New species and records of Tricholoma (Agaricales: Tricholomataceae) sections Genuina and Megatricholoma from Costa Rica and United States

Species of Tricholoma sections Genuina and Megatricholoma are characterized by having pilei that are some shade of brown, and by white or yellow lamellae that also discolor some shade of brown. From Costa Rica in sect. Genuina, we describe as new T. luteopallidum, T. cacumense, and T. talamancense, and confirm the occurrence of Tricholoma stans, and from the United States describe as new Tricholoma brunneoluteum. Tricholoma roseoacerbum from sect. Megatricholoma is con-firmed for Costa Rica. Morphology as well as ITS sequences are employed to confirm the species’ identifications or circumscription of the new species.

First Report of Powdery Mildew Caused by Erysiphe cruciferarum on Camelina sativa in Montana

Camelina sativa (L.) Crantz, also known as false flax, is an annual flowering plant in the family Brassicaceae and originated in Europe and Asia. In recent years, it is cultivated as an important biofuel crop in Europe, Canada, and the northwest of the United States. In June of 2021, severe powdery mildew was observed on C. sativa ‘Suneson’ plants under greenhouse conditions (temperature 18.3°C/22.2°C, night/day) in Bozeman, Montana (45°40'N, 111°2'W). The disease incidence was 80.67% (150 pots, one plant per pot). White ectophytic powdery mildew including mycelia and conidia were observed on the upper leaves, usually developed from bottom tissues to top parts, also present on stems and siliques. Mycelia on leaves were amphigenous and in patches, often spreading to become effused. These typical symptoms were similar to a previous report of powdery mildew on Broccoli raab (Koike and Saenz 1997). Appressoria are lobed, and foot cells are cylindrical with size 18 to 26 × 7 to 10 μm. Conidia are cylindrical and produced singly, with a size of 35 to 50 × 12 to 21 μm and a length : width ratio greater than two (Koike and Saenz 1997). No chasmothecia were observed under the greenhouse conditions. The symptoms and fungal microscopic characters are typical of Pseudoidium anamorph of Erysiphe (Braun 1995). The specific measurements and characteristics are consistent with previous records of Erysiphe cruciferarum Opiz ex L. Junell (Braun and Cook 2012; Vellios et al. 2017). To identify the pathogen, the partial internal transcribed spacer (ITS) region of rDNA of sample CPD-1 was amplified using primers ITS1 and ITS4 (White et al. 1990). The amplicons were sequenced, and the resulting 559-bp sequence was deposited in GenBank (CPD-1, Accession number: OK160719). A GenBank BLAST search of the ITS sequences showed an exact match (100% query cover, E-value 0, and 100% identity 559/559 bp) with those of E. cruciferarum on hosts Brassica sp. (KY660929.1), B. juncea from Vietnam (KM260718.1) and China (KT957424.1). A phylogenetic tree was generated with the CPD-1 ITS sequence with several of ITS sequences of close species with different hosts obtained from the GenBank. Isolate CPD-1 was grouped with pathogens from Brassica hosts rather than the holotype strain (KU672364.1) from papaveraceous hosts. To fulfill Koch's postulates, pathogenicity was confirmed through inoculation by dusting conidia onto leaves of seven healthy, potted, 14-day-old C. sativa seedlings (cv. Suneson). Seven non-inoculated plants served as a control treatment. The plants were incubated in a greenhouse with a temperature of 18°C (night) to 22°C (day). The inoculated plants developed similar symptoms after 7 days, whereas the control plants remained symptomless. The fungus on the inoculated plants was morphologically identical to that was originally observed on the diseased plants. Though many Brassica spp. have been known to be infected by E. cruciferarum throughout the world, powdery mildew of C. sativa cultivar Crantz in natural conditions by E. cruciferarum has been reported only in the province of Domokos in Central Greece (Vellios et al. 2017). To our knowledge, this is the first report of powdery mildew caused by E. cruciferarum on C. sativa in Montana. Though the powdery mildew on C. sativa was observed in the greenhouse conditions in this work, it poses a potential threat to the production of this biofuel crop in the northwest of the United States.

Discovery of Root-Lesion Nematode (Pratylenchus scribneri) on Corn in Hainan Province of China

Corn (Zea mays L.) is a very important cereal crop and serves as food, feed, and industrial material (Liu et al. 2016). The root-lesion nematode (RLN) is considered one of the most important plant-parasitic nematodes and can cause economic losses in agriculture worldwide (Jones et al. 2013). In January 2020, five samples were collected from a corn field in Lingshui Lizu Autonomous County, Hainan Province, China. The collected corn plants (cv. Denghai 685) were growing poorly and roots showed distinct lesions and rot. Corn fields with symptoms of stunted plants, and brown lesions on roots were widespread. This corn disease was severe in Lingshui Lizu Autonomous County. RLN were extracted from soil samples by the modified Baermann funnel (Hooper et al. 2005). All the samples contained RLN ranging from 9 to 82 (average 39) RLN per 100 cm3 of soil and 113 to 257 (average 194) RLN per 5 g roots. The extracted RLN were sterilized and cultured on carrot disks at 25°C for 90 days. Afterwards, seeds of corn (cv. Denghai 685) were sown in pots containing 1.8 liters of sterilized soil. Eight plants, one per pot, were infested with 1,000 RLN, eight pots of noninfested corn plants were used as controls, and plants were kept in a greenhouse at 25°C. At 75 days after inoculation, symptoms were like those initially observed in corn fields, whereas no symptoms were observed in the control plants. Nematodes in the soil and roots were extracted using the same method as previously described (Hooper et al. 2005). The average number of RLN per pot was approximately 4,250 in soil and 820 in roots, the reproduction factor (final number of nematodes/initial number of nematodes) was 5.07, no RLN were found in the control. The experiment was conducted twice. The morphological and molecular studies of RLN were examined to confirm species identification. The main morphological measurements of adult females (n = 15) included body length = 526.0 μm ± 17.1 (standard error) (range = 498.0 to 560.5 μm), stylet = 16.0 μm ± 0.3 (15.5 to 16.5 μm), tail length = 29.0 μm ± 1.5 (26.5 to 31.0 μm), a = 23.6 ± 0.6 (22.6 to 24.4), b = 5.6 ± 0.3 (5.2 to 6.0), c = 18.3 ± 0.9 (16.4 to 19.7), V = 78.2% ± 0.6 (77.4 to 79.2%), lip region with two annules. No males were found in the samples. This population was identified as Pratylenchus scribneri, based on the morphological characters (Castillo and Vovlas, 2007). DNA was isolated from individual nematodes followed by proteinase K-based lysis (Wang et al. 2011). The D2/D3 expansion region of the 28S rRNA gene, rDNA-internal transcribed spacer (ITS) region, and mitochondrial cytochrome oxidase I (mtDNA-COI) gene were amplified with primers D2A/-D3B (De Ley et al. 1999), TW81/ AB28 (Vovlas et al. 2011) and JB3/ JB5 (Liu et al. 2018), respectively. The PCR products were purified and ligated into pJET 1.2/blunt cloning vectors and transformed to Escherichia coli strain DH5α, and then sequenced. The obtained 28S rRNA gene D2/D3 region sequences (785bp), ITS sequences (886 bp) and mtDNA-COI (447bp) in this study were submitted to GenBank. The D2/D3 region of the 28S rRNA sequences of the RLN collected in Lingshui (GenBank accession no. MZ701843) showed 99.75% identity with P. scribneri sequences available in the GenBank (KX842628 and KX842625). The ITS sequences of the RLN collected in this study (MZ701842) showed the highest identity of 97.06% with P. scribneri sequences available in the GenBank (KX842626). The mtDNA-COI sequences of the RLN collected in this study (OK040228) showed 100% identity with P. scribneri (MN366409). Both morphological and molecular data confirmed the identity of P. scribneri. P. scribneri has been reported on corn in Inner Mongolia, Hebei, Shanxi, Shandong, Henan, Jiangsu, and Liaoning provinces of China (Li et al. 2019). As far as we know, this is the first report of P. scribneri on corn in Hainan Province, China. Since the RLN can cause considerable damage to corn, strategic measures should be taken to prevent the spread of P. scribneri to other regions in China.

First Report of Watermelon Charcoal Rot (Macrophomina phaseolina) in China

In June 2018 and 2019, charcoal rot-like symptoms and black microsclerotia suggestive of Macrophomina phaseolina infection were observed on the basal stems of citrullus lanatus cv. ‘Zaojia’, causing premature death. About 1 hectare of ‘Zaojia’ had been investigated, disease incidence rates were almost 50%, resulting in a 40% yield loss in a single field in Shanghai, China (31°23′N , 121°33′E). A fungus was consistently isolated from infected watermelon tissues. In total, 30 cuttings from 10 infected seedlings were surface disinfected with 3% sodium hypochlorite for 3 min, washed thrice with sterile distilled water, air dried, and transferred onto potato dextrose agar (PDA). Dishes were incubated for 3 days at 27°C in the dark. Twenty four single hypha subcultures were obtained from these samples and were cultured for an additional 5 days at 27°C. Colonies were initially white, and then became grey black (Fig.1A). During the more advanced stages of infection, black microsclerotia were produced that were spherical or ovoid in shape (Fig.1B). No sexual structures and conidia developed during culture on PDA. Isolate pathogenicity was assessed both in vitro and in vivo. Watermelon plants (cv. ‘Zaojia’) were grown in growth chambers at 28°C (day) and 23°C (night), with a 16 h photoperiod. When seedlings were 20 days old, they were inoculated. Briefly, a needle was used to puncture watermelon stems, and 5 mm agar plugs containing actively growing mycelia were placed on these needle wounds, followed by culture for 72 h at 27°C in a dark, humid chamber. In total, 10 seedlings were inoculated with 5 mm blank PDA, and the experiment was repeated three times, with the treatment being perfomed as described above. Seedling stems were inoculated 1-2 cm above the ground by puncturing them with a needle and then transferring 5 mm agar plugs containing fungi onto the wound sites. Seedlings were kept 75% humidity and then grown for 5 days at 27°C. Ten seedlings were inoculated per experiment. As a control, 10 seedlings were inoculated with 5 mm blank PDA plugs. Experiments were repeated three times. Necrotic spots around the wounds were evident on inoculated stems at 72 h (Fig.1C). Similarly, vascular tissue necrosis and the collapse of the surrounding pith and epidermis were observed on the residual parts of seedling stems after 5 days (Fig.1E), whereas control stems did not exhibit any disease related symptoms (Fig.1D, 1F). The same pathogen was then successfully re-isolated and was successfully regrown in pure culture, thus fulfilling Koch’s postulates. To identify the causative pathogen, total mycelial DNA was isolated via the CTAB method (Brandfass & Karlovsky, 2008), and the internal transcribed spacer (ITS) rDNA regions were amplified using the ITS1/ITS4 primers (White, 1990), the genus-specific MPKFI//span>MpKRI primers were used for further amplification (Babu et al., 2007). M. phaseolina ITS sequences in this study shared 100% similarity with the ITS sequences of M. phaseolina from Chickpea (MK757624.1). Genus-specific sequences from this isolate shared 100% homology with other M. phaseolina isolates (MT645816.1 and MN263167.1). As such, M. phaseolina was confirmed to be the pathogen responsible for watermelon charcoal rot in the present report, which resulted in the death of infected watermelons before maturity, causing fruits to lose their commodity value. This report is the first to our knowledge to identify M. phaseolina as a causal pathogen of watermelon charcoal rot in China.

Systematics of Scleranthus (Caryophyllaceae)

<p>Scleranthus is a genus of about 12 species of herbaceous flowering plants or small shrubs with a disjunct Eurasian/Australasian distribution. Monophyly of the genus is supported by the close similarity of gynoecial development of all species and consistent with nuclear ITS DNA sequence analysis. Traditionally the genus had been divided into two sections, section Scleranthus and section Mniarum. Section Mniarum is exclusively Australasian while section Scleranthus has been circumscribed to contain exclusively European species or a combination of European and Australasian species. Pollen and floral characters align the species into Australasian and Eurasian groups also supported by nuclear ITS DNA sequence analysis. Section Scleranthus as more broadly defined (i.e., sensu West and Garnock-Jones, 1986) is therefore at least paraphyletic or at worst polypyhyletic. Phylogenetic reconstructions based on morphological characters differ from those based on ITS sequences in supporting different relationships within the Australasian species of Scleranthus. Hybridisation and introgression within the genus are discussed and suggested as the cause of discordance between morphology and DNA sequence based trees. Low sequence divergence among Scleranthus ITS sequences suggests that the European and Australasian clades within the genus diverged within the last l0 million years. Biogeographic implications of these dating and competing hypotheses explaining the disjunct North-South distribution of the genus are discussed. Nuclear ITS and chloroplast ndhF DNA sequences both suggest that Scleranthus belongs to a clade within the family Caryophyllaceae consisting of members of subfamilies Alsinoideae and Caryophylloideae. Phylogenetic relationships between genera belonging to the three subfamilies of Caryophyllaceae (Alsinoideae, Caryophyloideae, and Paronychioideae) are addressed in this thesis through ndhF sequence analysis, which provides no support for the monophyly of traditionally recognised groups. Morphological character data sets are likely to always encompass multiple incongruent data partitions (sensu Bull et al. 1993). It may therefore be appropriate to combine data from DNA sequence and morphology for parsimony analysis even where the two are significantly incongruent.</p>

Systematics of Scleranthus (Caryophyllaceae)

<p>Scleranthus is a genus of about 12 species of herbaceous flowering plants or small shrubs with a disjunct Eurasian/Australasian distribution. Monophyly of the genus is supported by the close similarity of gynoecial development of all species and consistent with nuclear ITS DNA sequence analysis. Traditionally the genus had been divided into two sections, section Scleranthus and section Mniarum. Section Mniarum is exclusively Australasian while section Scleranthus has been circumscribed to contain exclusively European species or a combination of European and Australasian species. Pollen and floral characters align the species into Australasian and Eurasian groups also supported by nuclear ITS DNA sequence analysis. Section Scleranthus as more broadly defined (i.e., sensu West and Garnock-Jones, 1986) is therefore at least paraphyletic or at worst polypyhyletic. Phylogenetic reconstructions based on morphological characters differ from those based on ITS sequences in supporting different relationships within the Australasian species of Scleranthus. Hybridisation and introgression within the genus are discussed and suggested as the cause of discordance between morphology and DNA sequence based trees. Low sequence divergence among Scleranthus ITS sequences suggests that the European and Australasian clades within the genus diverged within the last l0 million years. Biogeographic implications of these dating and competing hypotheses explaining the disjunct North-South distribution of the genus are discussed. Nuclear ITS and chloroplast ndhF DNA sequences both suggest that Scleranthus belongs to a clade within the family Caryophyllaceae consisting of members of subfamilies Alsinoideae and Caryophylloideae. Phylogenetic relationships between genera belonging to the three subfamilies of Caryophyllaceae (Alsinoideae, Caryophyloideae, and Paronychioideae) are addressed in this thesis through ndhF sequence analysis, which provides no support for the monophyly of traditionally recognised groups. Morphological character data sets are likely to always encompass multiple incongruent data partitions (sensu Bull et al. 1993). It may therefore be appropriate to combine data from DNA sequence and morphology for parsimony analysis even where the two are significantly incongruent.</p>

Haplodontium altunense (Bryaceae, Bryopsida), a new moss species from Northwest China

Haplodontium altunense X.R.Wang &amp; S.Mamtimin, a new moss species of the family Bryaceae from Xinjiang Uygur Autonomous Region, China is described and illustrated. Genetic analysis based on ITS sequences shows that this species is a member of the Bryaceae and in the same clade as Anomobryum. Particularly distinctive features of the new species include: double peristome; the exostome has raised and membranous chomata with united lamellae between two teeth proximally; the endostome is poorly developed and all the endostomial material tightly adherent to the exostome.

Taxonomic identity of Crepidiastrum ×nakaii recorded on Hongdo Island

The plant known as “Hong-do-go-deul-ppae-gi” on Hongdo Island, belonging to the genus Crepidiastrum, has been recorded as C. ×nakaii, a hybrid between C. denticulatum and C. platyphyllum. During a survey of the flora of Hongdo Island, we reexamined the taxonomic identity of “Hong-do-go-deul-ppae-gi”. The morphological traits, geographical distribution, and internal transcribed spacer (ITS) sequences of “Hong-do-go-deul-ppae-gi” were compared to related species within the genus. A morphological examination revealed that the plant was similar to C. lanceolatum in that it has subshrub stems with caudex stout, 8 inner involucrals bracts, and 10 to 12 florets. In contrast, similarities to C. denticulatum were observed in terms of radical leaves deciduous in the flowering period, cauline leaves membranous with acute serrations, and beaked achenes. Furthermore, C. denticulatum and C. lanceolatum grew on Hongdo Island and nearby areas, but C. platyphyllum was not distributed in these areas. The results of morphological and distributional investigations revealed that the Hongdo Island plant should be regarded as C. ×muratagenii, a hybrid between C. denticulatum and C. lanceolatum, not C. ×nakaii, which is a hybrid between C. denticulatum and C. platyphyllum. However, the resolution of the ITS sequences was insufficient such that we could not separate the examined species.

First Report of Pod and Stem Blight and Seed Decay caused by Diaporthe longicolla on Soybean in Western Canada

In Oct. 2019, soybean plants (Glycine max) (cv. 24-10RY, R7 growth stage) with dry rot, necrosis, reddish-brown lesions, and small black fruiting bodies in linear rows were collected from fields in Manitoba (Carman, St. Adolph, Dauphin), Canada. The pods and seeds were shrivelled, small and some seeds were covered with whitish mycelium. Symptoms began as brown lesions, which darkened, elongated, causing wilt of the above stems then plant death. Microscopy showed that the fruiting bodies were pycnidia. Symptomatic stems were cut into 1-2 cm pieces and seeds surface-sterilized in 0.5% NaOCl, rinsed twice in sterilized H2O, air-dried on sterilized filter paper, and plated on PDA medium amended with 100 mg/L streptomycin sulfate at room temperature with 12-h fluorescent light/12-h dark for 3 days. The emerging hyphae were transferred using the hyphal tip method to new PDA petri dishes and incubated for 21 days (room T°). Mycelia of 20 isolates were dense, white and floccose with occasional green-yellow areas. Black stromata in concentric patterns or scattered as large masses were visible on the cultures’ back. Pycnidia formed solely or aggregated after 4-5 weeks of incubation on PDA. Alpha conidia emanated from pycnidia in creamy-to-yellowish drops and were hyaline, non-septate, ellipsoid to fusiform, and biguttulate. The average length and width of Alpha conidia were 5.5 μm and 1.5 μm, respectively (n = 30). No perithecia were seen. The cultures’ morphology was consistent with Phomopsis longicolla’s description (Hobbs et al., 1985). Seven isolates were selected for molecular characterization to confirm their identity by amplifying the ITS region with universal primers ITS4/ITS5 (White et al. 1990). All PCR amplicons were analyzed by electrophoresis through 1.5 % agarose gels and the size of PCR amplicons estimated using 1-kb plus DNA ladder (Thermo Fisher Sci., ON, Canada). PCR amplicons (~650 bp) were purified and sequenced in two directions by Psomagen Inc. (Rockville, MD, USA). ITS sequences were identical for all isolates, and GenBank searches (BLASTn: Altschul et al. 1990) confirmed species identity. ITS sequences (accessions MW466183-MW466189) were deposited in GenBank and matched the type sequence of Diaporthe longicolla strain ATCC 60325 (accession NR_144924) from G. max in USA with identities = 473/475 (99.6%) and gaps = 0/475 (0%). To confirm the pathogenicity of the seven isolates, the stems of V4-stage (four open trifolilates) soybean plants (cv. 24-10RY) were excised using a sterile scalpel. Mycelial plugs (9 mm in diameter) from 1-week-old culture of each isolate were placed over the wounded stems (Abdelmagid et al., 2019). Sterile PDA plugs were used on control plants. Six plants were used per isolate and control. Plugs of both treatments were wrapped with parafilm to avoid drying. The plants were incubated in a humidity chamber for 4 days and then in a greenhouse at 24:16°C day/night, 13:11-h light/dark cycle, and 70-80% relative humidity, and were irrigated as needed. Symptoms similar to those observed in the field were seen on the stems and seeds of all artificially-infected plants approx. 8 weeks after inoculation. Pods and seeds of inoculated plants were shrivelled and small. No symptoms were observed on control plants. Diaporthe longicolla was re-isolated only from the diseased plants and seeds. To our knowledge, this is the first report following Koch’s postulates to identify the causal pathogen of soybean pod and stem blight and seed decay in Western Canada. This will be instrumental in determining the causes of stem decay and contribute in properly dealing with soybean seed issues in Western Canada in the future.

A fresh outlook on the smooth-spored species of Inocybe: type studies and 18 new species

On the basis of detailed morphological and molecular investigation, eighteen new species of Inocybe (I. alberichiana, I. beatifica, I. bellidiana, I. clandestina, I. drenthensis, I. dryadiana, I. gaiana, I. ghibliana, I. grusiana, I. knautiana, I. lampetiana, I. oetziana, I. orionis, I. plurabellae, I. rivierana, I. scolopacis, I. sitibunda and I. tiburtina) are described. All of them are smooth-spored, and most of them are pruinose only in the apical part of the stipe. The new species are compared to 40 type specimens, all of which are described here and for several of which (partial) ITS sequences have been generated. For eight species, epi-, lecto- or neotypes were selected, among these are I. geophylla, I. glabripes and I. tigrina. Based on these studies, we suggest twelve synonymies, i.e. that I. clarkii is synonymous with I. sindonia, I. conformata with I. cincinnata, I. elegans with I. griseolilacina, I. fuscidula with I. glabripes, I. griseotarda with I. psammobrunnea, I. obscurella with I. obscuroides, I. obscuromellea with I. semifulva, I. patibilis and I. tigrinella with I. tigrina, I. petroselinolens with I. tenuicystidiata and I. rubidofracta with I. pseudorubens and I. subporospora is synonymized with I. tjallingiorum. All of the new species are supported by phylogenetic analyses. Among the previously described species accepted here, sixteen are represented by types in the phylogenetic analyses and ten by own collections morphologically corresponding to the type. In summary, we here verify or provide morphological concepts associated with molecular data for 44 smooth-spored species of Inocybe.