Variation in host specificity and gene content in strains from genetically isolated lineages of the ectomycorrhizal fungus Paxillus involutus s. lat.

Ectomycorrhizal fungi are known to vary in host range. Some fungi can enter into symbiosis with multiple plant species, while others have restricted host ranges. The aim of this study was to examine variation in host specificity among strains from the basidiomycete Paxillus involutus s. lat. Recent studies have shown that this fungus consists of at least four genetically isolated lineages, phylogenetic species (PS) I (which corresponds to the morphological species Paxillus obscurosporus), PS II (P. involutus s. str.), PS III (Paxillus validus), and PS IV (not yet supported by any reference material). Thirty-five Paxillus strains of PS I to IV were examined in microcosms for their capacity to infect birch (Betula pendula) and spruce (Picea abies). Seventeen strains were compatible and formed mycorrhizae with both tree species. Seven strains were incompatible with both birch and spruce. The gene content in three pairs of incompatible and compatible strains PS I, II, and III were compared using microarray-based comparative genomic hybridizations. Of 4,113 P. involutus gene representatives analyzed, 390 varied in copy numbers in at least one of the three pairwise comparisons. Only three reporters showed significant changes in all three pairwise comparisons, and none of these were changed in a similar way in three comparisons. Our data indicate that changes in host range have occurred frequently and independently among strains in P. obscurosporus, P. involutus s. str., and P. validus. No evidence was obtained demonstrating that these changes have been associated with the gain or loss of similar genes in these three species.     

Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, a disease confined to Latin America and of marked importance in the endemic areas due to its frequency and severity. This species is considered to be clonal according to mycological criteria and has been shown to vary in virulence. To characterize natural genetic variation and reproductive mode in this fungus, we analyzed P. brasiliensis phylogenetically in search of cryptic species and possible recombination using concordance and nondiscordance of gene genealogies with respect to phylogenies of eight regions in five nuclear loci. Our data indicate that this fungus consists of at least three distinct, previously unrecognized species: S1 (species 1 with 38 isolates), PS2 (phylogenetic species 2 with six isolates), and PS3 (phylogenetic species 3 with 21 isolates). Genealogies of four of the regions studied strongly supported the PS2 clade, composed of five Brazilian and one Venezuelan isolate. The second clade, PS3, composed solely of 21 Colombian isolates, was strongly supported by the alpha-tubulin genealogy. The remaining 38 individuals formed S1. Two of the three lineages of P. brasiliensis, S1 and PS2, are sympatric across their range, suggesting barriers to gene flow other than geographic isolation. Our study provides the first evidence for possible sexual reproduction in P. brasiliensis S1, but does not rule it out in the other two species.     

The Phylogenetics of Mycotoxin and Sclerotium Production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called “S” and “L.” The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as “S” or “L” based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the “S” sclerotium phenotype, whereas group I strains consisted of both “S” and “L” isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

Gene genealogies reveal cryptic species and host preferences for the pine fungal pathogen Grosmannia clavigera

Grosmannia clavigera is a fungal pathogen of pine forests in western North America and a symbiotic associate of two sister bark beetles: Dendroctonus ponderosae and D. jeffreyi. This fungus and its beetle associate D. ponderosae are expanding in large epidemics in western North America. Using the fungal genome sequence and gene annotations, we assessed whether fungal isolates from the two beetles inhabiting different species of pine in epidemic regions of western Canada and the USA, as well as in localized populations outside of the current epidemic, represent different genetic lineages. We characterized nucleotide variations in 67 genomic regions and selected 15 for the phylogenetic analysis. Using concordance of gene genealogies and distinct ecological characteristics, we identified two sibling phylogenetic species: Gc and Gs. Where the closely related Pinus ponderosa and P. jeffreyi are infested by localized populations of their respective beetles, Gc is present. In contrast, Gs is an exclusive associate of D. ponderosae mainly present on its primary host‐tree P. contorta; however, in the current epidemic areas, it is also found in other pine species. These results suggest that the host‐tree species and the beetle population dynamics may be important factors associated with the genetic divergence and diversity of fungal partners in the beetle‐tree ecosystems. Gc represents the original G. clavigera holotype, and Gs should be described as a new species.     

Studies in tetrapartite symbioses

Alnus incana seedlings were successfully inoculated with an endomycorrhizal fungus (Glomus fasciculatus), an ectomycorrhizal fungus (Paxillus involutus) and an isolate ofFrankia (ACN1) simultaneously. The effects of the inoculation treatments on the growth performance of the seedlings were evaluated under controlled conditions.The overall growth performance of the seedlings inoculated with the three organisms was better than those inoculated withFrankia, G. fasciculatus andP. involutus individually or withFrankia+G. fasciculatus andFrankia+P. involutus combinations. The highest growth performance and mycorrhizal infection occurred when the seedlings were inoculated simultaneously withFrankia+G. fasciculatus+P. involutus.     

Study on morphology,pathogenicity and genetic diversity of Wilsonomyces carpophilus isolates,the causal agent of shot hole of stone fruit trees based on RAPD-PCR in Iran

Shot hole disease is one of the most important diseases of stone fruit trees in Iran. The disease is wide spread among orchards of Prunus spp. During spring and summer of 2007, 80 monoconidial isolates of the pathogen were recovered from infected leaves, fruits and twigs of different Prunus spp. in West Azerbaijan, Tehran, Ghazvin and Razavi Khorasan provinces of Iran and were studied taxonomically. Based on morphological and physiological characteristics and growth optimal temperature, all isolates were identified as Wilsonomyces carpophilus. Seedlings of stone fruits (apricot, almond, peach, nectarine, plum, sweet cherry and sour cherry) were used for pathogenicity tests. All seedlings were susceptible to the fungal isolates and showed disease symptoms on twigs, leaves, buds and petioles. Genetic diversity of 28 selected fungal isolates was investigated based on DNA fingerprinting by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), using four random primers. Based on cluster analysis of the PCR results from the four primers, 10 fingerprinting groups (clonal lineages) and 27 haplotypes were identified. Clonal lineages “C”, “D” and “E”, each with six haplotypes formed the biggest clonal lineages, but other clonal lineages (“B”, “F”, “G”, “H”, “I” and “J”) included only one isolate. No correlation was detected among clonal lineages with the location of selected isolates and their host species. A correlation was found between the substrate (fruit, twig or leaf) and clonal lineages, particularly in “C” clonal lineage. The results showed that the fungus population had high genetic diversity which is distributed among the different areas of Iran.     

<Emphasis Type="Italic">Colletotrichum echinochloae</Emphasis>, a new species on Japanese barnyard millet (<Emphasis Type="Italic">Echinochloa utilis</Emphasis>)

Five isolates of a species of Colletotrichum were collected from Japanese barnyard millet (Echinochloa utilis) in Japan. Although the fungus had once been identi-fied as C. graminicola sensu lato, it was clearly different from C. graminicola isolated from maize (Zea mays) in its falcate and short conidia, 18.0–22.2 μm in length, cultural characteristics, and specific pathogenicity to E. utilis. Moreover, molecular phylogenetic analyses using sequences of rDNA-ITS, HMG, and SOD2 indicated a monophyly of the isolates. A new species, Colletotrichum echinochloae, is then proposed based on the morphological, pathological, and molecular characteristics.     

Subtyping of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> isolates by <Emphasis Type="Italic">actA</Emphasis> gene sequencing,PCR-fingerprinting,and cell-invasion assay

Analysis of actA gene sequence polymorphism has been shown to be an effective and relatively inexpensive method for subtyping Listeria monocytogenes isolates, allowing the division of the population of this species into two deeply separate lineages. This sequence-based method as well as PCR-mediated fingerprinting were applied here for the differentiation of 49 isolates of food and clinical origin. Correlation between these two typing approaches was high. Both methods divided the isolates into two lineages, designated I (33 isolates) and II (16 isolates). All the 33 lineage I isolates were assigned to the same, or closely related, six clusters by both typing methods. For the lineage II isolates, PCR fingerprinting was found to be more discriminatory. The isolates were characterized by cell invasion assay. All highly invasive isolates were assigned to lineage I, which constituted a heterogeneous group also containing low-invasive isolates. High-invasive isolates were not found in the genetically determined lineage II. A particular actA cluster, designated Ha, contained all the isolates showing the lowest invasiveness. A common trait of the isolates belonging to this cluster was the presence of a threonine-441 of the deduced ActA sequence instead of the alanine-441 present in the remaining isolates. Thirteen human isolates were classified to lineage I and five to lineage II. A PCR-based method can therefore differentiate L. monocytogenes isolates in accordance with the current phylogenetic model of the evolution of this species.     

Nitrogen amendments reduce the growth of extramatrical ectomycorrhizal mycelium

The effect of three different nitrogen sources on the growth of external ectomycorrhizal mycelium was studied in Perspex micorocosms. Nonsterile peat was used as substrate. Five different fungal isolates growing in symbiosis with pine seedlings were investigated: two isolates of Paxillus involutus, one of Suillus bovinus and two unidentified ectomycorrhizal fungi isolated from ectomycorrhizal root tips. Three different nitrogen sources were used: ammonium as (NH₄)₂SO₄, nitrate as NaNO₃ and a complete nutrient solution (Ingestad 1979), and three different nitrogen concentrations, 1, 2 or 4 mg N/g dry wt. of peat. The mycelial growth of all fungi was found to be negatively affected by the nitrogen amendments, although the sensitivity to nitrogen varied between the isolates. One of the unidentified isolates was extremely sensitive and growth was completely inhibited by all nitrogen treatments. In contrast, the growth of one of the P. involutus isolates was only slightly reduced by the nitrogen amendments. The different nitrogen sources all reduced growth, and since no significant difference was found between the nitrogen sources or between the different nitrogen concentrations the results were pooled to give one value that summarized the effect of nitrogen on mycelial growth. Thus, the mycelial growth of one of the two P. involutus isolates was reduced to approximately 80% of the growth in the control, the other P. involutus and one of the unidentified fungi, vgk 2 89.10, were reduced to 40–50% of the control growth, S. bovinus to 30% of the control and the most sensitive fungus, the unidentified isolate vg 1 87.10, was reduced to 3% of the growth in the control treatment. In all experiments, the shoot to root ratio generally increased, mainly as a result of increased shoot growth.     

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus--Thysanophora penicillioides--based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the beta-tubulin and combined ITS and beta-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.     

NephrotoxigenicPenicillium species occurring on farm-stored cereal grains in western Canada

The incidence of nephrotoxigenicPenicillium species on farm-stored cereals in western Canada was determined by morphological and metabolite profile examination. Of the 142 isolates examined 102 were toxin producers with 61P. aurantiogriseum and 27P. freii. Other nephrotoxigenic species includedP. tricolor (6 isolates),P. verrucosum Chemotype II (4 isolates) andP. viridicatum Westling (4 isolates). The nephrotoxigenicPenicillium species profile for western Canada appears to differ from that of Denmark whereP. verrucosum,P. cyclopium,P. freii and, to a lesser extent,P. aurantiogriseum,P. polonicum, andP. viridicatum predominate.     

Pythium insidiosum complex hides a cryptic novel species: Pythium periculosum

Early phylogenetic analysis of Pythium insidiosum, the etiologic agent of pythiosis in mammals, showed the presence of a complex comprising three monophyletic clusters. Two included isolates recovered from cases of pythiosis in the Americas (Cluster I) and Asia (Cluster II), whereas the third cluster included four diverged isolates three from humans in Thailand and the USA, and one isolate from a USA spectacled bear (Cluster III). Thereafter, several phylogenetic analyses confirmed the presence of at least three monophyletic clusters, with most isolates placed in clusters I and II. Recent phylogenetic analyses using isolates from environmental sources and from human cases in India, Spain, Thailand, and dogs in the USA, however, showed the presence of two monophyletic groups each holding two sub-clusters. These studies revealed that P. insidiosum possesses different phylogenetic patterns to that described by early investigators. In this study, phylogenetic, population genetic and protein MALDI-TOF analyses of the P. insidiosum isolates in our culture collection, as well as those available in the database, showed members in the proposed cluster III and IV are phylogenetically different from that in clusters I and II. Our analyses of the complex showed a novel group holding two sub-clusters the USA (Cluster III) and the other from different world regions (Cluster IV). The data showed the original P. insidiosum cluster III is a cryptic novel species, now identified as P. periculosum. The finding of a novel species within P. insidiosum complex has direct implications in the epidemiology, diagnosis, and management of pythiosis in mammalian hosts.     

Effect of nitrogen level on acid phosphatase activity of eight isolates of ectomycorrhizal fungus Paxillus involutus cultured in vitro

The higher levels of nitrogen in ammonium form stimulated the growth of mycelia and increased the accessible as well as the total acid phosphatase activity of Paxillus involutus (Batsch) Fr. isolates grown in pure culture. Rates of mycelia growth and acid phosphatase activities varied widely from one isolate to another. Scots pine (Pinus sylvestris L.) seedlings were inoculated with different P. involutus isolates in axenic conditions. Shoots of pine seedlings with mycorrhizae contained more phosphorus than shoots of non-mycorrhizal seedlings. The relations between growth and phosphatase activity of P. involutus isolates and their efficiency in supplying the host plant with phosphate are discussed.     

Interaction between the Ectomycorrhizal Fungus Paxillus involutus, Damping-off Fungi and Pinus resinosa Seedlings

Paxillus involutus, an ectomycorrhizal fungus, had an inhibitory effect on the root pathogenic fungus Fusarium moniliforme and two isolates of F. oxysporum when grown in paired cultures on modified Melin Norkrans’ medium. In contrast, one isolate of F. oxysporum was not inhibited and another damping-off fungus, Cylindrocarpon destructans inhibited growth of Pax. involutus in similar paried cultures. Survival of Pinus resinosa (red pine) seedlings was increased significantly when they were grown in vitro concomitantly with either Pax. involutus and F. moniliforme or Pax. involutus and the three isolates of F. oxysporum, compared with seedlings inoculated with either F. moniliforme or F. oxysporum isolates alone. pax. involutus showed no protective effect against C. destructans. The number of colony forming units of Fusarium spp. was reduced significantly in the root extract and rhizosphere substrate of P. resinosa seedlings inoculated with Pax. involutus. Spore germination of Fusarium spp. was reduced significantly when treated with culture filtrate of Pax. involutus and root extract of P. resinosa seedlings inoculated with Pax. involutus. Neither colony forming units nor spore germination of C. destructans was affected either by culture filtrate of Pax. involutus or root extract of P. resinosa seedlings inoculated with Pax. involutus.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.     

Molecular identification and relative abundance of cryptic Lophodermium species in natural populations of Scots pine,Pinus sylvestris L.

The multi-locus phylogenetic species recognition approach and population genetic analysis of Amplified Fragment Length Polymorphism (AFLP) markers were used to delineate Lophodermium taxa inhabiting needles of Scots pine (Pinus sylvestris) in native pinewoods within Scotland. These analyses revealed three major lineages corresponding to the morphological species Lophodermium seditiosum and Lophodermium conigenum, fruiting on broken branches, and Lophodermium pinastri, fruiting on naturally fallen needles. Within L. pinastri three well supported sister clades were found representing cryptic taxa designated L. pinastri I, L. pinastri II, and L. pinastri III. Significant differences in mean growth rate in culture were found among the cryptic taxa. Taxon-specific primers based on ITS sequences were designed and used to classify over 500 Lophodermium isolates, derived from fallen needles of P. sylvestris in three Scottish and one French pinewood site, into the three L. pinastri cryptic taxa. Highly significant differences in the relative abundance of the three taxa were found among the Scottish pinewood sites, and between the French and all of the Scottish sites.     

Genetic characterisation of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22–8, c29–2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types.