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. 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.

Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics.

Forest disease management relies principally on a preventive approach in which epidemiological surveillance plays a crucial role. However, efficient and cost-effective surveillance methods are not currently available for large spatial scales. Nevertheless, aerobiological networks have been set up for several decades in many countries to monitor pollen dispersal and provide real-time assessments of allergenic risk. Here, we suggest that the same approach could be used for the surveillance of forest pathogens. Using molecular methods, we analysed samples from 12 sites of the French aerobiological network, at different dates. Both metabarcoding by high-throughput sequencing (using two markers and two different bioinformatics approaches) and real-time PCR targeting eight important forest pathogens were conducted. To validate the approach, temporal and spatial trends of spore detection were compared with field disease data. The metabarcoding approach demonstrated that many fungal plant pathogens could be found in aerobiological samples. Moreover, five of the eight targeted forest pathogens were detected by real-time PCR, with temporal and spatial trends of spore capture consistent with field data. In particular, Hymenoscyphus fraxineus was detected at high frequency in aerobiological samples in the areas where ash dieback has been present for the longest period of time, and at lower frequency in areas with more recent invasion. Spore detection of seasonal pathogens showed a temporal pattern similar to that of disease reports. Overall, our study provides a proof of concept that permanent aerobiological networks combined with molecular methods may provide a useful tool for large-scale surveillance of forest pathogens.

Brown rot is an economically important fungal disease affecting stone and pome fruit orchards, as well as harvested fruit during storage and on the market. Monilinia fructicola, M. laxa, and M. fructigena are the main causal agents of this disease and each have a different regulatory status depending on regional regulations. In this study, a new multiplex tool based on real-time polymerase chain reaction was developed to detect the three pathogenic fungi in a single reaction on fruit, twigs, and flowers of Prunus and Malus spp. Species-specific primer-hydrolysis probe combinations were designed to amplify a region located in a previously described MO368 sequenced characterized amplified region marker, and used in a quadruplex format coupled with the 18S Uni universal primer-probe test in order to check the quality of the DNA template. The assay was designed and optimized with the objective to provide high performance values. Experimental data supported its sensitivity, specificity, reproducibility, and robustness. In addition, a set of quality controls was implemented to minimize the risk of false-positive and false-negative results, thus making this new test fit for use in serial analyses and reliable in the framework of official controls.

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.

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.

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.

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