W. Dunstan

Global diversity and geography of soil fungi

Introduction The kingdom Fungi is one of the most diverse groups of organisms on Earth, and they are integral ecosystem agents that govern soil carbon cycling, plant nutrition, and pathology. Fungi are widely distributed in all terrestrial ecosystems, but the distribution of species, phyla, and functional groups has been poorly documented. On the basis of 365 global soil samples from natural ecosystems, we determined the main drivers and biogeographic patterns of fungal diversity and community composition. Direct and indirect effects of climatic and edaphic variables on plant and fungal richness. Line thickness corresponds to the relative strength of the relationships between the variables that affect species richness. Dashed lines indicate negative relationships. MAP, mean annual precipitation; Fire, time since last fire; Dist. equator, distance from the equator; Ca, soil calcium concentration; P, soil phosphorus concentration; pH, soil pH. Rationale We identified soil-inhabiting fungi using 454 Life Sciences (Branford, CN) pyrosequencing and through comparison against taxonomically and functionally annotated sequence databases. Multiple regression models were used to disentangle the roles of climatic, spatial, edaphic, and floristic parameters on fungal diversity and community composition. Structural equation models were used to determine the direct and indirect effects of climate on fungal diversity, soil chemistry, and vegetation. We also examined whether fungal biogeographic patterns matched paradigms derived from plants and animals—namely, that species’ latitudinal ranges increase toward the poles (Rapoport’s rule) and diversity increases toward the equator. Last, we sought group-specific global biogeographic links among major biogeographic regions and biomes using a network approach and area-based clustering. Results Metabarcoding analysis of global soils revealed fungal richness estimates approaching the number of species recorded to date. Distance from equator and mean annual precipitation had the strongest effects on richness of fungi, including most fungal taxonomic and functional groups. Diversity of most fungal groups peaked in tropical ecosystems, but ectomycorrhizal fungi and several fungal classes were most diverse in temperate or boreal ecosystems, and many fungal groups exhibited distinct preferences for specific edaphic conditions (such as pH, calcium, or phosphorus). Consistent with Rapoport’s rule, the geographic range of fungal taxa increased toward the poles. Fungal endemicity was particularly strong in tropical regions, but multiple fungal taxa had cosmopolitan distribution. Conclusions Climatic factors, followed by edaphic and spatial patterning, are the best predictors of soil fungal richness and community composition at the global scale. Richness of all fungi and functional groups is causally unrelated to plant diversity, with the exception of ectomycorrhizal root symbionts, suggesting that plant-soil feedbacks do not influence the diversity of soil fungi at the global scale. The plant-to-fungi richness ratio declined exponentially toward the poles, indicating that current predictions—assuming globally constant ratios—overestimate fungal richness by 1.5- to 2.5-fold. Fungi follow similar biogeographic patterns as plants and animals, with the exception of several major taxonomic and functional groups that run counter to overall patterns. Strong biogeographic links among distant continents reflect relatively efficient long-distance dispersal compared with macro-organisms. Fungi play major roles in ecosystem processes, but the determinants of fungal diversity and biogeographic patterns remain poorly understood. Using DNA metabarcoding data from hundreds of globally distributed soil samples, we demonstrate that fungal richness is decoupled from plant diversity. The plant-to-fungus richness ratio declines exponentially toward the poles. Climatic factors, followed by edaphic and spatial variables, constitute the best predictors of fungal richness and community composition at the global scale. Fungi show similar latitudinal diversity gradients to other organisms, with several notable exceptions. These findings advance our understanding of global fungal diversity patterns and permit integration of fungi into a general macroecological framework. Global metagenomics detects hotspots of fungal diversity and macroecological patterns and indicates that plant and fungal diversity are uncoupled. [Also see Perspective by Wardle and Lindahl] Assessing fungal diversity worldwide Fungi are hyperdiverse but poorly known, despite their ecological and economic impacts. Tedersoo et al. collected nearly 15,000 topsoil samples from 365 sites worldwide and sequenced their genomes (see the Perspective by Wardle and Lindahl). Overall, they found a striking decline in fungal species richness with distance from the equator. For some specialist groups though, diversity depended more on the abundance of host plants than host diversity or geography. The findings reveal a huge gap between known and described species and the actual numbers of distinct fungi in the worlds soils. Science, this issue 10.1126/science.1256688; see also p. 1052

Multiple new Phytophthora species from ITS Clade 6 associated with natural ecosystems in Australia: evolutionary and ecological implications

During surveys of dying vegetation in natural ecosystems and associated waterways in Australia many new taxa have been identified from Phytophthora ITS Clade 6. For representative isolates, the region spanning the internal transcribed spacer region of the ribosomal DNA, the nuclear gene encoding heat shock protein 90 and the mitochondrial cox1 gene were PCR amplified and sequenced. Based on phylogenetic analysis and morphological and physiological comparison, four species and one informally designated taxon have been described; Phytophthora gibbosa, P. gregata, P. litoralis, P. thermophila and P. taxon paludosa. Phytophthora gibbosa, P. gregata and P. taxon paludosa form a new cluster and share a common ancestor; they are homothallic and generally associated with dying vegetation in swampy or water-logged areas. Phytophthora thermophila and P. litoralis are sister species to each other and more distantly to P. gonapodyides. Both new species are common in waterways and cause scattered mortality within native vegetation. They are self-sterile and appear well adapted for survival in an aquatic environment and inundated soils, filling the niche occupied by P. gonapodyides and P. taxon salixsoil in the northern hemisphere. Currently the origin of these new taxa, their pathogenicity and their role in natural ecosystems are unknown. Following the precautionary principle, they should be regarded as a potential threat to native ecosystems and managed to minimise their further spread.

The diversity of ectomycorrhizal fungi associated with introduced Pinus spp. in the Southern Hemisphere, with particular reference to Western Australia

A device and method for collecting a liquid, such as urine, with little or no contamination thereof. The device when delivered for use includes a container member, an intermediate member secured to the container and a cap member encased in a closed bag and in turn secured to the intermediate member through the bag. During use the cap member is removed from the intermediate member and placed on a convenient surface, still in the closed bag. The intermediate member has a handle which then permits the user easily to hold the container in position for voiding urine into the container. The intermediate member is then removed from the container and discarded. The uncontaminated cap member is then removed from the closed bag and is threadably secured to the container so that the urine contained therein can be delivered for analysis in a substantially uncontaminated state.

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria spp. with Eucalyptus diversicolor seedlings. Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L. bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2–3 months).

Persistence of some Australian Pisolithus species introduced into eucalypt plantations in China

Some eucalypt plantations in south China grow poorly because of soil infertility and low diversity of compatible ectomycorrhizal fungi. One option to improve productivity of plantations is to introduce beneficial fungi. In order to evaluate persistence of introduced symbiotic fungi, there is a need to discriminate them from any indigenous related species. Eucalypt mycorrhizal trial sites, established for nutrition and survival studies, were used for this purpose. Seedlings of Eucalyptus camaldulensis and Eucalyptus urophylla had been inoculated with pure cultures of selected Australian ectomycorrhizal fungi, including three isolates of Pisolithus albus and one isolate each of two unnamed Pisolithus spp., and ectomycorrhizal seedlings had been out-planted at two sites in Guangzhou Province, Peoples Republic of China. Sequences of the internal transcribed spacers (ITSs) of ribosomal DNA (rDNA) from inoculant fungi and fungi re-isolated from sporocarps in the field were compared. Sequencing confirmed that an Australian isolate (H4111) of one unnamed Pisolithus spp. had persisted in competition with an indigenous Pisolithus spp. Selected isolates of Australian Pisolithus formed macro- and micro-morphologically typical ectomycorrhizas in pure culture syntheses and in the field. In comparison, the indigenous Chinese Pisolithus formed an incomplete association with a poorly developed mantle. Comparisons between ITS sequences from Pisolithus isolates associated with Eucalyptus spp. from elsewhere in the world and ITS sequences of Australian Pisolithus spp. indicate that the same unnamed Pisolithus spp. has also become established in Portugal, Brazil and South Africa. Because Pisolithus isolate H4111 produced sporocarps in south China under eucalypts and promoted tree growth, this fungus would be useful in spore orchards to provide spore inoculum for eucalypt nurseries. The local Chinese Pisolithus is not recommended for inoculation programs because it is ineffective in forming mycorrhizas with eucalypts. The isolate H4111 is from a Pisolithus that occurs naturally along the east coast of Australia. Although this Pisolithus has been widely dispersed with eucalypts in other parts of the world, it is not present in eucalypt plantations in east Asia.

Phytophthora inundata from native vegetation in Western Australia

Phytophthora inundata was found associated with a dead Xanthorrhoea preissii in Eucalyptus marginata (jarrah) forest in the south-west of Western Australia in January 2005. The isolate was sterile in pure culture and when paired with tester isolates of A1 and A2 mating types of other Phytophthora species. Mature sporangia were produced in 4 hours and discharged zoospores very soon after maturation. Some hyphal swellings were observed. Another isolate of P. inundata was isolated in 2005 from soil from Dandaragan on the sandplains north of Perth. Phytophthora isolates collected from dying native vegetation near the South Coast in 1986 and near Mount Barker in 1997 were also shown to be P. inundata. This is the first record of P. inundata from Australian natural ecosystems.

Distribution and diversity of Phytophthora across Australia

The introduction and subsequent impact of Phytophthora cinnamomi within native vegetation is one of the major conservation issues for biodiversity in Australia. Recently, many new Phytophthora species have been described from Australia’s native ecosystems; however, their distribution, origin, and potential impact remain unknown. Historical bias in Phytophthora detection has been towards sites showing symptoms of disease, and traditional isolation methods show variable effectiveness of detecting different Phytophthora species. However, we now have at our disposal new techniques based on the sampling of environmental DNA and metabarcoding through the use of high-throughput sequencing. Here, we report on the diversity and distribution of Phytophthora in Australia using metabarcoding of 640 soil samples and we compare the diversity detected using this technique with that available in curated databases. Phytophthora was detected in 65% of sites, and phylogenetic analysis revealed 68 distinct Phytophthora phylotypes. Of these, 21 were identified as potentially unique taxa and 25 were new detections in natural areas and/or new introductions to Australia. There are 66 Phytophthora taxa listed in Australian databases, 43 of which were also detected in this metabarcoding study. This study revealed high Phytophthora richness within native vegetation and the additional records provide a valuable baseline resource for future studies. Many of the Phytophthora species now uncovered in Australia’s native ecosystems are newly described and until more is known we need to be cautious with regard to the spread and conservation management of these new species in Australia’s unique ecosystems.

A new homothallic Phytophthora from the jarrah forest in Western Australia

A homothallic Phytophthora, isolated at irregular intervals since the 1980s from Western Australian jarrah (Eucalyptus marginata) forest sites, has been previously misidentified as P. citricola based on morphological characters. Based on rDNA sequencing, it has been shown to be an undescribed species that is provisionally designated ‘Phytophthora sp. WA2’ pending a full description. It has been associated with lower stem lesions and root necrosis on dying 1- to 2-year-old jarrah seedlings growing in rehabilitated open-cut bauxite mine pits.

Pathways to false-positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study

Abstract Phytophthora cinnamomi is one of the worlds most invasive plant pathogens affecting ornamental plants, horticultural crops and natural ecosystems. Accurate diagnosis is very important to determine the presence or absence of this pathogen in diseased and asymptomatic plants. In previous studies, P. cinnamomi species‐specific primers were designed and tested using various polymerase chain reaction (PCR) techniques including conventional PCR, nested PCR and quantitative real‐time PCR. In all cases, the primers were stated to be highly specific and sensitive to P. cinnamomi. However, few of these studies tested their primers against closely related Phytophthora species (Phytophthora clade 7). In this study, we tested these purported P. cinnamomi‐specific primer sets against 11 other species from clade 7 and determined their specificity; of the eight tested primer sets only three were specific to P. cinnamomi. This study demonstrated the importance of testing primers against closely related species within the same clade, and not just other species within the same genus. The findings of this study are relevant to all species‐specific microbial diagnosis.

An overview of Australia's Phytophthora species assemblage in natural ecosystems recovered from a survey in Victoria

Although Phytophthora species cause serious diseases worldwide, until recently the main focus on disease in natural ecosystems in southern Australia has been on the distribution and impact of P. cinnamomi. However, new Phytophthora pathogens have emerged from natural ecosystems, and there is a need to better understand the diversity and distribution of these species in our natural forests, woodlands and heathlands. From a survey along a 70 km pipeline easement in Victoria, Phytophthora species were isolated from 249 rhizosphere samples and 25 bait bags deployed in 21 stream, river, or wetland locations. Of the 186 Phytophthora isolates recovered, 130 were identified to species based on ITS sequence data. Ninety-five isolates corresponded to 13 described Phytophthora species while additionally 35 isolates were identified as Clade 6 hybrids. Phytophthora cinnamomi was the most common species isolated (31 %), followed by P. elongata (6 %), both species were only recovered from soil. Samples from sites with the highest soil moisture at the time of sampling had the highest yield of isolates. Consistent with other studies throughout the world, Clade 6 species and their hybrids dominated water samples, although many of these species were also recovered less frequently from soil samples. Many of the species recovered in this study have not previously been reported from eastern Australia, reinforcing that Phytophthora species are widespread, abundant and diverse in natural ecosystems. We have probably been underestimating Phytophthora diversity in Australia.