Nuclear Ribosomal Small Subunit Research Articles

First Report of Polygonatum kingianum Leaf Spot Caused by Alternaria alternata in Kunming, China.

Polygonatum kingianum Coll. et Hemsl., a Polygonatum species in the Asparagaceae family, plays an important role in Chinese herbal medicine (Zhao et al. 2018). P. kingianum is widely planted in the Southwestern China. In September 2023, we observed a leaf spot of P. kingianum with disease incidence of 100%, and disease index reached 60 in commercial plantings in Kunming, Yunnan province, China (24.3610°N, 102.3740°E). In the initial stage of infection, symptoms manifested as a small circular brown spot. As the spots gradually expanded, they formed oval to irregular shaped lesions with grayish-white or dark-brown borders. Progressively the entire leaf withered and died. For identification of the causal agent of the leaf spot, leaf sections (5×5 mm2) were cut from the margin of the lesion and soaked in 75% ethanol for 10 s, 1% sodium hypochlorite for 3 min, washed with sterile distilled water, dried on sterilized tissue paper and placed on potato dextrose agar (PDA). The Petri dishes were then incubated at 28℃ for 3 days with a 12-h photoperiod. A predominant fungus was isolated from 95% of the samples. Three monosporic isolates were screened using a single-spore isolation method. After 4 days of incubation the colonies were white, after 7 days turned yellow-white. Conidia were black-brown, oblong or fusiform, with 3-7 transverse septa and 0-3 longitudinal septa, with dimensions of 19.5 to 49.5 × 8.7 to 17.6 µm (n = 30). Total genomic DNA of these three isolates was extracted from mycelia by the cetyltrimethylammonium bromide (CTAB) protocol. The nucleotide sequences of the elongation factor 1-alpha (EF1α), nuclear ribosomal internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit rRNA gene (LSU), 18S nuclear ribosomal small subunit rRNA gene (SSU), and the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2) gene regions were amplified using the primer pairs EF1-728F/EF1-986R (Carbone and Kohn 1999), ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Schoch et al. 2012), NS1/NS4 (Schoch et al. 2012), and fRPB2-5F/fRPB2-7Cr (Liu et al. 1999), respectively. Amplicons were cloned in a pMDTM19-T vector (code no. 6013, Takara, Kusatsu, Japan) and bidirectionally sequenced. All three isolates had identical nucleotide sequences. Sequences from one isolate (PkF03) were deposited in GenBank. BLASTn analyses showed that sequences of EF1α (GenBank accession no. PP695240), ITS (PP694046), LSU (PP683406), SSU (PP683407), and RPB2 (PP695241) of isolate PkF03 were 99.6 (KP125134), 100 (KP124358), 100 (KP124510), 99.9 (KP124980), and 100% (KP124826), respectively, identical with Alternaria alternata (Fr.) Keissl. strain CBS 118815. Based on the nucleotide sequences of EF1α, ITS, LSU, SSU, and RPB2, a maximum likelihood phylogenetic tree was constructed using MEGAX with Tamura-Nei model. Isolate PkF03 was grouped in the same clade as A. alternata. According to the morphology and sequence analyses isolate PkF03 was identified as A. alternata (Woudenberg et al. 2013). To determine pathogenicity of isolate PkF03, a spore suspension (106 spores/mL) was sprayed on 1-year-old healthy leaves of P. kingianum. The control leaves were sprayed with sterile water. All plants were incubated at 28℃, 70% relative humidity, and a 12-h photoperiod. The pathogenicity tests were repeated three times with six plants in each treatment. Fifteen days post-inoculation, the inoculated leaves showed brown-yellow lesions, whereas the control leaves remained symptomless. A. alternata was reisolated from infected leaves. To our knowledge, this is the first report of A. alternata causing leaf spot on P. kingianum in Kunming, China. The results provide a scientific basis for prevention and control of the disease.

Calcarisporium yuanyangense sp. nov. (Calcarisporiaceae, Hypocreales), a fungal pathogen isolated from Ophiocordyceps nutans

In this study, a new species, Calcarisporium yuanyangense, was isolated from Ophiocordyceps nutans collected from Yuanyang County, Yunnan Province, China, and described by morphology and molecular phylogeny. Based on the combined dataset of five genes including the internal transcribed spacer gene region (ITS), the nuclear ribosomal small and large subunit gene regions (nrSSU and nrLSU), the gene encoding translation elongation factor 1-α (tef-1α), and the RNA polymerase II second largest subunit gene region (rpb2), phylogenetic analyses were performed by the maximum likelihood (ML) and the Bayesian inference (BI) methods to determine the phylogenetic position of C. yuanyangense in Calcarisporium. This species was clustered with C. cordycipiticola, C. xylariicola, and C. arbuscula to form a separate branch. Calcarisporium yuanyangense had the basic morphological characteristics of Calcarisporium, but still differed morphologically from the other species in the genus. This species was similar to C. arbuscula in conidia shape, but the conidia of C. yuanyangense were larger and had a higher length/width ratio than those of C. arbuscula. This study deepens the understanding of the host diversity and the distribution of this taxonomic unit, as well as provides important taxonomic data for further exploration of the genus Calcarisporium.

Assessment of the macroalgal diversity of Kuwait by using the Germling Emergence Method

Cryptic stages of diverse macroalgae present in natural substrata, “the bank of microscopic forms”, were isolated into clonal cultures and identified based on both morphological characteristics and DNA barcoding. Approximately 120 clonal isolates from 308 natural substratum samples were collected from the entire coastline of Kuwait. Amongst these isolates, 77 (64%) were identified through DNA barcoding using the nuclear ribosomal small subunit, RuBisCO spacer (ITS2, <i>tufa</i>, <i>rbc</i>L, <i>psa</i>A, and <i>psb</i>A) and sequencing. Twenty-six isolates (34%) were identified in the division Chlorophyta, 18 (23%) as Phaeophyceae, and 33 (43%) as Rhodophyta. For all DNA sequences in this study, species-level cut off applied was ≥98% homology which depend entirely on the markers used. Three putative new records of Chlorophyta new for the Arabian Gulf were made: <i>Cladophora laetevirens</i> (Dillwyn) Kützing, <i>Ulva torta</i> (Mertens) Trevisan and <i>Ulvella leptochaete</i> (Huber) R. Nielsen, C. J. O′Kelly & B. Wysor in Nielsen, while <i>Cladophora gracilis</i> Kützing and <i>Ulva ohnoi</i> M. Hiraoka & <i>S. Shimada</i> are new records for Kuwait. For Phaeophyceae, Ectocarpus subulatus Kützing and <i>Elachista stellaris</i> Areschoug were new records for the Gulf and Kuwait. In the Rhodophyta, <i>Acrochaetium secundatum</i> (Lyngbye) Nägeli in Nägeli & Cramer, <i>Ceramium affine</i> Setchell & N. L. Gardner, <i>Gelidium pusillum</i> var. <i>pakistanicum</i> Afaq-Husain & Shameel and <i>Dasya caraibica</i> Børgesen are new records for the Gulf and Kuwait, while the red alga <i>Stylonema alsidii</i> (Zanardini) K. Drew is a new record for Kuwait. Several isolates identified corresponded to genera not previously reported in Kuwait and / or the Arabian Gulf, such as <i>Porphyrostromium</i> Trevisan, a new genus from the Bangiales, and two unidentified species for the Planophilaceae Škaloud & Leliaert. The isolates cultivated from substrata enhance understanding of the marine macroalgal diversity in the region and confirmed that the Germling Emergence Method is suitable for determining the actual diversity of a given study area through isolation from cryptic life-history phases.

(2955) Proposal to conserve the name Didymium against Mucilago and Spumaria (Physarales, Myxomycetes)

(2955) Proposal to conserve the name <i>Didymium</i> against <i>Mucilago</i> and <i>Spumaria</i> (<i>Physarales</i>, <i>Myxomycetes</i>)

First Report of Aloe vera Rust Caused by Uromyces aloes in an Ornamental Nursery in the United States.

Aloe vera (L.) Burm. f. is a tropical evergreen perennial in the family Liliaceae. Native to the Arabian Peninsula, it is sold in Pennsylvania as an ornamental and for its medical and topical purposes due to its high levels of amino acids, anthraquinones, saponins, and vitamins A, B, C, E (Sahu et al. 2013). In February 2020, at an ornamental plant nursery in Lancaster County, Pennsylvania, 5 out of 15 mature A. vera plants in 15 cm pots showed symptoms and signs of rust on the leaves, exhibiting dark-brown erumpent pycnial spots with a chlorotic band surrounding the infected tissue that turned necrotic after three days of incubation at 20°C. Only the telial stage was present. Sori (n=25) were rounded, concentrically arranged, 0.2-3.7 mm, and covered by a brown epidermis. Teliospores (n=40) were amphigenous, orange-brown, globose to ellipsoidal, measuring (29.2) 30.4-36.1 (39.5) × (27.4) 27.6-30.1 (30.5) µm, with a wall thickness of 4-5 µm, and a persistent hyaline pedicel ranging from 5 to 57.1 µm in length and 5.2 to 9.3 µm in width. These measurements were comparable to the descriptions of Uromyces aloes previously reported from India (teliospore size 25-42.5 x 20-30 µm, wall thickness 3-5 µm, and pedicel size 25-95 x 5-6.25 µm), and South Africa (teliospore size 30-44 x 24-32 µm, wall thickness 4-6 µm, and pedicel size 6-20 µm) (Maier et al. 2007; Soni et al. 2011). Based on these morphological traits and the plant host, the causal agent was identified as Uromyces aloes (Cooke) Magnus (Pucciniaceae, Uredinales). The sample was also independently identified as U. aloes by the USDA APHIS PPQ Beltsville lab (Interception # APEMD200552555001) based on morphological characteristics. Teliospores were harvested with a sterile pin, transferred to a 1.5 ml tube with DNA extraction buffer (100 mM Tris-HCL, 10 mM EDTA, 1 M KCl, pH 8) and macerated using a plastic mini-pestle. The DNA was precipitated using isopropanol, washed with 70% ethanol, and reconstituted in 50 µl of PCR-grade water. The segment of the internal transcribed spacer region (ITS) was amplified using ITS4/ITS5 primers (White et al. 1990). The nuclear ribosomal small subunit (18S) was amplified with rust specific primers Rust18S-R (Aime 2006) and NS1 (White et al. 1990). The nuclear ribosomal large subunit (28S) was amplified with primers LR0R and LR7 (Vilgalys et al. 1990). Amplified PCR products were cleaned using ExoSap (Affymetrix, Santa Clara, CA) or QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and sequenced at Penn State Genomics Core Facility. The nucleotide sequences were trimmed, analyzed, and aligned using Geneious 11.1.5 software (Biomatters, Auckland, NZ). The resulting 692-bp segment of the ITS, 1,633-bp segment of the 18S, and the 1,324-bp segment of the 28S regions were deposited in the GenBank database under accession numbers MT136509, MZ146345, and MZ146342, respectively. Based on GenBank BLAST analysis, a 529-bp fragment of our 28S product was found to share 98.87% (523/529) identity with U. aloes isolate WM3290 (DQ917740) from South Africa, with three nucleotide differences and three gaps between the two strains. Comparisons among ITS and 18S sequences could not be made because no ITS or 18S sequence data from U. aloes has previously been deposited in GenBank. To our knowledge, this is the first report of U. aloes from A. vera in the United States. Infected plants were confined inside a greenhouse and have been destroyed. Since the plants were purchased from either Ontario, Canada or Florida, the extent of infection in the United States is unknown.

First report of Geotrichum candidum causing sour rot of peach in China.

Peach (Prunus persica L. Batsch) is one of the most important fruit crops in China (Wang et al. 2011). Yangshan Town of Jiangsu Province is one of the four major peach producing areas in China, with a growing area of 2,000 ha (Tian et al. 2018). During June 2020, a postharvest disease presenting with brown necrosis and rot occurred on peaches in Yangshan Town. The estimated damage was more than 10% of the total harvest. The symptoms included soft rot, and the lesion appeared sunken, accompanied with sour odor and white mycelia. Twelve peaches with representative symptom were sampled for pathogen isolation. Pieces (about 5 mm × 5 mm) from the lesion edge of symptomatic fruits were dissected and surface disinfected (3% NaClO for 10 s and 75% ethanol for 30 s), then rinsed three times with distilled water, dried on sterile filter paper and transferred to Potato Dextrose Agar (PDA) media plates supplemented with 150 ng/mL streptomycin sulfate. The plates were incubated at 28 ℃ for 3 days. Forty-eight isolations were obtained from the plates and isolates were single-spored. All isolates presented white, flat, milky yeast-like colonies with radial mycelia. Hyphae under microscope were septate, branched, disarticulating into arthroconidia measuring 3.39 to 9.27 × 2.05 to 7.71 μm. The morphological characteristics are consistent with Geotrichum candidum (De Hoog et al. 1986). Internal transcribed spacer (ITS) and 18s nuclear ribosomal small subunit (SSU) of the 48 isolates were amplified and sequenced using the primers ITS5/ITS4, and NS1/NS4 for molecular identification (Schoch et al. 2012). The resulted sequences showed no difference among all the isolates. Alignment by blastn showed the sequence of ITS and SSU were 100% (accession number. GQ376093) and 99.7% identical (accession number. KY977411.1) to Geotrichum candidum, respectively. The sequences of ITS (accession number MW493646) and SSU (accession number MW493648) were submitted to the GenBank. Commercial ripe peaches with the size of about 15 cm × 15 cm × 10 cm was used for pathogenicity test. Peaches were surface disinfected with 75% ethanol, then a wound with 4 mm in diameter and 5 mm in depth was made on the surface of each fruit. Ten peaches were inoculated with 10 μL (1×105 spores /mL) of the isolate suspension. Another ten peaches were inoculated with 10 μL sterile water as the control. Peaches were incubated individually at 28 ℃and a relative humidity of about 85%. After three days, large scale of pits and necrosis appeared on every peach inoculated, and the symptoms were consistent with the diseased peaches in Yangshan Town, while no symptoms non-inoculated on the control peaches were observed. The pathogen was re-isolated from the diseased fruit and was identified again by sequencing of ITS and SSU. All the tests were conducted three times. Considering the evidence, we identified the pathogen as G. candidum. This pathogen has been reported to cause sour rot was reported in kiwifruit, strawberry, melon and other fruits (Alonzo et al. 2020; Cheng et al. 2020; Halfeld-Vieira et al. 2020). To our knowledge, this is the first report of G. candidum causing sour rot of peach in China, which may cause a great loss to peach industry of China.

Character evolution of modern fly‐speck fungi and implications for interpreting thyriothecial fossils

PremiseFossils show that fly‐speck fungi have been reproducing with small, black thyriothecia on leaf surfaces for ~250 million years. We analyzed morphological characters of extant thyriothecial fungi to develop a phylogenetic framework for interpreting fossil taxa.MethodsWe placed 59 extant fly‐speck fungi in a phylogeny of 320 Ascomycota using nuclear ribosomal large and small subunit sequences, including newly determined sequences from nine taxa. We reconstructed ancestral character states using BayesTraits and maximum likelihood after coding 11 morphological characters based on original observations and literature. We analyzed the relationships of three previously published Mesozoic fossils using parsimony and our morphological character matrix, constrained by the molecular phylogeny.ResultsThyriothecia evolved convergently in multiple lineages of superficial, leaf‐inhabiting ascomycetes. The radiate and ostiolate scutellum organization is restricted to Dothideomycetes. Scutellum initiation by intercalary septation of a single hypha characterizes Asterinales and Asterotexiales, and initiation by coordinated growth of two or more adjacent hyphae characterizes Aulographaceae. Scutella in Microthyriales are initiated apically on a lateral hyphal branch. Patterns of hyphal branching in scutella contribute to distinguishing among orders. Parsimony resolves three fossil taxa as Dothideomycetes; one is further resolved as a member of a Microthyriales‐Zeloasperisporiales clade within Dothideomycetes.ConclusionsThis is the most comprehensive systematic study of thyriothecial fungi and their relatives to date. Parsimony analysis of the matrix of character states of modern taxa provides an objective basis for interpreting fossils, leading to insights into morphological evolution and geological ages of Dothideomycetes clades.

The Minute Alga Schizocladia ischiensis (Schizocladiophyceae, Ochrophyta) Isolated by Germling Emergence from 24 m Depth off Rhodes (Greece)

Substratum collected during diving surveys of sublittoral communities off the Greek island of Rhodes (Dodecanese, South-East Aegean) in late 2015 was incubated in the laboratory. Among the emerging macroalgal germlings, there was the second-ever record and isolate of the small benthic multicellular alga Schizocladia ischiensis of the poorly known monotypic Schizocladiophyceae, the sister group of the brown algae (Phaeophyceae). Its nuclear ribosomal small subunit, Rubisco spacer (rbcL, psaA, and psbC sequences (in total 5237 bp)) were similar to those of the only previous isolate of the species from Ischia, western Mediterranean. Our new strain formed branched upright thalli attached to the substratum by an amorphous substance secreted at the bottom of the basal cell. It is possible that S. ischiensis is a common member of the infralittoral and circalittoral communities in the Mediterranean and generally overlooked because of its minute size. Germling emergence appears to represent the method of choice to reveal benthic algae of this small size.

First Report of Neofusicoccum mangiferae Causing Shoot Blight of Sassafras tzumu in China

HomePlant DiseaseVol. 104, No. 7First Report of Neofusicoccum mangiferae Causing Shoot Blight of Sassafras tzumu in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Neofusicoccum mangiferae Causing Shoot Blight of Sassafras tzumu in ChinaYu Wan, Xin-Yu Wang, Long-Xi He, Wu Xu, and Li-Hua ZhuYu WanCollege of Forestry, Nanjing Forestry University, Nanjing, Jiangsu 210037, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing, Jiangsu 210037, ChinaSearch for more papers by this author, Xin-Yu WangCollege of Forestry, Nanjing Forestry University, Nanjing, Jiangsu 210037, ChinaCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing, Jiangsu 210037, ChinaSearch for more papers by this author, Long-Xi HeJiangxi Forest Pest Control and Quarantine Bureau, Nanchang, Jiangxi 330038, ChinaSearch for more papers by this author, Wu XuShanghai Qingpu Forestry Station, Shanghai, 201700, ChinaSearch for more papers by this author, and Li-Hua Zhu†Corresponding author: L.-H. Zhu; E-mail Address: lhzhu@njfu.com.cnhttp://orcid.org/0000-0003-2740-4980College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu 210037, ChinaSearch for more papers by this author AffiliationsAuthors and Affiliations Yu Wan1 2 Xin-Yu Wang1 2 Long-Xi He3 Wu Xu4 Li-Hua Zhu1 † 1College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu 210037, China 2Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing, Jiangsu 210037, China 3Jiangxi Forest Pest Control and Quarantine Bureau, Nanchang, Jiangxi 330038, China 4Shanghai Qingpu Forestry Station, Shanghai, 201700, China Published Online:7 May 2020https://doi.org/10.1094/PDIS-11-19-2521-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Sassafras tzumu Hemsl. is native to China and was widely planted in central and southwestern China (Wu et al. 2008). The wood of S. tzumu is used for boat and furniture making, and the roots and bark are used in traditional Chinese medicine. Since 2014, severe blight of branches has been observed on S. tzumu in Yichun, Jiangxi, China. Symptoms began as brown round or elliptical necrosis spots at the area where the petioles attached to the branch; the spot gradually expanded to form a lesion and eventually caused wilting of the leaves. Finally, the lesion girdled the branch and killed it. A large number of dark brown to black stromatic conidiomata developed on the lesions. Small pieces of symptomatic stems were surface sterilized in 75% ethanol for 30 s, 1% NaClO for 90 s, rinsed with sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Fungal colonies were observed, and monosporic cultures of those colonies were obtained. A representative isolate, Sa-8, was obtained and deposited in the China Forestry Culture Collection Center (CFCC 53952). On PDA, the isolate developed white-gray, circular colonies with aerial mycelia and scalloped margins. The colonies turned to dark-gray after 5 days of incubation. At the beginning, conidia were hyaline, ellipsoid or fusiform, and nonseptated. With time, conidia became brown, two-celled, and measured 13.5 ± 1.4 × 7.3 ± 0.5 μm. Conidia morphology was similar to descriptions of Neofusicoccum sp. (Phillips et al. 2013). For molecular identification, genomic DNA of Sa-8 was extracted and amplified with internal transcribed spacer (ITS) primers (ITS1 and ITS4) (White et al. 1990), with the nuclear ribosomal 18S RNA small subunit (SSU), large subunit (LSU), elongation factor 1-alpha (EF1-α), and β-tubulin (β-tub) genes being amplified with primers NS1/NS4 (White et al. 1990), LR0R/LR05 (Schoch et al. 2009), EF1-F/EF1-R, and βt2a/βt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (accession nos. MK757880 [ITS], MK757885 [LSU], MN413328 [EF1-α], MN413329 [β-tub], and MK757888 [SSU]). BLAST results showed high similarities with sequences of Neofusicoccum mangiferae (JN688956.1 [ITS], identities = 525/525 [100%]; NG055730.1 [LSU], identities = 864/864, [100%]; EU673153 [SSU], identities = 924/933 [99%]; and KY000153 [β-tub], identities = 392/397 [99%]) and Dothiorella guttulata (KY815020.1 [EF1-α], identities = 353/360 [98%]). Phylogenetic analyses based on the combined sequences placed Sa-8 in the clade of N. mangiferae with 88% bootstrap support. Based on the morphological and molecular data, the isolate was identified as N. mangiferae (Syd. & P. Syd.) (Crous et al. 2006). Pathogenicity tests were performed on ten 1-year-old seedlings of S. tzumu. The stems of seedlings were wounded with a sterile needle, and mycelial plugs of Sa-8 on PDA were placed on the wounds and covered with Parafilm. Seedlings inoculated with PDA plugs were used as controls. Seedlings were incubated in a greenhouse at 25°C and a 16-h photoperiod. All seedlings inoculated with Sa-8 developed brown necrotic spots 5 days after inoculation and were blighted in 12 days. No symptoms were observed on the controls. The same fungus was reisolated from symptomatic stems. This is the first report of N. mangiferae causing shoot blight on S. tzumu. The disease has caused mortality to thousands of trees and has become a threat to the survival of S. tzumu. These results provided information for developing control strategies for this disease.The author(s) declare no conflict of interest.

First Report of Fusarium oxysporum Causing Root Rot in Ophiopogon bodinieri in China

Ophiopogon bodinieri H. Lev. is an important ornamental groundcover widely used in urban gardens in southern China (Liu et al. 2011). In September 2017, a disease occurred on approximately 20% of O. bodinieri in 9 ha in Guangzhou, Guangdong province in China. Symptoms included etiolation in the leaves, wilt, root rot, and necrotic vascular systems. Three diseased plants were sampled for pathogen isolation. Portions (about 5 mm²) of symptomatic root tissues were dissected and surface disinfected (3% NaClO for 10 s and 70% ethanol for 30 s). Tissues were rinsed three times using sterile distilled water, dried on sterile filter paper, and transferred to Petri plates with potato dextrose agar (PDA) supplemented with streptomycin sulfate (150 µg/ml). Petri plates were incubated at 28°C for 5 days (Dita et al. 2010). Only one isolate was obtained from all the plates and was subcultured to new PDA plates. A single-spore isolate was obtained from a hyphal tip, and the culture characteristics and conidial morphology were studied on PDA and carnation leaf agar (CLA) (Neish 1983). The isolate grown on PDA formed abundant white-colored fungal colonies with radial mycelium in 5 days at 28°C. Microscopic observations from CLA medium revealed the curved macroconidia were usually three- to five-septate, with the size of 2.2 to 4.0 × 18.3 to 42.4 μm. Microconidia were kidney shaped with the size of 4.7 to 6.8 × 7.3 to 12.1 μm. Chlamydospores were single or in clusters, with the size of 9.1 to 11.0 μm in diameter. The elongation factor 1-alpha (EF1α) gene (accession no. MN026924, 686 bp), amplified and sequenced using primer pair EF-1/EF-2 (O’Donnell et al. 1998), showed 100% identification to a Fusarium oxysporum strain (accession no. KY508353.1) (Geiser et al. 2004). The molecular identification was confirmed via BLAST on the Fusarium ID and Fusarium MLST databases. Ten-week-old plants were used for pathogenicity tests. First, the plants were wounded by cutting off 1 cm of the roots. Then, 10 plants were inoculated by root dipping (30 min, 10⁴ spores/ml), and another 10 plants were treated with sterile water as a control. The plants were then repotted in potting mix and incubated at 28°C. The assay was conducted three times. After 15 days, the plants showed symptoms of leaf wilting, root rot, and necrosis in vascular tissues. After 40 days, all the inoculated plants were dead, whereas no symptoms were observed in the controls. Subsequently, the F. oxysporum isolate was successfully reisolated from the inoculated plants and was identified again by sequencing the EF1α. The pathogen was further identified by PCR amplification and sequencing the internal transcribed spacer (ITS) gene region using the primers ITS5/ITS4 and the 18s nuclear ribosomal small subunit (SSU) using the primers NS1/NS4 (Schoch et al. 2012). The isolate showed 100% and 99% identity to those of F. oxysporum (accession KF498869.1 for ITS and KU512835.1 for SSU). The sequences of ITS (accession no. MH752745.1), SSU (accession no. MH752591.1), and EF1α (accession no. MN026924) were deposited in GenBank. The first F. oxysporum causing disease on another Ophiopogon species (O. japonicus) was discovered in Florida in 1991 (Farr and Rossman 2019). To our knowledge, this is the first report of root rot disease of O. bodinieri caused by F. oxysporum in the world. This disease may pose a risk for urban landscapes in China.

Three new Coccomyces species from Shennongjia National Nature Reserve in China

Shennongjia National Nature Reserve is an old-growth forest in China with high biodiversity. But only three species in Rhytismataceae had been reported here. Based on morphological and molecular evidence, specimens of Coccomyces collected in Hubei were proposed here as three new species, named Coccomyces fagicola, C. quercicola, and C. shennongjiaensis. Phylogenetic analyses were inferred based on combined nuclear large subunit ribosomal DNA (nrLSU) and mitochondrial small subunit ribosomal DNA (mtSSU) as well as internal transcribed spacer (ITS).

First Report of Diplodia corticola Causing Stem and Branch Cankers on White Oak (Quercus alba) in Missouri, U.S.A.

An investigation of white oaks (Quercus alba) with thin crowns, branch dieback, or both was performed in Crawford and Washington counties, Missouri, during September 2015. Affected stem and branch segments were cut from each tree for examination. After debarking, isolations were performed by using a sterile scalpel to cut cubes (approximately 2 mm³) from tissues at the edges of cankers and underlying stained sapwood. Cubes were placed in quarter-strength potato dextrose agar (PDA) amended with chloramphenicol and streptomycin sulfate (100 mg/liter). A fungus frequently recovered from four of six symptomatic trees was suspected to be a Botryosphaeriaceae species based on its colony morphology, reproductive structures, and conidia (Alves et al. 2004). DNA was extracted from seven isolates, and the internal transcribed spacer (ITS) and the gene portions of nuclear ribosomal small subunit (SSU) regions were sequenced after amplification using ITS1F and ITS4 and NS1 and NS4 primers (White et al. 1990). The resulting sequences were deposited in GenBank (accession nos. MH041506 to MH041510, MH036328, and MH036329). Contigs had 100% homology with the ITS and SSU regions of multiple Diplodia corticola isolates (accession nos. KX595187.1, KT440896.1, KX463057.1, KF766239.1, and EU673206.1). Two isolates were chosen as inocula for a pathogenicity test. In a greenhouse trial, five 2-year-old seedlings were inoculated with isolate 25, and another five seedlings were inoculated with isolate 101. The fully leafed and healthy, potted seedlings were exposed to natural light. Inoculations were made by puncturing the bark of seedling stems 5 cm above the soil surface with a sterile 8-mm-diameter cork borer, peeling back the outer bark, and placing PDA bearing mycelium of either isolate into the wound. The bark was replaced and the wound wrapped with Parafilm. As a control treatment, a sterile plug of noncolonized PDA was wound inoculated 10 cm above the D. corticola inoculation site. The bark was peeled from the wounds at 8 weeks. Canker shapes were drawn on transparency film and photographed prior to measuring length and surface area with ImageJ 1.51j8 (from the National Institutes of Health). Stems were split in half and cankers measured lengthwise. Tissues were removed from the canker margin and placed in PDA for recovery of the pathogen. Vascular tissues were sampled at 0, 1, 2, and 3 cm above and below the control wound to determine if D. corticola had spread from the inoculation to the control wound. Cankers of variable sizes developed around wounds. Mean (and range) of canker areas were 4.5 cm² ± 1.4 cm² SE (2.3 to 9.1 cm²) for isolate 25 and 4.9 cm² ± 1.7 cm² SE (1.1 to 9.6 cm²) for isolate 101. The means for lengths of cankers induced by isolates 25 and 101 were 5.7 ± 0.6 and 4.6 ± 1.0 cm, respectively, when measured on stems split lengthwise. Xylem was discolored to the pith for four of five and two of five wounds inoculated with isolates 25 and 101, respectively. Cankers did not develop on wounded controls. D. corticola was recovered from canker edges from three of five seedlings for each isolate but not from sampled tissues 0, 1, 2, or 3 cm from control wounds. Hence, the pathogen did not progress far from the inoculation point. The identities of the recovered isolates were confirmed by sequencing the ITS region. This is the first report of D. corticola-caused cankers on oaks in Missouri. Earlier reports have shown that D. corticola causes cankers on Q. alba and other oak species also abundant in Missouri’s oak-dominated forests (Acimovic et al. 2016; Munck et al. 2017; Smith and Stanosz 2018). The role of D. corticola in oak dieback and mortality bears further study to understand any potential impacts on oaks that are of predominant importance in Missouri forests.

Molecules and morphology reveal a new aberrant harvestman genus of Ortholasmatinae (Opiliones, Dypsnoi, Nemastomatidae) from Mexico

Ortholasmatinae is one of the most striking groups of Opiliones, because its members exhibit a peculiar dorsal ornamentation pattern of cells, formed by fusion of anvil-shaped tubercles. During the systematic revision of several specimens of ortholasmatines, we found specimens with shared morphological characters with two genera Trilasma Goodnight & Goodnight, 1942 and Dendrolasma Banks, 1894. In order to investigate their phylogenetic relationships, we performed model-based phylogenetic analyses using the mitochondrial protein-encoding cytochrome c oxidase subunit 1 (CO1) and the complete nuclear ribosomal small subunit 18S, including members of the five genera of this subfamily. The results support the creation of Cryptolasma Cruz-López, Cruz-Bonilla & Francke, gen. nov., and two new species: Cryptolasma aberrante Cruz-López, Cruz-Bonilla & Francke, sp. nov. (type species) and Cryptolasma citlaltepetl Cruz-López, Cruz-Bonilla & Francke, sp. nov., which are from Xalapa and Orizaba Volcano, Veracruz, respectively. Additionally, our results suggest Ruaxphilos petrukenvitchou Goodnight & Goodnight, 1945 is a nomina dubia. This study represents the first molecular hypothesis of Ortholasmatinae relationships, with Cryptolasma as the sister of the remaining ortholasmatine.www.zoobank.org/urn:lsid:zoobank.org:pub:3730EE48-9802-4B06-9DDF-EC1AFE9A3B61

Utility of taxon-specific molecular markers for the species identification of herbarium specimens: an example from Desmarestia japonica (Phaeophyceae, Desmarestiales) in Korea

Desmarestia japonica (Phaeophyceae, Desmarestiales) was recently established from the Japanese ligulate Desmarestia and is morphologically similar to D. ligulata. This species has been reported only from Japan. However, the taxonomic reports based on additional regional distributions are needed to clarify this taxonomic entity and its species boundaries. Because Desmarestia species have restricted distributions in Korea, we reexamined herbarium specimens of D. ligulata deposited at the National Institute of Biological Resources (South Korea). To improve the amplification efficiency of the polymerase chain reaction and avoid contamination by the DNA of other organisms, we developed taxon-specific molecular markers suitable for DNA barcoding of Desmarestia species. Nuclear ribosomal small subunit RNA (18S rDNA) and mitochondrial cytochrome c oxidase 1 (cox1) regions were selected as target DNA. As a result, both were successfully isolated from herbarium specimens of D. japonica acquired over 10 years. These molecular markers provide useful genetic information for herbarium specimens for which conventional molecular analysis is challenging.

Endophytic and endolichenic fungal diversity in maritime Antarctica based on cultured material and their evolutionary position among Dikarya.

Fungal endophytes comprise one of the most ubiquitous groups of plant symbionts. They live asymptomatically within vascular plants, bryophytes and also in close association with algal photobionts inside lichen thalli. While endophytic diversity in land plants has been well studied, their diversity in lichens and bryophytes are poorly understood. Here, we compare the endolichenic and endophytic fungal communities isolated from lichens and bryophytes in the Barton Peninsula, King George Island, Antarctica. A total of 93 fungal isolates were collected from lichens and bryophytes. In order to determine their identities and evolutionary relationships, DNA sequences of the nuclear internal transcribed spacer (ITS), nuclear ribosomal small subunit (nuSSU), nuclear large subunit (nuLSU), and mitochondrial SSU (mtSSU) rDNA were obtained and protein coding markers of the two largest subunit of RNA polymerase II (RPB1 and RPB2) were generated. Multilocus phylogenetic analyses revealed that most of the fungal isolates were distributed in the following six classes in the phylum Ascomycota: Dothideomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, Pezizomycetes and Sordariomycetes. For the first time we report the presence of subphylum Mortierellomycotina that may belong to an undescribed order in endophytic fungi. Taken together, our results imply that lichens and bryophytes provide similar niches and harbour a selection of these fungi, indicating generalists within the framework of evolutionary adaptation.

Phylogeny and a new tribal classification of Opiliaceae (Santalales) based on molecular and morphological evidence

AbstractOpiliaceae are a pantropical family of the Santalales mainly distributed in the Old World with only one genus in the neotropics. Opiliaceae have remained taxonomically unresolved and the generic relationships within the family have been disputed. Here we present molecular phylogenetic analyses of the family and its close relatives using a combined dataset of the nuclear ribosomal (small subunit rDNA and large subunit rDNA) and the chloroplast rbcL, matK, and trnL‐F regions. We also carried out a morphological phylogenetic analysis using 24 characters for all the species of Opiliaceae and three species in Santalaceae s.l. and Strombosiaceae as outgroups. Molecular analyses strongly supported the monophyly of Opiliaceae. Agonandra Miers ex Benth. &amp; Hook. f. is sister to all the other genera of Opiliaceae. The remaining genera form two major clades: the Opilia clade (including Cansjera Juss., Lepionurus Blume, Opilia Roxb., Pentarhopalopilia (Engl.) Hiepko, Rhopalopilia Pierre, and Urobotrya Stapf.), and the other consisting of Anthobolus plus the Champereia clade (including Champereia Griff., Melientha Pierre, and Yunnanopilia C. Y. Wu &amp; D. Z. Li). We propose a new classification of Opiliaceae, recognizing 12 genera and four tribes, with the description of a new tribe, Champereieae Bing Liu, C. T. Le, L. M. Lu &amp; Z. D. Chen.

Botryosphaeriaceae from Eucalyptus plantations and adjacent plants in China.

The Botryosphaeriaceae is a species-rich family that includes pathogens of a wide variety of plants, including species of Eucalyptus. Recently, during disease surveys in China, diseased samples associated with species of Botryosphaeriaceae were collected from plantation Eucalyptus and other plants, including Cunninghamina lanceolata, Dimocarpus longan, Melastoma sanguineum and Phoenix hanceana, which were growing adjacent to Eucalyptus. In addition, few samples from Araucaria cunninghamii and Cedrus deodara in two gardens were also included in this study. Disease symptoms observed mainly included stem canker, shoot and twig blight. In this study, 105 isolates of Botryosphaeriaceae were collected from six provinces, of which 81 isolates were from Eucalyptus trees. These isolates were identified based on comparisons of the DNA sequences of the internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS), and partial translation elongation factor 1-alpha (tef1), β-tubulin (tub), DNA-directed RNA polymerase II subunit (rpb2) and calmodulin (cmdA) genes, the nuclear ribosomal large subunit (LSU) and the nuclear ribosomal small subunit (SSU), and combined with their morphological characteristics. Results showed that these isolates represent 12 species of Botryosphaeriaceae, including Botryosphaeria fusispora, Cophinforma atrovirens, Lasiodiplodia brasiliense, L. pseudotheobromae, L. theobromae and Neofusicoccum parvum, and six previously undescribed species of Botryosphaeria and Neofusicoccum, namely B. pseudoramosa sp. nov., B. qingyuanensis sp. nov., B. wangensis sp. nov., N. hongkongense sp. nov., N. microconidium sp. nov. and N. sinoeucalypti sp. nov. Aside from B. wangensis, C. atrovirens and N. hongkongense, the other nine Botryosphaeriaceae species were isolated from Eucalyptus trees in South China. Botryosphaeria fusispora (26 % of the isolates from Eucalyptus) is the dominant species, followed by L. pseudotheobromae (23 % of the isolates from Eucalyptus). In addition to species found on Eucalyptus trees, we also found B. pseudoramosa on M. sanguineum; B. wangensis on C. deodara; C. atrovirens on D. longan; L. theobromae on C. lanceolata, D. longan and P. hanceana; and N. hongkongense on A. cunninghamii. Pathogenicity tests showed that the 12 species of Botryosphaeriaceae are pathogenic to three Eucalyptus clones and that Lasiodiplodia species are the most aggressive. The results of our study suggest that many more species of the Botryosphaeriaceae remain to be discovered in China. This study also provides confirmation for the wide host range of Botryosphaeriaceae species on different plants.

Molecular phylogenetic analysis to support the identification of samples DL0038A &amp; DL0038B belonging to Cordyceps genus

In our previous publication, we have tentatively concluded that our fungal specimen DL0038A and DL0038B are Cordyceps takaomontana. In order to further support the identification, we continued to analyse the nrSSU (nuclear ribosomal small subunit) and rpb1 (largest subunit of RNA polymerase II) genes, as well as combined analysis the nrLSU (largest subunit of RNA polymerase II) together with nrSSU and rpb2 genes on these specimens in order to support the morphological identification of those fungi. The results show that we successfully amplified all genes. Sequencing method was then adopted and proofread before molecular phylogenetic analysis was applied with reference sequences obtained from the publication of Sung et al. (2007). Once again, this analysis strongly supports the DL0038A and DL0038B specimen as Cordyceps takaomontana.

Characterisation of Pseudosuccinea columella and Radix natalensis (Gastropoda: Lymnaeidae) in Egypt using shell and molecular data

ABSTRACTPseudosuccinea columella and Radix natalensis live in the same habitat in Egypt and are important intermediate hosts of Fasciola hepatica and F. gigantica. Our study aimed to characterise both snail species using molecular analysis and shell measurements. The ranges of morphometric parameters overlapped in the two lymnaeids, indicating that they do not clearly differentiate the two species. PCR-sequence analysis of the nuclear ribosomal small subunit rRNA and the polymorphic mitochondrial cytochrome oxidase subunit 1 (CO1) genes were used to determine the genetic identity and the potential diversity of the snails. Little intrasequence variations were detected in the sequences of both gene loci, indicating the potential homogeneity of lymnaeid populations in Egypt. Generated sequences of the mitochondrial CO1 gene locus for R. natalensis showed obvious heterogeneity compared to other sequences in GenBank. Molecular characterisation of these lymnaeids might help to understand the snails’ biodiversity in a bid to control these populations and their related diseases.

Global biogeography of marine fungi is shaped by the environment

Abstract Fungi are essential components of marine ecosystems, yet very little is known about their global distribution and diversity in the marine environment. In this study, we analyzed marine fungal community structure at a global scale using the International Census of Marine Microbes dataset. Marine fungal communities sampled from both the water column and sediments were compared. Based on the sequences of the nuclear ribosomal small subunit V9 region, 2200 operational taxonomic units (OTUs) were identified at 97% similarity. There was a significant distinction between the pelagic and benthic communities, with 15.4% OTUs shared between the two realms. Environmental factors, particularly sample depth, oxygen, and nitrate, strongly correlated with the fungal community composition and explained more variance than did geographic distance. This study represents the first global-scale analysis of marine fungal community structure, and highlights potential opportunities for research in marine fungal ecology and biogeography.