (85 days after germination). There was no clear pattern in survival rates of the P. sylvestris seedlings from other populations treated with 1000 or 1 million spores ml-1 due to death of all emerged seedlings within a short period. Variations in susceptibility of different populations of P. sylvestris to F. circinatum may be used in future selection and breeding programmes to reduce the impact of the pathogen as it spreads over wider areas in Europe and Eurasia.

Worldwide diversity of endophytic fungi and insects associated with dormant tree twigs.

International trade in plants and climate change are two of the main factors causing damaging tree pests (i.e. fungi and insects) to spread into new areas. To mitigate these risks, a large-scale assessment of tree-associated fungi and insects is needed. We present records of endophytic fungi and insects in twigs of 17 angiosperm and gymnosperm genera, from 51 locations in 32 countries worldwide. Endophytic fungi were characterized by high-throughput sequencing of 352 samples from 145 tree species in 28 countries. Insects were reared from 227 samples of 109 tree species in 18 countries and sorted into taxonomic orders and feeding guilds. Herbivorous insects were grouped into morphospecies and were identified using molecular and morphological approaches. This dataset reveals the diversity of tree-associated taxa, as it contains 12,721 fungal Amplicon Sequence Variants and 208 herbivorous insect morphospecies, sampled across broad geographic and climatic gradients and for many tree species. This dataset will facilitate applied and fundamental studies on the distribution of fungal endophytes and insects in trees.

Co-Infections by Fusarium circinatum and Phytophthora spp. on Pinus radiata: Complex Phenotypic and Molecular Interactions.

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.

Emergency Thrombolysis During Cardiac Arrest Due to Pulmonary Thromboembolism: Our Experience Over 6 Years.

INTRODUCTION: Cardiac arrest (CA) is one of the leading causes of death worldwide. Among patients with CA, pulmonary embolism (PE) accounts for approximately 10% of all cases. OBJECTIVE: To compare the outcomes after cardiopulmonary-cerebral resuscitation (CCPR) with and without thrombolytic therapy (TT) in patients with CA secondary to PE. METHODS: We included patients older than 17 years admitted to our hospital between 2013 and 2017 with a diagnosis of CA with confirmed or highly suspected PE who received CCPR with or without TT. Measures of central tendency were used to depict the data. RESULTS: The study comprised 16 patients, 8 of whom received CCPR and thrombolysis with alteplase, whereas the remaining patients received CCPR without TT. The most frequent rhythm of CA in both groups was pulseless electrical activity. Return of spontaneous circulation (ROSC) occurred in 100% of patients who received TT and in 88% of non-thrombolysed patients. The mortality rate of patients who received TT and non-thrombolysed patients at 24 hours was 25% and 50%, respectively. However, at the time of hospital discharge, the mortality was the same in both groups (62%). In patients who received TT, mortality was related to sepsis and hemorrhage whereas in non-thrombolysed patients, mortality was due to myocardial dysfunction. CONCLUSION: Intra-arrest thrombolysis resulted in a higher likelihood of ROSC and a higher 24-hour survival in adults with CA secondary to acute PE. Overall, the survival at hospital discharge was the same in the two groups.

Real-time loop-mediated isothermal amplification assay for rapid detection of Fusarium circinatum.

Fusarium circinatum is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since F. circinatum is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for F. circinatum using a field-deployable portable instrument. The assay was able to recognize the pathogen in host tissues in just 30 min, and the sensitivity of the assay made it possible to detect even small amounts of F. circinatum DNA (as low as 0.5 pg/µl). The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.