Pantoea bathycoeliae sp. nov and Sodalis sp. are core gut microbiome symbionts of the two-spotted stink bug.

Stink bug species (Pentatomoidea superfamily) have developed an interdependence with obligate bacterial gut symbionts in specialized midgut crypts (M4 sub-region). Species of the Enterobacteriaceae family (predominantly Pantoea) are vertically transferred to their offspring and provide nutrients that cannot be obtained from plant sap food sources. However, the bacteria in the other gut compartments of stink bugs have rarely been investigated. The two-spotted stink bug, Bathycoelia distincta, is a serious pest of macadamias in South Africa. Nothing is currently known regarding its gut microbiome or how symbionts are transferred between insect generations. In this study, the consistency of B. distincta gut bacteria across geographic locations and life stages was determined with 16S rRNA metabarcoding, considering both the M4 and other gut compartments. A novel Pantoea species was found to be the primary M4 gut symbiont and is vertically transferred to the offspring. The other gut compartments had a low bacterial diversity and genera varied between stink bug populations but a Sodalis species was prominent in all populations. Sequence data of the M4 compartment were used to produce high-quality metagenome-assembled genomes (MAGs) for the Pantoea and Sodalis species. Functional analyses suggested a similar role in nutrient provision for the host, yet also unique metabolites produced by each species. The Sodalis sp. also had additional traits, such as secretion systems, that likely allowed it to establish itself in the host. The Pantoea species was described as Pantoea bathycoeliae sp. nov based on the rules of the SeqCode.

Characterisation of the Alarm Pheromone of Bathycoelia Distincta (Pentatomidae).

Bathycoelia distincta (Pentatomidae) is the dominant pest in South African macadamia orchards, where adults are responsible for causing severe yield losses. Similar to other hemipterans, B. distincta release volatile compounds from scent glands that can deter natural enemies and act as an alarm signal among conspecifics. The overall aim of this study was to characterise the alarm pheromone of B. distincta. We: (i) analysed the scent gland contents of individual adult B. distincta by gas chromatography-mass spectrometry (GC-MS), (ii) quantified volatiles released from live stink bugs after stress, and (iii) evaluated the electrophysiological and behavioural activity of alarm pheromone compounds with dose-response experiments. A blend of fourteen compounds was identified in the scent gland extracts of adult stink bugs. Of these, six compounds were detected in the effluvia of live stressed stink bugs [(E)-2-hexenal, (E)-2-decenal, tridecane, dodecane, (E)-4-oxohex-2-enal and (E)-2-decenyl acetate]. No qualitative or quantitative differences were observed between sexes. Tridecane was the most abundant compound, comprising ∼50% of total secretions. Only (E)-2-hexenal, (E)-2-decenal, and (E)-4-oxohex-2-enal elicited an antennal response in both sexes. Finally, exposure to a mixture of (E)-2-hexenal, (E)-2-decenal, and (E)-4-oxohex-2-enal resulted in an increase in the speed and distance travelled by walking bugs and a decrease in time spent resting compared to unexposed bugs. Our results show that the blend of (E)-2-hexenal, (E)-2-decenal, and (E)-4-oxohex-2-enal can induce an alarm response in B. distincta.

Diversity and Molecular Barcoding of Stink Bugs (Hemiptera: Pentatomidae) Associated with Macadamia in South Africa.

Stink bugs are major pests of macadamia in South Africa. Accurate identification and knowledge of species composition are important to inform management practices. The overall aims of this study were to identify stink bug species from macadamia orchards in South Africa using morphology, and to establish a DNA database based on the cytochrome c oxidase subunit 1 gene region. A total of 21 stink bug species were found in macadamia orchards in KwaZulu-Natal, Limpopo and Mpumalanga provinces. Bathycoelia distincta Distant, 1878, was the dominant species throughout all three growing regions. Two unidentified species of Boerias Kirkaldy, 1909, here designated as Boerias sp. 1 and Boerias sp. 2, were the second and third most abundant species found in KwaZulu-Natal. No species of Boerias has previously been reported in association with macadamia. Evidence of a cryptic third species of Boerias was also found. Species composition fluctuated over three growing seasons in Limpopo and differed between the three growing regions during the 2019-2020 season, highlighting the need for ongoing monitoring of these important pest species. The DNA barcode database developed in this study will be valuable for future monitoring and identifications, including cryptic or polymorphic stink bug species and different life stages.

Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa.

The South African macadamia industry is severely affected by a complex of stink bugs, dominated by the two-spotted stink bug, Bathycoelia distincta Distant (Pentatomidae). This species was first discovered during the spring of 1984 in the Limpopo province. Although considerable effort has been spent trying to manage this pest, it continues to be a pest of concern for the macadamia industry. Information on the genetic diversity of this species is lacking, despite the potential relevance of such information for management strategies. The present study aimed to characterise the genetic diversity of B. distincta populations in South Africa. The Cytochrome c Oxidase Subunit 1 (COI) and cytochrome b (Cytb) gene regions were sequenced from individuals collected from the three main regions of macadamia production over three different seasons (2018-2020). An overall high haplotype diversity (COI = 0.744, Cytb = 0.549 and COI+Cytb = 0.875) was observed. Pairwise mean genetic distance between populations from each region varied from 0.2-0.4% in both datasets, which suggests the absence of cryptic species. The median joining network for both datasets consisted of one or two central haplotypes shared between the regions in addition to unique haplotypes observed in each region. Finally, low genetic differentiation (FST < 0.1), high gene flow (Nm > 1) and the absence of a correlation between genetic and geographic distance were estimated among populations. Overall, these results suggest that the B. distincta populations are not structured among the areas of macadamia production in South Africa. This might be due to its ability to feed and reproduce on various plants and its high dispersal (airborne) between the different growing regions of the country along with the rapid expansion of macadamia plantations in South Africa.

A Detection Assay to Identify Alternative Food Sources of the Two-Spotted Stink Bug, Bathycoelia distincta (Hemiptera: Pentatomidae).

The two-spotted stink bug, Bathycoelia distincta Distant (Hemiptera: Pentatomidae), is a serious pest in South African macadamia orchards. This pest is predominantly controlled using insecticides, thus alternative control methods are essential. The stink bugs arrive as adults in the orchards, during the early nut set season, but little is known about their alternative plant hosts before their arrival. The aim of this study was to develop a PCR-based metabarcoding assay to identify plant material in the gut of B. distincta. Thereafter, the persistence of plant DNA in the gut, after switching food sources, was determined by rearing the stink bugs on Zea mays L. (Cyperales: Poaceae), transferring them to Macadamia sp. and then collecting insects at different time points. As a proof of concept, the assay was tested on insects collected from commercial macadamia orchards to determine if it can identify alternative food sources. The chloroplast gene markers, trnL and trnF, were most successful for plant DNA amplification. The time trial suggested that plant material can be detected 24 h after switching to the alternate food source and one of the samples still contained Z. mays DNA after five days. Various plant species were detected from the orchard collected samples, including known food sources of other stink bugs, such as tea plants (Camellia sinensis L. (Ericales:Theaceae)) and sunflowers (Helianthus annuus L. (Asterales: Asteraceae)). This study provides the first indication of potential alternative food sources of B. distincta. The assay developed in this study can now be implemented for large-scale field surveys to contribute to future integrated pest management strategies.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

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.

The synergistic effect of concatenation in phylogenomics: the case in Pantoea.

With the increased availability of genome sequences for bacteria, it has become routine practice to construct genome-based phylogenies. These phylogenies have formed the basis for various taxonomic decisions, especially for resolving problematic relationships between taxa. Despite the popularity of concatenating shared genes to obtain well-supported phylogenies, various issues regarding this combined-evidence approach have been raised. These include the introduction of phylogenetic error into datasets, as well as incongruence due to organism-level evolutionary processes, particularly horizontal gene transfer and incomplete lineage sorting. Because of the huge effect that this could have on phylogenies, we evaluated the impact of phylogenetic conflict caused by organism-level evolutionary processes on the established species phylogeny for Pantoea, a member of the Enterobacterales. We explored the presence and distribution of phylogenetic conflict at the gene partition and nucleotide levels, by identifying putative inter-lineage recombination events that might have contributed to such conflict. Furthermore, we determined whether smaller, randomly constructed datasets had sufficient signal to reconstruct the current species tree hypothesis or if they would be overshadowed by phylogenetic incongruence. We found that no individual gene tree was fully congruent with the species phylogeny of Pantoea, although many of the expected nodes were supported by various individual genes across the genome. Evidence of recombination was found across all lineages within Pantoea, and provides support for organism-level evolutionary processes as a potential source of phylogenetic conflict. The phylogenetic signal from at least 70 random genes recovered robust, well-supported phylogenies for the backbone and most species relationships of Pantoea, and was unaffected by phylogenetic conflict within the dataset. Furthermore, despite providing limited resolution among taxa at the level of single gene trees, concatenated analyses of genes that were identified as having no signal resulted in a phylogeny that resembled the species phylogeny of Pantoea. This distribution of signal and noise across the genome presents the ideal situation for phylogenetic inference, as the topology from a ≥70-gene concatenated species phylogeny is not driven by single genes, and our data suggests that this finding may also hold true for smaller datasets. We thus argue that, by using a concatenation-based approach in phylogenomics, one can obtain robust phylogenies due to the synergistic effect of the combined signal obtained from multiple genes.

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.

Architecture and Distribution of Introns in Core Genes of Four Fusarium Species.

Removal of introns from transcribed RNA represents a crucial step during the production of mRNA in eukaryotes. Available whole-genome sequences and expressed sequence tags (ESTs) have increased our knowledge of this process and revealed various commonalities among eukaryotes. However, certain aspects of intron structure and diversity are taxon-specific, which can complicate the accuracy of in silico gene prediction methods. Using core genes, we evaluated the distribution and architecture of Fusarium circinatum spliceosomal introns, and linked these characteristics to the accuracy of the predicted gene models of the genome of this fungus. We also evaluated intron distribution and architecture in F. verticillioides, F. oxysporum, and F. graminearum, and made comparisons with F. circinatum Results indicated that F. circinatum and the three other Fusarium species have canonical 5' and 3' splice sites, but with subtle differences that are apparently not shared with those of other fungal genera. The polypyrimidine tract of Fusarium introns was also found to be highly divergent among species and genes. Furthermore, the conserved adenosine nucleoside required during the first step of splicing is contained within unique branch site motifs in certain Fusarium introns. Data generated here show that introns of F. circinatum, as well as F. verticillioides, F. oxysporum, and F. graminearum, are characterized by a number of unique features such as the CTHAH and ACCAT motifs of the branch site. Incorporation of such information into genome annotation software will undoubtedly improve the accuracy of gene prediction methods used for Fusarium species and related fungi.

The distribution and host range of the banana Fusarium wilt fungus, Fusarium oxysporum f. sp. cubense, in Asia.

Fusarium oxysporum formae specialis cubense (Foc) is a soil-borne fungus that causes Fusarium wilt, which is considered to be the most destructive disease of bananas. The fungus is believed to have evolved with its host in the Indo-Malayan region, and from there it was spread to other banana-growing areas with infected planting material. The diversity and distribution of Foc in Asia was investigated. A total of 594 F. oxysporum isolates collected in ten Asian countries were identified by vegetative compatibility groups (VCGs) analysis. To simplify the identification process, the isolates were first divided into DNA lineages using PCR-RFLP analysis. Six lineages and 14 VCGs, representing three Foc races, were identified in this study. The VCG complex 0124/5 was most common in the Indian subcontinent, Vietnam and Cambodia; whereas the VCG complex 01213/16 dominated in the rest of Asia. Sixty-nine F. oxysporum isolates in this study did not match any of the known VCG tester strains. In this study, Foc VCG diversity in Bangladesh, Cambodia and Sri Lanka was determined for the first time and VCGs 01221 and 01222 were first reported from Cambodia and Vietnam. New associations of Foc VCGs and banana cultivars were recorded in all the countries where the fungus was collected. Information obtained in this study could help Asian countries to develop and implement regulatory measures to prevent the incursion of Foc into areas where it does not yet occur. It could also facilitate the deployment of disease resistant banana varieties in infested areas.

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

Genome-wide macrosynteny among Fusarium species in the Gibberella fujikuroi complex revealed by amplified fragment length polymorphisms.

The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.

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.

An integrated hypnotherapeutic model for the treatment of childhood sexual trauma: a case study.

Sexual abuse appears to constitute a major risk factor for a variety of problems in adult life. The effects of abuse on adult living are not uniform therefore intervention strategies should be individualized to address unique symptom constellations. The purpose of this paper is to introduce an integrated Ericksonian and Ego state therapy approach, based on a strengths perspective for the treatment of survivors of childhood sexual abuse. The theoretical foundation for this model is described, followed by a case study. The case study demonstrates how application of this model enabled the client to resolve the experience of sexual abuse, as well as to enhance her sense of general psychological well-being.

Evolutionary relationships among the Fusarium oxysporum f. sp. cubense vegetative compatibility groups.

Fusarium oxysporum f. sp. cubense, the causal agent of fusarium wilt of banana (Musa spp.), is one of the most destructive strains of the vascular wilt fungus F. oxysporum. Genetic relatedness among and within vegetative compatibility groups (VCGs) of F. oxysporum f. sp. cubense was studied by sequencing two nuclear and two mitochondrial DNA regions in a collection of 70 F. oxysporum isolates that include representatives of 20 VCGs of F. oxysporum f. sp. cubense, other formae speciales, and nonpathogens. To determine the ability of F. oxysporum f. sp. cubense to sexually recombine, crosses were made between isolates of opposite mating types. Phylogenetic analysis separated the F. oxysporum isolates into two clades and eight lineages. Phylogenetic relationships between F. oxysporum f. sp. cubense and other formae speciales of F. oxysporum and the relationships among VCGs and races of F. oxysporum f. sp. cubense clearly showed that F. oxysporum f. sp. cubense's ability to cause disease on banana has emerged multiple times, independently, and that the ability to cause disease to a specific banana cultivar is also a polyphyletic trait. These analyses further suggest that both coevolution with the host and horizontal gene transfer may have played important roles in the evolutionary history of the pathogen. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system should sexual reproduction occur. Although, no sexual structures were observed, some lineages of F. oxysporum f. sp. cubense harbored MAT-1 and MAT-2 isolates, suggesting a potential that these lineages have a sexual origin that might be more recent than initially anticipated.

Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea.

Botryosphaeria lutea (anamorph Fusicoccum luteum) most easily is distinguished from other Botryosphaeria spp. by a yellow pigment that is formed in young cultures. This fungus has been reported from a number of cultivated hosts in New Zealand and Portugal. During a survey of Botryosphaeria fungi that occur on native Acacia species in Australia, a yellow pigment was observed in some cultures. These isolates were morphologically similar to B. lutea, but the pigment differed slightly from the one formed by authentic B. lutea isolates. Preliminary data also revealed small differences in ITS rDNA sequence data. The aim of this study was to determine whether these small differences were indicative of separate species or merely variations within B. lutea. Anamorph, teleomorph and culture morphology were compared between B. lutea and Acacia isolates from Australia. Sequence data of two other genome regions, namely the ß-tubulin and EF1-α gene and intron regions, were combined with ITS rDNA sequence data to determine the phylogenetic relationship between these isolates. Isolates of B. lutea and those from Australian Acacia species were not significantly different in spore morphology. The yellow pigment, however, was much more distinct in cultures of B. lutea than in cultures from Acacia. There were only a few base pair variations in each of the analyzed gene regions, but these variations were fixed in the two groups in all regions. By combining these data it was clear that B. lutea and the isolates from Acacia were distinct species, albeit very closely related. We, therefore, propose the new epithet B. australis for the fungus from Australia. Botryosphaeria australis also was isolated in this study from exotic Sequoiadendron trees in Australia. Re-analyses of GenBank data in this study showed that B. australis also occurs on other native Australian hosts, namely a Banksia sp. and a Eucalyptus sp., as well as a native Protea sp. in South Africa and on Pistachio in Italy. These records from GenBank have been identified previously as B. lutea. The common occurrence of B. australis on a variety of native hosts across Australia suggests that this fungus is native to this area.