Environmental DNA methods for biosecurity and invasion biology in terrestrial ecosystems: Progress, pitfalls, and prospects.

Analysis of environmental DNA (eDNA) enables indirect detection of species without the need to directly observe and sample them. For biosecurity and invasion biology, eDNA-based methods are useful to address biological invasions at all phases, from detecting arrivals to confirming eradication of past invasions. We conducted a systematic review of the literature and found that in biosecurity and invasion biology, eDNA has primarily been used to detect new incursions and monitor spread in marine and freshwater ecosystems, with much slower uptake in terrestrial ecosystems, reflecting a broader trend common to the usage of eDNA tools. In terrestrial ecosystems, eDNA research has mostly focussed on the use of eDNA metabarcoding to characterise biodiversity, rather than targeting biosecurity threats or non-native populations. We discuss how eDNA-based methods are being applied to terrestrial ecosystems for biosecurity and managing non-native populations at each phase of the invasion continuum: transport, introduction, establishment, and spread; across different management options: containment, control, and eradication; and for detecting the impact of non-native organisms. Finally, we address some of the current technical issues and caveats of eDNA-based methods, particularly for terrestrial ecosystems, and how these might be solved. As eDNA-based methods improve, they will play an increasingly important role in the early detection and adaptive management of biological invasions, and the implementation of effective biosecurity controls.

Can natural gene drives be part of future fungal pathogen control strategies in plants?

Globally, fungal pathogens cause enormous crop losses and current control practices are not always effective, economical or environmentally sustainable. Tools enabling genetic management of wild pathogen populations could potentially solve many problems associated with plant diseases. A natural gene drive from a heterologous species can be used in the globally important cereal pathogen Fusarium graminearum to remove pathogenic traits from contained populations of the fungus. The gene drive element became fixed in a freely crossing population in only three generations. Repeat-induced point mutation (RIP), a natural genome defence mechanism in fungi that causes C to T mutations during meiosis in highly similar sequences, may be useful to recall the gene drive following release, should a failsafe mechanism be required. We propose that gene drive technology is a potential tool to control plant pathogens once its efficacy is demonstrated under natural settings.

Evaluation of the rust fungus Puccinia rapipes for biological control of Lycium ferocissimum (African boxthorn) in Australia: Life cycle, taxonomy and pathogenicity.

Fungal plant pathogens are increasingly recognised as being among the most effective and safe agents in classical weed biological control programs worldwide. Suitability of the rust fungus P. rapipes as a classical biological control agent for Lycium ferocissimum (African boxthorn) in Australia was assessed using a streamlined agent selection framework. Studies with P. rapipes were undertaken to elucidate its life cycle, confirm its taxonomic placement and determine its pathogenicity to L. ferocissimum and seven closely-related Solanaceae species that occur in Australia. Field surveys in the native range of South Africa, experiments in a containment facility in Australia and DNA sequencing confirmed that P. rapipes is macrocyclic and autoecious, producing all five spore stages on L. ferocissimum. The stages not previously encountered, spermogonia and aecia, are described. Sequencing also confirmed that P. rapipes is sister to Puccinia afra, in the 'Old World Lineage' of Puccinia species on Lycieae. Two purified isolates of the fungus, representing the Eastern and Western Cape distributions of P. rapipes in South Africa, were cultured in the containment facility for use in pathogenicity testing. L. ferocissimum and all of the Lycium species of Eurasian origin tested ‒ Lycium barbarum (goji berry), Lycium chinense (goji berry 'chinense') and Lycium ruthenicum (black goji berry) - were susceptible to both isolates of P. rapipes. The Australian native L. australe and three more distantly related species in different genera tested were resistant to both isolates. The isolate from the Western Cape was significantly more pathogenic on L. ferocissimum from Australia, than the Eastern Cape isolate. Our results indicate that P. rapipes may be sufficiently host specific to pursue as a biological control agent in an Australian context, should regulators be willing to accept damage to the Eurasian goji berries being grown, albeit to a limited extent, in Australia.

Monitoring infected dogs after a canine brucellosis outbreak.

Many episodes of canine brucellosis in dog kennels have been reported but recently an outbreak that involved pets and their owners has been described. The purpose of this study was to confirm that the outbreak had a common source and evaluate the evolution of 4 dogs involved in this outbreak after the measures implemented that included a survey of 41 animals from the same area. The variable number of tandem repeat (VNTR) analysis indicated that the B. canis isolated from the human clustered together with the isolates collected from the canine pups. Two dogs continued with bacteremia after the first antibiotic therapy and from one of them B. canis was also isolated from urine showing the importance of the later in the infection dissemination. In an effort to protect the public, stray dogs should be controlled and educational programs about the risk of this zoonotic disease should be implemented.

Micro- and macrospatial scale analyses illustrates mixed mating strategies and extensive geneflow in populations of an invasive haploid pathogen.

Sexual reproduction in fungi involves either a single individual (selfing) or two individuals (outcrossing). To investigate the roles that these two strategies play in the establishment of an invasive alien pathogen, the Eucalyptus leaf-infecting fungus, Teratosphaeria (Mycosphaerella) nubilosa was studied. Specifically, the genetic diversity of the pathogen was investigated at micro and macrospatial scales. Interestingly, while data obtained at microspatial scales show clearly that selfing is the main reproductive strategy, at macrospatial scales the population genetic structure was consistent with a genetically outcrossing organism. Additional analyses were performed to explore these apparently discordant results at different spatial scales and to quantify the contribution of selfing vs. outcrossing to the genotypic diversity. The results clearly show that the fungus has a mixed mating strategy. While selfing is the predominant form of mating, outcrosses must have occurred in the pathogen that increased the genotypic diversity of the fungus over time. This mating strategy, coupled with the high levels of geneflow between distant populations of the pathogen, has created an even distribution of maximum diversity from the smallest (leaf) to largest scales (>500 km), which will make breeding for resistance difficult. These data illustrate the evolutionary potential and danger of the introduction of multiple genotypes of a potentially outcrossing pathogen, especially when it has a high dispersal potential.

Teratosphaeria nubilosa, a serious leaf disease pathogen of Eucalyptus spp. in native and introduced areas.

BACKGROUND: Teratosphaeria nubilosa is a serious leaf pathogen of several Eucalyptus spp. This review considers the taxonomic history, epidemiology, host associations and molecular biology of T. nubilosa. TAXONOMY: Kingdom Fungi; Phylum Ascomycota; Class Dothideomycetes; Order Capnodiales; Family Teratosphaeriaceae; genus Teratosphaeria; species nubilosa. IDENTIFICATION: Pseudothecia hypophyllous, less so amphigenous, ascomata black, globose becoming erumpent, asci aparaphysate, fasciculate, bitunicate, obovoid to ellipsoid, straight or incurved, eight-spored, ascospores hyaline, non-guttulate, thin walled, straight to slightly curved, obovoid with obtuse ends, medially one-septate, slightly constricted at the median septum, tapering to both ends, ascospore germination type F, germinating from both ends, germ tubes growing parallel to the long axis of the spore with distortion of the primary ascospore cell. HOST RANGE: Teratosphaeria nubilosa is a primary pathogen of several Eucalyptus spp., including E. botryoides, E. bicostata, E. bridgesiana, E. cypellocarpa, E. dunnii, E. globulus ssp. bicostata, E. globulus ssp. globulus, E. globulus ssp. maidenii, E. globulus ssp. pseudoglobulus, E. grandis, E. gunnii, E. nitens, E. pilularis, E. quadrangulata, E. viminalis, E. grandis x E. resinifera and E. urophylla x E. globulus. DISEASE SYMPTOMS: Leaf spots predominantly occur on juvenile Eucalyptus foliage; however, T. nubilosa has also recently been found on mature Eucalyptus foliage. Leaf spots are amphigenous, varying in size from small spots that are round to irregular. Lesions enlarge and coalesce to form larger blotches over the leaf surface. Initial lesions appear as pale-green spots surrounded by purple margins and, once mature, are generally yellow to pale brown with dark-brown raised borders. USEFUL WEBSITES: Mycobank, http://www.mycobank.org; Mycosphaerella identification website, http://www.cbs.knaw.nl/mycosphaerella/BioloMICS.aspx.

A multi-gene phylogeny for species of Mycosphaerella occurring on Eucalyptus leaves.

Species of the ascomycete genus Mycosphaerella are regarded as some of the most destructive leaf pathogens of a large number of economically important crop plants. Amongst these, approximately 60 Mycosphaerella spp. have been identified from various Eucalyptus spp. where they cause leaf diseases collectively known as Mycosphaerella Leaf Disease (MLD). Species concepts for this group of fungi remain confused, and hence their species identification is notoriously difficult. Thus, the introduction of DNA sequence comparisons has become the definitive characteristic used to distinguish species of Mycosphaerella. Sequences of the Internal Transcribed Spacer (ITS) region of the ribosomal RNA operon have most commonly been used to consider species boundaries in Mycosphaerella. However, sequences for this gene region do not always provide sufficient resolution for cryptic taxa. The aim of this study was, therefore, to use DNA sequences for three loci, ITS, Elongation factor 1-alpha (EF-1alpha) and Actin (ACT) to reconsider species boundaries for Mycosphaerella spp. from Eucalyptus. A further aim was to study the anamorph concepts and resolve the deeper nodes of Mycosphaerella, for which part of the Large Subunit (LSU) of the nuclear rRNA operon was sequenced. The ITS and EF-1alpha gene regions were found to be useful, but the ACT gene region did not provide species-level resolution in Mycosphaerella. A phylogeny of the combined DNA datasets showed that species of Mycosphaerella from Eucalyptus cluster in two distinct groups, which might ultimately represent discrete genera.

Mycosphaerella species causing leaf disease in South African Eucalyptus plantations.

Commercial Eucalyptus plantations provide an important source of hardwood for forestry industries, worldwide. Several species of Mycosphaerella are associated with a destructive Eucalyptus leaf disease known as Mycosphaerella Leaf Blotch (MLB). During 2000, a survey was undertaken in several commercial Eucalyptus growing areas of South Africa to determine the identity of the Mycosphaerella spp. contributing to outbreaks of MLB. Symptomatic leaf samples were collected from three major Eucalyptus growing areas and the Mycosphaerella spp. were isolated. Isolates were identified using morphology, ascospore germination patterns and sequence data from the ribosomal DNA operon. Six species, namely M. ellipsoidea, M. irregulariramosa, M. juvenis, M. lateralis, M. marksii, M. nubilosa, as well as a new species, described here as M. fori sp. nov., were recognized. Mycosphaerella nubilosa was the most common species isolated from commercial plantations, particularly on E. nitens, and appears to be the dominant species contributing to MLB. Data obtained in this study show that MLB is caused by a complex of species contributing to disease outbreaks in South Africa.