ITS Gene Regions Research Articles

Etiology of Botryosphaeria Panicle and Shoot Blight of Pistachio (Pistacia vera) Caused by Botryosphaeriaceae in Italy.

Pistachio (Pistacia vera) is an important crop in Italy, traditionally cultivated in Sicily (southern Italy) for several decades now. In recent years, new orchards have been planted in new areas of the island. Field surveys conducted in 2019 revealed the presence of symptomatic trees showing shoot dieback, cankers, fruit spots, and leaf lesions. Isolations from symptomatic samples consistently yielded fungal species in the Botryosphaeriaceae family. Identification of collected isolates was conducted using morphological and molecular analyses. Morphological characterization was based on conidia measurements of representative isolates and also effects of temperatures on mycelial growth was evaluated. DNA data derived from sequencing the ITS, tef1-α, and tub2 gene regions were analyzed via phylogenetic analyses (maximum parsimony and maximum likelihood). Results of the analyses confirmed the identity of Botryosphaeria dothidea, Neofusicoccum hellenicum, and N. mediterraneum. Pathogenicity tests were conducted on detached twigs and in the fields both on shoots as well as on fruit clusters using the mycelial plug technique. The inoculation experiments revealed that among the Botryosphaeriaceae species identified in this study N. hellenicum (occasionally detected) and N. mediterraneum were the most aggressive based on lesion length on shoots and fruits. N. mediterraneum was the most widespread among the orchards while B. dothidea can be considered a minor pathogen involved in this complex disease of pistachio. Moreover, to our knowledge, this is the first report of N. hellenicum in Italy.

A New Disease for Europe of Ficus microcarpa Caused by Botryosphaeriaceae Species.

The Indian laurel-leaf fig (Ficus microcarpa) is an important ornamental tree widely distributed in the urban areas of Italy. Surveys conducted in 2019 and 2020 on several tree-lined streets, squares, and public parks in Catania and Siracusa provinces (Sicily, southern Italy) revealed the presence of a new disease on mature trees. About 9% of approximately 450 mature plants showed extensive branch cankers and dieback. Isolations from woody tissues obtained from ten symptomatic plants consistently yielded species belonging to the Botryosphaeriaceae family. The identification of the recovered fungal isolates was based on a multi-loci phylogenetic (maximum parsimony and maximum likelihood) approach of the ITS, tef1-α, and tub2 gene regions. The results of the analyses confirmed the presence of three species: Botryosphaeria dothidea, Neofusicoccum mediterraneum, and N. parvum. Pathogenicity tests were conducted on potted, healthy, 4-year-old trees using the mycelial plug technique. The inoculation experiments revealed that all the Botryosphaeriaceae species identified in this study were pathogenic to this host. Previous studies conducted in California showed similar disease caused by Botryosphaeriaceae spp., and the pathogenic role of these fungi was demonstrated. To our knowledge, this is the first report of Botryosphaeriaceae affecting Ficus microcarpa in Europe.

A new species of Neoroussoella peltophora in Roussoellaceae, from Thailand

In this study, we introduce an asexual morph of a novel species Neoroussoella peltophora (Roussoellaceae) on Peltophorum vogelianum (Fabaceae) collected from Chiang Rai Province, Thailand. Morphologically the new species resembles Neoroussoella species. Phylogenetic analyses of concatenated LSU, SSU, ITS, tef1-α and rpb2 sequence data revealed that (MFLU 21–0113) is phylogenetically distinct (91% MLBS/1.00 BYPP). Neoroussoella peltophora differs from N. fulvicomae in having thinner peridium and differs from N. alishanense in having thicker peridium. Further, N. peltophora is distinct from N. alishanense in having shorter conidiogenous cells and differs from N. fulvicomae in having longer conidiogenous cells. The base pair comparison of ITS, tef1-α and rpb2 gene regions also support that our isolate is distinctive from the other species in Neoroussoella.

Colletotrichum fructicola associated with fruit anthracnose of persimmon

AbstractAnthracnose, a major problem for persimmon exporters worldwide, reduces fruit yield and quality. In this study, a pathogen causing anthracnose of an imported persimmon fruit from an unknown country of origin was identified using a polyphasic approach. The pathogenicity of two fungal isolates, MBPER001A and MBPER002, was assessed by wound inoculation on healthy persimmon fruits. Anthracnose symptoms were observed 14 days after inoculation. Koch's postulates were fulfilled after the same fungus was re‐isolated from the inoculated fruits. Morphologies of both MBPER001A and MBPER002 isolates, original and re‐isolated, showed characteristics similar to Colletotrichum species in the gloeosporioides species complex. Multi‐locus analysis of the partial ITS, ACT, GAPDH and TUB2 gene regions showed MBPER001A and MBPER002 grouped within the Colletotrichum fructicola clade. Results from this study demonstrate the ability of C. fructicola to cause anthracnose in wounded persimmon fruits. Preventing wounding and damaging that expose the fruit during postharvest handling is therefore important to reduce infection. This study is the first report of C. fructicola in persimmon in the Philippines.

The Morphological Characteristics and Phylogenetic Analyses Revealed an Additional Taxon in Heteroradulum (Auriculariales)

Auriculariales is diverse, embracing a number of corticioid, poroid, and hydnoid genera. The present study covers a new wood-inhabiting fungal species of Heteroradulum niveum sp. nov that is proposed on the basis of a combination of morphological features and molecular evidence. The species is characterized by the resupinate basidiomata, a monomitic hyphal system with generative hyphae with clamp connections; tubular cystidia; two- to four-celled basidia; and allantoid, colorless, thin-walled, smooth, IKI–, CB–, basidiospores (6.5–13.5 × 2.7–5.5 µm). Sequences of ITS and nLSU rRNA gene regions of the specimens were generated, and phylogenetic analyses were carried out with methods of maximum parsimony, maximum likelihood, and Bayesian inference. These phylogenetic analyses inferred from ITS+nLSU indicated that H. niveum is nested in Heteroradulum within Auriculariales. Further study within Heteroradulum on the basis of ITS+nLSU dataset revealed that it formed a monophyletic lineage with a strong support (100% BS, 100% BP, 1.00 BPP) and then grouped with H. yunnanensis.

Supplementary files

Fig. S1. Phylogenetic tree resulting from ML analysis of the ITS gene region alignment. The confidence values are indicated at the nodes in the same way as in Fig. 7. Genus clades are discriminated with boxes in different colours and clades containing thermophilic species are highlighted with an orange background. The scale bar shows the expected number of changes per site. The tree is rooted with Pseudoechria longicollis and Schizotheciaceae, in the Schizotheciaceae.<br />Fig. S2. Phylogenetic tree resulting from ML analysis of the partial tub2 gene region alignment. The confidence values are indicated at the nodes in the same way as in Fig. 7. Genus clades are discriminated with boxes in different colours and clades containing thermophilic species are highlighted with an orange background. The scale bar shows the expected number of changes per site. The tree is rooted with Pseudoechria longicollis and Schizotheciaceae, in the Schizotheciaceae, Sordariales.<br />Fig. S3. Phylogenetic tree resulting from ML analysis of the partial rpb2 gene region alignment. The confidence values are indicated at the nodes in the same way as in Fig. 7. Genus clades are discriminated with boxes in different colours and clades containing thermophilic species are highlighted with an orange background. The scale bar shows the expected number of changes per site. The tree is rooted with Pseudoechria longicollis and Pseudoechria prolifica, in the Schizotheciaceae.<br />Fig. S4. Phylogenetic location of “Humicola koreana” based on the separate analyses of partial gene sequences of ITS (A) and LSU (B). The confidence values are indicated at the nodes in the same way as in Fig. 7. The scale bar shows the expected number of changes per site. The tree is rooted with Achaetomium strumarium.<br />Table S1. Comparison ascospore sizes measured in lactic acid (present study) and water (literature) as mounting medium.<br />Table S2. Overview species and strains used in phylogenetic analysis.

Supplementary files

Fig. S1. Phylogenetic tree derived from maximum likelihood analysis of the LSU gene region. The dataset consisted of 233 isolates and 859 characters before and after Gblocks treatment. Bootstrap values above 60 % are shown. Purple blocks indicate existing genera, yellow blocks new genera described in this study, green blocks genera that we redefine here, and blue blocks indicate genera that we here re-instate and re-define. <br />Fig. S2. Phylogenetic tree derived from maximum likelihood analysis of the ITS gene region. The dataset consisted of 235 isolates and 169 characters after Gblocks treatment (1 171 characters prior to Gblocks, including gaps). ML values above 60 % are shown. Purple blocks indicate existing genera, yellow blocks new genera described in this study, green blocks genera that we redefine here, and blue blocks indicate genera that we here re-instate and re-define.<br />Fig. S3. Phylogenetic tree derived from maximum likelihood analysis of the TEF1-α gene region. The dataset consisted of 207 isolates and 380 characters after Gblocks treatment (655 characters prior to Gblocks, including gaps). ML values above 60 % are shown. Purple blocks indicate existing genera, yellow blocks new genera described in this study, green blocks genera that we redefine here, and blue blocks indicate genera that we here re-instate and re-define.<br />Fig. S4. Phylogenetic tree derived from maximum likelihood analysis of the RPBII gene region. The dataset consisted of 171 isolates and 952 characters after Gblocks treatment (1 108 characters prior to Gblocks, including gaps). ML values above 60 % are shown. Purple blocks indicate existing genera, yellow blocks new genera described in this study, green blocks genera that we redefine here, and blue blocks indicate genera that we here re-instate and re-define.<br />Table S1. Taxa included in the phylogenomic analyses and their genome sequence statistics

Two new species and a new record of Astrocystis from Southwestern China

In this study, three species of Astrocystis were collected from bamboo culms in Guizhou and Yunnan Provinces in China. Morphological characteristics and phylogenetic analyses (based on ITS, rpb2, βtubulin and α-actin gene regions) supported the proposal of two new species viz., Astrocystis pseudomirabilis sp. nov. and A. tessellati sp. nov. and a new record of A. sublimbata from China. Astrocystis pseudomirabilis can be distinguished from A. mirabilis by its larger stromata (0.64–0.83 × 0.38–0.52 mm vs. 0.4–0.6 × 0.2–0.5 mm) and larger ascospores (11–14 × 6–8 μm vs. 10–12 × 4.5–6 μm). Astrocystis tessellati differs from A. multiloculata by its smaller ascospores (16.2–19.2 × 7.2–9 μm vs. 19–25 × 7–11 μm). Astrocystis pseudomirabilis and A. tessellati form two distinct clades within Astrocystis.

Molecular and morphological investigation of Cortinarius bulliardii from Tokat (Turkey)

A macrofungi species, Cortinarius bulliardii is collected from Tokat city province during field trips in 2019. It has a smooth, finely light fibrous, brownish-red pileus, whitish to light lilac-brownish flesh, broadly ellipsoidal spores, light brown when young, then dark brown lamellae with age. In addition to morphological analysis, molecular sequence data based on the ITS and LSU rDNA gene regions indicated that the studied species is Cortinarius bulliardii.

Characterization and pathogenicity of Rhizoctonia solani AG‐5 isolates obtained from chickpea in Turkey

AbstractSeventy‐three isolates of Rhizoctonia were obtained from chickpea‐growing areas of Uşak, Kütahya, Isparta and Denizli provinces in Turkey. All the isolates were multinucleate (MN) and 50 of them showed hyphal fusion with the reference isolate of Rhizoctonia solani anastomosis group 5 (AG‐5). To confirm the identification, ITS gene regions of the isolates were sequenced with the ITS1 and ITS4 primers and they were found to be most similar to R. solani AG‐5 to 91.0–100.0% identity. The isolates were examined for their cultural characteristics. All the isolates were found to be highly virulent on chickpea, and the disease severity values of them were between 56.1–99.0% under in vitro conditions. In the pot trial, IS23, one of the most virulent isolates, caused pre‐emerged damping‐off with the value of 98.0% disease severity. This is the first study in which morphological and pathogenic variabilities of R. solani AG‐5 isolates were determined in chickpea in Turkey.

Soybean Roots and Soil From High- and Low-Yielding Field Sites Have Different Microbiome Composition.

The occurrence of high- (H) and low- (L) yielding field sites within a farm is a commonly observed phenomenon in soybean cultivation. Site topography, soil physical and chemical attributes, and soil/root-associated microbial composition can contribute to this phenomenon. In order to better understand the microbial dynamics associated with each site type (H/L), we collected bulk soil (BS), rhizosphere soil (RS), and soybean root (R) samples from historically high and low yield sites across eight Pennsylvania farms at V1 (first trifoliate) and R8 (maturity) soybean growth stages (SGS). We extracted DNA extracted from collected samples and performed high-throughput sequencing of PCR amplicons from both the fungal ITS and prokaryotic 16S rRNA gene regions. Sequences were then grouped into amplicon sequence variants (ASVs) and subjected to network analysis. Based on both ITS and 16S rRNA gene data, a greater network size and edges were observed for all sample types from H-sites compared to L-sites at both SGS. Network analysis suggested that the number of potential microbial interactions/associations were greater in samples from H-sites compared to L-sites. Diversity analyses indicated that site-type was not a main driver of alpha and beta diversity in soybean-associated microbial communities. L-sites contained a greater percentage of fungal phytopathogens (ex: Fusarium, Macrophomina, Septoria), while H-sites contained a greater percentage of mycoparasitic (ex: Trichoderma) and entomopathogenic (ex: Metarhizium) fungal genera. Furthermore, roots from H-sites possessed a greater percentage of Bradyrhizobium and genera known to contain plant growth promoting bacteria (ex: Flavobacterium, Duganella). Overall, our results revealed that there were differences in microbial composition in soil and roots from H- and L-sites across a variety of soybean farms. Based on our findings, we hypothesize that differences in microbial composition could have a causative relationship with observed within-farm variability in soybean yield.

Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in the Netherlands.

Diaporthe (Diaporthaceae, Diaporthales) is a common fungal genus inhabiting plant tissues as endophytes, pathogens and saprobes. Some species are reported from tree branches associated with canker diseases. In the present study, Diaporthe samples were collected from Alnusglutinosa, Fraxinusexcelsior and Quercusrobur in Utrecht, the Netherlands. They were identified to species based on a polyphasic approach including morphology, pure culture characters, and phylogenetic analyses of a combined matrix of partial ITS, cal, his3, tef1 and tub2 gene regions. As a result, four species (viz. Diaporthepseudoalnea sp. nov. from Alnusglutinosa, Diaporthesilvicola sp. nov. from Fraxinusexcelsior, D.foeniculacea and D.rudis from Quercusrobur) were revealed from tree branches in the Netherlands. Diaporthepseudoalnea differs from D.eres (syn. D.alnea) by its longer conidiophores. Diaporthesilvicola is distinguished from D.fraxinicola and D.fraxini-angustifoliae by larger alpha conidia.

Morphological and phylogenetic characterization of fungi within Hymenochaetales: introducing two new species from southern China

Two new wood‐inhabiting fungal species, Lyomyces niveus and L. ochraceoalbus spp. nov. are proposed based on a combination of morphological and molecular evidence. Lyomyces ochraceoalbus is characterised by resupinate basidiomata with tuberculate, cracking hymenial surface, clavate basidia with a median constriction, gathering numerous irregular crystals, and thin‐walled basidiospores with one or two globules. Lyomyces niveus is characterised by resupinate basidiomata, smooth to grandinioid hymenial surface, presence of three kinds of cystidia, and broadly ellipsoid basidiospores. Sequences of the ITS and nLSU gene regions were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and Bayesian inference methods. The phylogenetic reconstruction of Schizoporaceae based on a concatenated ITS+nLSU dataset showed that the two new species are nested in Lyomyces. A second phylogenetic analysis, of the genus Lyomyces, demonstrated that multiple isolates of L. niveus forms a monophyletic lineage which is sister to a clade comprising L. crustosus, L. juniperi, L. ochraceoalbus and L. vietnamensis. Moreover, L. ochraceoalbus is closely related to L. crustosus.

Population Divergence and Evolution of the Hawaiian Endemic Sesbania tomentosa (Fabaceae)

Sesbania tomentosa (Fabaceae) is an endemic flowering plant primarily adapted to coastal strand and dry lowland habitat in the Hawaiian Islands. Previous studies have attempted to delineate distinct taxa from among the extant populations. In the most recent treatment of Hawaiian Fabaceae, however, S. tomentosa was recognized as a single polymorphic species with two forms. To readdress issues of taxonomy, the present study utilized microsatellite marker variation and DNA sequencing to discriminate Hawaiian Sesbania populations. While there was no DNA sequence variation among species for ITS or TRPT gene regions, microsatellite analysis found a highly differentiated population structure at the nine loci sampled. Bayesian genetic clustering assignments and associated private alleles occurred in a distinct phylogeographic pattern. As a result, three distinct genetic groups were identified corresponding to islands of origin: populations from Nihoa, Kaua‘i, and O‘ahu (group 1), populations from Maui, Kaho‘olawe, and Moloka‘i (group 2), and populations from Hawai‘i Island (group 3). The extremely varied yet randomly overlapping morphologies among the populations suggest widespread convergent evolution has occurred and precludes any subspecific taxonomic designations. We interpret the high levels of genetic marker and morphologic differentiation exhibited by Hawaiian Sesbania to be a product of the plant’s reproductive ecology and its influence in sub-structuring populations on a small geographic scale. Efforts toward preservation of the wide range of distinctive appearing morphotypes should continue, facilitated by the natural tendency of the plant to maintain a more or less fixed state of certain traits within populations.

Botryosphaeria dothidea causes stem canker on Fatsia japonica in Iran

In February 2020, stem cankers and necrotic lesions were observed on Fatsia japonica in the Khuzestan province, Iran. Four identical isolates were obtained from symptomatic tissue and identified as Botryosphaeria dothidea based on morphology and phylogenetic analysis of the ITS and tef1 gene regions. The pathogenicity of the isolates was confirmed by inoculating healthy shrubs of F. japonica.

Characterization of Diaporthe species on Camelliaoleifera in Hunan Province, with descriptions of two new species.

Tea-oil tree (Camelliaoleifera Abel.) is an important edible oil woody plant with a planting area over 3,800,000 hectares in southern China. Species of Diaporthe inhabit a wide range of plant hosts as plant pathogens, endophytes and saprobes. At present, relatively little is known about the taxonomy and genetic diversity of Diaporthe on C.oleifera. Here, we conducted an extensive field survey in Hunan Province in China to identify and characterise Diaporthe species associated with tea-oil leaf spots. As a result, eleven isolates of Diaporthe were obtained from symptomatic C.oleifera leaves. These isolates were studied by applying a polyphasic approach including morphological and phylogenetic analyses of partial ITS, cal, his3, tef1 and tub2 gene regions. Two new Diaporthe species (D.camelliae-oleiferae and D.hunanensis) were proposed and described herein, and C.oleifera was revealed to be new host records of D.hubeiensis and D.sojae. This study indicated there is a potential of more undiscovered Diaporthe species from C.oleifera in China.

Opening the Gap: Rare Lichens With Rare Cyanobionts - Unexpected Cyanobiont Diversity in Cyanobacterial Lichens of the Order Lichinales.

The last decades of research led to a change in understanding of lichens that are now seen as self-sustaining micro-ecosystems, harboring diverse microbial organisms in tight but yet not fully understood relationships. Among the diverse interdependencies, the relationship between the myco- and photobiont is the most crucial, determining the shape, and ecophysiological properties of the symbiotic consortium. Roughly 10% of lichens associate with cyanobacteria as their primary photobiont, termed cyanolichens. Up to now, the diversity of cyanobionts of bipartite lichens resolved by modern phylogenetic approaches is restricted to the filamentous and heterocytous genera of the order Nostocales. Unicellular photobionts were placed in the orders Chroococcales, Pleurocapsales, and Chroococcidiopsidales. However, especially the phylogeny and taxonomy of the Chroococcidiopsidales genera remained rather unclear. Here we present new data on the identity and phylogeny of photobionts from cyanolichens of the genera Gonohymenia, Lichinella, Peccania, and Peltula from a broad geographical range. A polyphasic approach was used, combining morphological and cultivation-depending characteristics (microscopy, staining techniques, life cycle observation, baeocyte motility, and nitrogen fixation test) with phylogenetic analyses of the 16S rRNA and 16S–23S ITS gene region. We found an unexpectedly high cyanobiont diversity in the cyanobacterial lichens of the order Lichinales, including two new genera and seven new species, all of which were not previously perceived as lichen symbionts. As a result, we describe the novel unicellular Chroococcidiopsidales genera Pseudocyanosarcina gen. nov. with the species Pseudocyanosarcina phycocyania sp. nov. (from Peltula clavata, Australia) and Compactococcus gen. nov. with the species Compactococcus sarcinoides sp. nov. (from Gonohymenia sp., Australia) and the new Chroococcidiopsidales species Aliterella compacta sp. nov. (from Peltula clavata, Australia), Aliterella gigantea sp. nov. (from Peltula capensis; South Africa), Sinocapsa ellipsoidea sp. nov. (from Peccania cerebriformis, Austria), as well as the two new Nostocales species Komarekiella gloeocapsoidea sp. nov. (from Gonohymenia sp., Czechia) and Komarekiella globosa sp. nov. (from Lichinella cribellifera, Canary Islands, Spain). Our study highlights the role of cyanolichens acting as a key in untangling cyanobacterial taxonomy and diversity. With this study, we hope to stimulate further research on photobionts, especially of rare cyanolichens.

Jackfruit (Artocarpus heterophyllus), a new host plant of Ceratocystis wilt in South Sumatra, Indonesia

In 2019, wilt and sudden death were observed on Artocarpus heterophyllus (jackfruit). Identification was performed by sequence analysis of the concatenated β-tubulin and ITS gene regions. Sequencing of the PCR product confirmed this pathogen was Ceratocystis fimbriata sensu stricto. This is the first report of C. fimbriata causing sudden death disease in A. heterophyllus in Indonesia and worldwide.

Insight into the Systematics of Novel Entomopathogenic Fungi Associated with Armored Scale Insect, Kuwanaspis howardi (Hemiptera: Diaspididae) in China.

This study led to the discovery of three entomopathogenic fungi associated with Kuwanaspis howardi, a scale insect on Phyllostachys heteroclada (fishscale bamboo) and Pleioblastus amarus (bitter bamboo) in China. Two of these species belong to Podonectria: P. kuwanaspidis X.L. Xu & C.L. Yang sp. nov. and P. novae-zelandiae Dingley. The new species P. kuwanaspidis has wider and thicker setae, longer and wider asci, longer ascospores, and more septa as compared with similar Podonectria species. The morphs of extant species P. novae-zelandiae is confirmed based on sexual and asexual morphologies. Maximum likelihood and Bayesian inference analyses of ITS, LSU, SSU, tef1-α, and rpb2 sequence data provide further evidence for the validity of the two species and their placement in Podonectriaceae (Pleosporales). The second new species, Microcera kuwanaspidis X.L. Xu & C.L. Yang sp. nov., is established based on DNA sequence data from ITS, LSU, SSU, tef1-α, rpb1, rpb2, acl1, act, cmdA, and his3 gene regions, and it is characterized by morphological differences in septum numbers and single conidial mass.

Hyphodermella zixishanensis (Polyporales, Basidiomycota), a new species with reddish hymenial surface

A new corticioid fungal species, Hyphodermella zixishanensis, is described based on a combination of morphological features and molecular evidence. The species is characterized by an annual growth habit, resupinate basidiomata with reddish hymenial surface, a monomitic hyphal system with generative hyphae bearing simple septa, IKI–, CB– and ellipsoid, colorless, thin‐walled, smooth basidiospores. Sequences of ITS and nLSU gene regions were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and Bayesian inference methods. The phylogenetic analyses showed that the species belongs to Hyphodermella and is placed as sister to H. aurantiaca.