Plant Parasitic Nematode Identification in Complex Samples with Deep Learning.

Plant parasitic nematodes are significant contributors to yield loss worldwide, causing devastating losses to every crop species, in every climate. Mitigating these losses requires swift and informed management strategies, centered on identification and quantification of field populations. Current plant parasitic nematode identification methods rely heavily on manual analyses of microscope images by a highly trained nematologist. This mode is not only expensive and time consuming, but often excludes the possibility of widely sharing and disseminating results to inform regional trends and potential emergent issues. This work presents a new public dataset containing annotated images of plant parasitic nematodes from heterologous soil extractions. This dataset serves to propagate new automated methodologies or speedier plant parasitic nematode identification using multiple deep learning object detection models and offers a path towards widely shared tools, results, and meta-analyses.

First Description of the Nuclear and Mitochondrial Genomes and Associated Host Preference of Trichopoda pennipes, a Parasitoid of Nezara viridula.

Trichopoda pennipes is a tachinid parasitoid of several significant heteropteran agricultural pests, including the southern green stink bug, Nezara viridula, and leaf-footed bug, Leptoglossus phyllopus. To be used successfully as a biological control agent, the fly must selectively parasitize the target host species. Differences in the host preference of T. pennipes were assessed by assembling the nuclear and mitochondrial genomes of 38 flies reared from field-collected N. viridula and L. phyllopus. High-quality de novo draft genomes of T. pennipes were assembled using long-read sequencing. The assembly totaled 672 MB distributed among 561 contigs, having an N50 of 11.9 MB and a GC of 31.7%, with the longest contig at 28 MB. The genome was assessed for completeness using BUSCO in the Insecta dataset, resulting in a score of 99.4%, and 97.4% of the genes were single copy-loci. The mitochondrial genomes of the 38 T. pennipes flies were sequenced and compared to identify possible host-determined sibling species. The assembled circular genomes ranged from 15,345 bp to 16,390 bp and encode 22 tRNAs, two rRNAs, and 13 protein-coding genes (PCGs). There were no differences in the architecture of these genomes. Phylogenetic analyses using sequence information from 13 PCGs and the two rRNAs individually or as a combined dataset resolved the parasitoids into two distinct lineages: T. pennipes that parasitized both N. viridula and L. phyllopus, and others that parasitized only L. phyllopus.

Morphometric and Molecular Diversity among Seven European Isolates of Pratylenchus penetrans.

Pratylenchus penetrans is an economically important root-lesion nematode species that affects agronomic and ornamental plants. Understanding its diversity is of paramount importance to develop effective control and management strategies. This study aimed to characterize the morphological and genetic diversity among seven European isolates. An isolate from the USA was included in the molecular analyses for comparative purposes. Morphometrics of the European P. penetrans isolates generally were within the range of the original descriptions for this species. However, multiple morphometric characteristics, including body length, maximum body width, tail length and length of the post-vulval uterine sac showed discrepancies when compared to other populations. Nucleotide sequence-based analyses revealed a high level of intraspecific diversity among the isolates. We observed no correlation between D2-D3 rDNA- and COXI-based phylogenetic similarities and geographic origin. Our phylogenetic analyses including selected GenBank sequences also suggest that the controversy surrounding the distinction between P. penetrans and P. fallax remains.

Nematode Identification Techniques and Recent Advances.

Nematodes are among the most diverse but least studied organisms. The classic morphology-based identification has proved insufficient to the study of nematode identification and diversity, mainly for lack of sufficient morphological variations among closely related taxa. Different molecular methods have been used to supplement morphology-based methods and/or circumvent these problems with various degrees of success. These methods range from fingerprint to sequence analyses of DNA- and/or protein-based information. Image analyses techniques have also contributed towards this success. In this review, we highlight what each of these methods entail and provide examples where more recent advances of these techniques have been employed in nematode identification. Wherever possible, emphasis has been given to nematodes of agricultural significance. We show that these alternative methods have aided nematode identification and raised our understanding of nematode diversity and phylogeny. We discuss the pros and cons of these methods and conclude that no one method by itself provides all the answers; the choice of method depends on the question at hand, the nature of the samples, and the availability of resources.

Mitochondrial introgression and interspecies recombination in the Fusarium fujikuroi species complex.

The Fusarium fujikuroi species complex (FFSC) is an economically important monophyletic lineage in the genus Fusarium. Incongruence observed among mitochondrial gene trees, as well as the multiple non-orthologous copies of the internal transcribed spacer region of the ribosomal RNA genes, suggests that the origin and history of this complex likely involved interspecies gene flow. Based on this hypothesis, the mitochondrial genomes of non-conspecific species should harbour signatures of introgression or introgressive hybridization. The aim of this study was therefore to search for recombination between the mitochondrial genomes of different species in the FFSC. Using methods based on mt genome sequence similarity, five significant recombinant regions in both gene and intergenic regions were detected. Using coalescent-based methods and the sequences for individual mt genes, various ancestral recombination events between different lineages of the FFSC were also detected. These findings suggest that interspecies gene flow and introgression are likely to have played key roles in the evolution of the FFSC at both ancient and more recent time scales.

Evidence for inter-specific recombination among the mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex.

BACKGROUND: The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called "African", "Asian" and "American" Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex. RESULTS: Overall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi ("Asian" Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets. CONCLUSION: The mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.

Molecular phylogeny of Boliniales (Sordariomycetes) with an assessment of the systematics of Apiorhynchostoma, Endoxyla and Pseudovalsaria.

The systematics of the ascomycete genera Apiorhynchostoma, Endoxyla and Pseudovalsaria are reevaluated based on the comparison of cultural characteristics, teleomorph morphology and DNA sequence data. Analyses of sequences of the internal transcribed spacer (ITS) of the ribosomal DNA operon and the large subunit (LSU) of the nuclear ribosomal DNA gene resolve Boliniales as a robustly supported lineage comprising Apiorhynchostoma, Camarops, Camaropella, Cornipulvina, Endoxyla and Pseudovalsaria. Within Boliniales, species of Endoxyla form a strongly supported lineage. Apiorhynchostoma curreyi and Pseudovalsaria ferruginea group with Cornipulvina ellipsoides. Species of Camarops are paraphyletic and comprise two clades, one of which includes Camaropella. Boliniaceae is emended, Endoxyla mallochii is described as new and Apiorhynchostoma trabicola is considered a synonym of Apiorhynchostoma altipetum. We also propose the combinations Endoxyla occulta, Endoxylina luteobasis and Jobellisia peckii. Keys to genera included in the Boliniaceae and to species of Apiocamarops, Apiorhynchostoma and Endoxyla are provided.

A new species of Heliocephala from Mexico with an assessment of the systematic positions of the anamorph genera Heliocephala and Holubovaniella.

During a study of the microfungi on decaying leaves collected in México we encountered a fungus sharing features of the anamorph genera Heliocephala and Holubovaniella. This hyphomycete resembled Hol. elegans in possessing obclavate to fusiform, three-septate, rostrate phragmoconidia, but it can be distinguished by its much shorter, branched, determinate conidiophores. Unlike members of genus Heliocephala, which also possess determinant conidiophores, the phragmoconidia of this taxon are short-rostrate and have not been observed to germinate iteratively. Prompted by the discovery of this hyphomycete, we investigated the phylogenetic relationships among species of Heliocephala and Holubovaniella. Analysis of large subunit rDNA gene sequences positioned species of Heliocephala and Holubovaniella in the Dothideomycetes and identified Stomiopeltis betulae (Micropeltididaceae) as their closest relative. These results also indicated that Heliocephala and Holubovaniella are closely related taxa. Based on these findings and the similarity of the developmental and morphological characters of species of Heliocephala and Holubovaniella, we recognize these genera as synonymous and describe a new species, Heliocephala triseptata. A dichotomous key to the species of Heliocephala is provided.

Systematics of Catenulifera (anamorphic Hyaloscyphaceae) with an assessment of the phylogenetic position of Phialophora hyalina.

Catenulifera, typified by C. rhodogena (=Scopulariopsis rhodogena), was established to accommodate the anamorphs of Hyphodiscus (Ascomycota, Helotiales) and to delimit these taxa from members of Phialophora section Catenulatae. Catenulifera rhodogena has been inferred as monophyletic, but its relationship to ascomycetes with poorly differentiated phialidic anamorphs remains enigmatic. To test the hypothesis that C. rhodogena is closely related to morphologically similar species of Phialophora and to further explore the systematics of Catenulifera, we analyzed nuclear rDNA and ß-tubulin gene sequences of isolates identified as C. rhodogena, Hyphodiscus hymeniophilus, P. brachyconia, P. brevicollaris and P. hyalina. ITS-LSU and ITS-LSU-ß-tubulin phylogenies positioned all isolates except P. hyalina in a single, well-supported clade that consisted of three subclades. Subclade 1 included fungicolous isolates of C. rhodogena and H. hymeniophilus that did not fluoresce when exposed to long-wave UV light. Subclade 2 contained fungicolous and lignicolous strains of C. rhodogena that produced fluorescent colonies and possessed a 366bp indel in the LSU rRNA gene. Neither lineage encompassed the ex-type strains of Cistella rubescens (=H. hymeniophilus) or S. rhodogena, but the former isolate was inferred as sister to Subclade 2. Subclade 3 included all isolates of P. brachyconia, a species recognized here as C. brachyconia comb. nov. A fourth isolate of P. brachyconia that was extralimital to Subclade 3 is described as C. luxurians sp. nov. The positions of C. brevicollaris comb. nov., a species based on P. brevicollaris, and C. luxurians were not resolved in the ITS-LSU phylogeny. P. hyalina is not closely related to Catenulifera.

Species-specific primers for Fusarium redolens and a PCR-RFLP technique to distinguish among three clades of Fusarium oxysporum.

The currently available morphological and molecular diagnostic techniques for Fusarium redolens and the three phylogenetic clades of Fusarium oxysporum are problematic. Aligned translation elongation factor 1 alpha (TEF-1 alpha) gene sequences from these species and their close relatives were used to design F. redolens-specific primers, and to identify restriction sites that discriminate among the three clades of F. oxysporum. The F. redolens-specific primers distinguished this species from all others included in the study. There were three TEF-1 alpha-RFLP patterns among formae speciales of F. oxysporum. These PCR-RFLP patterns corresponded with the three clades. These techniques provide simple and inexpensive diagnostic methods for the identification of F. redolens and members of the three clades of F. oxysporum.