Unidirectional mating-type switching is underpinned by a conserved MAT1 locus architecture.

Unidirectional mating-type switching is a form of homothallic reproduction known only in a small number of filamentous ascomycetes. Their ascospores can give rise to either self-sterile isolates that require compatible partners for subsequent sexual reproduction, or self-fertile individuals capable of completing this process in isolation. The limited studies previously conducted in these fungi suggest that the differences in mating specificity are determined by the architecture of the MAT1 locus. In self-fertile isolates that have not undergone unidirectional mating-type switching, the locus contains both MAT1-1 and MAT1-2 mating-type genes, typical of primary homothallism. In the self-sterile isolates produced after a switching event, the MAT1-2 genes are lacking from the locus, likely due to a recombination-mediated deletion of the MAT1-2 gene information. To determine whether these arrangements of the MAT1 locus support unidirectional mating-type switching in the Ceratocystidaceae, the largest known fungal assemblage capable of this reproduction strategy, a combination of genetic and genomic approaches were used. The MAT1 locus was annotated in representative species of Ceratocystis, Endoconidiophora, and Davidsoniella. In all cases, MAT1-2 genes interrupted the MAT1-1-1 gene in self-fertile isolates. The MAT1-2 genes were flanked by two copies of a direct repeat that accurately predicted the boundaries of the deletion event that would yield the MAT1 locus of self-sterile isolates. Although the relative position of the MAT1-2 gene region differed among species, it always disrupted the MAT1-1-1 gene and/or its expression in the self-fertile MAT1 locus. Following switching, this gene and/or its expression was restored in the self-sterile arrangement of the locus. This mirrors what has been reported in other species capable of unidirectional mating-type switching, providing the strongest support for a conserved MAT1 locus structure that is associated with this process. This study contributes to our understanding of the evolution of unidirectional mating-type switching.

Characterization of the Fusarium circinatum biofilm environmental response role.

The capacity to form biofilms is a common trait among many microorganisms present on Earth. In this study, we demonstrate for the first time that the fatal pine pitch canker agent, Fusarium circinatum, can lead a biofilm-like lifestyle with aggregated hyphal bundles wrapped in extracellular matrix (ECM). Our research shows F. circinatum's ability to adapt to environmental changes by assuming a biofilm-like lifestyle. This was demonstrated by varying metabolic activities exhibited by the biofilms in response to factors like temperature and pH. Further analysis revealed that while planktonic cells produced small amounts of ECM per unit of the biomass, heat- and azole-exposed biofilms produced significantly more ECM than nonexposed biofilms, further demonstrating the adaptability of F. circinatum to changing environments. The increased synthesis of ECM triggered by these abiotic factors highlights the link between ECM production in biofilm and resistance to abiotic stress. This suggests that ECM-mediated response may be one of the key survival strategies of F. circinatum biofilms in response to changing environments. Interestingly, azole exposure also led to biofilms that were resistant to DNase, which typically uncouples biofilms by penetrating the biofilm and degrading its extracellular DNA; we propose that DNases were likely hindered from reaching target cells by the ECM barricade. The interplay between antifungal treatment and DNase enzyme suggests a complex relationship between eDNA, ECM, and antifungal agents in F. circinatum biofilms. Therefore, our results show how a phytopathogen's sessile (biofilm) lifestyle could influence its response to the surrounding environment.

A LAMP Assay for Rapid Detection of the Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen Fusarium circinatum is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by F. circinatum can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to F. circinatum. Using a globally representative collection of F. circinatum isolates and other closely related species, we have demonstrated that the assay can be used to identify F. circinatum across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.

Novel mating-type-associated genes and gene fragments in the genomes of Mycosphaerellaceae and Teratosphaeriaceae fungi.

The mating-type (MAT1) locus encodes transcription factors essential for the onset of the sexual cycle in ascomycete fungi. This locus has been characterised in only a few heterothallic, plant pathogenic Mycosphaerellaceae and Teratosphaeriaceae. We used available genome sequences for Mycosphaerellales species to investigate the presence of two unique mating-type-associated features. The accessory MAT1 genes, MAT1-1-10 (MATORF2) and MAT1-2-12 (MATORF1), typically occurred in both MAT idiomorphs of Mycosphaerellaceae species. In contrast, they were associated with only one idiomorph in Teratosphaeriaceae species. In Pseudocercospora, phylogenetic analyses showed that homologs present in different idiomorphs were paralogous and subject to different selective pressures, indicating that their evolution is linked to mating type. In almost half of the investigated Mycosphaerellales genomes, numerous short fragment sequences, almost identical to portions of the MAT1-1-1 and MAT1-2-1 genes, were present in multiple areas outside of the MAT1 locus. Aligned to the MAT1 genes, these sequences resembled an mRNA transcript. Fragment sequences were similar among species groups and occurred at the same genomic positions, implying that monophyletic groups have the same origins of these sequences. Although the functions of the MAT fragment sequences and accessory MAT1 genes remain unknown, both were expressed in the representative Mycosphaerellaceae and Teratosphaeriaceae species that were investigated.

Bradyrhizobium altum sp. nov., Bradyrhizobium oropedii sp. nov. and Bradyrhizobium acaciae sp. nov. from South Africa show locally restricted and pantropical nodA phylogeographic patterns.

Africa is known for its rich legume diversity with a significant number of endemic species originating in South Africa. Many of these legumes associate with rhizobial symbionts of the genus Bradyrhizobium, of which most represent new species. Yet, none of the Bradyrhizobium species from South Africa have been described. In this study, phylogenetic analysis of 16S rRNA gene sequences of fourteen strains isolated in southern Africa from root nodules of diverse legumes (i.e., from the tribes Crotalarieae, Acacieae, Genisteae, Phaseoleae and Cassieae) revealed that they belong to the Bradyrhizobium elkanii supergroup. The taxonomic position and possible novelty of these strains were further interrogated using genealogical concordance of five housekeeping genes (atpD, dnaK, glnII, gyrB and rpoB). These phylogenies consistently recovered four monophyletic groups and one singleton within Bradyrhizobium. Of these groups, two were conspecific with Bradyrhizobium brasilense UFLA 03-321T and Bradyrhizobium ivorense CI-1BT, while the remaining three represented novel taxa. Their existence was further supported with genome data, as well as metabolic and physiological traits. Analysis of nodA gene sequences further showed that the evolution of these bacteria likely involved adapting to local legume hosts and environmental conditions through the acquisition, via horizontal gene transfer, of optimal symbiotic loci. We accordingly propose the following names Bradyrhizobium acaciae sp. nov. 10BBT (SARCC 730T = LMG 31409T), Bradyrhizobium oropedii sp. nov. Pear76T (SARCC 731T = LMG 31408T), and Bradyrhizobium altum sp. nov. Pear77T (SARCC 754T = LMG 31407T) to accommodate three novel species, all of which are symbionts of legumes in South Africa.

Genomic characterization of a novel potyvirus infecting Barleria repens in South Africa.

The complete RNA genome sequence of a novel member of the genus Potyvirus infecting Barleria repens has been determined. A plant showing symptoms of virus-like leaf mosaic was sampled in the Western Cape province of South Africa, and the associated virus has been tentatively named "Barleria repens mottle virus" (BaRMoV). The genome of BaRMoV consists of 9,561 nucleotides and encodes a typical potyvirus polyprotein that is 3,105 amino acids long. Pairwise comparisons showed that the BaRMoV genome shared an average of 70% nucleotide sequence identity with that of yam mosaic virus (YMV), and the corresponding polyprotein shared an average of 76.6% amino acid sequence identity with that of YMV. Phylogenetic analysis of the BaRMoV polyprotein amino acid sequence and those of other extant potyviruses confirmed the relationship between BarMoV and YMV. This is the first time that a member of the family Potyviridae has been identified in a member of the species Barleria.

Genomic characterization of soybean blotchy mosaic virus, a cytorhabdovirus from South Africa.

Samples showing blotchy mottle symptoms were collected from soybeans in North-West province, South Africa. The assembly of high-throughput sequencing data from three samples yielded contigs of 13,426 to 13,435nt, which represent the first complete genome sequences of soybean blotchy mosaic virus (SbBMV). SbBMV shows a typical cytorhabdovirus gene organization (3'-N-P-P3-M-G-L-5'), with each putative gene product being most similar, but with only 49.1-71.1% sequence identity, to those of cucurbit cytorhabdovirus 1. Given the species demarcation thresholds for rhabdoviruses, SbBMV is thus a distinct member of the genus Cytorhabdovirus.

All ANIs are not created equal: implications for prokaryotic species boundaries and integration of ANIs into polyphasic taxonomy.

In prokaryotic taxonomy, a set of criteria is commonly used to delineate species. These criteria are generally based on cohesion at the phylogenetic, phenotypic and genomic levels. One such criterion shown to have promise in the genomic era is average nucleotide identity (ANI), which provides an average measure of similarity across homologous regions shared by a pair of genomes. However, despite the popularity and relative ease of using this metric, ANI has undergone numerous refinements, with variations in genome fragmentation, homologue detection parameters and search algorithms. To test the robustness of a 95-96 % species cut-off range across all the commonly used ANI approaches, seven different methods were used to calculate ANI values for intra- and interspecies datasets representing three classes in the Proteobacteria. As a reference point, these methods were all compared to the widely used blast-based ANI (i.e. ANIb as implemented in JSpecies), and regression analyses were performed to investigate the correlation of these methods to ANIb with more than 130000 individual data points. From these analyses, it was clear that ANI methods did not provide consistent results regarding the conspecificity of isolates. Most of the methods investigated did not correlate perfectly with ANIb, particularly between 90 and 100% identity, which includes the proposed species boundary. There was also a difference in the correlation of methods for the different taxon sets. Our study thus suggests that the specific approach employed needs to be considered when ANI is used to delineate prokaryotic species. We furthermore suggest that one would first need to determine an appropriate cut-off value for a specific taxon set, based on the intraspecific diversity of that group, before conclusions on conspecificity of isolates can be made, and that the resulting species hypotheses be confirmed with analyses based on evolutionary history as part of the polyphasic approach to taxonomy.

The mating system of the Eucalyptus canker pathogen Chrysoporthe austroafricana and closely related species.

Fungi in the genus Chrysoporthe are economically important canker pathogens of commercially grown Eucalyptus species and native Myrtales. Before the current study, homothallism was widely accepted as the mating system of these species, but this hypothesis could not be fully tested. Using whole genome sequences, we characterized the MAT locus of two C. austroafricana isolates and its sibling species, C. cubensis and C. deuterocubensis. A unique MAT1-2 idiomorph containing a truncated MAT1-1-1 gene, and a MAT1-1-2 gene, was identified in one isolate of C. austroafricana and a MAT1-1 idiomorph was found in the other. The presence of a single idiomorph in each isolate suggests that this fungus is heterothallic. Screening for MAT genes in 65 C. austroafricana isolates revealed a bias towards MAT1-2 idiomorphs suggesting a recent introduction in Eucalyptus species. Chrysoporthe cubensis and C. deuterocubensis are apparently homothallic since all the expected MAT genes were identified in their genome sequences. These findings were corroborated by the expression profiles of pheromone genes and their receptors, which conformed to the expected patterns observed in heterothallic and homothallic isolates. Long terminal repeat sequences (LTRs) and specifically retrotransposons were identified in the MAT locus of C. deuterocubensis and C. cubensis, indicating that the evolution of mating systems in Chrysoporthe species could be mediated by these elements.

Trinickia dabaoshanensis sp. nov., a new name for a lost species.

"Burkholderia dabaoshanensis" was described in 2012. Although the name was effectively published, it could not be validly published, because the description provided in the original paper did not comply with the Rule 27 (2) (c) of the Bacterial Code. The Code requiresthat the properties of the taxon form part of the protologue. As the name of this species does not have standing in nomenclature, the recently published new combination Trinickia dabaoshanensis could also not be validly published. The current proposal attempts to rectify the situation by providing the information required to meet the criteria stipulated in Rule 27 for valid publication.

Minimal standards for the description of new genera and species of rhizobia and agrobacteria.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

Paraburkholderia strydomiana sp. nov. and Paraburkholderia steynii sp. nov.: rhizobial symbionts of the fynbos legume Hypocalyptus sophoroides.

Twelve nodulating Paraburkholderia strains isolated from indigenous South African fynbos legume Hypocalyptus sophoroides were investigated to determine their taxonomic status. Genealogical concordance analysis, based on six loci (16S rRNA, atpD, recA, rpoB, lepA and gltB), revealed that they separate into two consistent and exclusive groups. Average nucleotide identity and DNA-DNA hybridisation comparisons indicated that they were sufficiently divergent from their closest known phylogenetic relatives (Paraburkholderia caledonica and Paraburkholderia terrae, respectively) to be regarded as novel species. This was also supported by the results of fatty acid analysis and metabolic characterisation. For these two isolate groups, we accordingly propose the new species Paraburkholderia strydomiana sp. nov. with WK1.1fT (= LMG 28731T = SARCC1213T) as its type strain and Paraburkholderia steynii sp. nov. with HC1.1baT (= LMG 28730T = SARCC696T) as its type strain. Our data thus showed that H. sophoroides may be considered a promiscuous symbiotic partner due to its ability to associate with multiple species of Paraburkholderia.

Unexpected placement of the MAT1-1-2 gene in the MAT1-2 idiomorph of Thielaviopsis.

Sexual reproduction in the Ascomycota is controlled by genes encoded at the mating-type or MAT1 locus. The two allelic versions of this locus in heterothallic species, referred to as idiomorphs, are defined by the MAT1-1-1 (for the MAT1-1 idiomorph) and MAT1-2-1 (for the MAT1-2 idiomorph) genes. Both idiomorphs can contain additional genes, although the contents of each is typically specific to and conserved within particular Pezizomycotina lineages. Using full genome sequences, complemented with conventional PCR and Sanger sequencing, we compared the mating-type idiomorphs in heterothallic species of Thielaviopsis (Ceratocystidaceae). The analyses showed that the MAT1-1 idiomorph of T. punctulata, T. paradoxa, T. euricoi, T. ethacetica and T. musarum harboured only the expected MAT1-1-1 gene. In contrast, the MAT1-2 idiomorph of T. punctulata, T. paradoxa and T. euricoi encoded the MAT1-2-1, MAT1-2-7 and MAT1-1-2 genes. Of these, MAT1-2-1 and MAT1-2-7 are genes previously reported in this idiomorph, while MAT1-1-2 is known only in the MAT1-1 idiomorph. Phylogenetic analysis showed that the Thielaviopsis MAT1-1-2 groups with the known homologues of this gene in other Microascales, thus confirming its annotation. Previous work suggests that MAT1-1-2 is involved in fruiting body development, a role that would be unaffected by its idiomorphic position. This notion is supported by our findings for the MAT1 locus structure in Thielaviopsis species. This also serves as the first example of a MAT1-1-specific gene restricted to only the MAT1-2 idiomorph.

Mixta gen. nov., a new genus in the Erwiniaceae.

The Erwiniaceae contain many species of agricultural and clinical importance. Although relationships among most of the genera in this family are relatively well resolved, the phylogenetic placement of several taxa remains ambiguous. In this study, we aimed to address these uncertainties by using a combination of phylogenetic and genomic approaches. Our multilocus sequence analysis and genome-based maximum-likelihood phylogenies revealed that the arsenate-reducing strain IMH and plant-associated strain ATCC 700886, both previously presumptively identified as members of Pantoea, represent novel species of Erwinia. Our data also showed that the taxonomy of Erwinia teleogrylli requires revision as it is clearly excluded from Erwinia and the other genera of the family. Most strikingly, however, five species of Pantoea formed a distinct clade within the Erwiniaceae, where it had a sister group relationship with the Pantoea + Tatumella clade. By making use of gene content comparisons, this new clade is further predicted to encode a range of characters that it shares with or distinguishes it from related genera. We thus propose recognition of this clade as a distinct genus and suggest the name Mixta in reference to the diverse habitats from which its species were obtained, including plants, humans and food products. Accordingly, a description for Mixta gen. nov. is provided to accommodate the four species Mixta calida comb. nov., M. gaviniae comb. nov., M. intestinalis comb. nov. and M. theicola comb. nov., with M. calida as the type species for the genus.

Practically delineating bacterial species with genealogical concordance.

Bacterial species are commonly defined by applying a set of predetermined criteria, including DNA-DNA hybridization values, 16S rRNA gene sequence similarity, phenotypic data as well as genome-based criteria such as average nucleotide identity or digital DNA-DNA hybridization. These criteria mostly allow for the delimitation of taxa that resemble typical bacterial species. Their application is often complicated when the objective is to delineate new species that are characterized by significant population-level diversity or recent speciation. However, we believe that these complexities and limitations can be easily circumvented by recognizing that bacterial species represent unique and exclusive assemblages of diversity. Within such a framework, methods that account for the population processes involved in species evolution are used to infer species boundaries. A method such as genealogical concordance analysis is well suited to delineate a putative species. The existence of the new taxon is then interrogated using an array of traditional and genome-based characters. By making use of taxa in the genera Pantoea, Paraburkholderia and Escherichia we demonstrate in a step-wise process how genealogical concordance can be used to delimit a bacterial species. Genetic, phenotypic and biological criteria were used to provide independent lines of evidence for the existence of that taxon. Our six-step approach to species recognition is straightforward and applicable to bacterial species especially in the post-genomic era, with increased availability of whole genome sequences. In fact, our results indicated that a combined genome-based comparative and evolutionary approach would be the preferred alternative for delineating coherent bacterial taxa.

Phylogenomic resolution of the bacterial genus Pantoea and its relationship with Erwinia and Tatumella.

Investigation of the evolutionary relationships between related bacterial species and genera with a variety of lifestyles have gained popularity in recent years. For analysing the evolution of specific traits, however, a robust phylogeny is essential. In this study we examined the evolutionary relationships among the closely related genera Erwinia, Tatumella and Pantoea, and also attempted to resolve the species relationships within Pantoea. To accomplish this, we used the whole genome sequence data for 35 different strains belonging to these three genera, as well as nine outgroup taxa. Multigene datasets consisting of the 1039 genes shared by these 44 strains were then generated and subjected to maximum likelihood phylogenetic analyses, after which the results were compared to those using conventional multi-locus sequence analysis (MLSA) and ribosomal MLSA (rMLSA) approaches. The robustness of the respective phylogenies was then explored by considering the factors typically responsible for destabilizing phylogenetic trees. We found that the nucleotide datasets employed in the MLSA, rMLSA and 1039-gene datasets contained significant levels of homoplasy, substitution saturation and differential codon usage, all of which likely gave rise to the observed lineage specific rate heterogeneity. The effects of these factors were much less pronounced in the amino acid dataset for the 1039 genes, which allowed reconstruction of a fully supported and resolved phylogeny. The robustness of this amino acid tree was also supported by different subsets of the 1039 genes. In contrast to the smaller datasets (MLSA and rMLSA), the 1039 amino acid tree was also not as sensitive to long-branch attraction. The robust and well-supported evolutionary hypothesis for the three genera, which confidently resolved their various inter- and intrageneric relationships, represents a valuable resource for future studies. It will form the basis for studies aiming to understand the forces driving the divergence and maintenance of lineages, species and biological traits in this important group of bacteria.

Genetic basis for high population diversity in Protea-associated Knoxdaviesia.

Sexual reproduction is necessary to generate genetic diversity and, in ascomycete fungi, this process is controlled by a mating type (MAT) locus with two complementary idiomorphs. Knoxdaviesia capensis and K. proteae (Sordariomycetes; Microascales; Gondwanamycetaceae) are host-specific saprophytic fungi that show high population diversity within their Protea plant hosts in the Cape Floristic Region of South Africa. We hypothesise that this diversity is the result of outcrossing driven by a heterothallic mating system and sought to describe the MAT1 loci of both species. The available genome assembly of each isolate contained only one of the MAT1 idiomorphs necessary for sexual reproduction, implying that both species are heterothallic. Idiomorph segregation during meiosis, a 1:1 ratio of idiomorphs in natural populations and mating experiments also supported heterothallism as a sexual strategy. Long-range PCR and shot-gun sequencing to identify the opposite idiomorph in each species revealed no sequence similarity between MAT1-1 and MAT1-2 idiomorphs, but the homologous idiomorphs between the species were almost identical. The MAT1-1 idiomorph contained the characteristic MAT1-1-1 and MAT1-1-2 genes, whereas the MAT1-2 idiomorph consisted of the genes MAT1-2-7 and MAT1-2-1. This gene content was similar to that of the three species in the Ceratocystidaceae (Microascales) with characterized MAT loci. The Knoxdaviesia MAT1-2-7 protein contained and alpha domain and predicted intron, which suggests that this gene arose from MAT1-1-1 during a recombination event. In contrast to the Ceratocystidaceae species, Knoxdaviesia conformed to the ancestral Sordariomycete arrangement of flanking genes and is, therefore, a closer reflection of the structure of this locus in the Microascalean ancestor.

Crotalarieae and Genisteae of the South African Great Escarpment are nodulated by novel Bradyrhizobium species with unique and diverse symbiotic loci.

The genus Bradyrhizobium contains predominantly nitrogen-fixing legume symbionts. Phylogenetic analysis of the genes responsible for their symbiotic abilities (i.e., those encoded on the nodulation [nod] and nitrogen-fixation [nif] loci) has facilitated the development of an extensive phylogeographic framework for the genus. This framework however contains only a few nodulating isolates from Africa. Here we focused on nodulating Bradyrhizobium isolates associated with native southern African legumes in the tribes Genisteae and Crotalarieae found along the Great Escarpment in the Mpumalanga Province of South Africa. The aims of this study were to: (1) obtain rhizobial isolates from legumes in the Genisteae and Crotalarieae; (2) verify their nodulation ability; (3) characterize them to species level based on phylogenetic analyses of several protein coding gene regions (atpD, dnaK, glnII, recA, rpoB and gyrB) and (4) determine their placement in the phylogeographic framework inferred from the sequences of the symbiotic loci nodA and nifD. Twenty of the 21 Bradyrhizobium isolates belonged to six novel species, while one was conspecific with the recently described B. arachidis. Among these isolates, the nodA phylogeny revealed several new clades, with 18 of our isolates found in Clades XIV and XV, and only three forming part of the cosmopolitan Clade III. These strains formed predominantly the same groups in the nifD phylogeny although with slight differences; indicating that both vertical and horizontal inheritance of the symbiotic loci occurred. These findings suggest that the largely unexplored diversity of indigenous African rhizobia are characterized by unique ancestries that might mirror the distribution of their hosts and the environmental factors driving their evolution.

Three novel lineages of 'Candidatus Liberibacter africanus' associated with native rutaceous hosts of Trioza erytreae in South Africa.

Greening disease of citrus in South Africa is associated with 'Candidatus Liberibacter africanus' (Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae). Despite the implementation of control strategies, this disease remains problematic, suggesting the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum capense were collected throughout the natural distribution of these trees in South Africa. Total DNA was extracted from samples and tested for the presence of liberibacters by a generic Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of tree host species from which liberibacter sequences were obtained was verified by sequencing host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10 specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences identical to that of 'Candidatus Liberibacter africanus subsp. capensis' (LafC), whereas those from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene regions revealed unique clusters for liberibacters associated with each tree species. We propose the following names for these novel liberibacters: 'Candidatus Liberibacter africanus subsp. clausenae' (LafCl), 'Candidatus Liberibacter africanus subsp. vepridis' (LafV) and 'Candidatus Liberibacter africanus subsp. zanthoxyli' (LafZ). This study did not find any natural hosts of Laf associated with greening of citrus. While native citrus relatives were shown to be infected with Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources of Laf to citrus orchards, per se.

Correction: Hough et al. Fungal Viruses Unveiled: A Comprehensive Review of Mycoviruses. Viruses 2023, 15, 1202.

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