Natural infection of white grubs (Coleoptera: Scarabaeidae) with entomopathogenic nematodes in the KwaZulu-Natal province of South Africa.

White grubs are root feeding larvae of beetles (Coleoptera: Scarabaeidae) that are sporadic pests in agriculture and can lead to economic damage. The grubs feed on the roots of plants, while the adult beetle can bore into underground stems, as well as cause defoliation of plants. Sporadic incidence of larvae with symptoms of nematode infections were detected in wattle and sugarcane plantations in the KwaZulu-Natal province of South Africa. The larvae with infection symptoms were isolated, washed, and put on water traps to collect infective juveniles of possible nematode infections. Three species of entomopathogenic nematodes (EPNs) were isolated from the white grub larvae. These included Steinernema bertusi isolated from Maladera sp. 4., Oscheius myriophila from Maladera sp. 4 and Schizonchya affinis, and Steinernema fabii isolated from Maladera sp. 4., Pegylis sommeri, and S. affinis. Of these S. fabii was the most common species in the sample (87%). This is the first report of such a high diversity of locally occurring EPNs found naturally associated with white grub species in this region of South Africa.

Confronting the constraints of morphological taxonomy in the Botryosphaeriales.

Identification of fungi and the International Code of Nomenclature underpinning this process, rests strongly on the characterisation of morphological structures. Yet, the value of these characters to define species in many groups has become questionable or even superfluous. This has emerged as DNA-based techniques have increasingly revealed cryptic species and species complexes. This problem is vividly illustrated in the present study where 105 isolates of the Botryosphaeriales were recovered from both healthy and diseased woody tissues of native Acacia spp. in Namibia and South Africa. Thirteen phylogenetically distinct groups were identified based on Internal Transcribed Spacer (ITS) rDNA PCR-RFLP and translation elongation factor 1-α (TEF1-α) sequence data, two loci that are known to be reliable markers to distinguish species in the Botryosphaeriales. Four of these groups could be linked reliably to sequence data for formerly described species, including Botryosphaeria dothidea, Dothiorella dulcispinae, Lasiodiplodia pseudotheobromae and Spencermartinsia viticola. Nine groups, however, could not be linked to any other species known from culture and for which sequence data are available. These groups are, therefore, described as Aplosporella africana, A. papillata, Botryosphaeria auasmontanum, Dothiorella capri-amissi, Do. oblonga, Lasiodiplodia pyriformis, Spencermartinsia rosulata, Sphaeropsis variabilis and an undescribed Neofusicoccum sp. The species described here could not be reliably compared with the thousands of taxa described in these genera from other hosts and regions, where only morphological data are available. Such comparison would be possible only if all previously described taxa are epitypified, which is not a viable objective for the two families, Botryosphaeriaceae and Aplosporellaceae, in the Botryosphaeriales identified here. The extent of diversity of the Botryosphaeriales revealed in this and other recent studies is expected to reflect that of other undersampled regions and hosts, and illustrates the urgency to find more effective ways to describe species in this, and indeed other, groups of fungi.

Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework.

The order Botryosphaeriales represents several ecologically diverse fungal families that are commonly isolated as endophytes or pathogens from various woody hosts. The taxonomy of members of this order has been strongly influenced by sequence-based phylogenetics, and the abandonment of dual nomenclature. In this study, the phylogenetic relationships of the genera known from culture are evaluated based on DNA sequence data for six loci (SSU, LSU, ITS, EF1, BT, mtSSU). The results make it possible to recognise a total of six families. Other than the Botryosphaeriaceae (17 genera), Phyllostictaceae (Phyllosticta) and Planistromellaceae (Kellermania), newly introduced families include Aplosporellaceae (Aplosporella and Bagnisiella), Melanopsaceae (Melanops), and Saccharataceae (Saccharata). Furthermore, the evolution of morphological characters in the Botryosphaeriaceae were investigated via analysis of phylogeny-trait association. None of the traits presented a significant phylogenetic signal, suggesting that conidial and ascospore pigmentation, septation and appendages evolved more than once in the family. Molecular clock dating on radiations within the Botryosphaeriales based on estimated mutation rates of the rDNA SSU locus, suggests that the order originated in the Cretaceous period around 103 (45-188) mya, with most of the diversification in the Tertiary period. This coincides with important periods of radiation and spread of the main group of plants that these fungi infect, namely woody Angiosperms. The resulting host-associations and distribution could have influenced the diversification of these fungi. TAXONOMIC NOVELTIES: New families - Aplosporellaceae Slippers, Boissin & Crous, Melanopsaceae Phillips, Slippers, Boissin & Crous, Saccharataceae Slippers, Boissin & Crous.

The Botryosphaeriaceae: genera and species known from culture.

In this paper we give an account of the genera and species in the Botryosphaeriaceae. We consider morphological characters alone as inadequate to define genera or identify species, given the confusion it has repeatedly introduced in the past, their variation during development, and inevitable overlap as representation grows. Thus it seems likely that all of the older taxa linked to the Botryosphaeriaceae, and for which cultures or DNA sequence data are not available, cannot be linked to the species in this family that are known from culture. Such older taxa will have to be disregarded for future use unless they are epitypified. We therefore focus this paper on the 17 genera that can now be recognised phylogenetically, which concentrates on the species that are presently known from culture. Included is a historical overview of the family, the morphological features that define the genera and species and detailed descriptions of the 17 genera and 110 species. Keys to the genera and species are also provided. Phylogenetic relationships of the genera are given in a multi-locus tree based on combined SSU, ITS, LSU, EF1-α and ß-tubulin sequences. The morphological descriptions are supplemented by phylogenetic trees (ITS alone or ITS + EF1-α) for the species in each genus. TAXONOMIC NOVELTIES: New species - Neofusicoccum batangarum Begoude, Jol. Roux & Slippers. New combinations - Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves, Botryosphaeria ramosa (Pavlic, T.I. Burgess, M.J. Wingf.) A.J.L. Phillips & A. Alves, Cophinforma atrovirens (Mehl & Slippers) A. Alves & A.J.L. Phillips, Cophinforma mamane (D.E. Gardner) A.J.L. Phillips & A. Alves, Dothiorella pretoriensis (Jami, Gryzenh., Slippers & M.J. Wingf.) Abdollahz. & A.J.L. Phillips, Dothiorella thailandica (D.Q. Dai., J.K. Liu & K.D. Hyde) Abdollahz., A.J.L. Phillips & A. Alves, Dothiorella uruguayensis (C.A. Pérez, Blanchette, Slippers & M.J. Wingf.) Abdollahz. & A.J.L. Phillips, Lasiodiplodia lignicola (Ariyawansa, J.K. Liu & K.D. Hyde) A.J.L. Phillips, A. Alves & Abdollahz., Neoscytalidium hyalinum (C.K. Campb. & J.L. Mulder) A.J.L. Phillips, Groenewald & Crous, Sphaeropsis citrigena (A.J.L. Phillips, P.R. Johnst. & Pennycook) A.J.L. Phillips & A. Alves, Sphaeropsis eucalypticola (Doilom, J.K. Liu, & K.D. Hyde) A.J.L. Phillips, Sphaeropsis porosa (Van Niekerk & Crous) A.J.L. Phillips & A. Alves. Epitypification (basionym) - Sphaeria sapinea Fries. Neotypifications (basionyms) - Botryodiplodia theobromae Pat., Physalospora agaves Henn, Sphaeria atrovirens var. visci Alb. & Schwein.

Fungal Planet description sheets: 154-213.

Novel species of microfungi described in the present study include the following from South Africa: Camarosporium aloes, Phaeococcomyces aloes and Phoma aloes from Aloe, C. psoraleae, Diaporthe psoraleae and D. psoraleae-pinnatae from Psoralea, Colletotrichum euphorbiae from Euphorbia, Coniothyrium prosopidis and Peyronellaea prosopidis from Prosopis, Diaporthe cassines from Cassine, D. diospyricola from Diospyros, Diaporthe maytenicola from Maytenus, Harknessia proteae from Protea, Neofusicoccum ursorum and N. cryptoaustrale from Eucalyptus, Ochrocladosporium adansoniae from Adansonia, Pilidium pseudoconcavum from Greyia radlkoferi, Stagonospora pseudopaludosa from Phragmites and Toxicocladosporium ficiniae from Ficinia. Several species were also described from Thailand, namely: Chaetopsina pini and C. pinicola from Pinus spp., Myrmecridium thailandicum from reed litter, Passalora pseudotithoniae from Tithonia, Pallidocercospora ventilago from Ventilago, Pyricularia bothriochloae from Bothriochloa and Sphaerulina rhododendricola from Rhododendron. Novelties from Spain include Cladophialophora multiseptata, Knufia tsunedae and Pleuroascus rectipilus from soil and Cyphellophora catalaunica from river sediments. Species from the USA include Bipolaris drechsleri from Microstegium, Calonectria blephiliae from Blephilia, Kellermania macrospora (epitype) and K. pseudoyuccigena from Yucca. Three new species are described from Mexico, namely Neophaeosphaeria agaves and K. agaves from Agave and Phytophthora ipomoeae from Ipomoea. Other African species include Calonectria mossambicensis from Eucalyptus (Mozambique), Harzia cameroonensis from an unknown creeper (Cameroon), Mastigosporella anisophylleae from Anisophyllea (Zambia) and Teratosphaeria terminaliae from Terminalia (Zimbabwe). Species from Europe include Auxarthron longisporum from forest soil (Portugal), Discosia pseudoartocreas from Tilia (Austria), Paraconiothyrium polonense and P. lycopodinum from Lycopodium (Poland) and Stachybotrys oleronensis from Iris (France). Two species of Chrysosporium are described from Antarctica, namely C. magnasporum and C. oceanitesii. Finally, Licea xanthospora is described from Australia, Hypochnicium huinayensis from Chile and Custingophora blanchettei from Uruguay. Novel genera of Ascomycetes include Neomycosphaerella from Pseudopentameris macrantha (South Africa), and Paramycosphaerella from Brachystegia sp. (Zimbabwe). Novel hyphomycete genera include Pseudocatenomycopsis from Rothmannia (Zambia), Neopseudocercospora from Terminalia (Zambia) and Neodeightoniella from Phragmites (South Africa), while Dimorphiopsis from Brachystegia (Zambia) represents a novel coelomycetous genus. Furthermore, Alanphillipsia is introduced as a new genus in the Botryosphaeriaceae with four species, A. aloes, A. aloeigena and A. aloetica from Aloe spp. and A. euphorbiae from Euphorbia sp. (South Africa). A new combination is also proposed for Brachysporium torulosum (Deightoniella black tip of banana) as Corynespora torulosa. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Retracing the routes of introduction of invasive species: the case of the Sirex noctilio woodwasp.

Understanding the evolutionary histories of invasive species is critical to adopt appropriate management strategies, but this process can be exceedingly complex to unravel. As illustrated in this study of the worldwide invasion of the woodwasp Sirex noctilio, population genetic analyses using coalescent-based scenario testing together with Bayesian clustering and historical records provide opportunities to address this problem. The pest spread from its native Eurasian range to the Southern Hemisphere in the 1900s and recently to Northern America, where it poses economic and potentially ecological threats to planted and native Pinus spp. To investigate the origins and pathways of invasion, samples from five continents were analysed using microsatellite and sequence data. The results of clustering analysis and scenario testing suggest that the invasion history is much more complex than previously believed, with most of the populations being admixtures resulting from independent introductions from Europe and subsequent spread among the invaded areas. Clustering analyses revealed two major source gene pools, one of which the scenario testing suggests is an as yet unsampled source. Results also shed light on the microevolutionary processes occurring during introductions, and showed that only few specimens gave rise to some of the populations. Analyses of microsatellites using clustering and scenario testing considered against historical data drastically altered our understanding of the invasion history of S. noctilio and will have important implications for the strategies employed to fight its spread. This study illustrates the value of combining clustering and ABC methods in a comprehensive framework to dissect the complex patterns of spread of global invaders.

Gene expression associated with vegetative incompatibility in Amylostereum areolatum.

In filamentous fungi, vegetative compatibility among individuals of the same species is determined by the genes encoded at the heterokaryon incompatibility (het) loci. The hyphae of genetically similar individuals that share the same allelic specificities at their het loci are able to fuse and intermingle, while different allelic specificities at the het loci result in cell death of the interacting hyphae. In this study, suppression subtractive hybridization (SSH) followed by pyrosequencing and quantitative reverse transcription PCR were used to identify genes that are selectively expressed when vegetatively incompatible individuals of Amylostereum areolatum interact. The SSH library contained genes associated with various cellular processes, including cell-cell adhesion, stress and defence responses, as well as cell death. Some of the transcripts encoded proteins that were previously implicated in the stress and defence responses associated with vegetative incompatibility. Other transcripts encoded proteins known to be associated with programmed cell death, but have not previously been linked with vegetative incompatibility. Results of this study have considerably increased our knowledge of the processes underlying vegetative incompatibility in Basidiomycetes in general and A. areolatum in particular.

Genetic linkage map for Amylostereum areolatum reveals an association between vegetative growth and sexual and self-recognition.

Amylostereum areolatum is a filamentous fungus that grows through tip extension, branching and hyphal fusion. In the homokaryotic phase, the hyphae of different individuals are capable of fusing followed by heterokaryon formation, only if they have dissimilar allelic specificities at their mating-type (mat) loci. In turn, hyphal fusion between heterokaryons persists only when they share the same alleles at all of their heterokaryon incompatibility (het) loci. In this study we present the first genetic linkage map for A. areolatum, onto which the mat and het loci, as well as quantitative trait loci (QTLs) for mycelial growth rate are mapped. The recognition loci (mat-A and het-A) are positioned near QTLs associated with mycelial growth, suggesting that the genetic determinants influencing recognition and growth rate in A. areolatum are closely associated. This was confirmed when isolates associated with specific mat and het loci displayed significantly different mycelial growth rates. Although the link between growth and sexual recognition has previously been observed in other fungi, this is the first time that an association between growth and self-recognition has been shown.

Levels of specificity of Xylaria species associated with fungus-growing termites: a phylogenetic approach.

Fungus-growing termites live in obligate mutualistic symbiosis with species of the basidiomycete genus Termitomyces, which are cultivated on a substrate of dead plant material. When the termite colony dies, or when nest material is incubated without termites in the laboratory, fruiting bodies of the ascomycete genus Xylaria appear and rapidly cover the fungus garden. This raises the question whether certain Xylaria species are specialised in occupying termite nests or whether they are just occasional visitors. We tested Xylaria specificity at four levels: (1) fungus-growing termites, (2) termite genera, (3) termite species, and (4) colonies. In South Africa, 108 colonies of eight termite species from three termite genera were sampled for Xylaria. Xylaria was isolated from 69% of the sampled nests and from 57% of the incubated fungus comb samples, confirming high prevalence. Phylogenetic analysis of the ITS region revealed 16 operational taxonomic units of Xylaria, indicating high levels of Xylaria species richness. Not much of this variation was explained by termite genus, species, or colony; thus, at level 2-4 the specificity is low. Analysis of the large subunit rDNA region, showed that all termite-associated Xylaria belong to a single clade, together with only three of the 26 non-termite-associated strains. Termite-associated Xylaria thus show specificity for fungus-growing termites (level 1). We did not find evidence for geographic or temporal structuring in these Xylaria phylogenies. Based on our results, we conclude that termite-associated Xylaria are specific for fungus-growing termites, without having specificity for lower taxonomic levels.

Planted forest health: The need for a global strategy.

Several key tree genera are used in planted forests worldwide, and these represent valuable global resources. Planted forests are increasingly threatened by insects and microbial pathogens, which are introduced accidentally and/or have adapted to new host trees. Globalization has hastened tree pest emergence, despite a growing awareness of the problem, improved understanding of the costs, and an increased focus on the importance of quarantine. To protect the value and potential of planted forests, innovative solutions and a better-coordinated global approach are needed. Mitigation strategies that are effective only in wealthy countries fail to contain invasions elsewhere in the world, ultimately leading to global impacts. Solutions to forest pest problems in the future should mainly focus on integrating management approaches globally, rather than single-country strategies. A global strategy to manage pest issues is vitally important and urgently needed.

A class-wide phylogenetic assessment of Dothideomycetes.

We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidae is expanded to include the aquatic order Jahnulales. An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.

DNA sequence and RFLP data reflect geographical spread and relationships of Amylostereum areolatum and its insect vectors.

The white rot fungus, Amylostereum areolatum (Basidiomycetes), is best known for its symbiotic relationship with various siricid wood wasp species. In this study, the relationship between isolates of A. areolatum associated with two wood wasp species, Sirex noctilio and S. juvencus, are considered to identify possible intraspecific groups. Isolates from the northern (native) and southern (exotic) hemispheres are included to determine patterns of geographical spread and origin of introductions into the southern hemisphere. The phylogenetic relationships of these isolates to authentic isolates of A. chailletii, A. laevigatum and A. ferreum were also investigated. Sequence and restriction fragment length polymorphism (RFLP) analyses of the variable nuc-IGS-rDNA region provided markers to distinguish intraspecific groups within A. areolatum. Isolates of A. areolatum associated with S. noctilio and S. juvencus contained four heterogenic sequences in the DNA region analysed. These sequences occurred in one of five combinations in each isolate. Some of these sequences were unique to isolates of A. areolatum from either wasp species, while others were present in both groups. This shows the ancient and specialized evolutionary relationship that exists between these insects and fungi. Isolates from the southern hemisphere all share the same sequence group. This supports previous hypotheses that S. noctilio has spread between countries and continents of this region. At the interspecific level, the IGS-rDNA sequence analysis showed that A. ferreum and A. laevigatum are closely related to each other, and they in turn are related to A. chailletii. Amylostereum areolatum was the most distinctly defined species in the genus. This can be attributed to the obligate relationship between A. areolatum and its insect vectors. Polymerase chain reaction-RFLP analysis was also shown to be an effective tool to distinguish between the different species of Amylostereum.