Lecanosticta pharomachri and Its Newly Discovered Sexual State Causing a Serious Needle Disease of Pinus spp. in Colombia.

Brown spot needle blight (BSNB), caused by the fungal pathogen Lecanosticta acicola, is a well-known disease of Pinus spp. in several northern hemisphere countries. In the southern hemisphere, this disease has been reported only in Colombia and, apart from a single report of severe defoliation of Pinus radiata plantations in the early 1980s, has not caused serious damage in this country. An outbreak of a disease resembling BSNB on Mesoamerican Pinus spp. grown in Colombia has raised concern that L. acicola may have reemerged as a pathogen. DNA sequence-based analyses for the internal transcribed spacers, translation elongation factor 1-α and RNA polymerase II second largest subunit regions showed that the outbreaks were caused by L. pharomachri, a species distinct from, but closely related to, L. acicola. The discovery of L. pharomachri in Colombia is the first incidence of the pathogen causing a serious disease problem and the first occurrence on the hosts P. patula and P. maximinoi. A sexual state for L. pharomachri was discovered for the first time, and the description of the species has thus been emended.

Population genetic structure and migration patterns of the maize pathogenic fungus, Cercospora zeina in East and Southern Africa.

Cercospora zeina is a causal pathogen of gray leaf spot (GLS) disease of maize in Africa. This fungal pathogen exhibits a high genetic diversity in South Africa. However, little is known about the pathogen's population structure in the rest of Africa. In this study, we aimed to assess the diversity and gene flow of the pathogen between major maize producing countries in East and Southern Africa (Kenya, Uganda, Zambia, Zimbabwe, and South Africa). A total of 964 single-spore isolates were made from GLS lesions and confirmed as C.zeina using PCR diagnostics. The other causal agent of GLS, Cercospora zeae-maydis, was absent. Genotyping all the C.zeina isolates with 11 microsatellite markers and a mating-type gene diagnostic revealed (i) high genetic diversity with some population structure between the five African countries, (ii) cryptic sexual recombination, (iii) that South Africa and Kenya were the greatest donors of migrants, and (iv) that Zambia had a distinct population. We noted evidence of human-mediated long-distance dispersal, since four haplotypes from one South African site were also present at five sites in Kenya and Uganda. There was no evidence for a single-entry point of the pathogen into Africa. South Africa was the most probable origin of the populations in Kenya, Uganda, and Zimbabwe. Continuous annual maize production in the tropics (Kenya and Uganda) did not result in greater genetic diversity than a single maize season (Southern Africa). Our results will underpin future management of GLS in Africa through effective monitoring of virulent C.zeina strains.

Signatures of Post-Glacial Genetic Isolation and Human-Driven Migration in the Dothistroma Needle Blight Pathogen in Western Canada.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Global Genetic Diversity and Mating Type Distribution of Calonectria pauciramosa: An Important Wide-Host-Range Plant Pathogen.

The fungal pathogen, Calonectria pauciramosa, has caused serious diseases of many important plants worldwide. Understanding the genetic diversity and mating type distribution of this pathogen provides an essential step toward the development of disease control measures. In this study, we designed 15 polymorphic microsatellite markers by using genome sequences of two Ca. pauciramosa isolates having opposite mating type and from different countries. These markers were used to determine the genetic diversity of 145 isolates representing 13 different hosts (12 plant hosts residing in 12 genera, and soil) from 10 countries. In addition, mating type genes were amplified to investigate the reproduction mode of the pathogens in these populations by using mating type primers designed for Calonectria spp. Results revealed that a single dominant genotype, isolated from 11 plant genera residing in eight families, was present in seven countries across five continents. Only mating type MAT1-1 or MAT1-2 was amplified in each of the isolates, confirming that Ca. pauciramosa is heterothallic. Both mating types were detected in isolates from Eucalyptus in South Africa and Uruguay. The MAT1-2 phenotype was widely distributed in isolates from 12 different hosts (11 plant hosts and soil) collected in 10 countries. Overall, the results suggest that there has been substantial global movement of Ca. pauciramosa and that this has shaped its current population structure.

Phylogenomic incongruence in Ceratocystis: a clue to speciation?

BACKGROUND: The taxonomic history of Ceratocystis, a genus in the Ceratocystidaceae, has been beset with questions and debate. This is due to many of the commonly used species recognition concepts (e.g., morphological and biological species concepts) providing different bases for interpretation of taxonomic boundaries. Species delineation in Ceratocystis primarily relied on genealogical concordance phylogenetic species recognition (GCPSR) using multiple standard molecular markers. RESULTS: Questions have arisen regarding the utility of these markers e.g., ITS, BT and TEF1-α due to evidence of intragenomic variation in the ITS, as well as genealogical incongruence, especially for isolates residing in a group referred to as the Latin-American clade (LAC) of the species. This study applied a phylogenomics approach to investigate the extent of phylogenetic incongruence in Ceratocystis. Phylogenomic analyses of a total of 1121 shared BUSCO genes revealed widespread incongruence within Ceratocystis, particularly within the LAC, which was typified by three equally represented topologies. Comparative analyses of the individual gene trees revealed evolutionary patterns indicative of hybridization. The maximum likelihood phylogenetic tree generated from the concatenated dataset comprised of 1069 shared BUSCO genes provided improved phylogenetic resolution suggesting the need for multiple gene markers in the phylogeny of Ceratocystis. CONCLUSION: The incongruence observed among single gene phylogenies in this study call into question the utility of single or a few molecular markers for species delineation. Although this study provides evidence of interspecific hybridization, the role of hybridization as the source of discordance will require further research because the results could also be explained by high levels of shared ancestral polymorphism in this recently diverged lineage. This study also highlights the utility of BUSCO genes as a set of multiple orthologous genes for phylogenomic studies.

Genome comparisons suggest an association between Ceratocystis host adaptations and effector clusters in unique transposable element families.

Ceratocystis fimbriata is a host specific fungal pathogen of sweet potato (Ipomoea batatas). The closely related species, C. manginecans, is an important pathogen of trees (e.g. Acacia mangium and Mangifera indica) but has never been isolated from tuber crops. The genetic factors that determine the host range and host specificity of these species have not been determined. The aim of this study was to compare the genomes of C. fimbriata and C. manginecans in order to identify species-specific genetic differences that could be associated with host specificity. This included whole-genome alignments as well as comparisons of gene content and transposable elements (TEs). The genomes of the two species were found to be very similar, sharing similar catalogues of CAZymes, peptidases and lipases. However, the genomes of the two species also varied, harbouring species-specific genes (e.g. small secreted effectors, nutrient processing proteins and stress response proteins). A portion of the TEs identified (17%) had a unique distribution in each species. Transposable elements appeared to have played a prominent role in the divergence of the two species because they were strongly associated with chromosomal translocations and inversions as well as with unique genomic regions containing species-specific genes. Two large effector clusters, with unique TEs in each species, were identified. These effectors displayed non-synonymous mutations and deletions, conserved within a species, and could serve as mutational hot-spots for the development of host specificity in the two species.

DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine.

The detrimental effect of fungal pathogens on forest trees is an increasingly important problem that has implications for the health of our planet. Despite this, the study of molecular plant-microbe interactions in forest trees is in its infancy, and very little is known about the roles of effector molecules from forest pathogens. Dothistroma septosporum causes a devastating needle blight disease of pines, and intriguingly, is closely related to Cladosporium fulvum, a tomato pathogen in which pioneering effector biology studies have been carried out. Here, we studied D. septosporum effectors that are shared with C. fulvum, by comparing gene sequences from global isolates of D. septosporum and assessing effector function in both host and non-host plants. Many of the effectors were predicted to be non-functional in D. septosporum due to their pseudogenization or low expression in planta, suggesting adaptation to lifestyle and host. Effector sequences were polymorphic among a global collection of D. septosporum isolates, but there was no evidence for positive selection. The DsEcp2-1 effector elicited cell death in the non-host plant Nicotiana tabacum, whilst D. septosporum DsEcp2-1 mutants showed increased colonization of pine needles. Together these results suggest that DsEcp2-1 might be recognized by an immune receptor in both angiosperm and gymnosperm plants. This work may lead to the identification of plant targets for DsEcp2-1 that will provide much needed information on the molecular basis of gymnosperm-pathogen interactions in forests, and may also lead to novel methods of disease control.

Euwallacea perbrevis (Coleoptera: Curculionidae: Scolytinae), a confirmed pest on Acacia crassicarpa in Riau, Indonesia, and a new fungal symbiont; Fusarium rekanum sp. nov.

Non-native Acacia plantations in Indonesia were first reported to be infested by a native ambrosia beetle species, identified as Euwallacea fornicatus in 1993. Recently the level of infestation in these plantations by ambrosia beetles has steadily increased. The recent redefinition of the taxonomic parameters of the Euwallacea fornicatus species complex has resulted in the identity of the ambrosia beetle species in these plantations becoming unclear. This is also true for their obligate fungal associates. Therefore, the aim of this study was to identify the ambrosia beetle species, as well as its corresponding fungal associate/s, infesting Acacia crassicarpa plantations in Riau, Indonesia. Morphological identification and phylogenetic analysis of the mitochondrial cytochrome oxidase c subunit I (COI) gene, revealed that the beetles are E. perbrevis, previously a synonym of E. fornicatus and commonly referred to as the Tea Shot Hole Borer A (TSHBa). Multi-locus phylogenetic analyses of the fungal associate of E. perbrevis revealed a Fusarium sp. that is among members of the Ambrosia Fusarium Clade (AFC), but that is genetically distinct from other previously identified Fusarium symbionts of Euwallacea species. This novel fungal species is described here as Fusarium rekanum sp. nov.

Influence of farming practices on the population genetics of the maize pathogen Cercospora zeina in South Africa.

Gray leaf spot (GLS) is an important foliar disease of maize. This disease, caused by Cercospora zeina, is prevalent in both smallholder and commercial maize farms in South Africa. Notably, smallholder practices are geared towards conservation agriculture, planting diverse maize genotypes within a field and avoiding chemical control. This study examined the population genetic structure of 129 C. zeina isolates from three smallholder farm sites in KwaZulu-Natal in South Africa using 13 microsatellite markers. These were analysed, together with 239 isolates previously analysed from four commercial farms in the same province, to determine whether farming systems influence the genetic diversity of C.zeina. In addition, we wanted to determine whether the smallholder farming system harboured a greater diversity of C.zeina haplotypes due to lack of chemical spraying of these crops. Overall, farming systems exhibited partial, but significant, population differentiation, contributing 10% of the genetic variation observed. A 16% genetic variation conferred between KwaNxamalala (smallholder) and Cedara (commercial) areas that are in close proximity, confirmed this. Private alleles accounted for 29% of the 52 alleles observed in smallholder farms. Smallholder farms harboured a higher gene and genotypic diversity, with a clonal fraction of only 13% compared to 32% in commercial farms. Mating type ratios indicative of sexual recombination and lower linkage disequilibrium in most smallholder populations were consistent with higher levels of diversity. This study suggests that commercial farming practices, such as fungicides and monoculture crop planting, may result in a narrower genetic diversity of the pathogen that is then propagated by asexual reproduction. In contrast, management of GLS disease in smallholder farms should consider the greater diversity of pathogen genotypes, especially if future research shows that this equates to a greater diversity of pathogenicity alleles.

QTL mapping of mycelial growth and aggressiveness to distinct hosts in Ceratocystis pathogens.

Some species of Ceratocystis display strong host specificity, such as C. fimbriata sensu stricto that is restricted to sweet potato (Ipomoea batatas) as host. In contrast, the closely related C. manginecans, infects Acacia mangium and Mangifera indica but is not pathogenic to I. batatas. Despite the economic importance of these fungi, knowledge regarding the genetic factors that influence their pathogenicity and host specificity is limited. A recent inheritance study, based on an interspecific cross between C. fimbriata and C. manginecans and the resultant 70 F1 progeny, confirmed that traits such as mycelial growth rate, spore production and aggressiveness on A. mangium and I. batatas are regulated by multiple genes. In the present study, a quantitative trait locus (QTL) analysis was performed to determine the genomic loci associated with these traits. All 70 progeny isolates were genotyped with SNP markers and a linkage map was constructed. The map contained 467 SNPs, distributed across nine linkage groups, with a total length of 1203 cm. Using the progeny genotypes and phenotypes, one QTL was identified on the linkage map for mycelial growth rate, one for aggressiveness to A. mangium and two for aggressiveness to I. batatas (P < 0.05). Two candidate genes, likely associated with mycelial growth rate, were identified in the QTL region. The three QTLs associated with aggressiveness to different hosts contained candidate genes involved in protein processing, detoxification and regions with effector genes and high transposable element density. The results provide a foundation for studies considering the function of genes regulating various quantitative traits in Ceratocystis.

Looking for relationships between the populations of Dothistroma septosporum in northern Europe and Asia.

Dothistroma septosporum, a notorious pine needle pathogen with an unknown historical geographic origin and poorly known distribution pathways, is nowadays found almost in all areas inhabited by pines (Pinus spp.). The main aim of this study was to determine the relationship between North European and East Asian populations. In total, 238 Eurasian D. septosporum isolates from 11 countries, including 211 isolates from northern Europe, 16 isolates from Russian Far East and 11 isolates from Bhutan were analysed using 11 species-specific microsatellite and mating type markers. The most diverse populations were found in northern Europe, including the Baltic countries, Finland and European Russia. Notably, D. septosporum has not caused heavy damage to P. sylvestris in northern Europe, which may suggest a long co-existence of the host and the pathogen. No indication was obtained that the Russian Far East or Bhutan could be the indigenous area of D. septosporum, as the genetic diversity of the fungus there was low and evidence suggests gene flow from northern Europe to Russian Far East. On the western coast of Norway, a unique genetic pattern was observed, which differed from haplotypes dominating other Fennoscandian populations. As an agent of dothistroma needle blight, only D. septosporum was documented in northern Europe and Asia, while D. pini was found in Ukraine and Serbia.

Evolution of polyketide synthesis in a Dothideomycete forest pathogen.

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.

Lack of Population Structure and Mixed Reproduction Modes in Exserohilum turcicum from South Africa.

Exserohilum turcicum is the causal agent of northern corn leaf blight, a destructive foliar disease of maize that results in yield losses worldwide. In South Africa, typical yield losses range from 15 to 30%. Previous studies found high haplotypic diversity with evidence for sexual recombination in E. turcicum populations from tropical climates such as Kenya. However, the population genetic structure and method of reproduction of E. turcicum in South Africa is unknown and, therefore, was investigated. Twelve polymorphic microsatellite markers were screened on 258 E. turcicum isolates from maize collected during 2012 and 2013 from three maize fields in South Africa. A multiplex polymerase chain reaction assay amplifying both mating type idiomorphs was applied to investigate the distribution of mating types. No distinct genetic clusters were observed. Shared haplotypes were identified between isolates separated by distances of up to 762 km, which provided evidence of migration. High haplotypic diversity indicated that sexual reproduction is occurring among E. turcicum isolates, although mating type ratios and linkage disequilibrium analyses did not support the hypothesis of random mating. The population genetic structure of E. turcicum in South Africa is likely due to the direct movement and spread of isolates undergoing a mixed reproductive lifecycle.

Genetic Analyses Suggest Separate Introductions of the Pine Pathogen Lecanosticta acicola Into Europe.

Lecanosticta acicola is a heterothallic ascomycete that causes brown spot needle blight on native and nonnative Pinus spp. in many regions of the world. In this study we investigated the origin of European L. acicola populations and estimated the level of random mating of the pathogen in affected areas. Part of the elongation factor 1-α gene was sequenced, 11 microsatellite regions were screened, and the mating type idiomorphs were determined for 201 isolates of L. acicola collected from three continents and 17 host species. The isolates from Mexico and Guatemala were unique, highly diverse and could represent cryptic species of Lecanosticta. The isolates from East Asia formed a uniform and discrete group. Two distinct populations were identified in both North America and Europe. Approximate Bayesian computation analyses strongly suggest independent introductions of two populations from North America into Europe. Microsatellite data and mating type distributions indicated random recombination in the populations of North America and Europe. Its intercontinental introduction can most likely be explained as a consequence of the movement of infected plant material. In contrast, the spread of L. acicola within Europe appears to be primarily due to conidial dispersion and probably also ascospore dissemination.

Cercospora zeina from Maize in South Africa Exhibits High Genetic Diversity and Lack of Regional Population Differentiation.

South Africa is one of the leading maize-producing countries in sub-Saharan Africa. Since the 1980s, Cercospora zeina, a causal agent of gray leaf spot of maize, has become endemic in South Africa, and is responsible for substantial yield reductions. To assess genetic diversity and population structure of C. zeina in South Africa, 369 isolates were collected from commercial maize farms in three provinces (KwaZulu-Natal, Mpumalanga, and North West). These isolates were evaluated with 14 microsatellite markers and species-specific mating type markers that were designed from draft genome sequences of C. zeina isolates from Africa (CMW 25467) and the United States (USPA-4). Sixty alleles were identified across 14 loci, and gene diversity values within each province ranged from 0.18 to 0.35. High levels of gene flow were observed (Nm = 5.51), and in a few cases, identical multilocus haplotypes were found in different provinces. Overall, 242 unique multilocus haplotypes were identified with a low clonal fraction of 34%. No distinct population clusters were identified using STRUCTURE, principal coordinate analysis, or Weir's theta θ statistic. The lack of population differentiation was supported by analysis of molecular variance tests, which indicated that only 2% of the variation was attributed to variability between populations from each province. Mating type ratios of MAT1-1 and MAT1-2 idiomorphs from 335 isolates were not significantly different from a 1:1 ratio in all provinces, which provided evidence for sexual reproduction. The draft genome of C. zeina CMW 25467 exhibited a complete genomic copy of the MAT1-1 idiomorph as well as exonic fragments of MAT genes from both idiomorphs. The high level of gene diversity, shared haplotypes at different geographical locations within South Africa, and presence of both MAT idiomorphs at all sites indicates widespread dispersal of C. zeina between maize fields in the country as well as evidence for sexual recombination. The outcomes of this genome-enabled study are important for disease management since the high diversity has implications for dispersal of fungicide resistance should it emerge and the need for diversified resistance breeding.

New Ceratocystis species from Eucalyptus and Cunninghamia in South China.

During routine surveys for possible fungal pathogens in the rapidly expanding plantations of Eucalyptus and Cunninghamia lanceolata in China, numerous isolates of unknown species in the genus Ceratocystis (Microascales) were obtained from tree wounds. In this study we identified the Ceratocystis isolates from Eucalyptus and Cunninghamia in the GuangDong, GuangXi, FuJian and HaiNan Provinces of South China based on morphology and through comparisons of DNA sequence data for the ITS, partial ß-tubulin and TEF-1α gene regions. Morphological and DNA sequence comparisons revealed two previously unknown species residing in the Indo-Pacific Clade. These are described here as Ceratocystis cercfabiensis sp. nov. and Ceratocystis collisensis sp. nov. Isolates of Ceratocystis cercfabiensis showed intragenomic variation in their ITS sequences and four strains were selected for cloning of the ITS gene region. Twelve ITS haplotypes were obtained from 17 clones selected for sequencing, differing in up to seven base positions and representing two separate phylogenetic groups. This is the first evidence of multiple ITS types in isolates of Ceratocystis residing in the Indo-Pacific Clade. Caution should thus be exercised when using the ITS gene region as a barcoding marker for Ceratocystis species in this clade. This study also represents the first record of a species of Ceratocystis from Cunninghamia.

IMA Genome - F19 : A genome assembly and annotation guide to empower mycologists, including annotated draft genome sequences of Ceratocystis pirilliformis, Diaporthe australafricana, Fusarium ophioides, Paecilomyces lecythidis, and Sporothrix stenoceras.

The pace at which Next Generation Sequence data is being produced continues to accelerate as technology improves. As a result, such data are increasingly becoming accessible to biologists outside of the field of bioinformatics. In contrast, access to training in the methods of genome assembly and annotation are not growing at a similar rate. In this issue, we report on a Genome Assembly Workshop for Mycologists that was held at the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria, South Africa and make available the 12 draft genome sequences emanating from the event. With the aim of making the process of genome assembly and annotation more accessible to biologists, we provide a step-by-step guide to both genome assembly and annotation, intended to encourage and empower mycologists to use genome data in their research.

New Resinogalea species from Araucaria araucana resin in Chile and reclassification of the genus in the Cryptocaliciomycetidae.

Araucaria araucana is an ancient conifer, native to the mountain ranges in Chile and Argentina. These trees host a large number of organisms, mainly insects, strongly or even exclusively associated with them. The recent emergence of a novel canker disease on A. araucana has emphasised the importance of fungi associated with these iconic trees and has resulted in the discovery of various new species. In this study, we considered the identity of an unknown calicioid fungus consistently found on resin on the branches of A. araucana. Preliminary phylogenetic analyses placed isolates in the recently described sub-class Cryptocaliciomycetidae, closest to Cryptocalicium blascoi. However, the morphology of the ascomata and its occurrence in a unique niche suggested that the closest relative could be Resinogalea humboldtensis (Bruceomycetaceae, incertae sedis), a fungus with similar sporing structures found on resin of Araucaria humboldtensis in New Caledonia. There are no living cultures or sequence data available for either R. humboldtensis or its supposed closest relative, Bruceomyces castoris, precluding sequence-based comparisons. Morphological comparisons of the sporing structures on A. araucana confirmed that the ascomatal morphology of our unknown calicioid fungus and R. humboldtensis are almost identical and resemble each other more so than B. castoris or Cr. blascoi. A phylogenetic analysis based on the small subunit (SSU), internal transcribed spacer (ITS) and large subunit (LSU) rDNA regions resolved our strains into two clades with Cr. blascoi as its closest relative. Further analyses applying the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) based on ITS, mini chromosome maintenance protein complex (MCM7), RNA polymerase II second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF) gene regions, confirmed that strains represent two new species. Based on our morphological observations and phylogenetic analyses, we introduce two new Resinogalea species, R. araucana and R. tapulicola, and reclassify the genus in the subclass Cryptocaliciomycetidae.

Genetic Diversity of Lecanosticta acicola in Pinus Ecosystems in Northern Spain.

Lecanosticta acicola is one of the most damaging species affecting Pinus radiata plantations in Spain. Favourable climatic conditions and unknown endogenous factors of the pathogen and host led to a situation of high incidence and severity of the disease in these ecosystems. With the main aim of understanding the factors intrinsic to this pathogenic species, a study of the population structure in new established plantations with respect to older plantations was implemented. The genetic diversity, population structure and the ability of the pathogen to spread was determined in Northern Spain (Basque Country), where two thirds of the total Pinus radiata plantations of Spain are located. From a total of 153 Lecanosticta acicola isolates analysed, two lineages were present; the southern lineage, which was prevalent, and the northern lineage, which was scarce. A total of 22 multilocus genotypes were detected with a balanced composition of both mating types and evidence for sexual reproduction. In addition to the changing environmental conditions enhancing disease expression, the complexity and diversity of the pathogen will make it difficult to control and to maintain the wood productive system fundamentally based on this forest species.