Lepiota lilacea (Agaricales, Basidiomycota), a New Record from Pakistan

The Rhizopus strains in the Universitas Indonesia Culture Collection (UICC) were isolated from tempeh from various regions in Indonesia. Five strains of Rhizopus (UICC 10, UICC 85, UICC 119, UICC 120, and UICC 135) were previously identified based on phenotypic characters (morphology and physiology) as R. oryzae. The aim of this study was to clarify the identities of five Rhizopus strains based on internal transcribed spacer (ITS) regions of ribosomal DNA sequence data. Molecular phylogeny was carried out to establish the identities of these five strains. Primer set ITS5 and ITS4 were used for amplification and sequencing of the ITS regions of rDNA. The sequence data was sent to genBank for homology search using basic local alignment search tool (BLAST) program. Construction of phylogenetic tree was conducted using Neighbor-Joining method with Kimura’s two-parameter model. The results on gel electrophoresis showed that sizes of the ITS regions of rDNA fragment lengths of all strains were approximately 650 bp. The BLAST homology search results of five strains showed 99.8% homology to R. oryzae CBS 112.07T type strain. The phylogenetic tree clearly indicated that these five strains were included in a monophyletic group with R. oryzae CBS 112.07T type strain, and it was supported by high bootstrap value (84%). Morphological observation of all strains showed the characters corresponded to R. oryzae. The ITS regions of rDNA sequences and morphological characters have confirmed the strains as R. oryzae.

Molecular profIles of PCR-RFLP and sequence analysis of internal transcribed spacer (ITS) regions of rDNA were compared between morphologically distinguishable species of Trichoderma isolated from substrates of oyster mushroom in Korea, T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum, T. virens, and two unidentified species, Trichoderma sp. 1 and 2. PCR­RFLP analysis divided the Trichoderma spp. into six RFLP groups, A, B, C, D, E, and F. The RFLP groups were generally agreed with described morphological species, except that the RFLP group A containing the two unidentified species. A neighbor-joining tree based on ITS sequences well supported RFLP groups observed by RFLP analysis of ITS regions of rDNA. Additionally, the two unidentified species, Trichoderma sp. 1 and 2, which could not be distinguished by PCR­RFLP analysis, were separated in sequence analysis of ITS regions of rDNA.

Using a sedimentation method, the prevalence of the nodular worm Oesophagostomum stephanostomum (Nematoda: Strongylida) in western lowland gorillas at Moukalaba-Doudou National Park (MDNP), Gabon, was determined in fecal samples collected between January 2007 and October 2011, along with their coprocultures. Concurrently, possible zoonotic Oesophagostomum infections in villagers living near MDNP were assessed from their fecal samples collected during October and November of 2011. In the gorillas, strongylid (Oesophagostomum and/or hookworm) eggs were found in 47 of 235 fecal samples (20.0 %) and Oesophagostomum larvae were detected in 101 of 229 coprocultures (44.1 %). In the villagers, strongylid eggs were found in 9 of 71 fecal samples (12.7 %), but no Oesophagostomum larvae were detected in coprocultures. The internal transcribed spacer (ITS) region of ribosomal RNA gene (rDNA) and cytochrome c oxidase subunit-1 (cox-1) region of mitochondrial DNA (mtDNA) of coprocultured Oesophagostomum larvae were amplified using parasite DNA extracted from 7–25 larvae/sample, cloned into Escherichia coli, and sequenced. Sequenced rDNA contained 353/354-bp long ITS1, 151-bp long 5.8S rDNA, and 227-bp long ITS2. Parts of clones showed variations at 1–3 bases in the ITS1 region at a frequency of 24/68 (35.3 %) and at 1–2 bases in the ITS2 region at a frequency of 7/68 (10.3 %), whereas the 5.8S rDNA was essentially identical. Sequenced cox-1 gene of the parasites, 849 bp in length, showed a higher number of nucleotide variations, mainly at the third nucleotide position of the codon. The majority of clones (27/41 (65.9 %)) had an identical amino acid sequence. These results suggest that at MDNP, Gabon, only a single population of O. stephanostomum with a degree of genetic diversity is prevalent in western lowland gorillas, without zoonotic complication in local inhabitants. The possible genetic variations in the ITS region of rDNA and cox-1gene of mtDNA presented here may be valuable when only a limited amount of material is available for the molecular species diagnosis of O. stephanostomum.

MenispermumdauricumDC. is an ornamental plant used in traditional Chinese medicine. (Tang et al. 1992). In September 2019, a leaf spot on M. dauricum DC. was first found in a medicinal plant plantation in Harbin city (45.80°N, 126.53°E), Heilongjiang Province, China. The incidence was 76-90% on the 0.02 ha plantation. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to blight. Fresh leaf samples from ten M. dauricum plants with typical symptoms were collected. The areas of leafbetween symptomatic and healthy tissue (5㎜×5㎜)were cut and surface disinfeated in 75% ethanol for 2 min, and with 1% HgCl2 for 1min, and then rinsed three times with sterile water. Small lesion pieces were incubated on potato dextrose agar (PDA) for 7 days at 25℃, in the dark. Ten fungal isolates were obtained and transferred onto new PDA and potato carrot agar (PCA) plates to establish pure cultures. After 8 days, the colonies on PDA were 75-86㎜ in diameter, circular, with distinct concentric rings and a whitish aerial-mycelium margin, cottony, light gray to dark bluish brown. The colonies on the PCA were olive-green and bordered by white aerial hyphae. A total of 150 conidia were single or in short chains, obclavate, oval or inverted pear, light brown to brown, smooth or slightly spiny, with 1 to 6 transverse septa, 0 to 4 longitudinal or oblique septa, not narrow or slightly narrowed at the separation, 22.5-42.5×7.5-15.5㎛, and rostrate. Conidiophores were simple, erect, or ascending, dark brown, geniculate, septate, and with one or several conidial scars, 32.5-77.5×3.0-5.0㎛. Beaks were columnar or conical, 7.5-22.5×2.5-3.5㎛. Morphologically, all ten isolates were most similar to Alternaria alternata (Simmons 2007). For further identification of the fungus at the molecular level, internal transcribed spacer rDNA regions (ITS), RNApolymerase second largest subunit gene(RPB2) and Alternariamajorallergen(Alt a 1) were amplified and sequenced using the primers ITS1 and ITS4, RPB2-5F2 andRPB2-7CR, Alt-forandAlt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS: MT995193, MZ150794, RPB2: MT999483, MZ170963, Alt a 1: MT802122, MZ170962). BLAST search of these sequences showed 99%-100% homology with the ITS (FJ196306), RPB2 (KC584375) and Alt a 1 (KT315515) of the type strain CBS 916.96 of A. alternata, respectively. Thus, the fungus was identified as A. alternata based on the morphology and molecular analysis. For the pathogenicity test, spore suspensions (1×106 spores/mL) of the representative isolates BFG001 and BFG002 were sprayed onto the leaves of six healthy plants, separately. As a control, six plants were treated with sterile distilled water. The plants used in the experiment were covered with plastic bags and incubated at 25℃ for 10 days. Eight days after inoculation, irregular, slightly sunken black leaf spots appeared at the leaf margin. The experiment was repeated three times with the same method. The same fungus was successfully re-isolated from the leaves of the inoculated plants, fulfilling Koch's postulates. No symptoms were observed on control plants. To our knowledge, this is the first report of leaf spot disease on M.dauricum DC. caused by A. alternata in the world. The appearance of leaf spot disease reduces the yield and quality of Chinese medicinal materials. This report has laid the foundation for the further research and control of leaf spot disease.

PCR primers for selective detection of intra-species variations in the bloom-forming cyanobacterium, Microcystis

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.

Fomitiporia is a genus of wood-inhabiting Hymenochaetaceae (Agaricomycetes) that includes pathogens and decomposers occurring on a wide array of plant substrates. Some species decay culms of woody bamboos, a poorly known biotic interaction. Four bambusicolous species of Fomitiporia are currently known. However, no studies concerning their phylogenetic relationships have been performed. In order to assess species boundaries and their relationships to each other, we conducted molecular phylogenetic analyses of nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and portions of nuc 28S rDNA (28S), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1), as well as morphological analyses. Four species that occur on culms of woody bamboos, F. bambusarum, F. spinescens, F. uncinata, and the new species F. bambusipileata, grouped together in an exclusive clade within a primarily Neotropical lineage. The new species differs from all other species in the group by the pileate basidiomata. Hypotheses regarding host-exclusivity are discussed.

ABSTRACTIn this paper, species of the genus Morchella are investigated in China. Based on morphological characteristics and molecular phylogenetic analyses of the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and the combined data set ITS + nuclear large subunit rDNA (28S) + the translation elongation factor 1-α (TEF1) gene + RNA polymerase II first largest subunit (RPB1) + RNA polymerase II second largest subunit (RPB2), six new phylogenetic species are illustrated and described: M. clivicola, M. confusa, M. odonnellii, M. owneri, M. yangii, and M. yishuica. Furthermore, two new record species, M. dunensis and M. palazonii, which were only known in Europe, are now reported for the first time from Asia. New species of morels will provide additional information on species diversity and genetic resource candidates for improving the cultivation of this economically important fungus.

Sequences of the ribosomal DNA (rDNA) internal transcribed spacer (ITS) regions were examined to infer a molecular phylogeny of small-spored Phomopsis isolates, designated W-type (mainly white colony, weakly virulent, bearing both alpha and beta conidia at 25°C on PDA) and G-type (mainly gray colony, highly virulent, bearing only alpha conidia at 25°C on PDA), and P. amygdali from fruit trees. Phomopsis G-type and P. amygdali were a monophyletic group distinct from the W-type. The W-type isolates were divided into two monophyletic groups. Diaporthe citri, D. tanakae, P. asparagi, P. viticola, P. vitimegaspora and D. nomurai, which are morphologically distinguishable from W- and G-types, differed from the W- and G-types in molecular phylogenetic analyses. PCR-RFLP analysis of rDNA ITS regions was useful to distinguish each of the Phomopsis species and groups using three restriction enzymes. In mating tests, W-type isolates from fruit trees were heterothallic and inter-fertile even between isolates belonging to the different monophyletic groups. Isolates of the G-type and P. amygdali collected in Japan were cross-fertile. Some isolates from Lunaria annua, Ulmus glabra and Juglans regia belonged to one of the two monophyletic groups of the W-type and were cross-fertile with W-type isolates from Rosaceous fruit trees.

The taxonomy of the umbelliferous species Angelica amurensis and its allies was reviewed on the basis of molecular phylogenies derived from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) regions. Strict consensus of six minimal length 119-step trees derived from equally weighted maximum parsimony analysis of combined nuclear rDNA ITS1 and ITS2 sequences from 29 accessions of Angelica and outgroups indicated that Angelica purpuraefolia, known to be endemic to Korea, is the same species as A. amurensis. Comparisons of sequence pairs across both spacer regions revealed identity or 1-2 bp differences between A. purpuraefolia and A. amurensis. These results indicated that the two taxa are not distinguished taxonomically. Also, nuclear rDNA ITS regions are discussed as potential barcoding loci for identifying Korean Angelica.

Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4–6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and ¼ potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.

Agaric fungi of the southern Appalachian Mountains including Great Smoky Mountains National Park are often heterozygous for the rDNA internal transcribed spacer region (ITS) with >42% of collections showing some heterozygosity for indels and/or base-pair substitutions. For these collections, intra-individual haplotype divergence is typically less than 2%, but for 3% of these collections intra-individual haplotype divergence exceeds that figure. We hypothesize that high intra-individual haplotype divergence is due to hybridization between agaric fungi with divergent haplotypes, possibly migrants from geographically isolated glacial refugia. Four species with relatively high haplotype divergence were examined: Armillaria mellea, Amanita citrina f. lavendula, Gymnopus dichrous and the Hygrocybe flavescens/chlorophana complex. The ITS region was sequenced, haplotypes of heterozygotes were resolved through cloning, and phylogenetic analyses were used to determine the outcome of hybridization events. Within Armillaria mellea and Amanita citrina f. lavendula, we found evidence of interbreeding and recombination. Within G. dichrous and H. flavescens/chlorophana, hybrids were identified but there was no evidence for F2 or higher progeny in natural populations suggesting that the hybrid fruitbodies might be an evolutionary dead end and that the genetically divergent Mendelian populations from which they were derived are, in fact, different species. The association between ITS haplotype divergence of less than 5% (Armillaria mellea = 2.6% excluding gaps; Amanita citrina f. lavendula = 3.3%) with the presence of putative recombinants and greater than 5% (Gymnopus dichrous = 5.7%; Hygrocybe flavescens/chlorophana = 14.1%) with apparent failure of F1 hybrids to produce F2 or higher progeny in populations may suggest a correlation between genetic distance and reproductive isolation.

Gummy stem blight is a major foliar disease of muskmelon (Cucumis melo L.). In this study, morphological characteristics and rDNA internal transcribed spacer (ITS) sequences were analyzed to identify the causal organism of this disease. Morphological examination of the Jeonbuk isolate revealed that the percentage of monoseptal conidia ranged from 0% to 10%, and the average length × width of the conidia was 70 (± 0.96) × 32.0 (± 0.15) μm on potato dextrose agar. The BLAST analysis showed nucleotide gaps of 1/494, 2/492, and 1/478 with identities of 485/492 (98%), 492/494 (99%), 491/494 (99%), and 476/478 (99%). The similarity in sequence identity between the rDNA ITS region of the Jeonbuk isolate and other Didymella bryoniae from BLAST searches of GenBank was 100% and was 95.0% within the group. Nucleotide sequences of the rDNA ITS region from pure culture ranged from 98.2% to 99.8%. Phylogenetic analysis with related species of D. bryoniae revealed that D. bryoniae is a monophyletic group distinguishable from other Didymella spp., including Ascochyta pinodes, Mycosphaerella pinodes, M. zeae-maydis, D. pinodes, D. applanata, D. exigua, D. rabiei, D. lentis, D. fabae, and D. vitalbina. Phylogenetic analysis, based on rDNA ITS sequence, clearly distinguished D. bryoniae and Didymella spp. from the 10 other species studied. This study identified the Jeonbuk isolate to be D. bryoniae.

From 2018 to 2020, powdery mildew-like signs and symptoms were observed on chayote (Sechium edule var. virens levis) in a commercial field located in Santa María del Río, San Luis Potosí, Mexico. Signs appeared as whitish powdery masses on both sides of leaves and stems. Disease incidence was about 30% and signs covered up to 70% of leaf surface. Ten samples were collected and analyzed. Mycelium was amphigenous, persistent, white, in dense patches. Hyphal appressoria were lobed and solitary. Conidiophores (n = 30) were hyaline, erect, straight, and 62 to 101 μm long. Foot cells were cylindrical and straight, followed by 1-3 shorter cells, and forming conidia in short chains. Conidia (n = 100) were hyaline, surface striate, cylindrical-ellipsoid, doliiform or ovoid, 25.7 to 37.6 × 11.9 to 18.4 μm, without fibrosin bodies, and with germ tubes terminal or subterminal. Conidial appressoria were lobed. Chasmothecia were not observed. The morphological characters were consistent with those of the anamorphic state of Neoerysiphe sechii (Gregorio-Cipriano et al. 2020). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH192. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium, and the internal transcribed spacer (ITS) region and part of the 28S gene were amplified by PCR and sequenced. The ITS region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000) and NL1/TW14 (Mori et al. 2000) for the first and second reactions, respectively. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods, including ITS and 28S sequences of isolates of Neoerysiphe spp. were performed and confirmed the results obtained in the morphological analysis. The isolate UACH192 grouped in a clade with isolates of N. sechii. The ITS + 28S sequence was deposited in GenBank under accession number MZ468642. Pathogenicity was confirmed by gently dusting conidia from infected leaves onto ten leaves of healthy chayote plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 60 to 70%. All inoculated leaves developed similar symptoms to the original observation after 8 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. The pathogenicity test was repeated twice with similar results. Based on morphological data and phylogenetic analysis, as well as pathogenicity test, the fungus was identified as N. sechii. This pathogen has been previously reported causing powdery mildew on S. edule and S. mexicanum in Veracruz, Mexico (Gregorio-Cipriano et al. 2020). However, to our knowledge, this is the first report of N. sechii causing powdery mildew on chayote in San Luis Potosí (Central Mexico). This pathogen represents a serious threat to chayote production and disease management strategies should be developed.

Sonchus oleraceus, common sow thistle, is native to Europe, Northern Africa, and Western Asia. This plant has become a common weed throughout the world. In Mexico, this weed has become widely naturalized by replacing indigenous plants and invading many agricultural areas. During the spring of 2018 and 2019, common sow thistle plants showing typical symptoms and signs of powdery mildew, were collected from agricultural fields in Ahome, Sinaloa, Mexico. As much as 30% of plants were diseased and 60 to 95% of the foliage was affected. Mycelium was conspicuous and white-gray, and on stems and both surfaces of leaves. Appressoria were nipple-shaped to crenulate. Conidiophores (n= 30) were hyaline, cylindrical, erect, and up to 150 μm long. Foot-cells (n= 30) were distinctly curved, 47 to 75 × 10 to 13 μm, slightly constricted, followed by 1-3 shorter cells and formed conidia in chains. Conidia (n= 100) were ellipsoid to doliiform to subcylindrical, 28 to 37 × 14 to 19 μm, lacked fibrosin bodies, and germinated from the apex. Chasmothecia were not observed. The morphological characters were consistent with those of the anamorphic state of Golovinomyces sonchicola (Braun and Cook 2012, Jakše et al. 2019). A voucher specimen (accession no. FAVF215) was deposited in the Herbarium of the Faculty of Agriculture of El Fuerte Valley at the Autonomous University of Sinaloa (Juan Jose Rios, Sinaloa, Mexico). To confirm the morphological identification, genomic DNA was extracted from mycelium and conidia, and the internal transcribed spacer (ITS) region and part of the 28S gene were amplified by PCR and sequenced. The ITS region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000) and NL1/TW14 (Mori et al. 2000) for the first and second reactions, respectively. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods (Braun et al. 2019), including ITS and 28S sequences of isolates of Golovinomyces spp. were performed and confirmed the results obtained from the morphological analysis. Isolate FAVF215 grouped in a clade with the other isolates of G. sonchicola. The ITS and 28S sequences were deposited in GenBank under accession numbers MW425872 and MW442972, respectively. Pathogenicity was demonstrated by gently dusting conidia from infected leaves onto leaves of 20 healthy plants and covered with plastic bags for 24 h. Ten non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25 to 35ºC. All inoculated plants developed similar symptoms to those observed in the field from natural infections after 12 days, whereas powdery mildew symptoms and signs were not observed on control plants. The morphology asexual structures of fungus on inoculated plants were identical to those on naturally infected plants, fulfilling Koch's postulates. Inoculation tests were repeated twice with identical results. Based on the morphological data and phylogenetic analysis, the fungus was identified as G. sonchicola. This fungus has been reported causing powdery mildew on S. oleraceus in Germany, The Netherlands, Slovenia, and The United Kingdom (Farr and Rossman 2021). To the best of our knowledge, this is the first report of G. sonchicola causing powdery mildew on S. oleraceus in Mexico. This powdery mildew pathogen may represent an option for the biological control of common sow thistle.