RPB2 Gene Research Articles

Dominikia bonfanteae and Glomus atlanticum, two new species in the Glomeraceae (phylum Glomeromycota) with molecular phylogenies reconstructed from two unlinked loci

Examination of morphological characters of two arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), which produced clusters with glomoid spores in single-species cultures, suggested that they were undescribed species, one belonging to the genera Dominikia, Microdominikia or Kamienskia, and one to the genus Glomus. Phylogenetic analyses of the nuc rDNA small subunit (18S), internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) and large subunit (28S) (= 18S-ITS-28S) region, of the largest subunit of RNA polymerase II (RPB1) gene, as well as of concatenated sequences of these two loci (18S-ITS-28S + RPB1) confirmed our hypotheses and proved that both fungi were new species, here described as Dominikia bonfanteae sp. nov. and Glomus atlanticum sp. nov., respectively. In nature, D. bonfanteae was found in a plant community on the shore of Kokotek pond II (Lubliniec in the Silesian Upland, Poland). Glomus atlanticum originated from the Dunes of Aguda Park (Northern Portugal). The phylogenetic position of these new species and their close family relatives within the Glomeraceae were compared and discussed depending on the sequence alignment analysed. This paper for the first time shows the phylogeny of AMF reconstructed from analyses of two concatenated unlinked loci: 18S-ITS-28S plus RPB1. The partial RPB1 gene of D. bonfanteae was amplified using newly designed primers.

Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces.

The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr ≤ 1.65%, R2 values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ≥5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches.

Evaluating species in Botryosphaeriales.

The Botryosphaeriales (Dothideomycetes) includes numerous endophytic, saprobic, and plant pathogenic species associated with a wide range of symptoms, most commonly on woody plants. In a recent phylogenetic treatment of 499 isolates in the culture collection (CBS) of the Westerdijk Institute, we evaluated the families and genera accommodated in this order of important fungi. The present study presents multigene phylogenetic analyses for an additional 230 isolates, using ITS, tef1, tub2, LSU and rpb2 loci, in combination with morphological data. Based on these data, 58 species are reduced to synonymy, and eight novel species are described. They include Diplodia afrocarpi (Afrocarpus, South Africa), Dothiorella diospyricola (Diospyros, South Africa), Lasiodiplodia acaciae (Acacia, Indonesia), Neofusicoccum podocarpi (Podocarpus, South Africa), N. rapaneae (Rapanea, South Africa), Phaeobotryon ulmi (Ulmus, Germany), Saccharata grevilleae (Grevillea, Australia) and S. hakeiphila (Hakea, Australia). The results have clarified the identity of numerous isolates that lacked Latin binomials or had been deposited under incorrect names in the CBS collection in the past. They also provide a solid foundation for more in-depth future studies on taxa in the order. Sequences of the tef1, tub2 and rpb2 genes proved to be the most reliable markers. At the species level, results showed that the most informative genes were inconsistent, but that a combination of four candidate barcodes (ITS, tef1, tub2 and rpb2) provided reliable resolution. Furthermore, given the large number of additional isolates included in this study, and newly generated multigene DNA datasets, several species could also be reduced to synonymy. The study illustrates the value of reassessing the identity of older collections in culture collections utilising modern taxonomic frameworks and methods. Citation: Zhang W, Groenewald JZ, Lombard L, et al. 2021. Evaluating species in Botryosphaeriales. Persoonia 46: 63-115. https://doi.org/10.3767/persoonia.2021.46.03.

Redefining genera of cereal pathogens: Oculimacula, Rhynchosporium and Spermospora.

The taxonomy of Oculimacula, Rhynchosporium and Spermospora is re-evaluated, along with that of phylogenetically related genera. Isolates are identified using comparisons of DNA sequences of the internal transcribed spacer ribosomal RNA locus (ITS), partial translation elongation factor 1-alpha (tef1), actin (act), DNA-directed RNA polymerase II largest (rpb1) and second largest subunit (rpb2) genes, and the nuclear ribosomal large subunit (LSU), combined with their morphological characteristics. Oculimacula is restricted to two species, O. acuformis and O. yallundae, with O. aestiva placed in Cyphellophora, and O. anguioides accommodated in a new genus, Helgardiomyces. Rhynchosporium s. str. is restricted to species with 1-septate conidia and hooked apical beaks, while Rhynchobrunnera is introduced for species with 1-3-septate, straight conidia, lacking any apical beak. Rhynchosporium graminicola is proposed to replace the name R. commune applied to the barley scald pathogen based on nomenclatural priority. Spermospora is shown to be paraphyletic, representing Spermospora (type: S. subulata), with three new species, S. arrhenatheri, S. loliiphila and S. zeae, and Neospermospora gen. nov. (type: N. avenae). Ypsilina (type: Y. graminea), is shown to be monophyletic, but appears to be of minor importance on cereals. Finally, Vanderaaea gen. nov. (type: V. ammophilae), is introduced as a new coelomycetous fungus occurring on dead leaves of Ammophila arenaria. Citation: Crous PW, Braun U, McDonald BA, Lennox CL, Edwards J, Mann RC, Zaveri A, Linde CC, Dyer PS, Groenewald JZ (2020). Redefining genera of cereal pathogens: Oculimacula, Rhynchosporium and Spermospora. Fungal Systematics and Evolution 7: 67-98. doi: 10.3114/fuse.2021.07.04.

Three Novel Species of Russula Pers. Subg. Compactae (Fr.) Bon from Dinghushan Biosphere Reserve in Southern China

Three novel species of Russula Pers. subg. Compactae (Fr.) Bon from southern China are described with morphological evidence and phylogenetic analyses based on ITS and the concatenated partial LSU, mtSSU, rpb1, rpb2 and tef1 gene sequences. All three species possess sparse and broad lamellae. Russula latolamellata Y.Song & L.H.Qiu, sp. nov., is characterized by its cracking, black-tan pileus, scarlet-turning context on bruising and absence of pileocystidia and caulocystidia. Russula nigrocarpa S.Y.Zhou, Y.Song & L.H.Qiu, sp. nov., can be distinguished by its off-white lamellae, small basidiospores, hymenial cystidia and pileocystidia of various forms, often with forked apices and by its gelatinous pileipellis. Russula ochrobrunnea S.Y.Zhou, Y.Song & L.H.Qiu, sp. nov., is characterized by its cracking, grayish brown pileus with striate margin, light-brown lamellae whose edges turn dark-brown when mature, small basidiospores and slightly flexuous ormoniliform hymenial cystidia. Their detailed morphological features and phylogenetic positions are discussed and compared among closely related species.

Variation in Botryosphaeriaceae from Eucalyptus plantations in YunNan Province in southwestern China across a climatic gradient

ABSTRACTThe Botryosphaeriaceae accommodates many important pathogens of woody plants, including Eucalyptus. Recently, Botryosphaeriaceae were isolated from diseased plant parts from surveys of Eucalyptus plantations in the YunNan Province, China. The aims of this study were to identify these Botryosphaeriaceae isolates and to evaluate their pathogenicity to Eucalyptus. A total of 166 isolates of Botryosphaeriaceae were obtained from six regions in the YunNan Province, of which 76 were from Eucalyptus urophylla × E. grandis hybrids, 49 from E. globulus trees, and 41 isolates were from other unknown Eucalyptus species or hybrids. Isolates were identified by comparing DNA sequences of the internal transcribed spacer ribosomal RNA locus (ITS), partial translation elongation factor 1-alpha (tef1), β-tubulin 2 (tub2) and DNA-directed RNA polymerase II subunit (rpb2) genes, and combined with their morphological characteristics. Eleven species were identified, including Botryosphaeria fusispora, B. wangensis, Lasiodiplodia pseudotheobromae, Neofusicoccum kwambonambiense, N. parvum, and six novel species described as B. puerensis, N. dianense, N. magniconidium, N. ningerense, N. parviconidium and N. yunnanense. The dominant species across the regions were N. yunnanense, N. parvum and B. wangensis, representing 31.3, 25.3 and 19.9% of the total isolates, respectively. Species diversity and composition changed across the different climatic zones, despite their relatively close geographic proximity and the fact that some of the species have a global distribution. All the Botryosphaeriaceae species were pathogenic to one-year-old plants of an E. urophylla × E. grandis clone and E. globulus seed-derived plants, but showed significant inter- and intra-species variation in aggressiveness amongst isolates. The study provides a foundation for monitoring and management of Botryosphaeriaceae through selection and breeding of Eucalyptus in the YunNan Province of southwestern China.

First Report of Aspergillus niger causing Black rot of Grapes in Pakistan.

In June 2015 & 2016, a postharvest survey of table grapes (Vitis vinifera) cv. King's Ruby, was carried out in five different commercial fruit markets of Rawalpindi (33°38'19.2″N, 73°01'45.0″E) district, Punjab Province. Symptoms appeared as brownish lesions with black sporulation on grapes berries. The incidence of these symptoms on bunches ranged from 12 to 17% at all sites. Symptomatic tissue pieces were surface-sterilized with 0.1% sodium hypochlorite (NaOCl) for 30 seconds, rinsed three times with sterile distilled water, dried on filter paper for 45 seconds, and incubated on potato dextrose agar (PDA) at 25°C. After 3 days, dark brown to black mycelium were formed on PDA media. A total of 24 isolates were examined morphologically. The apex of the conidiophore was observed to be radiate. Vesicles were found to be spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose measured (3.14 μm ± 2.24 in averaged diameter: n=50), dark brown to black, with roughened cell walls. The conidiophores were also smooth-walled, hyaline, and became melanized toward the vesicle. These characteristics of the fungus were similar to those described for Aspergillus niger van Tiegh (de Hoog et al. 2000). For molecular identification, the internal transcribed spacer (ITS) region, beta-tubulin (Bt) gene and partial RNA polymerase II largest subunit (RPB2) gene of representative isolate (Asp.n02) was amplified using primers ITS1/ITS4, BT2a/BT2b and RPB2-6F/RPB2-7R respectively (White et al., 1990; Glass & Donaldson, 1995; Liu et al. 1999). Sequences were deposited in GenBank (ITS, MN658871; Bt2, MT117924; and RPB2, MT318289). Based on BLAST analysis, sequences of the ITS region, Bt2 genes, and RPB2 gene showed 99 to 100% similarity of isolate Asp.n02 to Aspergillus niger (Accession Nos. MK307680.1, MN195121.1, MF078661.1 for ITS gene, MN567299.1, MK451029.1, MK451020.1 for Bt2 gene, and MK450788.1, MK450790.1 for RPB2 gene). To complete Koch's postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of isolate: Asp.n 02 was pipetted onto three non-wounded and four wounded (5 mm diam) asymptomatic grape berries cv. King's Ruby (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries similaries to serve as a negative control (Ghuffar et al. 2018; Jayawardena et al. 2018). Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice. Brownish lesions leading to black sporulation similar to the original symptoms were observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control. The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Aspergillus niger was reported previously in Europe and Israel causing mycotoxin (Ochratoxin A) OTA production on Table grapes (Bau et al. 2006). To our knowledge, this is the first report of Aspergillus niger causing black rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against the black rot of grapes in Pakistan.

First Report of Fusarium xylarioides Causing Root and Stem Rot on Aloe vera in China.

Aloe vera (L.) Burm f. is a perennial herb belonging to the family liliaceae. It is widely grown for medicinal, cosmetic and vegetable use. In 2018 and 2019, a root rot disease occurred on potted A. vera plants in a nursery in the Hunan Province of China. Symptoms of the disease include water soaking lesions, brown spots on taproot or basal part of the stem. The plants were easy to pull out when the taproot is rotten or necrotic. As the disease progressed upward, leaves in the basal part of stems became red-brown and gradually fell off. In severe cases, the whole plants became rotten and wilted. For isolation purposes, diseased tissues were excised from the lesion margins, surface disinfested with 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile water thrice, and then placed on potato dextrose agar (PDA) and incubated at 26°C for 3 days in the dark. When cultured on PDA, fungal strains with similar morphology were consistently isolated and purified by single spore isolation. Colonies showed thick, pink aerial mycelium with a growth rate of 1.3 cm /day. The pigmentation was more intense in the colony center and became pale orange and white at the edge of colony. When cultured on SNA (Spezieller Nährstoffarmer agar), the fungus showed less pigmentation and thinner hyphae. Microconidia were abundantly produced, clavate and oval to kidney shaped, 7.1 to 15.2 μm × 2.5 to 5.1 μm, with 0 to 1 transverse septa. Macroconidia were sickle shaped, slender, slightly incurved in apical cell and foot-shaped in the basal cell, measured 27.9 to 53.2 μm × 2.5 to 3.5 μm, with 3 to 5 septa. These morphological characteristics were similar with those of Fusarium spp. (Booth 1971). For molecular identification, genomic DNA of the fungus was extracted by cetyl trimethyl ammonium bromide method. A portion of EF-1α (translation elongation factor 1-α) and RPB1 (the largest subunit of RNA polymerase) genes were amplified and directly sequenced using the EF-1/EF-2 and Fa/G2R primers (O'Donnell et al. 2010). The EF-1α and RPB1 were deposited in the GenBank with accession numbers MT755386 and MT755387. The EF-1α and RPB1 had 97.14% (ID FD_01334) and 99.62% identity (FD_03853), respectively, to F. xylarioides strains in the Fusarium-ID database (Geiser et al. 2004). In addition, the EF1-a showed 96.825% identity to the F. lateritium CBS 119871(AM295281) (a synonym of F. xylarioides), and the RPB1 showed 99.623% identity to the F. xylarioides NRRL 25486 (JX171517.1). Accordingly, the fungus was putatively identified to be F. xylarioides. For pathogenicity assay, A.vera seedlings were pot planted using sterilized nursery soil and inoculated with conidia suspension (1 × 105 conidia/ml), which were eluted from 7-day-old PDA cultures with sterilized water, according to the method described previously (Vakalounakis et al. 2015). The collar of each potted plant was poured with 20 ml of conidia suspensions. Plants mock inoculated with sterile water were used as control. All the inoculated plants were placed in a growth chamber at 25°C under 12/12 h light/dark cycle. The inoculation assays were carried out twice, with each one had three replicated plants. After 30 days, rot symptoms seen from the roots and basal part of stems were observed on the inoculated plants, but no visible symptoms were observed on control plants. The fungus was re-isolated from the inoculated plants and identified to be F. xylarioides by morphological and molecular characteristics, thus confirming Koch's postulates. As we know, many Fusarium species have been reported to cause root and stem rot disease in A.vera such as the F. oxysporum (Ji et al. 2007) and F. solani (Vakalounakis et al. 2015). However, to the best of our knowledge, this is the first report of F. xylarioides causing root and stem rot disease of A.vera in China. The identification of the pathogen fungus might provide a foundation for taking appropriate control strategies to this disease.

Three new Ophiocordyceps species in the Ophiocordyceps pseudoacicularis species complex on Lepidoptera larvae in Southeast Asia

Three new Ophiocordyceps species with superficial perithecia on Lepidoptera larvae are described: O. campes, O. longistromata, and O. phuwiangensis. Ophiocordyceps longistromata is characterized by long stromata and is closely related to O. campes based on morphological characters and phylogenetic position. Ophiocordyceps phuwiangensis is distinguished from closely related species by producing small ovoid superficial perithecia and multiseptate whole ascospores. Molecular phylogenies based on LSU, TEF, RPB1, and RPB2 gene loci also confirmed the distinctiveness of these new species and clustered with other specimens that produced the Hirsutella asexual morph with helical twisting of the phialide necks and are phylogenetically distinct from other known species characters in this clade.

<p><strong><em>Pleurotus sirindhorniae </em></strong><strong>(Pleurotaceae, Agaricales), a new species from northern Thailand</strong></p>

A new saprophytic fungus, Pleurotus sirindhorniae, is described based on collections from northern Thailand. This species is characterized by greyish yellow to olive brown pileus, ellipsoid to elongate basidiospores and clavate to mucronate cheilocystidia. It is distinguished from the previously described Pleuotus species by its distinct morphological characteristics. Molecular phylogenetic analyses of the large subunit (LSU) and internal transcribed spacers (ITS) of the nuclear ribosomal DNA (rDNA), the translation elongation factor 1-alpha (tef1) and the RNA polymerase II largest subunit (rpb1) genes also support P. sirindhorniae as a distinct new species within the genus Pleurotus. A full description, color photographs, illustrations and a phylogenetic tree to show the position of P. sirindhorniae are provided.

Phylogeny and species delimitation in Silene sect. Arenosae (Caryophyllaceae): a new section.

A putatively monophyletic group of annual Silene species is revised taxonomically and described as the new section S. sect. Arenosae. The species of this section were previously treated as a part of a widely circumscribed and polyphyletic S. sect. Rigidulae. Silene sect. Arenosae as circumscribed here consists of nine species. Members of the section show a predominantly E Mediterranean to SW Asian distribution pattern from Turkey southward to Egypt and eastward to Iran and Pakistan, although most of the species have a limited distribution range. The species of S. sect. Arenosae are characterized by narrowly lanceolate calyx teeth, which are often highly polymorphic, and lanceolate to oblanceolate (non-spathulate) basal leaves. The provided taxonomic revision is based on morphological characters and supported by phylogenetic analyses of two nuclear loci (nrITS and an intron of the RPB2 gene) and one chloroplast locus (the intron of the rps16 gene). The species descriptions are formalized using a novel implementation of the Prometheus Description Model.

First report of brown leaf spot of rice caused by Bipolaris zeicola in Pakistan.

Rice (Oryza sativa L.) is one of the highly consumed cereal grain crops in Pakistan. In September 2017, leaf samples of cultivar Basmati-385 showing brown to dark brown spots (5 to 9 mm in diameter) that were oval or cylindrical in shape with a chlorotic yellow halo and grayish tan centers were collected from fields near the University of Agriculture, Faisalabad (31.43633 N 73.05981 E). Average disease incidence was 69% in six rice fields that were sampled for diseased plants with visible symptoms. To isolate the pathogen, from 20 diseased leaves, 5 mm2 segments from the margins of lesions were cut, rinsed with sterile distilled water (SDW), surface disinfected by 70% ethanol and again rinsed with SDW. The samples were dried on sterilized filter paper discs, plated on potato dextrose agar (PDA) and incubated at 27°C for 5 to 7 days. Twelve isolates were sub-cultured and single-sporing was performed to obtain pure cultures. Fungal isolates with light to dark gray in color, thick or fluffy aerial mycelium, circular and smooth margins were obtained after 7 days of incubation. Conidia were 47-83 μm × 10-17 μm (n=100), with 4 to 10 distosepta, dark or olivaceous brown, straight or moderately curved, and the cells at the ends occasionally looked paler than those in the middle. Conidiophore of the fungus were simple, smooth, cylindrical, septate, and straight to flexuous. These characteristics resembled those of Bipolaris zeicola (Stout) Shoemaker (Manamgoda et al. 2014). For molecular identification, genomic DNA (isolate SU-11) was extracted and the internal transcribed spacer (ITS) region, large subunit (LSU) of ribosomal DNA, translation elongation factor (tef), glyceraldehyde 3-phosphate dehydrogenase (gpd), and RNA polymerase II second largest subunit (rpb2) genes were amplified and sequenced by using the primers ITS1-F/ITS4-R (White et al. 1990), LROR-F/LR5-R (Schoch et al. 2012), EF1-983F/EF1-2218R (Rehner and Buckley 2005), GPD1F/GPD2R (Berbee et al. 1999), and 5F2/7CR (O'Donnell et al. 2007), respectively. BLASTn searches showed 100% homology with the LSU and rpb2 sequences of B. zeicola (GenBank Accession Nos. MH876201 and HF934842) and 98-99% similarity with ITS, tef, and gpd sequences of B. zeicola (GenBank Accession Nos. KM230398, KM093752 and KM034815). The sequences of ITS, LSU, tef, gpd, and rpb2 were deposited in GenBank with accession numbers MN871712, MN877767, MN867685, MN904511 and MT349837, respectively. To fulfill Koch's postulates, 25 greenhouse-grown rice plants (cv. Basmati-385) at 2- to 3-leaf stage were spray inoculated with a spore suspension (105 spores/ml; isolate SU-11) prepared in SDW. Plants were covered with plastic wraps to maintain humid conditions for 24 hours and incubated at 27°C for one week. Similarly, ten non-inoculated plants sprayed with SDW served as controls. After one week, observed symptoms were similar to those from natural infections and no disease symptoms were observed on the non-inoculated plants. The experiment was repeated twice and the pathogen was re-isolated from the infected leaves and characterized morphologically. Globally, B. zeicola has also been reported to cause the leaf spot of rice and maize plants (Sivanesan 1987; Kang et al. 2018). To our information, this is the first report of B. zeicola causing brown leaf spot of rice in Pakistan. The increasing risk of this fungal pathogen in the rice-growing areas of Pakistan need a rigorous exploration and outreach effort to develop effective management practices.

First Report of Mycoleptodiscus terrestris Causing Crown and Root Rot of Alfalfa (Medicago sativa) in Minnesota.

Mycoleptodiscus terrestris (Gerd.) Ostaz. has been studied extensively as a potential mycoherbicide against aquatic weeds since the early 1970s (Mathur and Gehlot 2018; Shearer 1998). However, it is also a pathogen on many legumes including alfalfa (Gerdemann 1953; Smith et al. 1998). In August 2019, alfalfa plants with stunted yellow foliage and rotted stems at the base of the crown were observed in a variety trial planted in spring 2018 at the University of Minnesota, St. Paul, Minnesota. Affected plants were in patches across the field with approximately 20% of plants showing symptoms. Plants from patches had few lateral and fibrous roots and dark lesions appeared in the crown and root tissue. Tissue samples (5 mm2) from 10 plants with symptomatic crown and root tissues were surface disinfested with 70% ethanol for 5 min, followed by 30 s in 10% NaOCl, and then rinsed three times in sterile distilled water. Samples were air-dried and placed on water agar (WA) amended with 25 µg/ml rifampicin. Plates were incubated at room temperature for 3 days and fungal hyphal tips from tissue samples were transferred to potato dextrose agar (PDA). After 7 days, isolates were tentatively identified by morphological characteristics as M. terrestris (Ostazeski 1967). The hyaline mycelia turned from olive-gray to dark gray with age. Abundant dark microsclerotia formed 5 days after incubation that varied in size and shape, measuring 300-860 x 270-600 µm. Molecular identification of two representative isolates (DAZ5 and DAZ9) was done by sequencing the internal transcribed spacer (ITS) regions of the rDNA, the translation elongation factor 1α (TEF1), and the second largest subunit of RNA polymerase II (RPB2) genes. For both isolates the ITS, TEF1, and RPB2 genes were amplified and sequenced with primers ITS1/4 (White et al. 1990), 983F/1567R (Rehner and Buckley 2005), and fRPB2-7cR/RPB-5F2 (Liu et al. 1999; Sung et al. 2007), respectively. ITS amplification conditions followed White et al. (1990), the TEF and the partial RPB2 gene was obtained by using a touchdown PCR protocol as described in Rehner and Buckley (2005) and Woudenberg et al. (2017), respectively. Based on a BLAST search of the NCBI nucleotide database, the ITS sequences (GenBank MN851265.1 and MN851266.1) had 100% identity with M. terrestris strain CBS 231.53 (MK487754.1). The TEF1 (MN873019, MN873020) and RPB2 sequences (MN873021, MN873022) had 100% identity with M. terrestris strain IMI 159038 (MK495977.1, MK492735.1). A pathogenicity test was performed by inoculating five 3-week-old alfalfa plants per cultivar (cv. DKA44-16RR, Vernal, 53V52, and Agate) with isolates DAZ5 and DAZ9. Plants were inoculated around the exposed stem base with three 5 mm diameter PDA plugs from a culture of M. terrestris covered with microsclerotia. Control plants were inoculated with sterile PDA plugs. After inoculation plants were incubated at 25 0C with a 16-h photoperiod in a growth chamber. The first symptoms appeared two months after inoculation on all cultivars as a dark lesion at the stem base followed by yellowing and rotting of stems. Inoculated plants had more fibrous roots than the controls. The control plants were symptomless with healthy root development. The pathogen was re-isolated from all infected alfalfa cultivars and had the same morphology as isolates DAZ5 and DAZ9, fulfilling Koch's postulates. To our knowledge, this is the first report of M. terrestris causing crown and root rot disease of alfalfa in Minnesota.

A new isolate of Nosema fumiferanae (Microsporidia: Nosematidae) from the date moth Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839 (Lepidoptera: Pyralidae).

In this study, a microsporidian pathogen of the date moth (Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839) also known as the carob moth, is described based on light microscopy, ultrastructural characteristics and comparative molecular analysis. The pathogen infects the gut and hemolymph of A. ceratoniae. All development stages are in direct contact with the host cell cytoplasm. Fresh spores with nuclei arranged in a diplokaryon are oval and measured 3.29 ± 0.23 μm (4.18-3.03 μm, n = 200) in length and 1.91 ± 0.23 μm (2.98-1.66 μm, n = 200) in width. Spores stained with Giemsa's stain measured 3.11 ± 0.31 μm (3.72-2.41 μm, n = 150) in length and 1.76 ± 0.23 μm (2.16-1.25 μm, n = 150) in width. Spores have an isofilar polar filament with 10-12 coils. An 1110 bp long alignment of the current microsporidium showed an SSU rRNA gene difference of only 0.0009, corresponding to >99.91% sequence similarity with Nosema fumiferanae, while RPB1 gene sequences were 98.03% similar within an alignment of 969 bp. All morphological, ultrastructural and molecular features indicate that the microsporidian pathogen of A. ceratoniae is the new isolate of the N. fumiferanae and is named here as Nosema fumiferanae TY61.

First report of stem and root rot of coriander caused by Fusarium equiseti in China.

Coriander (Coriandrum sativum L.) is one of the most important vegetables used as a seasoning in China. During the summer of 2019 and 2020, two-month-old coriander plants with stem and root rot were observed in commercial fields located in Tianjin, China. Symptoms were first observed when temperatures were about 24°C. Diseased plants had chlorotic lower leaves, were stunted, had rotted roots and stems, and eventually wilted and died (Fig. S1). In severe cases, disease incidence was approximately 85%. Plant samples with the same symptoms were collected from five fields by five-point sampling method (4 plants/point; 20 plants/field), out of which twenty plants were arbitrarily selected for pathogen isolation. Root tissue fragments (3 mm2; 3 fragements/plant) at the boundary of the symptomatic area were excised, washed in 1% NaClO for 2 min, rinsed in sterile distilled water (SDW), and incubated on PDA containing 50 mg/liter of streptomycin sulfate. Plates were incubated at 25°C for 5 days in the dark. 25% of the isolates were tentatively identified as Fusarium equiseti according to cultural and morphological characteristics (Leslie and Summerell, 2006). Colonies developed abundant white aerial mycelium with pale brown pigments on PDA. Microconidia were single-celled, hyaline, non-septate and ovoid, and ranged from 4.6 to 17.6 × 1.5 to 3.8 μm (n = 40). Macroconidia were mostly five-septate, slightly curved at apex, and ranged from 13.3 to 34.6 × 2.2 to 4.3 μm (n = 40). Chlamydospores with thick, roughened walls were abundant in clumps or chains, ellipsoidal or subglobose. Diseased-plant samples and pathogen isolates were deposited to China General Microbiological Culuture Collection Center (CGMCC). Species identity was confirmed by amplifying and sequencing the ITS, TEF1-α, mtSSU and RPB2 (Carbone and Kohn 1999; Li et al. 1994; Miller and Huhndorf 2005). BLASTn analyses of ITS amplicon (Genbank No. MT579854), TEF1-α (MT586131), mtSSU (MT587798) and RPB2 (MT648497) genes obtained with cognate sequences available in GenBank showed 99.8% (498 bp out of 499 bp), 100% (493 bp out of 493 bp), 99.7% (625 bp out of 627 bp) and 98.9% (656 bp out of 663 bp) identity to F. equiseti (LS479418 [ITS], KU939020 [TEF1-α], MF667972 [mtSSU] and MG839490 [RPB2]) in NCBI databases, respectively. Pathogenicity tests were conducted with each F. equiseti isolate. Plants were grown in sterilized 10-cm diameter plastic pots containing steamed peat substrate, and three replicated pots (five plants/pot) were included in each treatment. Fifteen-day-old coriander plants were inoculated with 5 mL of 1×106 conidia/mL suspension by root-drenching method. Control plants were inoculated with SDW. Each treatment was incubated in a greenhouse at 29/22°C (12h/12h, light/dark), and watered every other day to keep high humidity. After 4 days, symptoms similar to those observed in the field were observed on inoculated plants (Fig. S2), whereas control plants remained symptomless. Reisolation of F. equiseti from inoculated plants fulfilled Koch's postulates, and identification was confirmed by morphological and molecular methods. To our knowledge, this is the first report of F. equiseti causing coriander stem and root rot in China. This pathogen poses a threat to coriander production, and its accurate identification is necessary to develop effective management strategies.

Species diversity of Pleosporalean taxa associated with Camellia sinensis (L.) Kuntze in Taiwan

Pleosporales species are important plant pathogens, saprobes, and endophytes on a wide range of economically important plant hosts. The classification of Pleosporales has undergone various modifications in recent years due to the addition of many families described from multiple habitats with a high level of morphological deviation. Numerous asexual genera have been described in Pleosporales that can be either hyphomyceteous or coelomycetous. Phoma- or coniothyrium-like species are common and have been revealed as polyphyletic in the order Pleosporales and linked with several sexual genera. A total of 31 pleosporalean strains were isolated in different regions of Taiwan between 2017 and 2018 from the leaves of Camellia sinensis plants with symptoms of leaf spot disease. These strains were evaluated morphologically and genotypically using multi-locus sequence analyses of the ITS, LSU, SSU, rpb2, tef1 and tub2 genes. The results demonstrated the affiliation of these strains with the various families in Pleosporales and revealed the presence of one new genus (Neoshiraia) and eight new species (Alloconiothyrium camelliae, Amorocoelophoma camelliae, Leucaenicola camelliae, L. taiwanensis, Neoshiraia camelliae, N. taiwanensis, Paraconiothyrium camelliae and Paraphaeosphaeria camelliae). Furthermore, to the best of our understanding, Didymella segeticola, Ectophoma pomi and Roussoella mexican were reported for the first time from C. sinensis in Taiwan.

Diversity of Nematode Microbial Antagonists from Algeria Shows Occurrence of Nematotoxic Trichoderma spp.

Fungi and bacteria associated to phytoparasitic nematodes Globodera rostochiensis and Meloidogyne spp. in Algeria were identified and characterized. Trichoderma spp. showed the highest prevalence in the cysts of G. rostochiensis. A number of isolates were identified through PCR amplification and the sequencing of the internal transcribed spacer (ITS)1-2 and Rpb2 gene regions. The most represented species were T. harzianum and T. afroharzianum. The latter and T. hirsutum were reported for the first time in Algeria. Fusarium spp., including F. oxysporum and F. solani, comprised a second group of fungi found in cysts. Taxa associated to females of Meloidogyne spp. included T. harzianum, Fusarium spp. and other hyphomycetes. To assess the efficacy of Trichoderma spp., two assays were carried out in vitro with the culture filtrates of two T. afroharzianum and T. harzianum isolates, to check their toxicity versus the second stage juveniles of M. incognita. After 24–48 h exposure, a mortality significantly higher than the control was observed for both filtrates at 1% dilutions. The TRI genes involved in the production of trichothecenes were also amplified with the PCR from some Trichoderma spp. isolates and sequenced, supporting a putative role in nematode toxicity. Bacteria isolated from the cysts of G. rostochiensis included Brucella, Rhizobium, Stenotrophomonas and Bacillus spp., identified through 16S rRNA gene sequencing. The potential of the microbial isolates identified and their mechanisms of action are discussed, as part of a sustainable nematode management strategy.

Tree inhabiting gnomoniaceous species from China, with Cryphogonomonia gen. nov. proposed.

Species of Gnomoniaceae are commonly associated with leaf spot diseases of a wide range of plant hosts worldwide. During our investigation of fungi associated with tree diseases in China, several gnomoniaceous isolates were recovered from symptomatic branches and leaves on different woody plants in the Fagaceae, Pinaceae, and Salicaceae families. These isolates were studied by applying a polyphasic approach including morphological, cultural data, and phylogenetic analyses of partial ITS, LSU, tef1, rpb2 and tub2 gene sequences. As a result, three species were identified with characters fitting into the family Gnomoniaceae. One of these species is described herein as Cryphognomonia pinigen. et sp. nov., characterized by developed pseudostromata and ascospores with obvious hyaline sheath; Gnomoniopsis xunwuensissp. nov. is illustrated showing sympodially branched conidiophore, oval or fusiform conidia; and one known species, Plagiostoma populinum. The current study improves the understanding of gnomoniaceous species causing diebacks and leaf spot on ecological and economic forest trees.

New section and species in Talaromyces.

Talaromyces is a monophyletic genus containing seven sections. The number of species in Talaromyces grows rapidly due to reliable and complete sequence data contributed from all over the world. In this study agricultural soil samples from Fujiang, Guangdong, Jiangxi, Shandong, Tibet and Zhejiang provinces of China were collected and analyzed for fungal diversity. Based on a polyphasic approach including phylogenetic analysis of partial ITS, BenA, CaM and RPB2 gene sequences, macro- and micro-morphological analyses, six of them could not be assigned to any described species, and one cannot be assigned to any known sections. Morphological characters as well as their phylogenetic relationship with other Talaromyces species are presented for these putative new species. Penicillium resedanum is combined in Talaromyces section Subinflati as T. resedanus.

Botrytis prunorum Associated to Vitis vinifera Blossom Blight in Chile.

Table grapes are highly susceptible to Botrytis cinerea infections during the bloom period. After reaching the flower development stage, B. cinerea remains quiescent until berry ripening or gives rise to blossom blight under specific climate conditions. A research study was conducted on the Chilean Central Valley during the 2018-2019 growing season. Flowers of Vitis vinifera cv. Thompson Seedless were collected and B. cinerea was isolated together to a second and morphologically different species, characterized by white mycelium and low to no sporulation (11.4% of total isolates). Three randomly selected isolates within this population were genetically examined and identified as Botrytis prunorum based on a phylogenetic multilocus approach using partial regions of genes RPB2, HSP60, and G3PDH or NEP1 and NEP2. Pathogenicity tests showed that B. prunorum infects and causes wilting in healthy table grape flowers. B. prunorum isolates were able to infect Thompson Seedless berries, inducing lesions between 13.11 and 41.53% with respect to the lesion diameter generated by B. cinerea B05.10. The fungicide sensitivity was evaluated. The three genetically characterized isolates were sensitive to boscalid and to cyprodinil/fludioxonil mixture with a mean EC50 value of 5.5 µg/ml and 0.065 µg/ml, respectively. However, loss of sensitivity to fenhexamid was determined, with a mean EC50 value of 5.13 µg/ml. Our understanding about blossom blight in V. vinifera has been limited to B. cinerea. Here we associated B. prunorum as a second causal agent of this disease in Chile. This data represents a first approach to the epidemiological characteristics of B. prunorum associated with blossom blight in table grapes.