Palm Fungi and Their Key Role in Biodiversity Surveys: A Review.

Over the past three decades, a wealth of studies has shown that palm trees (Arecaceae) are a diverse habitat with intense fungal colonisation, making them an important substratum to explore fungal diversity. Palm trees are perennial, monocotyledonous plants mainly restricted to the tropics that include economically important crops and highly valued ornamental plants worldwide. The extensive research conducted in Southeast Asia and Australasia indicates that palm fungi are undoubtedly a taxonomically diverse assemblage from which a remarkable number of new species is continuously being reported. Despite this wealth of data, no recent comprehensive review on palm fungi exists to date. In this regard, we present here a historical account and discussion of the research on the palm fungi to reflect on their importance as a diverse and understudied assemblage. The taxonomic structure of palm fungi is also outlined, along with comments on the need for further studies to place them within modern DNA sequence-based classifications. Palm trees can be considered model plants for studying fungal biodiversity and, therefore, the key role of palm fungi in biodiversity surveys is discussed. The close association and intrinsic relationship between palm hosts and palm fungi, coupled with a high fungal diversity, suggest that the diversity of palm fungi is still far from being fully understood. The figures suggested in the literature for the diversity of palm fungi have been revisited and updated here. As a result, it is estimated that there are about 76,000 species of palm fungi worldwide, of which more than 2500 are currently known. This review emphasises that research on palm fungi may provide answers to a number of current fungal biodiversity challenges.

Diaporthe Species on Palms: Molecular Re-Assessment and Species Boundaries Delimitation in the D. arecae Species Complex.

Due to cryptic diversification, phenotypic plasticity and host associations, multilocus phylogenetic analyses have become the most important tool in accurately identifying and circumscribing species in the Diaporthe genus. However, the application of the genealogical concordance criterion has often been overlooked, ultimately leading to an exponential increase in novel Diaporthe spp. Due to the large number of species, many lineages remain poorly understood under the so-called species complexes. For this reason, a robust delimitation of the species boundaries in Diaporthe is still an ongoing challenge. Therefore, the present study aimed to resolve the species boundaries of the Diaporthe arecae species complex (DASC) by implementing an integrative taxonomic approach. The Genealogical Phylogenetic Species Recognition (GCPSR) principle revealed incongruences between the individual gene genealogies. Moreover, the Poisson Tree Processes' (PTPs) coalescent-based species delimitation models identified three well-delimited subclades represented by the species D. arecae, D. chiangmaiensis and D. smilacicola. These results evidence that all species previously described in the D. arecae subclade are conspecific, which is coherent with the morphological indistinctiveness observed and the absence of reproductive isolation and barriers to gene flow. Thus, 52 Diaporthe spp. are reduced to synonymy under D. arecae. Recent population expansion and the possibility of incomplete lineage sorting suggested that the D. arecae subclade may be considered as ongoing evolving lineages under active divergence and speciation. Hence, the genetic diversity and intraspecific variability of D. arecae in the context of current global climate change and the role of D. arecae as a pathogen on palm trees and other hosts are also discussed. This study illustrates that species in Diaporthe are highly overestimated, and highlights the relevance of applying an integrative taxonomic approach to accurately circumscribe the species boundaries in the genus Diaporthe.

Re-Evaluating Botryosphaeriales: Ancestral State Reconstructions of Selected Characters and Evolution of Nutritional Modes.

Botryosphaeriales (Dothideomycetes, Ascomycota) occur in a wide range of habitats as endophytes, saprobes, and pathogens. The order Botryosphaeriales has not been subjected to evaluation since 2019 by Phillips and co-authors using phylogenetic and evolutionary analyses. Subsequently, many studies introduced novel taxa into the order and revised several families separately. In addition, no ancestral character studies have been conducted for this order. Therefore, in this study, we re-evaluated the character evolution and taxonomic placements of Botryosphaeriales species based on ancestral character evolution, divergence time estimation, and phylogenetic relationships, including all the novel taxa that have been introduced so far. Maximum likelihood, maximum parsimony, and Bayesian inference analyses were conducted on a combined LSU and ITS sequence alignment. Ancestral state reconstruction was carried out for conidial colour, septation, and nutritional mode. Divergence times estimates revealed that Botryosphaeriales originated around 109 Mya in the early epoch of the Cretaceous period. All six families in Botryosphaeriales evolved in the late epoch of the Cretaceous period (66-100 Mya), during which Angiosperms also appeared, rapidly diversified and became dominant on land. Families of Botryosphaeriales diversified during the Paleogene and Neogene periods in the Cenozoic era. The order comprises the families Aplosporellaceae, Botryosphaeriaceae, Melanopsaceae, Phyllostictaceae, Planistromellaceae and Saccharataceae. Furthermore, current study assessed two hypotheses; the first one being "All Botryosphaeriales species originated as endophytes and then switched into saprobes when their hosts died or into pathogens when their hosts were under stress"; the second hypothesis states that "There is a link between the conidial colour and nutritional mode in botryosphaerialean taxa". Ancestral state reconstruction and nutritional mode analyses revealed a pathogenic/saprobic nutritional mode as the ancestral character. However, we could not provide strong evidence for the first hypothesis mainly due to the significantly low number of studies reporting the endophytic botryosphaerialean taxa. Results also showed that hyaline and aseptate conidia were ancestral characters in Botryosphaeriales and supported the relationship between conidial pigmentation and the pathogenicity of Botryosphaeriales species.

Identification and Characterization of Colletotrichum Species Associated with Cherry Leaf Spot Disease in China.

Leaf spot is a common and serious disease of sweet cherry worldwide and has become a major concern in China. From 2018 to 2020, disease investigations were carried out in Beijing City, Sichuan, Shandong, and Liaoning Provinces in China, and 105 Colletotrichum isolates were obtained from diseased samples. Isolates were identified by morphological characterization coupled with multigene phylogenetic analyses based on six loci (internal transcribed spacer region, glyceraldehyde 3-phosphate dehydrogenase, calmodulin, actin, chitin synthase, and ß-tubulin). A total of 13 Colletotrichum species were identified, namely Colletotrichum aenigma, C. gloeosporioides, C. fructicola, C. siamense, C. temperatum, C. conoides, C. hebeiense, C. sojae, C. plurivorum, C. karsti, C. truncatum, C. incanum, and C. dematium. Among these, C. aenigma (25.7%) was the most prominent species isolated from diseased leaves, followed by C. gloeosporioides (19.0%) and C. fructicola (12.4%). Pathogenicity was tested on detached leaves of cv. 'Tieton' and 'Summit' and young seedlings of cv. 'Brooks' under greenhouse conditions. All 13 species were pathogenic to cherry leaves, and C. aenigma, C. conoides, and C. dematium showed high levels of virulence. Seedlings inoculated with the isolates developed similar symptoms to those seen in the orchards. This study provides the first reports for 11 of the 13 Colletotrichum species on sweet cherry in the world, excluding C. aenigma and C. fructicola. This is the first comprehensive study of Colletotrichum species associated with cherry leaf spot in China, and the results will provide basic knowledge to develop sustainable control measures for cherry leaf spot.

Taxonomy and Multigene Phylogeny of Diaporthales in Guizhou Province, China.

In a study of fungi isolated from plant material in Guizhou Province, China, we identified 23 strains of Diaporthales belonging to nine species. These are identified from multigene phylogenetic analyses of ITS, LSU, rpb2, tef1, and tub2 gene sequence data coupled with morphological studies. The fungi include a new genus (Pseudomastigosporella) in Foliocryphiaceae isolated from Acer palmatum and Hypericum patulum, a new species of Chrysofolia isolated from Coriaria nepalensis, and five new species of Diaporthe isolated from Juglans regia, Eucommia ulmoides, and Hypericum patulum. Gnomoniopsis rosae and Coniella quercicola are newly recorded species for China.

Lasiodiplodia fici sp. nov., Causing Leaf Spot on Ficus altissima in China.

High temperatures and the seasonality in tropical ecosystems favours plant pathogens, which result in many fungal diseases. Among these, diseases caused by Botryosphaeriaceae species are prominent as dieback, canker and leaf spots. In this research, we isolated one leaf-spot-causing Botryosphaeriaceae species from Ficus altissima leaves, which were collected in Guangzhou, Guangdong Province, China. Isolation and identification of the pathogen were based on morphological and molecular aspects. Based on multigene phylogenetic analysis of combined internal transcribed spacer (ITS), translation elongation factor 1-α gene (tef1) and beta-tubulin gene (tub2), the fungus associated with leaf spots on F. altissima is described as Lasiodiplodia fici, a novel species. Pathogenicity assays were conducted by inoculating the fungus onto detached shoots and plants under controlled environmental conditions. The results revealed that the L. fici isolates can infect the plant tissues under stress conditions by developing disease symptoms on detached shoots within three days. However, when it was inoculated onto the leaves of the host and grown in natural conditions, the progression of the disease was slow. The putative pathogen was re-isolated, and Koch's assumptions were satisfied. This is the first report of Lasiodiplodia species causing disease on Ficus altissima. Results from the present study will provide additional knowledge on fungal pathogens associated with forest and ornamental plant species.

Caveats of the internal transcribed spacer region as a barcode to resolve species boundaries in Diaporthe.

Species in Diaporthe are largely reported as important plant pathogens. Identification of species in this genus has been complemented by morphological and molecular features. However, one important factor delaying this process is the struggle to formulate robust species concepts to create adequate international phytosanitary measures. Regardless of the wide use of the internal transcribed spacer (ITS) rDNA region, established as the primary DNA barcode for fungi, the tendency for intraspecific variation has been reported, misleading interpretation of phylogenetic analyses. Therefore, the present study aimed to illustrate, using specific examples, how the ITS region may be problematic for species delimitation. We showed that the ITS region is highly variable, with strains of Diaporthe malorum and Diaporthe novem falling into more than one clade, which if analyzed on their own, would be likely recognized as distinct taxa. Divergent ITS paralogs were also proven to coexist within the genome of D. novem. We also suggest that ITS may have escaped from concerted evolution or has undergone a duplication event. Furthermore, this study reports for the first time the existence of a putative hybrid in the genus Diaporthe. Our findings offer new clues towards the intraspecific and intragenomic variation in the ITS region, raising questions about its value for barcoding, i.e., identifying species in the genus Diaporthe. Therefore, we recommend that the ITS region be analyzed cautiously and always compared for congruence prior to description of novel taxa.

Two new Botryosphaeria (Botryosphaeriales, Botryosphaeriaceae) species in China.

Five ascomycetous strains were isolated from dead branches and leaves of Salix (Salicaceae) and Osmanthusfragrans (Oleaceae), respectively. BLAST searches with ITS sequences in GenBank suggested a high degree of similarity to Botryosphaeriadothidea. To accurately identify these strains, we further analysed their morphological characteristics of asci, ascospores, all conidiophore cells and conidia. Phylogenetic relationships, based on ITS, rpb2, tef1 and tub2 gene sequences, confirmed our strains represented two novel species, which are introduced here as B.salicicola and B.osmanthuse spp. nov.

Fungal diversity notes 1387-1511: taxonomic and phylogenetic contributions on genera and species of fungal taxa.

This article is the 13th contribution in the Fungal Diversity Notes series, wherein 125 taxa from four phyla, ten classes, 31 orders, 69 families, 92 genera and three genera incertae sedis are treated, demonstrating worldwide and geographic distribution. Fungal taxa described and illustrated in the present study include three new genera, 69 new species, one new combination, one reference specimen and 51 new records on new hosts and new geographical distributions. Three new genera, Cylindrotorula (Torulaceae), Scolecoleotia (Leotiales genus incertae sedis) and Xenovaginatispora (Lindomycetaceae) are introduced based on distinct phylogenetic lineages and unique morphologies. Newly described species are Aspergillus lannaensis, Cercophora dulciaquae, Cladophialophora aquatica, Coprinellus punjabensis, Cortinarius alutarius, C. mammillatus, C. quercoflocculosus, Coryneum fagi, Cruentomycena uttarakhandina, Cryptocoryneum rosae, Cyathus uniperidiolus, Cylindrotorula indica, Diaporthe chamaeropicola, Didymella azollae, Diplodia alanphillipsii, Dothiora coronicola, Efibula rodriguezarmasiae, Erysiphe salicicola, Fusarium queenslandicum, Geastrum gorgonicum, G. hansagiense, Helicosporium sexualis, Helminthosporium chiangraiensis, Hongkongmyces kokensis, Hydrophilomyces hydraenae, Hygrocybe boertmannii, Hyphoderma australosetigerum, Hyphodontia yunnanensis, Khaleijomyces umikazeana, Laboulbenia divisa, Laboulbenia triarthronis, Laccaria populina, Lactarius pallidozonarius, Lepidosphaeria strobelii, Longipedicellata megafusiformis, Lophiotrema lincangensis, Marasmius benghalensis, M. jinfoshanensis, M. subtropicus, Mariannaea camelliae, Melanographium smilaxii, Microbotryum polycnemoides, Mimeomyces digitatus, Minutisphaera thailandensis, Mortierella solitaria, Mucor harpali, Nigrograna jinghongensis, Odontia huanrenensis, O. parvispina, Paraconiothyrium ajrekarii, Parafuscosporella niloticus, Phaeocytostroma yomensis, Phaeoisaria synnematicus, Phanerochaete hainanensis, Pleopunctum thailandicum, Pleurotheciella dimorphospora, Pseudochaetosphaeronema chiangraiense, Pseudodactylaria albicolonia, Rhexoacrodictys nigrospora, Russula paravioleipes, Scolecoleotia eriocamporesi, Seriascoma honghense, Synandromyces makranczyi, Thyridaria aureobrunnea, Torula lancangjiangensis, Tubeufia longihelicospora, Wicklowia fusiformispora, Xenovaginatispora phichaiensis and Xylaria apiospora. One new combination, Pseudobactrodesmium stilboideus is proposed. A reference specimen of Comoclathris permunda is designated. New host or distribution records are provided for Acrocalymma fici, Aliquandostipite khaoyaiensis, Camarosporidiella laburni, Canalisporium caribense, Chaetoscutula juniperi, Chlorophyllum demangei, C. globosum, C. hortense, Cladophialophora abundans, Dendryphion hydei, Diaporthe foeniculina, D. pseudophoenicicola, D. pyracanthae, Dictyosporium pandanicola, Dyfrolomyces distoseptatus, Ernakulamia tanakae, Eutypa flavovirens, E. lata, Favolus septatus, Fusarium atrovinosum, F. clavum, Helicosporium luteosporum, Hermatomyces nabanheensis, Hermatomyces sphaericoides, Longipedicellata aquatica, Lophiostoma caudata, L. clematidis-vitalbae, Lophiotrema hydei, L. neoarundinaria, Marasmiellus palmivorus, Megacapitula villosa, Micropsalliota globocystis, M. gracilis, Montagnula thailandica, Neohelicosporium irregulare, N. parisporum, Paradictyoarthrinium diffractum, Phaeoisaria aquatica, Poaceascoma taiwanense, Saproamanita manicata, Spegazzinia camelliae, Submersispora variabilis, Thyronectria caudata, T. mackenziei, Tubeufia chiangmaiensis, T. roseohelicospora, Vaginatispora nypae, Wicklowia submersa, Xanthagaricus necopinatus and Xylaria haemorrhoidalis. The data presented herein are based on morphological examination of fresh specimens, coupled with analysis of phylogenetic sequence data to better integrate taxa into appropriate taxonomic ranks and infer their evolutionary relationships.

https://botryosphaeriales.org/, an online platform for up-to-date classification and account of taxa of Botryosphaeriales.

Fungi are eukaryotes that inhabit various ecosystems worldwide and have a decomposing effect that other organisms cannot replace. Fungi are divided into two main groups depending on how their sexual spores are formed, viz. Ascomycota and Basidiomycota. The members of Botryosphaeriales (Dothideomycetes, Ascomycota) are ubiquitous. They are pathogenic on a wide range of hosts, causing diverse diseases including dieback, canker, leaf spots and root rots and are also reported as saprobes and endophytes worldwide. As an important fungal group, of which most are plant pathogens, it is necessary to organize data and information on Botryosphaeriales so that scientific literature can be used effectively. For this purpose, a new website, https://botryosphaeriales.org is established to gather all published data together with updates on the present taxonomy of Botryosphaeriales. The website consists of an easy-to-operate searching system and provides an up-to-date classification together with accounts of Botryosphaeriales taxa, including colour illustrations, descriptions, notes and numbers of species in each genus, as well as their classification. Thus, readers will be able to obtain information on botryosphaerialean taxa through this platform. Database URL: https://botryosphaeriales.org/.

Importance of Molecular Data to Identify Fungal Plant Pathogens and Guidelines for Pathogenicity Testing Based on Koch's Postulates.

Fungi are an essential component of any ecosystem, but they can also cause mild and severe plant diseases. Plant diseases are caused by a wide array of fungal groups that affect a diverse range of hosts with different tissue specificities. Fungi were previously named based only on morphology and, in many cases, host association, which has led to superfluous species names and synonyms. Morphology-based identification represents an important method for genus level identification and molecular data are important to accurately identify species. Accurate identification of fungal pathogens is vital as the scientific name links the knowledge concerning a species including the biology, host range, distribution, and potential risk of the pathogen, which are vital for effective control measures. Thus, in the modern era, a polyphasic approach is recommended when identifying fungal pathogens. It is also important to determine if the organism is capable of causing host damage, which usually relies on the application of Koch's postulates for fungal plant pathogens. The importance and the challenges of applying Koch's postulates are discussed. Bradford Hill criteria, which are generally used in establishing the cause of human disease, are briefly introduced. We provide guidelines for pathogenicity testing based on the implementation of modified Koch's postulates incorporating biological gradient, consistency, and plausibility criteria from Bradford Hill. We provide a set of protocols for fungal pathogenicity testing along with a severity score guide, which takes into consideration the depth of lesions. The application of a standard protocol for fungal pathogenicity testing and disease assessment in plants will enable inter-studies comparison, thus improving accuracy. When introducing novel plant pathogenic fungal species without proving the taxon is the causal agent using Koch's postulates, we advise the use of the term associated with the "disease symptoms" of "the host plant". Where possible, details of disease symptoms should be clearly articulated.

Molecular and Morphological Assessment of Septoria Species Associated with Ornamental Plants in Yunnan Province, China.

The Karst landform is the main geographic characteristic in South China. Such areas are rich in vegetation and especially suitable for growth of shrubs and herbaceous plants. In this study, 11 Septoria strains were obtained from different plants' leaves collected in the Kunming Botanical Garden, Yunnan Province, China. Based on single-gene and multi-gene analyses of five gene loci (tef1, rpb2, tub2, ITS, and LSU) and four gene regions (without LSU), these strains were found to belong to three independent phylogenetic lineages representing five species, including four novel taxa, and one new record for China. Five single gene trees were also provided to evaluate the effectiveness of each gene for discriminating the species, as a result of which tub2 was found to have the most suitable DNA barcode for rapid identification. Morphological descriptions, illustrations, and comparisons are provided for a more comprehensive assessment. Genealogical Concordance Phylogenetic Species Recognition (GCPSR) with a pairwise homoplasy index (PHI) test was used to evaluate the conclusions of the phylogenetic analyses.

Fungi vs. Fungi in Biocontrol: An Overview of Fungal Antagonists Applied Against Fungal Plant Pathogens.

Plant pathogens cause severe losses or damage to crops worldwide and thereby significantly reduce the quality and quantity of agricultural commodities. World tendencies are shifting towards reducing the usage of chemically synthesized pesticides, while various biocontrol methods, strategies and approaches are being used in plant disease management. Fungal antagonists play a significant role in controlling plant pathogens and diseases and they are used as Biocontrol Agents (BCAs) throughout the world. This review provides a comprehensive list of fungal BCAs used against fungal plant pathogens according to modern taxonomic concepts, and clarifies their phylogenetic relationships because thewrong names are frequently used in the literature of biocontrol. Details of approximately 300 fungal antagonists belonging to 13 classes and 113 genera are listed together with the target pathogens and corresponding plant diseases. Trichoderma is identified as the genus with greatest potential comprising 25 biocontrol agents that have been used against a number of plant fungal diseases. In addition to Trichoderma, nine genera are recognized as significant comprising five or more known antagonistic species, namely, Alternaria, Aspergillus, Candida, Fusarium, Penicillium, Pichia, Pythium, Talaromyces, and Verticillium. A phylogenetic analysis based on partial sequences of the 28S nrRNA gene (LSU) of fungal antagonists was performed to establish their phylogenetic relationships.

Nigrospora Species Associated with Various Hosts from Shandong Peninsula, China.

Nigrospora is a monophyletic genus belonging to Apiosporaceae. Species in this genus are phytopathogenic, endophytic, and saprobic on different hosts. In this study, leaf specimens with disease symptoms were collected from host plants from the Shandong Peninsula, China. The fungal taxa associated with these leaf spots were studied using morphology and phylogeny based on ITS, TEF1, and TUB2 gene regions. In this article, we report on the genus Nigrospora with N. gorlenkoana, N. oryzae, N. osmanthi, N. rubi, and N. sphaerica identified with 13 novel host associations including crops with economic importance such as bamboo and Chinese rose.

Endophytic Diaporthe Associated With Citrus grandis cv. Tomentosa in China.

Diaporthe species are associated with Citrus as endophytes, pathogens, and saprobes worldwide. However, little is known about Diaporthe as endophytes in Citrus grandis in China. In this study, 24 endophytic Diaporthe isolates were obtained from cultivated C. grandis cv. "Tomentosa" in Huazhou, Guangdong Province in 2019. The nuclear ribosomal internal transcribed spacer (ITS), partial sequences of translation elongation factor 1-α (tef1), ß-tubulin (tub2), and partial calmodulin (cal) gene regions were sequenced and employed to construct phylogenetic trees. Based on morphology and combined multigene phylogeny, eleven Diaporthe species were identified including two new species, Diaporthe endocitricola and D. guangdongensis. These are the first report of D. apiculata, D. aquatica, D. arecae, D. biconispora, D. limonicola, D. masirevicii, D. passifloricola, D. perseae, and D. sennae on C. grandis. This study provides the first intensive study of endophytic Diaporthe species on C. grandis cv. tomentosa in China. These results will improve the current knowledge of Diaporthe species associated with C. grandis. The results obtained in this study will also help to understand the potential pathogens and biocontrol agents and to develop a platform in disease management.

Phylogeny and morphology of Lasiodiplodia species associated with Magnolia forest plants.

Two new species of Lasiodiplodia (Lasiodiplodia endophytica and Lasiodiplodia magnoliae) are described and illustrated from Magnolia forests in Yunnan, China. Endophytic and saprobic Lasiodiplodia pseudotheobromae and endophytic L. thailandica are new records from this host. The internal transcribed spacers (ITS), part of the translation elongation factor-1α (tef1) and partial ß-tubulin (tub2) sequence data were analyzed to investigate the phylogenetic relationships of the new species with other Lasiodiplodia species. Lasiodiplodia magnoliae is phylogenetically sister to L. mahajangana and L. pandanicola but morphologically distinct from L. mahajangana in having larger conidia. Lasiodiplodia endophytica is most closely related to L. iraniensis and L. thailandica and the three species can be distinguished from one another by 2 base pair differences in ITS and three or four base pair differences in tef1. The new collections suggest that Magnolia forest plants are good hosts for Lasiodiplodia species with endophytic and saprobic life-styles.

Tzeananiaceae, a new pleosporalean family associated with Ophiocordycepsmacroacicularis fruiting bodies in Taiwan.

The order Pleosporales comprises a miscellaneous group of fungi and is considered to be the largest order of the class Dothideomycetes. The circumscription of Pleosporales has undergone numerous changes in recent years due to the addition of large numbers of families reported from various habitats and with a large amount of morphological variation. Many asexual genera have been reported in Pleosporales and can be either hyphomycetes or coelomycetes. Phoma-like taxa are common and have been shown to be polyphyletic within the order and allied with several sexual genera. During the exploration of biodiversity of pleosporalean fungi in Taiwan, a fungal strain was isolated from mycelium growing on the fruiting body of an Ophiocordyceps species. Fruiting structures that developed on PDA were morphologically similar to Phoma and its relatives in having pycnidial conidiomata with hyaline conidia. The fungus is characterised by holoblastic, cylindrical, aseptate conidiogenous cells and cylindrical, hyaline, aseptate, guttulated, thin-walled conidia. Phylogenetic analysis based on six genes, ITS, LSU, rpb2, SSU, tef1 and tub2, produced a phylogenetic tree with the newly generated sequences grouping in a distinct clade separate from all of the known families. Therefore, a new pleosporalean family Tzeananiaceae is established to accommodate the monotypic genus Tzeanania and the species T.taiwanensis in Pleosporales, Dothideomycetes. The Ophiocordyceps species was identified as O.macroacicularis and this is a new record in Taiwan.

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species.

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.

Comparative genome and transcriptome analyses reveal adaptations to opportunistic infections in woody plant degrading pathogens of Botryosphaeriaceae.

Botryosphaeriaceae are an important fungal family that cause woody plant diseases worldwide. Recent studies have established a correlation between environmental factors and disease expression; however, less is known about factors that trigger these diseases. The current study reports on the 43.3 Mb de novo genome of Lasiodiplodia theobromae and five other genomes of Botryosphaeriaceae pathogens. Botryosphaeriaceous genomes showed an expansion of gene families associated with cell wall degradation, nutrient uptake, secondary metabolism and membrane transport, which contribute to adaptations for wood degradation. Transcriptome analysis revealed that genes involved in carbohydrate catabolism, pectin, starch and sucrose metabolism, and pentose and glucuronate interconversion pathways were induced during infection. Furthermore, genes in carbohydrate-binding modules, lysine motif domain and the glycosyl hydrolase gene families were induced by high temperature. Among these genes, overexpression of two selected putative lignocellulase genes led to increased virulence in the transformants. These results demonstrate the importance of high temperatures in opportunistic infections. This study also presents a set of Botryosphaeriaceae-specific effectors responsible for the identification of virulence-related pathogen-associated molecular patterns and demonstrates their active participation in suppressing hypersensitive responses. Together, these findings significantly expand our understanding of the determinants of pathogenicity or virulence in Botryosphaeriaceae and provide new insights for developing management strategies against them.

Mating type genes in the genus Neofusicoccum: Mating strategies and usefulness in species delimitation.

The genus Neofusicoccum includes species with wide geographical and plant host distribution, some of them of economic importance. The genus currently comprises 27 species that are difficult to identify based on morphological features alone. Thus, species differentiation is based on phylogenetic species recognition using multigene genealogies. In this study, we characterised the mating type genes of Neofusicoccum species. Specific primers were designed to amplify and sequence MAT genes in several species and a PCR-based mating type diagnostic assay was developed. Homothallism was the predominant mating strategy among the species tested. Furthermore, the potential of mating type gene sequences for species delimitation was evaluated. Phylogenetic analyses were performed on both MAT genes and compared with multigene genealogies using sequences of the ribosomal internal transcribed spacer region, translation elongation factor 1-alpha and beta-tubulin. Phylogenies based on mating type genes could discriminate between the species analysed and are in concordance with the results obtained with the more conventional multilocus phylogenetic analysis approach. Thus, MAT genes represent a powerful tool to delimit cryptic species in the genus Neofusicoccum.