Comparative study of Epicoccum sorghinum in Southern Africa

The Coelomycetous genus, Phoma, is defined as filamentous fungi that produce pycnidial conidiomata with monophialidic, doliiform to flask-shaped conidiogenous cells. Host specificity was regarded as an important characteristic in identifying Phoma and this Saccardoan system, together with only minor differences in morphological characteristics between species, led to the description of a high number of species with no true taxonomic relevance. Species were extensively revised by Boerema and co-authors in 2004 and reduced to 223 taxa divided into nine sections, although not all species were considered. Experience was still required to accurately differentiate between species. Phoma section Peyronellaea was characterised by alternarioid dictyochlamydospores, epicoccoid shaped chlamydospores and/or unicellular chlamydospores that looked like pseudosclerotia. This section was later dissolved and the genus Peyronellaea re-instated. Phoma sorghina belonged to this section, and has a worldwide distribution. It is considered as a weak secondary parasite of plants that produce metabolites such as mycotoxins, phytotoxins and anthraquinones. Since its first description in 1878 by Saccardo as Phyllosticta sorghina until 1973, when it was named Phoma sorghina, it has been renamed numerous times based on morphological characteristics. It was moved to Epicoccum based on phylogenetic and morphological characteristics in 2010. The aim of this review is to discuss the complexity of the taxonomic challenges in the genus, Phoma, with special reference to Epicoccum sorghinum. In addition, an attempt is also made to demonstrate the importance of E. sorghinum as a plant pathogen and the threat it poses to human health.Dissertation (MSc)--University of Pretoria, 2014.National Research Foundation (NRF)Microbiology and Plant PathologyMScUnrestricte

Population structure and genetic diversity suggest recent introductions of Dothistroma pini in Slovakia

Dothistroma pini is one of two pathogens causing Dothistroma needle blight (DNB), a foliar disease of pines. The species was redefined in 2004 and subsequently recorded in several European countries. In Slovakia, the first report of the pathogen was in 2013. In this study, the population structure, genetic diversity, and reproductive mode of 105 isolates collected from 10 localities and seven hosts were determined in Slovakia. Species-specific mating type markers, ITS haplotype determination, and 16 microsatellite markers were used to characterize and genotype the isolates. Overall, 15 unique multilocus haplotypes (MLHs) based on microsatellite markers and three ITS haplotypes were identified. Three independent methods (DAPC, STRUCTURE, EDENetwork) separated the isolates into two distinct population clusters corresponding with ITS haplotypes. A high level of clonality was recorded suggesting that conidia are the primary source of pathogen dispersal. The low genetic diversity, predominantly asexual reproductive mode of the pathogen, and the fact that most isolates were collected from introduced tree species and native species in artificially planted urban greenery, supports the hypothesis that D. pini has been recently introduced into Slovakia.Vedecká Grantová Agentúra MŠVVaŠ SR a SAV and Tree Protection Co-operative Programme.http://www.wileyonlinelibrary.com/journal/ppahj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc

Population structure and diversity of the needle pathogen Dothistroma pini suggests human-mediated movement in Europe

Dothistroma needle blight (DNB) is an important disease of Pinus species that can be caused by one of two distinct but closely related pathogens; Dothistroma septosporum and Dothistroma pini. Dothistroma septosporum has a wide geographic distribution and is relatively well-known. In contrast, D. pini is known only from the United States and Europe, and there is a distinct lack of knowledge regarding its population structure and genetic diversity. The recent development of 16 microsatellite markers for D. pini provided an opportunity to investigate the diversity, structure, and mode of reproduction for populations collected over a period of 12 years, on eight different hosts in Europe. In total, 345 isolates from Belgium, the Czech Republic, France, Hungary, Romania, Western Russia, Serbia, Slovakia, Slovenia, Spain, Switzerland, and Ukraine were screened using microsatellite and species-specific mating type markers. A total of 109 unique multilocus haplotypes were identified and structure analyses suggested that the populations are influenced by location rather than host species. Populations from France and Spain displayed the highest levels of genetic diversity followed by the population in Ukraine. Both mating types were detected in most countries, with the exception of Hungary, Russia and Slovenia. Evidence for sexual recombination was supported only in the population from Spain. The observed population structure and several shared haplotypes between non-bordering countries provides good evidence that the movement of D. pini in Europe has been strongly influenced by human activity in Europe

Lecanosticta acicola : a growing threat to expanding global pine forests and plantations

Lecanosticta acicola causes brown spot needle blight (BSNB) of Pinus species. The pathogen occurs mostly in the Northern Hemisphere but has also been reported in Central America and Colombia. BSNB can lead to stunted growth and tree mortal-ity, and has resulted in severe damage to pine plantations in the past. There have been increasingly frequent new reports of this pathogen in Europe and in North America during the course of the past 10 years. This is despite the fact that quarantine prac-tices and eradication protocols are in place to prevent its spread. TAXONOMY: Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomy cotina; Class Dothideomycetes; Subclass Dothideomy-cetidae; Order Capniodales; Family Mycosphaerellaceae; Genus Lecanosticta. HOST RANGE AND DISTRIBUTION: Lecanosticta spp. occur on various Pinus species and are found in North America, Central America, South America (Colombia), Europe as well as Asia. DISEASE SYMPTOMS: Small yellow irregular spots appear on the infected pine needles that become brown over time. They can be surrounded by a yellow halo. These characteristic brown spots develop to form narrow brown bands that result in needle death from the tips down to the point of infection. Needles are prema-turely shed, leaving bare branches with tufts of new needles at the branch tips. Infection is usually most severe in the lower parts of the trees and progresses upwards into the canopies. USEFUL WEBSITES: The EPPO global database providing informa-tion on L. acicola (https ://gd.eppo.int/taxon/ SCIRAC)Reference genome of L. acicola available on GenBank (https ://www.ncbi.nlm.nih.gov/genom e/?term=Lecan ostic ta+acicola)JGI Gold Genome database information sheet of L. acicola sequenced genome (https ://gold.jgi.doe.gov/organ ism?xml:id=Go004 7147).The National Research Foundation of South Africa as well as members of the Tree Protection Cooperative Program.https://onlinelibrary.wiley.com/journal/13643703pm2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Biodiversity of Lecanosticta pine-needle blight pathogens suggests a Mesoamerican Centre of origin

Lecanosticta acicola causes the disease known as brown spot needle blight (BSNB), on Pinus species. The pathogen is thought to have a Central American centre of origin. This was based on the morphological variation between isolates believed to represent L. acicola from native Pinus spp. Two species of Lecanosticta, L. brevispora and L. guatemalensis, have recently been described from Mexico and Guatemala respectively based on morphology and sequence-derived phylogenetic inference. However, the putative native pathogen, L. acicola, was not found in those areas. In this study, the species diversity of a large collection of Lecanosticta isolates from Central America was considered. Phylogenetic analyses of the BT1, ITS, MS204, RPB2 and TEF1 gene regions revealed six species of Lecanosticta, four of which represented undescribed taxa. These are described here as Lecanosticta jani sp. nov. from Guatemala and Nicaragua, L. pharomachri sp. nov. from Guatemala and Honduras, L. tecunumanii sp. nov. from Guatemala and L. variabilis sp. nov. from Guatemala, Honduras, and Mexico. New host and country records were also found for the previously described L. brevispora and L. guatemalensis. Lecanosticta acicola was not found in any of the samples from Central America, and we hypothesize that it could be a northern hemisphere taxon. The high species diversity of Lecanosticta found in Mesoamerica suggests that this is a centre of diversity for the genus.Figure S1. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the ITS region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “Δ” next to the isolate indicates that the isolate exhibits Type 2 morphology but it groups with Subclade 1 or exhibits Type 1 morphology but groups with Subclade 2.Figure S2. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the BT1 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1.Figure S3. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the MS204 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma septosporum was used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “Δ” next to the isolate indicates that the isolate exhibits Type 2 morphology but it groups with Subclade 1 or exhibits Type 1 morphology but groups with Subclade 2.Figure S4. Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the RPB2 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “Δ” next to the isolate indicates that the isolate exhibits Type 2 morphology but it groups with Subclade 1 or exhibits Type 1 morphology but groups with Subclade 2.The National Research Foundation of South Africa (Thuthuka Grant no 80670, and Grant no 95875) as well as by members of the Tree Protection Cooperative Program (TPCP). AvdN was supported by a Scarce Skills Doctoral Scholarship (no 89086) provided by the National Research Foundation of South Africa.http://www.imafungus.orgam2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Rapid detection of pine pathogens Lecanosticta acicola, Dothistroma pini and D. septosporum on needles by probe-based lamp assays

Needle blights are serious needle fungal diseases affecting pines both in natural and productive forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics since they are regulated species in many countries and may require differential management measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species are hard to distinguish using morphological characteristics. The symptoms can also be confused with those caused by insects or abiotic agents. DNA-based detection is therefore recommended. However, the specific PCR assays that have been produced to date for the differential diagnosis of these pathogens can be applied only in a well-furnished laboratory and the procedure takes a relatively long execution time. Surveillance and forest protection would benefit from a faster diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each assay, targeting the beta tubulin ( -tub2) gene for the two Dothistroma species and the elongation factor (EF-1 ) region for L. acicola. Each reaction detected its respective pathogen rapidly and with high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected pine needles. These qualities and the compatibility with inexpensive portable instrumentation position these LAMP assays as an effective method for routine phytosanitary control of plant material in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.Part of this work was funded by the project “Holistic management of emerging forest pests and diseases” (HOMED) a European Union’s Horizon 2020 Programme for Research & Innovation under grant agreement No 771271 to LG. The rest of the work was funded by state and federal funds appropriated to the Warnell School of Forestry and Natural Resources, University of Georgia.https://www.mdpi.com/journal/forestsam2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Genetic Diversity of Lecanosticta acicola in Pinus Ecosystems in Northern Spain

14 Pág.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.This research was funded by Project RTA 2017-00063-C04-03 INIA, the Project Healthy Forest: LIFE14 ENV/ES/000179, the Department of Economic Development, Sustainability and Environment (Basque Government), grant reference: SANFOR2020. Forestry and Agricultural Biotechnology Institute, at the University of Pretoria (FABI).Peer reviewe

New hosts for Lecanosticta acicola and Dothistroma septosporum in newly established arboreta in Spain

A historical outbreak of needle blight disease was recorded during 2018 to 2019 in plantations of Pinus radiata and Pinus nigra in the North of Spain. The main pathogens involved in this historical outbreak were identified as Lecanosticta acicola and Dothistroma septosporum. Recently, a variety of tree species in three arboreta planted between 2011 and 2013 in the Basque Country as part of the European project REINFFORCE were showing symptoms of needle blight and defoliation. The aim of this study was to determine which pine species were affected with these pathogens. Tree species sampled included several provenances of P. brutia, P. elliottii, P. nigra, P. pinaster, P. pinea, P. ponderosa, P. sylvestris and P. taeda. Using molecular identification methods, Lecanosticta acicola was confirmed infecting Pinus brutia (Provenance: Alexandropolis, Greece and var. eldarica, Crimea) and represents a new host species for this pathogen. Pinus elliottii (Provenance: Georgia, USA) and P. ponderosa (Provenance: Central California, USA) are new host reports of L. acicola for Spain. Dothistroma septosporum was found for the first time on P. brutia (Provenance: Marmaris, Turkey) and P. ponderosa (Provenance: Oregon, USA) in Spain and was also detected infecting P. nigra (Provenance: Sologne Vayrières, France).Their respective institutions and by the Project RTA 2017-00063-C04-03 INIA and the Project: Healthy Forest: LIFE14 ENV/ES/000179.http://wileyonlinelibrary.com/journal/efphj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains

Dothistroma needle blight is one of the most devastating pine tree diseases worldwide. New and emerging epidemics have been frequent over the last 25 years, particularly in the Northern Hemisphere, where they are in part associated with changing weather patterns. One of the main Dothistroma needle blight pathogens, Dothistroma septosporum, has a global distribution but most molecular plant pathology research has been confined to Southern Hemisphere populations that have limited genetic diversity. Extensive genomic and transcriptomic data are available for a D. septosporum reference strain from New Zealand, where an introduced clonal population of the pathogen predominates. Due to the global importance of this pathogen, we determined whether the genome of this reference strain is representative of the species worldwide by sequencing the genomes of 18 strains sampled globally from different pine hosts. Genomic polymorphism shows substantial variation within the species, clustered into two distinct groups of strains with centres of diversity in Central and South America. A reciprocal chromosome translocation uniquely identifies the New Zealand strains. Globally, strains differ in their production of the virulence factor dothistromin, with extremely high production levels in strain ALP3 from Germany. Comparisons with the New Zealand reference revealed that several strains are aneuploids; for example, ALP3 has duplications of three chromosomes. Increased gene copy numbers therefore appear to contribute to increased production of dothistromin, emphasizing that studies of population structure are a necessary adjunct to functional analyses of genetic polymorphisms to identify the molecular basis of virulence in this important forest pathogen.Supplementary Material: Fig. S1 Predicted duplications and deletions in chromosomes 1‐14 for 18 strains of D. septosporum.Fig. S2 Initial evidence for a reciprocal chromosome translocation in the NZE10 genome. (A) Assembled contigs from the SLV genome were aligned with NZE10 reference chromosomes (scaffolds). Two contigs (circled) mapped to both chromosomes 5 and 13 of the NZE10 reference genome. This was found in many of the other genome sequences. (B) Visualisation of reads from the ALP3 genome mapped onto a region of chromosome 13 show a gap, in which mate pairs are mapped to chromosome 5.Fig. S3 A reciprocal translocation involving chromosomes 5 and 13 in the NZE10 genome. (A) The reciprocal translation was centred on an identical sequence (GCGCGGT) found at positions 1459800‐1459806 in NZE10 chromosome 5 and 717926‐717932 in chromosome 13. Chromosomes 5 and 13 are shaded grey and pale blue respectively with ends coloured to distinguish the two arms in each case. Coloured sequences surrounding the breakpoint indicate which arm they are from. (B) In strains from regions other than Australasia, the two long sections of NZE10 chromosomes 5 and 13 are joined to make a 2.2 Mb chromosome and two short sections to make a 1.4 Mb chromosome. Sequences around the common 7 bp sequence are shown for strain ALP3 as an example. (C, D) Pairs of divergently transcribed genes straddle the breakpoints on NZE10 chromosomes 5 (C) and 13 (D). A GC content of about 70% was seen at the breakpoint regions (50 bp sliding window) as shown by the %GC (blue) profiles.Fig. S4 Alignment of pathway regulator AflR from 19 D. septosporum strains. Amino acid changes compared to strain NZE10 are highlighted in blue (these sites are also variant between AflR sequences of D. septosporum , Cladosporium fulvum , Aspergillus parasiticus and Aspergillus nidulans ; (Chettri et al ., 2013)) or in green (at sites conserved between those four species). The Zn2Cys6 zinc binuclear domain is highlighted in pink; the linker sequence thought to determine DNA binding specificity in grey; the acidic glutamine rich motif in yellow and C terminal arginine residues implicated in AflJ binding in red.Fig. S5 Secondary structure predictions for AflR from D. septosporum NZE10 and ALP3. Pairwise alignment predicted by HHpred. The arrow indicates the location of the N349K polymorphism in ALP3.Table S1 Transposable elements in the Dothistroma septosporum genomes.Table S2 Genes deleted in the 18 genomes compared to Dothistroma septosporum NZE10.Table S3 Genes deleted from chromosome 14 and their expression levels in NZE10.Table S4 Single Nucleotide Polymorphisms (SNPs) in dothistromin genes, grouped by dothistromin gene loci.Table S5 Deleted genes on Dothistroma septosporum chromosome 12.Table S6 Gene duplications predicted by CNV (copy number variant) analysis.Table S7 (a) Polymerase Chain Reaction (PCR) primers used for verification of 5:13 translocation (b) Primers used for copy number variant (CNV) verification (quantitative PCR [qPCR]).The Bio‐Protection Research Centre of New Zealand, Scion and Massey University.https://onlinelibrary.wiley.com/journal/13643703pm2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog