First records of three Lepiota species (Agaricales, Basidiomycota) from Ukraine, with notes on a poorly known species, Lepiota subalba

New records of four species of the genus Lepiota (Agaricales, Basidiomycota) are reported from Ukraine. Three species, L. fuscovinacea, L. griseovirens, and L. roseolivida, are recorded in Ukraine for the first time, whereas a poorly known species, L. subalba, earlier known in Ukraine from a few records, is confirmed using molecular identification methods. All species reports are supplemented with original descriptions and drawings based on newly collected material, as well as data on general distribution, habitat, references to new collections and public databases. Original nucleotide sequence of the ITS region of ribosomal DNA obtained from our voucher specimen of L. subalba is provided.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Cucurbita argyrosperma in Mexico

Cucurbita argyrosperma, commonly named as winter or cushaw squash, is highly sought for its seeds, which have important uses in culinary arts. During the autumn 2021, powdery mildew-like signs and symptoms were observed on cushaw squash in several commercial fields located in Cocula, Guerrero, Mexico. Signs were initially appeared as whitish powdery patches on both sides of leaves and then covering entire leaves and causing premature senescence. The disease incidence was estimated to be 80% in about 1000 plants in two fields. The mycelium was amphigenous, persistent, white in color, and occurred in dense patches. A voucher specimen was deposited in the Herbarium of the Colegio Superior Agropecuario del Estado de Guerrero under the accession number CSAEG22. For the morphological characterization by light microscopy, fungal structures were mounted in a drop of lactic acid on a glass slide. Microscopic examination showed nipple-shaped hyphal appressoria. Conidiophores (n = 30) were straight, 100 to 190 × 10 to 12 μm and produced 2 to 6 conidia in chains. Foot-cells were cylindrical, 41 to 78 μm long, followed by 1 to 2 shorter cells. Conidia (n = 100) were ellipsoid-ovoid to barrel-shaped, 29.5 to 39.1 × 19.4 to 22.7 μm, and contained conspicuous fibrosin bodies. Germ tubes were produced from a lateral position on conidia. Chasmothecia were not observed during the growing season. The morphological characters were consistent with those of the anamorphic state of Podosphaera xanthii (Braun and Cook 2012). For further confirmation, total DNA was extracted from conidia and mycelia following the CTAB method (Doyle and Doyle 1990), 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 Likelihood method, including ITS and 28S sequences of isolates of Podosphaera spp. were performed and confirmed the results obtained in the morphological analysis. The isolate CSAEG22 grouped in a clade with isolates of Podosphaera xanthii. The ITS and 28S sequences were deposited in GenBank under accession numbers OL423329 and OL423343, respectively. Pathogenicity was confirmed by gently dusting conidia from infected leaves onto ten leaves of healthy C. argyrosperma plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 35 ºC, and relative humidity of 60 to 70%. All inoculated leaves developed similar signs to the original observation after 10 days, whereas control leaves remained symptomless. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed on diseased plants, fulfilling Koch’s postulates. Podosphaera xanthii has been previously reported on C. maxima, C. moschata, and C. pepo in Mexico (Yañez-Morales et al. 2009; Farr and Rossman 2021). To our knowledge, this is the first report of P. xanthii causing powdery mildew on C. argyrosperma in Mexico. This pathogen is a serious threat to C. argyrosperma production in Mexico and disease management strategies should be developed.

Development of Gas Chromatography-Mass Spectrometry Fingerprints for Warburgia Ugandensis Herbal Materials

Warbugia ugandensis (W. ugandensis) is among the 10 most utilised medicinal plants in East Africa. Stem bark and leaves are used as remedies for malaria, stomachache, coughs and skin diseases. Consequently, the plant is endangered because of uncontrolled harvest and lack of domestication. There is therefore fear of poor quality commercialised products due to lack of evaluation mechanisms. This study explored the chemical profiles that could be used to confirm its authenticity and purity. W. ugandensis used as reference during method development was harvested from Kenyatta University Medicinal Plant Research Garden (KUMPRG). Six other samples were obtained from different geographical locations in Kenya. The samples were identified by a botanist and a voucher specimen (MO/002- 008/2013) deposited in the East African Herbarium, National Museums of Kenya, Nairobi. Samples were harvested and processed by World Health Organization (WHO) recommended methods. Chromatographic profiles of the leaf and stem bark were established based on parameters arrived at iteratively. The study characterised over 100 compounds in the leaf and stem bark. Based on area percent and known medicinal value, 22 compounds from the leaf and 38 from the stem bark were selected as major chemical profiles. The compounds in the stem bark included gamma-sitosterol (1.0%–2.5%), squalene (0.2%–4.6%), isolongifolene (1.2%–2.8%), phenol 2-methoxy (0.8%–1.8%) and nerolidol (0.3%–1.5%). Those in the leaf included nerolidol 2 (0.3%–1.1%), phytol (0.6%–1.7%), 2-methoxy phenol (0.2%– 2.2%), gamma-tocopherol (0.2%–0.9%), vitamin E (0.4%–1.5%) and gamma-sitosterol (1.8%–4.9%). Most of these compounds were characterised in W. ugandensis for the first time. The profiles therefore can form fingerprints for use to evaluate its quality, purity and authenticity.

The loneliness of the long-distance ethnobotanist: a constructive critique of methods used in an ethnoveterinary study in Mongolia

Background Fieldwork plays an important role in research projects across a variety of fields, especially in the multidisciplinary setting of natural and social science research. As is the nature of fieldwork, things do not always work out as planned, and yet this is not often written about. In response to the need for honest and transparent accounts of fieldwork, the purpose of this article is to review the methods used during fieldwork for the first author’s dissertation research on ethnoveterinary knowledge. Methods To critically review and reflect on the fieldwork methods used for an ethnoveterinary study in Mongolia, we compare the theory underpinning each method with the practical reality of implementing the method in the field. From this comparison, we draw out and discuss a number of key themes. Results Eighteen methods and approaches used for the research project are reviewed and compared. From this, we distil and further discuss the following five overarching themes: reflections on specific data collection methods (free listing, semi-structured interviews with interpreters, voucher specimen collection); assumptions around involving local people; power dynamics; gender relations; and researcher well-being. Conclusion By juxtaposing the theory and practical reality of the methods used, we highlight many potential fieldwork challenges and, within this context, offer general pointers, especially for novice female researchers doing fieldwork in foreign countries. A critical review of this type, where the experience and use of various methods, techniques, and approaches are openly shared and evaluated, is a contribution to selecting, adapting, and fine-tuning the methods best suited to a particular research context.

Evaluation of Antimicrobial Activity of Solvent Extracts and Essential Oil of Leaves of Cipadessa baccifera (Roth.) Miq.

The present study was designed to conduct with the main purpose of evaluation of antimicrobial activity of solvent extracts and essential oil of leaves of Cipadessa baccifera (Roth.) Miq. The plant samples for investigation were collected from Thavarekere and Savandurga, Magadi Taluk, Bengaluru Rural district, and outskirts of Bengaluru, and identification was authenticated by National Ayurveda and Dietetics Research Institute, Bangalore; vide voucher specimen number, RRCBI-8971.The dried leaf samples were pulverized in an electric blender and the powdered material was stored in air tight containers for further analyses. The leaf samples were subjected to sequential extraction using Soxhlet apparatus, and extracted exhaustively in organic solvents such as, hexane, chloroform, methanol and water. The essential oil from leaf sample was extracted in Clevenger apparatus. The sequential extracts and essential oil of leaf parts of C. baccifera (Roth) Miq. were subjected to evaluation of antimicrobial activity against diarrhoea, skin, wound and oral infections causing selected pathogens, including 07 Gram positive, 06 Gram negative bacteria and 06 fungal strains. Results revealed that essential oils and crude solvent extracts leaves of Cipadessa baccifera showed significant anti-bacterial potential against diarrhoea, skin wound and oral pathogens inhibiting both Gram positive, Gram negative and fungal species. The broad spectrum of anti-bacterial activity of C. baccifera revealed in the present investigation gives scientific validity for its usage in treatment of dysentery, skin related disorders and wounds in traditional and folk medicines.

First Report of Sawadaea polyfida Causing Powdery Mildew of Acer palmatum in China

Acer palmatum Thunb. is an important colorful leaf ornamental tree species widely distributed in Japan, Korea and China (Carlos et al. 2016). In October 2019, powdery mildew was observed on leaves of A. palmatum planted at Qixia Mountain Park and the campus of Nanjing Forestry University, Nanjing, Jiangsu, China. The powdery mildew infected and colonized leaves, covering both leaf surfaces with white mycelia, giving affected plants an unsightly appearance. Nearly 17.4% of the plants (87/501) exhibited these signs and symptoms. Fresh specimens were collected and examined for the identification of the pathogen. Photos were taken with a ZEISS Axio Imager A2m microscope and a scanning electronic microscope. Chasmothecia were scattered or aggregated on the upper and lower surfaces of the leaves, blackish brown, oblate, 157.5 to 238.1 × 152.3 to 217.8 μm (n=30), with numerous appendages (100 to 200). Appendages were often (1−) 2 to 3 times branched from the middle of the stalk, uncinate to circinate at the apex, hyaline, aseptate, 30.0 to 70.8 × 4.1 to 8.2 μm (n=30). Asci were 11 to 21 per chasmothecium (n=30), long oval, oval, oblong, with short stalk or sessile, 80.6 ± 8.6 × 40.3 ± 4.0 um (n=30) in length, 6 to 8 spored (n=30). Ascospores were ovoid, 18.2 ± 1.6 × 11.1 ± 1.2 μm (n=30). Microconidiophores were 25 to 50 × 4.0 to 5.5 μm, producing microconidia in chains. Microconidia were ellipsoidal, subglobose, 8.7 ± 0.6 × 7.2 ± 0.6 μm (n=30). Macroconidia were not observed. Based on the morphological characteristics, the fungus was identified as Sawadaea polyfida (C.T. Wei) R.Y. Zheng & G. Q. Chen (Zheng and Yu 1987). To confirm the causative species identity, a representative voucher specimen collected and deposited at Nanjing Forestry University was used for a molecular analysis. Mycelia and conidia were collected from diseased leaves and genomic DNA of the pathogen was extracted and the internal transcribed spacer region (ITS) was amplified with primers ITS1/ITS4 (White et al. 1990). The resulting sequence of 461 bp was deposited in GenBank (accession no. MW255383). BLAST result showed that this sequence fully agreed with a sequence of S. polyfida [AB193381.1 (ITS), identities = 461/461 (100%)]. A maximum likelihood phylogenetic analyses using IQtree v. 1.6.8 with the ITS sequence placed this fungus in the S. polyfida clade. Based on the morphology and phylogeny, the fungus was identified as S. polyfida (Hirose et al. 2005; Zheng and Yu 1987). Pathogenicity was tested through inoculation by gently pressing the naturally infected leaves onto healthy ones of three potted A. palmatum seedlings wih five leaves. Healthy leaves from three other seedlings served as control. Inoculated and control seedlings were placed in separate growth chambers maintained at 20 ± 2°C, 70% humidity, with a 16 h/8 h light/dark period. Symptoms developed 8 days after inoculation. The powdery mildew developing on the inoculated seedlings was sequenced and confirmed as S. polyfida. The control leaves did not develop powdery mildew. S. polyfida has been reported on Acer catalpifolium in China (Zheng and Chen 1980), A. amoenum, A. australe, A. japonicum, A. palmatum, A. shirasawanum, and A. sieboldianum in Japan (Hirose et al. 2005; Meeboonet al. 2015), as well as A. takesimense in Korea (Lee et al. 2011). To the best of our knowledge, this is the first report of powdery mildew caused by S. polyfida on A. palmatum in China. These results form the basis for developing effective strategies for monitoring and managing this disease.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Verbena bonariensis in Korea

Verbena bonariensis L., named as purple-top vervain or Argentinian vervain, is native to tropical South America. It is cultivated worldwide as an ornamental plant. During summer and autumn of 2020, over 50% of the leaves of V. bonariensis were found infected with powdery mildew in a flower garden in Seoul (37°35'19"N 127°01'07"E), Korea. White, superficial mycelia developed initially on the leaves and subsequently covered surfaces of leaves and stems, are resulting in leaf discoloration, early defoliation, and shoots distortion. Heavily infected plants lost ornamental value. A representative voucher specimen was deposited in the Korea University herbarium (KUS-F32168). Morphological characterization and measurements of conidiophores and conidia were carried out using fresh samples. Microscopic observation showed that aAppressoria on the superficial hypha were nipple-shaped, but rarely found or nearly absent. Conidiophores (n = 30) were cylindrical, 110 to 220 × 10 to 12 µm, and produced 2 to 5 immature conidia in chains with a sinuate outline, followed by 2 to 3 short cells. Foot-cells of conidiophores were straight, cylindrical, and 46 to 90 μm long. Conidia (n = 30) were hyaline, ellipsoid to doliiform, 28 to 40 × 18 to 24 μm with a length/width ratio of 1.3 to 2.0, and contained small be like oil-like drops, but without distinct fibrosin bodies. Primary conidia were apically rounded and sub-truncate at the base. Germ tubes were produced at perihilar position of the conidia. Chasmothecia were not observed. These morphological characteristics were typical of the conidial stage of the genus Golovinomyces (Braun and Cook 2012, Qiu et al. 2020). To identify the fungus, rDNA was extracted from the voucher sample. PCR products were amplified using the primer pair ITS1F/PM6 for internal transcribed spacer (ITS), and PM3/TW14 for the large subunit (LSU) of the rDNA (Takamatsu and Kano 2001). The resulting sequences were registered to GenBank (MW599742 for ITS, and MW599743 for LSU). Using Blast’n search of GenBank, sequences showed 100% identity for ITS and LSU with G. ambrosiae (MT355557, KX987303, MH078047 for ITS, and AB769427, AB769426 for LSU), respectively. Thus, based on morphology and molecular analysis, the isolate on V. bonariensis in Korea was identified as G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook. Pathogenicity tests were carried out by touching an infected leaf onto healthy leaves of disease-free pot-grown plants using a replication of five plants, with five non-inoculated plants used as controls. After 7 days, typical powdery mildew colonies started to appear on the inoculated leaves. The fungus on inoculated leaves was morphologically identical to that originally observed in the field. All non-inoculated control leaves remained symptomless. On different global Verbena species, tThere have been many reports of Golovinomyces powdery mildews including G. cichoracearum s.lat., G. longipes, G. monardae, G. orontii s.lat., and G. verbenae (Farr and Rossman 2021). In China, G. verbenae was recorded on V.erbena phlogiflora (Liu et al. 2006). Golovinomyces powdery mildew has not been reported on Verbena spp. in Korea. Powdery mildew has been reported on V. bonariensis in California, but identity of the causal agent had not been reported. To our knowledge, this is the first report on the identity of the powdery mildew caused by G. ambrosiae on V. bonariensis in Korea. Since heavily infected plants lost ornamental value, appropriate control measures should be developed.

A re-examination of the molecular systematics and phylogeography of taxa of the Peromyscus aztecus species group, with comments on the distribution of P. winkelmanni

The objectives of this study are to examine the available molecular data from the mitochondrial cytochrome-b gene (Cytb) and a concatenated dataset with this gene and two nuclear introns (Adh-1-I2 and Fgb-I7) to reexamine the systematic and phylogeographic conclusions reached by Sullivan et al. (1997) concerning the Peromyscus aztecus species group. The divergence of samples of P. aztecus oaxacensis across the Isthmus of Tehuantepec are further examined and taxonomic revisions are suggested. In addition, this study reviews the sources of data that lead to the conclusion that P. winkelmanni occurred in the Sierra Madre del Sur in Guerrero including a morphometric examination of a reported voucher. Bayesian and maximum likelihood analyses were conducted on a dataset of 31 Cytb sequences of all taxa in the P. aztecus group except for P. a. cordillerae and a concatenated dataset including five individuals of this group. Representative taxa of the P. boylii, P. mexicanus, and P. truei groups were included in both analyses. Body and cranial measurements of the voucher of the P. winkelmanni from Guerrero from which a Cytb sequence is reported to have been obtained was compared with measurements from specimens taken from the vicinity of Dos Aguas, Michoacán, including the type locality. We identified seven instances involving problematic identifications in GenBank. Once these issues were addressed, well-supported monophyletic sister clades of the P. aztecus and P. boylii species groups were recovered from phylogenetic analyses of Cytb sequences (Fig 1). Phylogenetic analyses of the Cytb and the concatenated datasets recover similar topologies that support the relationships of taxa of the aztecus group proposed by an earlier molecular study. Populations of P. a. oaxacensis southeast of the Isthmus of Tehuantepec represent a distinct species. Measurements of the voucher from Guerrero identified as the source of a P. winkelmanni Cytb sequence are smaller than P. winkelmanni for several characters. The divergent populations of P. a. oaxacensis from southeast of the Isthmus of Tehuantepec are recognized as two subspecies of P. cordillerae, P. c. cordillerae and P. c. hondurensis, whereas those northwest of the Isthmus are retained as P. a. oaxacensis. The lack of genetic divergence observed between P. a. evides and P. a. oaxacensis questions whether these two taxa should continue to be recognized as separate subspecies. Northern and southern populations of P. spicilegus demonstrate moderate divergence and additional examination of morphological and molecular differentiation within this taxon is warranted. The distribution of P. winkelmanni should be restricted to the vicinity of Dos Aguas, Michoacán, due to the lack of a voucher specimen that would confirm its reported occurrence in Guerrero.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Benincasa hispida in Korea

Benincasa hispida (Thunb.) Cogn. (syn. B. cerifera Savi, Cucurbita hispida Thunb.), called wax gourd or ash gourd, is a cucurbitaceous vine grown for medicinal purposes and commercial values of its large fruits in Southeast Asia (Al-Snafi 2013). During the summer and autumn of 2020, leaves of wax gourd were observed to be affected by powdery mildew with 100% disease incidence in an experimental plot of Jeonbuk National University (35°50′55″N, 127°07′48″E), Korea. Fungal colonies were initially circular to irregular, forming white patches on both sides of the leaves and young stems, finally covering entire leaves and causing premature senescence of the leaves and poor growth. A representative voucher specimen was deposited in the Korea University herbarium (KUS-F32171). At least 30 measurements were taken for each asexual diagnostic features. Conidiophores arising from superficial hyphae were straight, 100 to 210 μm long, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 46 to 74 ×10 to 12 μm, followed by 1 to 2 shorter cells. Conidia were ellipsoid-ovoid to barrel-shaped, 30 to 40 × 18 to 23 μm with a length/width ratio of 1.4 to 2.0 and contained conspicuous fibrosin bodies. Germ tubes were produced from a lateral position on conidia. Sexual stage was not observed during the growing season. The morphological characteristics of the fungus were compatible with those of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012), a well-known cucurbitaceous powdery mildew. DNA was extracted from mycelium, and primer sets ITS1F/PM6 and PM3/TW14 were used for amplification of ITS1-5.8S-ITS2 regions and 5´-end of 28S rDNA gene, respectively (Takamatsu and Kano 2001). Sequences determined in this study were deposited to the GenBank under the accession numbers MW559231 and MW559420, respectively. The sequences for ITS regions and 28S rDNA gene showed 99.78% and 99.07% similarity respectively with those of P. xanthii (MH465242, MH465243, MT250855 for ITS, and MK357436, MT826247 for LSU). Pathogenicity was confirmed twice by pressing a diseased leaf onto young leaves of five wax gourd plants. Five non-inoculated plants were used as controls. Inoculated leaves developed symptoms after 5 days, whereas the control plants remained symptomless. The fungus present on the inoculated plants was identical morphologically to that originally observed on diseased plants. Sphaerotheca fuliginea (syn. P. xanthii) on B. hispida has been listed in Hungary, India, Japan, Singapore, and Taiwan so far (Farr and Rossman 2021). Recently, the identity of P. xanthii on B. hispida in Taiwan was confirmed with morphological examination and molecular analysis by Wu and Kirschner (2017). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on B. hispida in Korea. Since wax gourd production is only recently started on a commercial scale in the southern part of Korea, powdery mildew infections pose a serious threat to the safe production of the fruits, especially in organic farming where chemical control options are limited.

First Report of Golovinomyces sonchicola Causing Powdery Mildew on Sonchus oleraceus in Mexico

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.