Next-generation fungal identification using target enrichment and Nanopore sequencing.

BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

Comparative Genomics of Fusarium circinatum Isolates Used to Screen Southern Pines for Pitch Canker Resistance.

Pitch canker, caused by the fungal pathogen Fusarium circinatum, is a global disease affecting many Pinus spp. Often fatal, this disease causes significant mortality in both commercially grown and natural pine forests and is an issue of current and growing concern. F. circinatum isolates collected from three locations in the U.S. state of Florida were shown to be virulent on both slash and loblolly pine, with two of the isolates causing equivalent and significantly larger lesions than those caused by the third isolate during pathogenicity trials. In addition, significant genetic variation in lesion length in the pedigreed slash pine population was evident and rankings of parents for lesion length were similar across isolates. Experimental data demonstrate that both host and pathogen genetics contribute to disease severity. High-quality genomic assemblies of all three isolates were created and compared for structural differences and gene content. No major structural differences were observed among the isolates; however, missing or altered genes do contribute to genomic variation in the pathogen population. This work evaluates in planta virulence among three isolates of F. circinatum, provides genomic resources to facilitate study of this organism, and details comparative genomic methods that may be used to explore the pathogen's contribution to disease development.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Molecular Characterization of Laboratory Mutants of Fusarium oxysporum f. sp. niveum Resistant to Prothioconazole, a Demethylation Inhibitor (DMI) Fungicide.

Fusarium oxysporum f. sp. niveum (FON) is the causal agent of Fusarium wilt in watermelon, an international growth-limiting pathogen of watermelon cultivation. A single demethylation inhibitor (DMI) fungicide, prothioconazole, is registered to control this pathogen, so the risk of resistance arising in the field is high. To determine and predict the mechanism by which FON could develop resistance to prothioconazole, FON isolates were mutagenized using UV irradiation and subsequent fungicide exposure to create artificially resistant mutants. Isolates were then put into three groups based on the EC50 values: sensitive, intermediately resistant, and highly resistant. The mean EC50 values were 4.98 µg/mL for the sensitive, 31.77 µg/mL for the intermediately resistant, and 108.33 µg/mL for the highly resistant isolates. Isolates were then sequenced and analyzed for differences in both the coding and promoter regions. Two mutations were found that conferred amino acid changes in the target gene, CYP51A, in both intermediately and highly resistant mutants. An expression analysis for the gene CYP51A also showed a significant increase in the expression of the highly resistant mutants compared to the sensitive controls. In this study, we were able to identify two potential mechanisms of resistance to the DMI fungicide prothioconazole in FON isolates: gene overexpression and multiple point mutations. This research should expedite growers' and researchers' ability to detect and manage fungicide-resistant phytopathogens.

Fusarium oxysporum f. sp. niveum Molecular Diagnostics Past, Present and Future.

Watermelon is an important commercial crop in the Southeastern United States and around the world. However, production is significantly limited by biotic factors including fusarium wilt caused by the hemibiotrophic fungus Fusarium oxysporum forma specialis niveum (Fon). Unfortunately, this disease has increased significantly in its presence over the last several decades as races have emerged which can overcome the available commercial resistance. Management strategies include rotation, improved crop resistance, and chemical control, but early and accurate diagnostics are required for appropriate management. Accurate diagnostics require molecular and genomic strategies due to the near identical genomic sequences of the various races. Bioassays exist for evaluating both the pathogenicity and virulence of an isolate but are limited by the time and resources required. Molecular strategies are still imperfect but greatly reduce the time to complete the diagnosis. This article presents the current state of the research surrounding races, both how races have been detected and diagnosed in the past and future prospects for improving the system of differentiation. Additionally, the available Fon genomes were analyzed using a strategy previously described in separate formae speciales avirulence gene association studies in Fusarium oxysporum races.

Phylogenetic and phenotypic characterization of Fusarium oxysporum f. sp. niveum isolates from Florida-grown watermelon.

Fusarium wilt of watermelon (Citrullus lanatus) caused by Fusarium oxysporum f. sp. niveum (Fon), has become an increasing concern of farmers in the southeastern USA, especially in Florida. Management of this disease, most often through the use of resistant cultivars and crop rotation, requires an accurate understanding of an area's pathogen population structure and phenotypic characteristics. This study improved the understanding of the state's pathogen population by completing multilocus sequence analysis (MLSA) of two housekeeping genes (BT and TEF) and two loci (ITS and IGS), aggressiveness and race-determining bioassays on 72 isolates collected between 2011 and 2015 from major watermelon production areas in North, Central, and South Florida. Multilocus sequence analysis (MLSA) failed to group race 3 isolates into a single large clade; moreover, clade membership was not apparently correlated with aggressiveness (which varied both within and between clades), and only slightly with sampling location. The failure of multilocus sequence analysis using four highly conserved housekeeping genes and loci to clearly group and delineate known Fon races provides justification for future whole genome sequencing efforts whose more robust genomic comparisons will provide higher resolution of intra-species genetic distinctions. Consequently, these results suggest that identification of Fon isolates by race determination alone may fail to detect economically important phenotypic characteristics such as aggressiveness leading to inaccurate risk assessment.

Marker Development for Differentiation of Fusarium Oxysporum f. sp. Niveum Race 3 from Races 1 and 2.

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (FON), is pathogenic only to watermelon and has become one of the main limiting factors in watermelon production internationally. Detection methods for this pathogen are limited, with few published molecular assays available to differentiate FON from other formae speciales of F. oxysporum. FON has four known races that vary in virulence but are difficult and costly to differentiate using traditional inoculation methods and only race 2 can be differentiated molecularly. In this study, genomic and chromosomal comparisons facilitated the development of a conventional polymerase chain reaction (PCR) assay that could differentiate race 3 from races 1 and 2, and by using two other published PCR markers in unison with the new marker, the three races could be differentiated. The new PCR marker, FNR3-F/FNR3-R, amplified a 511 bp region on the "pathogenicity chromosome" of the FON genome that is absent in race 3. FNR3-F/FNR3-R detected genomic DNA down to 2.0 pg/µL. This marker, along with two previously published FON markers, was successfully applied to test over 160 pathogenic FON isolates from Florida, Georgia, and South Carolina. Together, these three FON primer sets worked well for differentiating races 1, 2, and 3 of FON. For each marker, a greater proportion (60 to 90%) of molecular results agreed with the traditional bioassay method of race differentiation compared to those that did not. The new PCR marker should be useful to differentiate FON races and improve Fusarium wilt research.

Draft Genome Sequences of Three Fusarium oxysporum f. sp. niveum Isolates Used in Designing Markers for Race Differentiation.

Here, we report the draft genome sequences of three Fusarium oxysporum f. sp. niveum isolates that were used to design markers for molecular race differentiation. The isolates were collected from watermelon fields in Georgia (USA) and were determined to be different races of F. oxysporum f. sp. niveum using a traditional bioassay.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification.

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen's spores (zoospores) and is later used to amplify the pathogen's DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Multilocus phylogeny of Acrospermaceae: New epibiotic species and placement of Gonatophragmium, Pseudovirgaria, and Phaeodactylium anamorphs.

Acrospermum is a poorly known genus of epibiotic and saprophytic species with a subcosmopolitan distribution. Here, we investigate the intriguing relationship between Acrospermum and its host plants in the fern family Polypodiaceae, where it occurs upon approximately 45 neotropical species. We conducted phylogenetic analyses using an eight-marker comprehensive ascomycete data set comprising 719 species representing all major lineages along with 23 new Acrospermum specimens sampled from ferns. We ask whether fern-dwelling Acrospermum are monophyletic, whether epibiotic Acrospermum have evolved independently from saprophytic ancestors, and identify anamorphic phases by incorporating sequences for all suspected taxa. Our results corroborate the placement of Acrospermales within the Dothideomycetes with strong support. However, the order remains incertae sedis due to weak support along the branches subtending the clade that includes the Acrospermales plus Dyfrolomycetales. Our results show a strong phylogenetic pattern in lifestyles but do not clearly identify an ancestral life history state. The first divergence in Acrospermaceae splits fungicolous taxa from taxa that inhabit plants; saprophytes and anamorphic phases found on angiosperms occur in both clades. Fungicolous species are monophyletic, whereas species with an epibiotic or necrotic life history upon plants are nonmonophyletic due to the position of the saprophyte A. longisporium. Previously, all Acrospermum collected from ferns were identified as A. maxonii. Our results indicate that this is not monophyletic due to the inclusion of Gonatophragmium triuniae. Two species are described herein as A. gorditum, sp. nov., and A. leucocephalum, sp. nov. We find no instances of co-cladogenesis; however, our ability to detect this is limited by the lack of resolution in the A. maxonii clade. Rather, we see that that the distribution of epibiotic Acrospermum is explained by the overlap between the ecological niche of the Acrospermum species and its host.

Comparative analysis of different molecular and serological methods for detection of Xylella fastidiosa in blueberry.

Bacterial leaf scorch, caused by Xylella fastidiosa, is a major threat to blueberry production in the southeastern United States. Management of this devastating disease is challenging and often requires early detection of the pathogen to reduce major loss. There are several different molecular and serological detection methods available to identify the pathogen. Knowing the efficiency and suitability of these detection techniques for application in both field and laboratory conditions is important when selecting the appropriate detection tool. Here, we compared the efficiency and the functionality of four different molecular detection techniques (PCR, real-time PCR, LAMP and AmplifyRP® Acceler8™) and one serological detection technique (DAS-ELISA). The most sensitive method was found to be real-time PCR with the detection limit of 25 fg of DNA molecules per reaction (≈9 genome copies), followed by LAMP at 250 fg per reaction (≈90 copies), AmplifyRP® Acceler8™ at 1 pg per reaction (≈350 copies), conventional PCR with nearly 1.25 pg per reaction (≈ 440 copies) and DAS-ELISA with 1x105 cfu/mL of Xylella fastidiosa. Validation between assays with 10 experimental samples gave consistent results beyond the variation of the detection limit. Considering robustness, portability, and cost, LAMP and AmplifyRP® Acceler8™ were not only the fastest methods but also portable to the field and didn't require any skilled labor to carry out. Among those two, AmplifyRP® Acceler8™ was faster but more expensive and less sensitive than LAMP. On the other hand, real-time PCR was the most sensitive assay and required comparatively lesser time than C-PCR and DAS-ELISA, which were the least sensitive assays in this study, but all three assays are not portable and needed skilled labor to proceed. These findings should enable growers, agents, and diagnosticians to make informed decisions regarding the selection of an appropriate diagnostic tool for X. fastidiosa on blueberry.