Maintenance of divergent lineages of the Rice Blast Fungus Pyricularia oryzae through niche separation, loss of sex and post-mating genetic incompatibilities.

Many species of fungal plant pathogens coexist as multiple lineages on the same host, but the factors underlying the origin and maintenance of population structure remain largely unknown. The rice blast fungus Pyricularia oryzae is a widespread model plant pathogen displaying population subdivision. However, most studies of natural variation in P. oryzae have been limited in genomic or geographic resolution, and host adaptation is the only factor that has been investigated extensively as a contributor to population subdivision. In an effort to complement previous studies, we analyzed genetic and phenotypic diversity in isolates of the rice blast fungus covering a broad geographical range. Using single-nucleotide polymorphism genotyping data for 886 isolates sampled from 152 sites in 51 countries, we showed that population subdivision of P. oryzae in one recombining and three clonal lineages with broad distributions persisted with deeper sampling. We also extended previous findings by showing further population subdivision of the recombining lineage into one international and three Asian clusters, and by providing evidence that the three clonal lineages of P. oryzae were found in areas with different prevailing environmental conditions, indicating niche separation. Pathogenicity tests and bioinformatic analyses using an extended set of isolates and rice varieties indicated that partial specialization to rice subgroups contributed to niche separation between lineages, and differences in repertoires of putative virulence effectors were consistent with differences in host range. Experimental crosses revealed that female sterility and early post-mating genetic incompatibilities acted as strong additional barriers to gene flow between clonal lineages. Our results demonstrate that the spread of a fungal pathogen across heterogeneous habitats and divergent populations of a crop species can lead to niche separation and reproductive isolation between distinct, widely distributed, lineages.

Correction: Maintenance of divergent lineages of the Rice Blast Fungus Pyricularia oryzae through niche separation, loss of sex and post-mating genetic incompatibilities.

[This corrects the article DOI: 10.1371/journal.ppat.1010687.].

Fast and reliable molecular methods to detect fungal pathogens in woody plants.

Plant diseases caused by pathogenic microorganisms represent a serious threat to plant productivity, food security, and natural ecosystems. An effective framework for early warning and rapid response is a crucial element to mitigate or prevent the impacts of biological invasions of plant pathogens. For these reasons, detection tools play an important role in monitoring plant health, surveillance, and quantitative pathogen risk assessment, thus improving best practices to mitigate and prevent microbial threats. The need to reduce the time of diagnosis has prompted plant pathologists to move towards more sensitive and rapid methods such as molecular techniques. Considering prevention to be the best strategy to protect plants from diseases, this review focuses on fast and reliable molecular methods to detect the presence of woody plant pathogens at early stage of disease development before symptoms occur in the host. A harmonized pool of novel technical, methodological, and conceptual solutions is needed to prevent entry and establishment of new diseases in a country and mitigate the impact of both invasive and indigenous organisms to agricultural and forest ecosystem biodiversity and productivity.

New multiplex conventional PCR and quadruplex real-time PCR assays for one-tube detection of Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis in Citrus: development and validation.

Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg µL-1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg µL-1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. KEY POINTS: • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

A Genomic Approach to Develop a New qPCR Test Enabling Detection of the Pyricularia oryzae Lineage Causing Wheat Blast.

Rapid detection is key to managing emerging diseases because it allows their spread around the world to be monitored and limited. The first major wheat blast epidemics were reported in 1985 in the Brazilian state of Paraná. Following this outbreak, the disease quickly spread to neighboring regions and countries and, in 2016, the first report of wheat blast disease outside South America was released. This Asian outbreak was due to the trade of infected South American seed, demonstrating the importance of detection tests in order to avoid importing contaminated biological material into regions free from the pathogen. Genomic analysis has revealed that one particular lineage within the fungal species Pyricularia oryzae is associated with this disease: the Triticum lineage. A comparison of 81 Pyricularia genomes highlighted polymorphisms specific to the Triticum lineage, and this study developed a real-time PCR test targeting one of these polymorphisms. The test's performance was then evaluated in order to measure its analytical specificity, analytical sensitivity, and robustness. The C17 quantitative PCR test detected isolates belonging to the Triticum lineage with high sensitivity, down to 13 plasmid copies or 1 pg of genomic DNA per reaction tube. The blast-based approach developed here to study P. oryzae can be transposed to other emerging diseases.

First report of Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) causing banana wilt in the Island of Mayotte.

Fusarium oxysporum f. sp. cubense (Foc) is a fungus causing Fusarium wilt of banana (Musa spp.). The fungus is divided into three races and 24 vegetative compatibility groups (VCG) of which VCG 01213/16, commonly known as Foc tropical race 4 (Foc TR4), is of particular concern. Foc TR4 severely affects Cavendish (AAA) bananas, which comprise about 50% of all bananas produced globally, as well as many varieties susceptible to the other races of Foc. The pathogen was restricted to Southeast Asia and Australia until 2012, where after it has been detected in the Middle East, Mozambique in Africa, and Colombia in South America (Viljoen et al. 2020). Here we report the first detection of Foc TR4 in the French department of Mayotte, located in the Indian Ocean. In September 2019, leaf yellowing and wilting symptoms were observed in individual plants of the banana subgroups Silk (AAB) (cv. "Kissoukari") and Bluggoe (ABB) (cv. "Baraboufaka"). The symptomatic individuals were found in private gardens in the village of Poroani in Southwest Mayotte (World Geodetic System [WGS] 12° 53' 31.83''S, 45° 8' 30.98" E). When the pseudostems of symptomatic plants were split open, dark red to brown vascular discoloration was observed. Pseudostem tissue samples were collected and identified as Foc TR4 with the real-time PCR assay developed by Aguayo et al. (2017). Sections of the pseudostem samples were surface sterilized and used to isolate the fungus on potato dextrose agar (PDA) medium. Isolates were identified as F. oxysporum based on cultural and morphological characteristics as described in Leslie and Summerell (2006), which included fluffy aerial mycelia on PDA and the presence of short monophialides conidigenous cells bearing microconidia arranged in false heads. Abundant chlamydospores were also produced on synthetic nutrient poor agar (SNA) media. Single-spored isolates were used to develop nit mutants for vegetative compatibility group (VCG) testing (Correll 1991; Puhalla 1985). The isolates were confirmed as VCG 01213/16 as formation of heterokaryons was obtained with the nit mutants of the universal Foc TR4 tester. Two VCG 01213/16 isolates were then selected for pathogenicity testing by inoculating 2-month-old tissue culture-derived Cavendish plants, using the method described by Viljoen et al. (2017). After 10 weeks, the Foc TR4-inoculated plants produced wilting symptoms and internal rhizome discoloration typical of Fusarium wilt. Fusarium oxysporum was re-isolated from the inoculated plants and identified as Foc TR4/VCG 01213/16 by PCR (Dita et al. 2010; Matthews et al. 2020), thereby fulfilling Koch's postulates. Local authorities have destroyed the infected plants, and have undertaken an extensive survey to determine the distribution of Foc TR4 on the island. Three additional positive cases, identified with the real-time PCR assay of Aguayo et al. (2017), were found in the localities of Koungou ([WGS] 12° 44' 03''S, 45° 12' 08" E) and Bouéni ([WGS] 12° 54' 25''S, 45° 04' 43" E). These included infected Cavendish banana (AAA) plants (cv. "Kontriké"). This is the first time that Foc TR4 has been found on a banana variety other than Cavendish when newly detected in a country. Considering the proximity of Mayotte to other islands of the Comoros archipelago, Madagascar and the East African coast, where banana is considered an important staple, this report describes a serious threat to banana production and the livelihoods of people in the region.

A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits.

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg µl-1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) µl-1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

Assessment of Passive Traps Combined with High-Throughput Sequencing To Study Airborne Fungal Communities.

Techniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results.IMPORTANCE The advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples.

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization. Symptoms closely resembling those due to F. circinatum can be instead associated with C. pinea. Since F. circinatum is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications, while C. pinea, even if now emerging, is not regulated in the EU nor in the USA. For this reason, a reliable and accurate diagnostic tool able to distinguish between the two organisms was considered a priority. In this study, we developed and standardized a duplex real-time PCR assay allowing the simultaneous recognition of C. pinea and F. circinatum DNA in pine tissue in a reasonably short time and for amounts as small as 0.06 pg/µl. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains (110 different isolates) collected in different regions of the world and host trees, and gave reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls. In addition, the duplex real-time PCR assay presented here is the first DNA-based method designed to detect C. pinea, which is becoming an increasing threat to pine stands both in North America and in Europe.

Mating type and microsatellite genotyping indicate that the Tunisian population of Phyllosticta citricarpa is clonal and thrives only asexually.

Citrus black spot (CBS) caused by Phyllosticta citricarpa was reported for the first time in Tunisia in 2019. This was also the first reported occurrence of the disease in a Mediterranean climate. In Tunisia, CBS is mainly found in lemon (Citrus limon) orchards, and is seldom observed on sweet orange (Citrus × sinensis). This recent finding in North Africa raises questions about how the disease has been able to spread under Mediterranean climatic conditions. In this work, 216 Phyllosticta strains collected from lemon orchards in 2021, 2022 and 2023 throughout the country's main citrus-growing provinces were characterised by species morphological and molecular identification, mating type and Simple Sequence Repeats (SSR) microsatellite genotyping (MLG). P. citricarpa was the only species found to be associated with CBS in Tunisia. Although P. citricarpa is a heterothallic fungal species, potentially able to reproduce both sexually and asexually, a single mating type (MAT 1-1-1) idiomorph was found in the population. In addition, three MLGs were observed, across ten microsatellite loci, one of which was massively represented (93 %), indicating a clonal population. The clonality observed suggests a single recent introduction of the pathogen into the country. These findings support the idea that in Tunisia, P. citricarpa only reproduces asexually by pycniospores, with a relatively limited dispersal potential. This is consistent with the absence of pseudothecia on the leaf litter. These results show that CBS is able to thrive under Mediterranean conditions, even in the absence of sexual reproduction. This should be taken into consideration for CBS risk assessment and management.

Harnessing the power of comparative genomics to support the distinction of sister species within Phyllosticta and development of highly specific detection of Phyllosticta citricarpa causing citrus black spot by real-time PCR.

Citrus crops are affected by many fungal diseases. Among them, Citrus Black Spot caused by the ascomycete Phyllosticta citricarpa is particularly economically damaging wherever it occurs. Many other species of Phyllosticta are described on Citrus, but only P. citricarpa is considered a quarantine pest on the European continent. In order to prevent the introduction of this species into Europe, it is essential to have a detection test which can reliably identify it, and not confuse it with other species present on citrus, notably P. paracitricarpa. The latter taxon has recently been described as very close to P. citricarpa, and most detection tests do not allow to distinguish the two species. In this work, we exploited the genomic data of 37 isolates of Phyllosticta spp. from citrus, firstly to assess their phylogenetic relationships, and secondly to search for genomic regions that allowed the definition of species-specific markers of P. citricarpa. Analysis of 51 concatenated genes separated P. citricarpa and P. paracitricarpa in two phylogenetic clades. A locus was selected to define a hydrolysis probe and primers combination that could be used in real-time PCR for the specific detection of the quarantine species, to the exclusion of all others present on Citrus. This test was then thoroughly validated on a set of strains covering a wide geographical diversity, and on numerous biological samples to demonstrate its reliability for regulatory control. The validation data highlighted the need to check the reliability of the test in advance, when a change of reagents was being considered.

An optimized duplex real-time PCR tool for sensitive detection of the quarantine oomycete Plasmopara halstedii in sunflower seeds.

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.

Molecular Detection of Wheat Blast Pathogen in Seeds.

Pyricularia oryzae is a fungal plant pathogen causing blast disease in several species of the Poaceae family. It encompasses several genetic lineages, including one that is pathogenic on wheat and belongs to the Triticum lineage of P. oryzae. The fungus spreads at short distances by its airborne and rain-splash dispersed spores, and at longer distances via cryptically infected wheat seeds, through trade. Here, we describe a practical method to detect P. oryzae Triticum lineage in wheat seeds, after a biological enrichment step, with various options for molecular testing involving several DNA-based technologies: polymerase chain reaction (PCR), real-time PCR, and loop-mediated isothermal amplification (LAMP). The array of available molecular assays is presented in this protocol, each of them targeting specific regions of the P. oryzae Triticum lineage and offering different levels in terms of sensitivity and specificity.

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystisplatani, the Agent of Canker Stain of Platanus spp.

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters-diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility-were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.

Identification and pathogenicity of Alternaria species associated with leaf blotch disease and premature defoliation in French apple orchards.

Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed.

Development, comparison, and validation of real-time and conventional PCR tools for the detection of the fungal pathogens causing brown spot and red band needle blights of pine.

Dothistroma pini, D. septosporum, and Lecanosticta acicola are fungal pathogens that cause severe foliage diseases in conifers. All three pathogens are listed as quarantine organisms in numerous countries throughout the world and, thus, are subject to specific monitoring. Detection and identification of these pathogens still often relies on cumbersome and unsatisfactory methods that are based upon the morphological characterization of fungal fruiting bodies and conidia. In this study, we present the development of several new molecular tools that enable a rapid and specific in planta detection of each of these pathogens. Several DNA extraction procedures starting from infected needles were compared and four commercial DNA extraction kits provided DNA of satisfactory quality for amplification by polymerase chain reaction (PCR). In addition, we developed several sets of conventional PCR primers, dual-labeled probes (DLPs), and duplex-scorpion probes (DSPs), all of which targeted each pathogen. Their ability to detect the pathogens in a series of naturally infected needle samples was compared. The quadruplex DLP real-time assay proved to be more sensitive than the DSP assay and conventional PCR but the two real-time probe formats yielded identical results in the naturally infected samples. Both real-time assays proved to be significantly superior to the technique of humid chamber incubation, which often failed to produce spores for the accurate identification of the pathogens.

A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds.

Wheat blast is a devastating disease caused by the pathogenic fungus Pyricularia oryzae. Wheat blast first emerged in South America before more recently reaching Bangladesh. Even though the pathogen can spread locally by air-dispersed spores, long-distance spread is likely to occur via infected wheat seed or grain. Wheat blast epidemics are caused by a genetic lineage of the fungus, called the Triticum lineage, only differing from the other P. oryzae lineages by less than 1% genetic divergence. In order to prevent further spread of this pathogen to other wheat-growing areas in the world, sensitive and specific detection tools are needed to test for contamination of traded seed lots by the P. oryzae Triticum lineage. In this study, we adopted a comparative genomics approach to identify new loci specific to the P. oryzae Triticum lineage and used them to design a set of new markers that can be used in conventional polymerase chain reaction (PCR), real-time PCR, or loop-mediated isothermal amplification (LAMP) for the detection of the pathogen, with improved inclusivity and specificity compared to currently available tests. A preliminary biological enrichment step of the seeds was shown to improve the sensitivity of the tests, which enabled the detection of the target at an infection rate as low as 0.25%. Combined with others, this new toolkit may be particularly beneficial in preventing the trade of contaminated seeds and in limiting the spread of the disease.

Sensitive detection of Fusarium circinatum in pine seed by combining an enrichment procedure with a real-time polymerase chain reaction using dual-labeled probe chemistry.

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

Metabarcoding targeting the EF1 alpha region to assess Fusarium diversity on cereals.

Fusarium head blight (FHB) is a major cereal disease caused by a complex of Fusarium species. These species vary in importance depending on climatic conditions, agronomic factors or host genotype. In addition, Fusarium species can release toxic secondary metabolites. These mycotoxins constitute a significant food safety concern as they have health implications in both humans and animals. The Fusarium species involved in FHB differ in their pathogenicity, ability to produce mycotoxins, and fungicide sensitivity. Accurate and exhaustive identification of Fusarium species in planta is therefore of great importance. In this study, using a new set of primers targeting the EF1α gene, the diversity of Fusarium species on cereals was evaluated using Illumina high-throughput sequencing. The PCR amplification parameters and bioinformatic pipeline were optimized with mock and artificially infected grain communities and further tested on 65 field samples. Fusarium species were retrieved from mock communities and good reproducibility between different runs or PCR cycle numbers was be observed. The method enabled the detection of as few as one single Fusarium-infected grain in 10,000. Up to 17 different Fusarium species were detected in field samples of barley, durum and soft wheat harvested in France. This new set of primers enables the assessment of Fusarium diversity by high-throughput sequencing on cereal samples. It provides a more exhaustive picture of the Fusarium community than the currently used techniques based on isolation or species-specific PCR detection. This new experimental approach may be used to show changes in the composition of the Fusarium complex or to detect the emergence of new Fusarium species as far as the EF1α sequence of these species show a sufficient amount of polymorphism in the portion of sequence analyzed. Information on the distribution and prevalence of the different Fusarium species in a given geographical area, and in response to various environmental factors, is of great interest for managing the disease and predicting mycotoxin contamination risks.

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.