Analysis Of Internal Transcribed Spacer Research Articles

Tuber cumberlandense and T. canirevelatum, two new edible Tuber species from eastern North America discovered by truffle-hunting dogs

ABSTRACT Ectomycorrhizal fungi in the genus Tuber form hypogeous fruiting bodies called truffles. Many Tuber species are highly prized due to their edible and aromatic ascomata. Historically, there has been attention on cultivating and selling European truffle species, but there is growing interest in cultivating, wild-harvesting, and selling species of truffles endemic to North America. North America has many endemic Tuber species that remain undescribed, including some that have favorable culinary qualities. Here, we describe two such Tuber species from eastern North America. Maximum likelihood and Bayesian phylogenetic analyses of ITS (internal transcribed spacer), tef1 (translation elongation factor 1-alpha), and rpb2 (second largest subunit of RNA polymerase II) sequences were used to place these species within a phylogenetic context. We coupled these data with morphological analyses and volatile analyses based on gas chromatography–mass spectrometry. Tuber cumberlandense, sp. nov. (previously referred to as Tuber sp. 66), is a member of the Rufum clade that has been opportunistically harvested for commercial sale from T. melanosporum orchards across eastern North America. Tuber canirevelatum, sp. nov. belongs in the Macrosporum clade and thus far is only known from eastern Tennessee, USA. Both new species were discovered with the assistance of trained truffle dogs. The volatile profiles of T. canirevelatum and T. cumberlandense were measured in order to characterize aromas based on the chemical compounds produced by these fungi. Ascomata from both species were enriched in acetone, dimethyl sulfide, 1-(methylthio)-1-propene, and 1-(methylthio)propane. In this work, we celebrate and encourage the use of trained truffle-hunting dogs for fungal biodiversity discovery and research.

First Report of Powdery mildew caused by Golovinomyces ambrosiae on Bidens pilosa in Guangdong Province, China.

Bidens pilosa L., an annual herbaceous plant with a wide distribution, possesses novel medicinal properties. In January 2021, a powdery mildew disease outbreak was documented on B. pilosa plants located in the roadside areas in Shenzhen, Guangdong Province, China, with 60 to 80% disease incidence. Initial symptoms manifested as small, irregular white powdery patches, primarily on the adaxial surfaces of leaves. Subsequently, the colonies expanded, forming coalescent colonies that spread across the leaves, petioles, and stems, eventually leading to the distortion and senescence of leaves. Hyphae are hyaline, flexuous to straight, septate, with thin walls and a width ranging from 2 to 8 μm. Hyphal appressoria are nipple-shaped. Conidophores are erect or slightly flexuous, ranging from 80 to 150 µm in length and 12 to 18 µm in width (n = 30). Typically, these conidophores bear chains of 2 to 5 immature conidia, displaying a sinuate outline. Foot-cells, located at the base of conidophores, are cylindrical and erect, approximately 33 to 100 µm in length and 6 to 10 µm in width (n = 30). Conidia are hyaline, ellipsoid-ovoid to barrel-shaped, and lack fibrosin bodies. Primary conidia are ellipsoid-ovoid in shape, characterized by a rounded apex and a subtruncate base, 25 to 40 µm × 15 to 22 µm in width. Secondary conidia are barrel-shaped with truncate or subtruncate ends, 27 to 35 µm × 15 to 20 µm in width. Germ tubes exhibit a longitubus pattern and are prominently produced at the perihilar or apical region of the conidia. No chasmothecia were observed in the collected samples. In order to conduct a molecular-level identification, mycelium and conidia were collected from B. pilosa leaves. Genomic DNA was subsequently extracted from these samples. The internal transcribed spacer (ITS), intergenic spacer (IGS) and beta-tubulin (tub2) sequences were performed using primer pairs ITS1/ITS4, IGS-12a/NS1R, and tub2, respectively (Carbone and Kohn 1999; Scholin et al. 1994; White et al.,1990). A 568-bp ITS, a 366-bp IGS, and a 354-bp tub2 sequences (GenBank accession nos. OR647592, OR978282 and OR978283) were obtained. The ITS sequence exhibited over 99.6% similarity to Golovinomyces ambrosiae (MT929773) and G. cichoracearum (MH590731). The IGS sequence displayed 100% similarity to G. ambrosiae (MK383490) and G. ambrosiae (OK349420). The tub2 sequence displayed 100% similarity to G. ambrosiae (MW981257) and G. ambrosiae (MW981256). Phylogenetic analysis of IGS, ITS and tub2 also grouped obtained sequences within the G. ambrosiae complex. Based on the analysis of morphological characteristics and sequence identity, the pathogen was identified as G. ambrosiae. In order to satisfy Koch's postulates, an infected leaf was carefully pressed onto leaves of six healthy young B. pilosa plants, each grown in a separate pot. Additionally, a control group consisted of six non-inoculated plants. All plants were placed in a greenhouse: 25°C, 14/10-h light/dark photoperiod, and relative humidity 50%. After 10 days, the inoculated leaves exhibited powdery mildew colonies similar to those observed in the original infected plants. At 16 days, the inoculated leaves exhibited discoloration and premature leaf drop. The pathogenicity test was conducted twice. Microscopic observation and sequencing confirmed that isolated fungus was identical to the original pathogen. G. ambrosiae has previously been documented on B. pilosa in Fuzhou, Fujian Province, China (Mukhtar et al., 2022). However, to the best of our knowledge, this study represents the first report of powdery mildew caused by G. ambrosiae on B. pilosa in Shenzhen, Guangdong Province, China.

First Report of Rust Caused by Puccinia sasicola on Corylopsis coreana in South Korea.

Korean winter hazel (Corylopsis coreana) is an endemic species of the South Korea (Seo et al. 2016; Kim et al. 2021), which is cultivated as an ornamental plant in this country, but also in China and Japan (Yoon et al. 2016). In July 2022, typical symptoms of a rust disease were observed on C. coreana at Jirisan National Park (35°22'07.7"N 127°34'57.7"E) in Namwon, South Korea. Spermogonia were epiphyllous, densely grouped, pale brown or orange-yellow, round, and 0.23 - 0.38 × 0.19 - 0.41 mm in size. Aecia were hypophyllous, mostly densely grouped, yellow or pale orange, resembling small wart-like galls, and 0.04 - 0.06 × 0.89 - 1.68 mm in size. Aeciospores were hyaline, mostly angularly globose, ellipsoid or oblong-ellipsoid, and 17.8 - 25.2 × 15 - 26.5 μm (average 19.2 × 19.1 μm; n=50) in size. Aeciospore walls were echinulate-verrucose, and 1.1 - 2.2 µm (average 1.7 µm; n=50) in thickness. In December 2022, dark brown telia were observed on the lower surface of Sasa borealis leaves near C. coreana. Telia were mostly scattered but often compacted, brown to dark brown, round, and 1.5 - 1.95 × 1.24 - 1.55 mm in size. Teliospores were either one- or two-celled with a long tapering apex, and light brown to brown in color. One-celled teliospores were globose, and 95.1 - 186.5 × 20.5 - 36.4 μm (average 136.4 × 27.7 μm; n=50) in size. Two-celled teliospores were ellipsoid-cylindrical, and 111.4 - 180.3 × 13.5 - 32.6 μm (average 149 × 21.1 μm; n=50) in size. Side walls of teliospores were golden and 2.2 - 5.5 µm thick (average 3.5 µm; n=50), and pedicels were hyaline, measuring 150 - 300 μm long. Uredinial stage was not observed. Disease symptomology and pathogen morphology were mostly consistent with that of Puccinia sasicola reported in Japan (Hino. 1955). For phylogenetic analysis, genomic DNA was extracted from the aeciospores collected from C. coreana and the teliospores collected from S. borealis. The internal transcribed spacer (ITS) rDNA and the large subunit (LSU) rDNA regions were amplified using ITS5u/ITS4rust (Pfunder and Schürch 2001) and LRust1R/LRust3 (Beenken et al. 2012) primers, respectively. Both sequences were identical for the spores collected from the two different hosts. The sequences were deposited in GenBank (PP171665, PP174211 [ITS], PP171709, PP174356 [LSU]). A GenBank BLAST search revealed 89.53% and 96.78% similarity with Puccinia kusanoi (KX610657) and Puccinia sp. (MT7298241) for ITS and LSU sequences, respectively. In maximum-likelihood phylogenetic analysis of ITS and LSU sequences, the isolates from C. coreana and S. borealis formed a separate clade from other Puccinia species. To test Koch's postulates, leaf disks with telia from S. borealis were directly attached to the adaxial surface of six healthy C. coreana leaves with tape. As controls, healthy S. borealis leaf disks were attached to the adaxial surface of six C. coreana leaves. After four weeks, four inoculated leaves developed small yellow wart-like galls on the abaxial surface, while the control leaves remained symptom-free. The isolates obtained from the inoculated leaves had identical sequences to the original isolate. There are no publicly available sequences for P. sasicola, nor did we find any sequences that match our Puccinia samples. Nevertheless, based on morphological characteristics and life cycle, our isolates closely matched with the previous description of P. sasicola by Hino (1955). To our knowledge, this is the first report of P. sasicola causing leaf rust in C. coreana in South Korea.

First Report of Leaf Spot Disease on Lonicera japonica Caused by Nigrospora oryzae in China.

Lonicera japonica Thunb. is a traditional Chinese medicinal plant, which widely cultivated in China, Japan and Korea. From August to October in 2021 and 2022, severe leaf spots symptoms were observed on L. japonica in medicinal botanical garden of Shandong University of Traditional Chinese Medicine (36°55'89"N, 116°79'91"E), Jinan, Shandong Province, China. The disease incidence was above 80% in the 25 acre cultivation area. Early symptoms were small brown spots on the leaves. Then the number of small spots gradually increased and spread over the entire leaves. The small brown spots seldom merge together to form larger lesions. Leaves with typical symptoms were collected from twenty individual plants, and cut into small 5×5 mm fragments in the junction of infected and healthy tissues. The fragments were sterilized in 75% ethanol for 30 s and 1% NaClO for 60 s, rinsed three times in sterile water, and then placed on potato dextrose agar (PDA). After 3 days of incubation at 25°C, fungal plugs along the edge of the colony were cut and transferred to new PDA for purification. A total number of 23 colonies with similar morphological characteristics were obtained, and three representative strains (Lj14, Lj18 and Lj20) were selected for subsequent study. The colonies grew rapidly on PDA and covered the entire petri dish in 4 days. Colonies had abundant aerial hyphae, initially white, round, later turning gray and black. Conidia were oblate or nearly spherical, single-celled, black, and measured in size from 9.6 to 13.2 μm × 7.9 to 16.1 μm in diameter (n=150) (Figure S1). The observed characteristics were close to those of Nigrospora spp. ( Wang et al. 2017). The genomic DNA was extracted, and PCR amplification of the rDNA internal transcribed spacer (ITS), β-tubulin gene (TUB), and translation elongation factor 1-alpha gene (TEF1) were completed by primers ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R (Carbone and Kohn, 1999). Sequences were deposited in GenBank (accession nos. OR936661, OR936662, OR936671 for ITS, OR947626, OR947627, OR947628 for TUB, and OR947629, OR947630, OR947631 for TEF1 sequences, respectively). BLAST analyses of ITS (OR936661), TUB(OR947626) and TEF1 (OR947629) sequences exhibited 100% (487 bp out of 487 bp), 99.48% (380 bp out of 382 bp), and 99.6% (248 bp out of 249 bp) similarity to the sequences of N. oryzae strains KoLRI_053384 (MZ855426), LC2991 (KY019496) and LC7307 (KY019409), respectively. Lj14, Lj18 and Lj20 formed a clade with N. oryzae LC6763 and LC2991 in phylogenetic tree (Figure S2). Based on morphological and molecular evidence, the pathogen was identified as N. oryzae (Berk. &Broome) Petch. To fulfill Koch's postulates, the pathogenicity was tested in vivo experiments. Thirty non-wounded healthy leaves of ten intact plants were inoculated with 10 µl spore suspension (106 spores/ml) of three strains, respectively. As negative control, thirty leaves of ten healthy plants were inoculated with sterile water. The inoculated plants were placed at 28°C in the growth chamber with high relative humidity. The pathogenicity tests were repeated three times. Distinct symptoms similar to that of natural conditions were observed on the leaves of inoculated plants after 4 to 7 days. The strain was reisolated from the lesions and identified as N. oryzae by morphological features and ITS sequence. The pathogen has been reported to cause leaf spot disease on tobacco (Wang et al. 2022) and asiatic dayflower (Qiu et al. 2022). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Lonicera japonica in China. The research will be helpful for leaf spot disease control.

First report of Stagonosporopsis cucurbitacearum Causing Gummy Stem Blight on Trichosanthes kirilowii Maxim. in China.

Trichosanthes kirilowii Maxim. (Cucurbitaceae), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease of cucurbits, was widespread in many T. kirilowii plantations. The initial symptoms on the naturally infected stems appeared as dark brown water-soaked lesions, and as the disease progressed, vines of T. kirilowii gradually withered. On leaves, brown water-soaked lesions were visible initially, and then lesions enlarged and coalesced, resulting in extensive necrosis of leaves. On fruit, lesions covered with the white mycelium were nearly circular and tan to brown initially. Subsequently, the diseased fruit turned black and rotten commonly known as fruit rot or black rot. A Stagonosporopsis-like organism was consistently isolated from symptomatic stems, leaves and fruits. Fungal isolates were initially white and later turned dark grey or black with woolly to floccose aerial mycelium on PDA medium. Twenty-four isolates from different plantations were selected for further morphological studies. Pycnidia and conidia were formed after inoculating on cucumber fruit for 3 days. Pycnidia were globose to sub-globose, brown, ostiolate and 106.7 to 213.6 μm (average 160.1 μm, n = 50) in diameter. Conidia were hyaline, ellipsoidal, aseptate or one-septate, slightly constricted at the septa, 6.1 to 13.6 × 3.5 to 4.8 μm (average 9.9 × 4.1 μm, n = 50), and contained two or more oil drops. Three different loci of the genomic DNA, including the nuclear ribosome DNA internal transcribed spacer (ITS), RNA polymerase II second-largest subunit (RPB2), and β-tubulin (TUB2) genes., were amplified using primers ITS1/ITS4 (White et al. 1990), RBP2DF/RBP2DR (Lawrence et al. 2013), and T1/β-Sandy-R (O' Donnell and Cigelnik 1997; Stukenbrock et al. 2012), respectively and sequenced. A phylogenetic tree was built based on analysis of ITS, RPB2, and TUB2 sequences that deposited in GenBank (MW485497-MW485502 for ITS, MW531661-MW531666 for RPB2, and MW531667-MW531672 for TUB2), using the maximum likelihood method. The phylogenetic tree showed that the isolates fell into a single clade with S. cucurbitacearum. On the basis of morphological and molecular characteristics, the isolates obtained from T. kirilowii were identified as Stagonosporopsis cucurbitacearum. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruit collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were similarly inoculated with agar plugs. The diameters of lesions were measured in two perpendicular directions. Re-isolations from the stem and leaf lesions were performed on the PDA medium. Stagonosporopsis cucurbitacearum, was re-identified based on its colony and conidial characteristics and, therefore, completed Koch's postulates. Gummy stem blight caused by S. cucurbitacearum has been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruits collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were treated similarly but inoculated with agar plugs. Diameters of lesions were measured in two mutually perpendicular directions. Reisolations from the lesions were performed on PDA medium, and was re-identified based on its colony and conidial characteristics to complete Koch's postulates. Gummy stem blight caused by S. cucurbitacearum have been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies.

First Report of Colletotrichum siamense Causing Anthracnose on Bixa orellana in China.

Annatto (Bixa orellana L.) is widely cultivated in China. Its seed is used as medicine and as an astringent antipyretic. Since 2019, anthracnose-type lesions have been observed on the annatto leaves in the field (about 30 hectares) in Zhanjiang (21˚18'12''N, 110˚17'22''E), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 3 ha). The early symptoms were yellow spots on the edge or tip of leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions (Supplemental Figure 1-A). Ten symptomatic leaves from 10 plants were sampled. The margins of the lesions were cut into 2 × 2 mm pieces and the surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec. After that, pieces were rinsed thrice in sterile water, placed on potato dextrose agar(PDA) medium, and incubated at 28 ℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (BOC-1, BOC-2, and BOC-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. Besides, the three isolates were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University. Colonies on PDA were white to gray with cottony mycelia after incubating in the dark for 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 9.6 to 18.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown, and they measured 6 to 9 µm × 4.5 to 8 µm (n = 30) (Supplemental Figure 1-D, E and F). These morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009;Sharma et al. 2013). For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates were amplified using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences were deposited in GenBank under nos. MZ047377-MZ047379 (ITS), MZ126934-MZ1269346 (GAPDH), MZ126904-MZ1269046 (CHS-1), and MZ126844-MZ1268446 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from ITS, GAPDH, CHS-1, and ACT sequences that clustered the three isolates with C. siamense (the type strain MFLU 090230), (Supplemental Figure 2). The pathogenicity of the three isolates was tested respectively in a greenhouse maintained at 25 to 29℃ and 80% relative humidity. Annatto seeding ( n =5, 2-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water.. The experient was repeated three times. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy (Supplemental Figure 1-G, and H). The same pathogen was re-isolated from all the inoculated leaves based on morphology and ITS analysis. C. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012; Wang et al. 2017; Liu et al. 2017; Zhuo et al. 2017 ), but not in B. orellana. To our knowledge, this is the first report of C. siamense causing anthracnose on B. orellana in China. Our study provides important reference information for controlling this disease.

First Report of Colletotrichum scovillei Causing Anthracnose Fruit Rot on Pepper in Ontario, Canada.

Anthracnose fruit rot affecting field peppers (Capsicum annuum L.) has been reported in Ontario, Canada, leading to significant crop losses of up to 80% over the past three years. Ten symptomatic fruits per field, exhibiting one or more soft, sunken lesions covered with salmon-colored spore masses (Fig. S1), were collected from one and two Banana pepper fields in August 2022 and 2023, respectively, all located in southwestern Ontario. Small sections of diseased tissue (0.5 cm in length) from lesion edges underwent surface sterilization and plated on 2% potato dextrose agar (PDA, Difco) supplemented with kanamycin (50 mg liter-1), neomycin sulfate (12 mg liter-1) and streptomycin sulfate (100 mg liter-1), and incubated at 22°C for 7 days in the dark. Fifteen fungal colonies were isolated and purified using the hyphal tipping method. All fungal isolates showed a pale gray colony morphology with a faint salmon tint on PDA (Fig. S1). Conidia, produced on PDA after incubating the 15 isolates at 22°C for 17 days in the dark, were hyaline, aseptate, smooth-walled, cylindrical with obtuse ends (Fig. S1), and measured 9.4 to 15.0 × 2.7 to 4.8 µm (mean ± standard deviation of 145 conidia = 11.3 ± 1.2 μm × 3.7 ± 0.5 μm), the typical morphology of Colletotrichum species (Damm et al. 2012). Internal transcribed spacer (ITS), actin (ACT), chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), histone H3 (HIS3) and beta-tubulin 2 (TUB2) gene regions of all isolates were amplified and sequenced with primers ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, GSF1/GSR1, CYLH3F/CYLH3R and Bt2a/Bt2b and deposited in GenBank (Accession Nos. ITS: PP060584 to PP060596; ACT, CHS-1, GAPDH, GS, HIS3 and TUB2: PP085919 to PP086005), respectively. The sequences were 100% identical to Colletotrichum scovillei strains from different hosts and countries (ITS: PP079643; ACT: MN718468; CHS-1: MN718466, GAPDH: MN718465.1, HIS3: MT592502, TUB2: MK462971). The maximum likelihood-based phylogenetic analysis of ITS, ACT, CHS-1, GAPDH, GS, HIS3, and TUB2 concatenated sequences was conducted using IQ-TREE 2.2.2.7 (Minh et al. 2020). All isolates from this study were grouped with high bootstrap support values with the holotype C. scovillei CBS 126529 (Fig. S2). Living cultures of these isolates were deposited in the Canadian Collection of Fungal Cultures (DAOMC 252833 to 252847). Pathogenicity was tested by inoculating 4 Banana (cv. Jumbo Stuff) and 4 Bell (cv. Archimedes) pepper fruits with 10 μl droplet of a 1 × 105 conidia ml-1 suspension of each isolate onto a wound made with a sterile pipette tip. Eight control fruits were mock-inoculated with sterilized water. Nine days post-inoculation, necrotic lesions measuring 24.7 ± 0.3 mm on Bell and 27.9 ± 0.2 mm on Banana peppers were observed. Colletotrichum scovillei was re-isolated from all symptomatic fruits, and its species identity was confirmed through morphology, fulfilling Koch's postulates. Control fruits remained symptom-free, and no fungi were isolated from them. This is the first report of C. scovillei in Canada. Previously identified as a pathogen causing anthracnose on peppers in eastern Asia, the United States, Brazil, and Kosovo (Farr and Rossman 2024; Xhemali et al. 2023), its emergence in Ontario raises significant concerns for pepper crops. Additional research is essential to better understand the epidemiology of the disease and develop effective phytosanitary strategies for control.

The comparative plastisphere microbial community profile at Kung Wiman beach unveils potential plastic-specific degrading microorganisms.

Plastic waste is a global environmental issue that impacts the well-being of humans, animals, plants, and microorganisms. Microplastic contamination has been previously reported at Kung Wiman Beach, located in Chanthaburi province along with the Eastern Gulf of Thailand. Our research aimed to study the microbial population of the sand and plastisphere and isolate microorganisms with potential plastic degradation activity. Plastic and sand samples were collected from Kung Wiman Beach for microbial isolation on agar plates. The plastic samples were identified by Fourier-transform infrared spectroscopy. Plastic degradation properties were evaluated by observing the halo zone on mineral salts medium (MSM) supplemented with emulsified plastics, including polystyrene (PS), polylactic acid (PLA), polyvinyl chloride (PVC), and bis (2-hydroxyethyl) terephthalate (BHET). Bacteria and fungi were identified by analyzing nucleotide sequence analysis of the 16S rRNA and internal transcribed spacer (ITS) regions, respectively. 16S and ITS microbiomes analysis was conducted on the total DNA extracted from each sample to assess the microbial communities. Of 16 plastic samples, five were identified as polypropylene (PP), four as polystyrene (PS), four as polyethylene terephthalate (PET), two as high-density polyethylene (HDPE), and one sample remained unidentified. Only 27 bacterial and 38 fungal isolates were found to have the ability to degrade PLA or BHET on MSM agar. However, none showed degradation capabilities for PS or PVC on MSM agar. Notably, Planococcus sp. PP5 showed the highest hydrolysis capacity of 1.64 ± 0.12. The 16S rRNA analysis revealed 13 bacterial genera, with seven showing plastic degradation abilities: Salipiger, Planococcus, Psychrobacter, Shewanella, Jonesia, Bacillus, and Kocuria. This study reports, for the first time of the BHET-degrading properties of the genera Planococcus and Jonesia. Additionally, The ITS analysis identified nine fungal genera, five of which demonstrated plastic degradation abilities: Aspergillus, Penicillium, Peacilomyces, Absidia, and Cochliobolus. Microbial community composition analysis and linear discriminant analysis effect size revealed certain dominant microbial groups in the plastic and sand samples that were absent under culture-dependent conditions. Furthermore, 16S and ITS amplicon microbiome analysis revealed microbial groups were significantly different in the plastic and sand samples collected. We reported on the microbial communities found on the plastisphere at Kung Wiman Beach and isolated and identified microbes with the capacity to degrade PLA and BHET.

First Report of Powdery Mildew on Viburnum chinshanense Caused by Erysiphe pseudoviburni in China.

Viburnum chinshanense, a deciduous shrub in the family Caprifoliaceae, is a dominant tree distributed mainly in the North-Central and South-Central regions of China (Zhu et al. 2023). Because of its lush white flowers and vibrant red fruits, V. chinshanense is used widely as ornamental tree in China. In May 2022, severe powdery mildew symptoms were observed on V. chinshanense on the Huaxi Campus of Guizhou Normal University, Guiyang, China. The incidence was approximately 75% among 80 V. chinshanense plants observed. White mycelia were present on both adaxial and abaxial leaf sides, but not on fruits, petioles, or stems. Infected leaves showed slight chlorosis and twisting. The mycelia were amphigenous, forming small-to-large patches, often sparse on the upper leaf surface, but mostly confluent on the lower leaf surface. Hyphae were hyaline, 4-7 μm wide. Hyphal appressoria were lobed to multilobed, in opposite pairs or solitary. Conidiophores were erect, straight, or somewhat flexuous, 60-130 µm long (n = 30). Foot cells were subcylindrical to slightly curved-sinuous at the base, 20-40 × 6-10 µm (n = 30) in size, followed by 1-3 shorter cells. Conidia formed singly, occasionally two to three in a chain. Conidia were ellipsoid to ovoid, cylindrical, and 24-40 × 16-20 µm (n = 50). No fibrosin bodies were observed on the conidia. Chasmothecia were subglobose, 56-115 µm in diameter. The appendages were 35-70 µm long. Based on these morphological characteristics, the powdery mildew fungus was identified as Erysiphe pseudoviburni (Bradshaw et al. 2020). To confirm the identification, the ribosomal DNA internal transcribed spacer (ITS) and the ribosomal large subunit (LSU) region were amplified and sequenced using the ITS1/ITS4 primer pair (White et al. 1990) and the NL1/NL4 primer pair (Ziemiecki et al. 1990), respectively. The obtained 643-bp ITS sequence (GenBank accession no. ON729292) had 99.84% identity with E. pseudoviburni strains KUS-F27310 (MN431595) and MUMH0001 (LC009904). The obtained 593-bp LSU sequence (ON729293) had 99.83% identity with E. pseudoviburni (LC009904 and MN431595). Based on the phylogenetic analysis of the combined ITS and LSU dataset (Bradshaw et al. 2020), the isolate (GZVD-1) was grouped in a clade with the E. pseudoviburni strains KUS-F27319, KUS-F27310, and MUMH0001. To fulfill Koch's postulates, leaves of three healthy potted V. chinshanense plants were inoculated by gently pressing with diseased leaves. Non-contact plants were used as controls. All plants were incubated in a greenhouse at 25 ± 2°C, 80% relative humidity. Similar powdery mildew symptoms were observed on the inoculated plants 12 days after inoculation, whereas the control plants remained symptomless. The reisolated fungus from the inoculated plants was morphologically identical to that on originally diseased plants. ITS and LSU sequences of the reisolated fungus showed 100% identity with ON729292 and ON729293, respectively. E. pseudoviburni has previously been reported to infect some Viburnum species, including V. sieboldii in Japan (Takamatsu et al. 2015) and V. odoratissimum in South Korea (Bradshaw et al. 2020). To the best of our knowledge, this is the first report of powdery mildew caused by E. pseudoviburni on V. chinshanense in China. This work expands the known host range of E. pseudoviburni in the Viburnum genus.

First report of Powdery Mildew Caused by Podosphaera xanthii on Siraitia grosvenorii in Guizhou, China.

Swingle (Siraitia grosvenorii), a member of the Cucurbitaceae family, stands out as a distinctive plant with both economic and medicinal significance. In October 2023, severe powdery mildew were observed on S. grosvenorii in Guiyang City (26.50°N; 106.66°E), Guizhou Province, China. About 80 % of the plants in the greenhouse showed powdery mildew symptoms. Three infected plant samples were selected for morphological and molecular analysis (GZAAS 23-0801, GZAAS 23-0802 and GZAAS 23-0803). The voucher specimens are deposited in the Key Laboratory of Agricultural Biotechnology of Guizhou Province. The symptoms initially manifested as irregular to nearly circular, small yellow spots, with distinct depressions as well as surfaces covered in white mycelium. Over time, these spots gradually expanded and merged patches. In the final stages, the entire leaves turned into yellow and withered. Microscopic observations showed that fungal hyphae were septate, branched, and flexuous to straight and 5 to 9 µm wide, and appressoria were indistinct to slightly nipple-shaped. Conidia were hyaline and ellipsoid to oval with fibrosin bodies and measured 31 to 43 × 18 to 24 μm (n = 50) with a length/width ratio of 1.3 to 2.3. Conidiophores were unbranched, straight, 120 to 268 × 14 to 22 µm (n = 30), producing two to five immature conidia in chains. Foot cells of conidiophores were cylindrical, 39 to 84 × 8 to 14 μm (n = 30), followed by one to three short cells. Short cells were cylindrical, 12 to 32 × 8 to 15 μm (n = 50). The morphological characteristics were identical with the previous description of Podosphaera xanthii (Braun and Cook, 2012). Total DNA was extracted from conidia and mycelia by the Chelex method (Walsh et al., 1991). The ribosomal DNA internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU) were amplified by using the primers ITS1/ITS4 (White et al., 1990) and LSU1/LSU2 (Scholin et al., 1994), respectively. The ITS (OR825802, OR825803 and OR825804, respectively) and LSU (OR825805, OR825806 and OR825807, respectively) sequences of three isolates, were deposited in GenBank. The BLAST results revealed that both the ITS and LSU region sequence were 100% identical to those of P. xanthii (ITS: MF043939, MG754404 and KJ698669; LSU: OQ061319, AB936277and OP218411). Phylogenetic analyses of ITS and LSU sequences showed that our three isolates were clustered with P. xanthii (KX842351, LC270782 and LC270779) with high statistical support (ML/MP/BI: 100%/97%/1.00). Combined with their morphological characteristics, these three isolates were identified as P. xanthii. Pathogenicity tests were performed by gently brushing conidia onto the leaves of five healthy S. grosvenorii plants. Five non-inoculated plants were used as the control. All plants were maintained in a greenhouse at 25 ± 2°C. One week after inoculation, similar symptoms were observed in the inoculated plants, whereas no symptoms occurred on the control plants. By microscopic observation, the fungus on the inoculated plants was morphologically identical to those on originally diseased plants. Powdery mildew caused by P. xanthii has been reported on Vernonia cinerea (Wu et al., 2023), Vigna unguiculata (Zhang et al., 2023), Cucumis melo (Meesam et al., 2023). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. grosvenorii in Guizhou, China. The occurrence of powdery mildew on S. grosvenorii may pose a potential threat to its large-scale cultivation. The pathogen could become a threat to other Cucurbitaceae members in the future.

First Report of Pseudocercospora oenotherae Causing Leaf Spot in Rhinacanthus nasutus in China

Rhinacanthus nasutus (L.) Kurz is a traditional Chinese medicine in China. In August 2022, leaf spots were observed on R. nasutus in a Chinese herbal garden in Zhanjiang, Guangdong Province, China (21°17′30″N, 110°18′25″E). Disease incidence was 90% (n = 100 investigated plants from about 800 plants). The yellow spots were round, gray in the center and scattered on the leaves. The coalescence of the individual spot eventually led to leaf wilt. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The tissue surface was disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-eight isolates were obtained (isolation frequency = 28/4 × 10 = 70%). Three representative single-spore isolates (RNPO-1, RNPO-2, and RNPO-3) by a single-spore isolation method (Fang. 1998) were used for further study. The colonies of isolates on PDA were olive green in 7 days at 28 °C. Conidiogenous cells were unbranched, geniculate–sinuous, tapered toward the apex, and 10 to 20 × 3 μm (n = 20). Conidia were solitary, smooth, straight or curved, pale brown, 3 to 8-septate, apex acute, base truncate, and 50.5 to 88.5 × 2.0 to 3.5 μm (n = 50). For molecular identification, the colony PCR method with Taq DNA polymerase and MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), actin (ACT) , and RNA polymerase II second largest subunit (RPB2) loci of the isolates using primer pairs ITS1/ITS4, EF1/EF2, ACT-512F/ACT-783R, and RPB2-7CF/fRPB2-11aR, respectively (O’Donnell et al. 1998; O’Donnell et al. 2010). Their sequences were deposited in GenBank under nos. OP963568 to OP963570 (ITS), OP998376 to OP998378 (TEF1), OP998373 to OP998375 (ACT), and OP998379 to OP998381 (RPB2). By using the Maximum Likelihood method, a phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, ACT, and RPB2 that clustered the isolates with P. oenotherae (the type strain CBS 131920). The fungus was thus identified as P. oenotherae basing on these morphological and molecular characteristics (Guo and Liu. 1992; Kirschner. 2015). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 28 °C to 30 °C. Healthy plants of R. nasutus were grown in pots, with one plant in each pot. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) of the isolates for about 15 s before they were adhered to the leaves for 3 days. Each isolate was inoculated with three plants (2 month old), and each plant was inoculated with five leaves. Sterile distilled water was as the control and same treatment. The test was performed three times. Symptom were found on the inoculated plants after 2 weeks with the disease incidence 100%, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and ITS analyses. No pathogen was isolated from the control plants. P. oenotherae caused leaf spot on Oenothera biennis L. (Guo and Liu. 1992). To the best of our knowledge, this is the first report that R. nasutus is the new host of P. oenotherae (Crous et al. 2013). Thus, this work provides an important reference for the control of this disease in the future.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

First Report of Colletotrichum truncatum Causing leaf spot on Clausena lansium in China.

Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, leaf spot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 2 ha). Leaf spots were round or irregular with a clear yellow halo around a brown, necrotic lesion. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed thrice in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C in the darkfor 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (CLCT-1, CLCT-2, and CLCT-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. The colonies on PDA were gray white at first, subsequently turning grayish to dark gray, with numerous black microsclerotia and setae. Conidia were hyaline, aseptate, falcate with pointed ends, and 16.5 to 22.3 × 2.5 to 3.2 μm (n = 30). Morphological characteristics of the isolates were consistent with the description of Colletotrichum truncatum (Schwein.) Andrus & W. D. Moore (Sawant et al. 2012). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequences were submitted to GenBank under accession numbers OP740964 to OP740966 (ITS), OP800837 to OP800839 (GAPDH), and OP800843 to OP800845 (ACT). The sequences of the three isolates were 100% identical (ITS, 547/547 bp; GAPDH, 290/290 bp; and ACT, 266/266 bp) with those of C.truncatum (accession nos. GU227869, GU228261, and GU227967) through BLAST analysis.. In addition, a phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that nested within the clade containing C. truncatum (the type strain CBS 112998) by the maximum likelihood method. From the combination of the morphological and molecular characteristics, the isolates were determined to be C.truncatum. A pathogenicity test was performed in a greenhouse at 24 to 30°C with 80% relative humidity. Wampee plants (cv. JIXIN, n =5, 1-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water. Leaf spots were observed on the inoculated plants after 10 days while the control ones remained healthy. The pathogen re-isolated from all the symptomatic leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C.truncatumhas also beenreportedto be thecausalagent of anthracnose disease in multiple crops (Diao et al. 2014;Villafana et al. 2018; Stella de et al. 2021), thus, this is the first to report C.truncatum causing leaf spot on C. lansium in China. This study provides an important reference for the control of the disease due to the high host range ofC.truncatum.

First report of Neophysopella vitis causing leaf rust on Parthenocissus semicordata in Guangdong Province, China.

In April 2022, leaf rust disease of Parthenocissus semicordata was discovered in the urban greenbelt in Guangzhou city, China (23.06°N, 113.16°E). The disease incidence was 70% and disease severity was 75%. Chlorotic spots and red-brown necrotic flecks were present on the upper surface of infected leaves, and orange uredinia were distributed on the lower surface (Fig. 1 a-c). Two representative disease plants were collected as voucher specimens and dried, then deposited in Mycological Herbarium of the Zhongkai University of Agriculture and Engineering (MHZU GR0413, MHZU GR0414). Microscopic examination of the pustules of the samples revealed the presence of uredial paraphyses and urediniospores (Fig.1 d-f). Uredial paraphyses were hyaline, incurved, length 20-25 μm and dorsal wall 4.5-8.5 μm thick. Urediniospores were subglobose to ovoid, 16-24.5 × 10.5-17 μm. The wall of the urediniospore was hyaline or pale yellow, echinulate, and 1.0-2.0 μm thick. Telial structures were not observed. The morphological characteristics of uredial paraphyses and urediniospores were consistent with Yoshitaka's description of Neophysopella vitis (Yoshitaka 2000). Ten uredinia were picked using a sterile tweezer and the FlaPure Plant DNA Extraction Kit (Genesand Biotech Co., Ltd) was used to extract genomic DNA. Primers ITS4-rust (5'-CAGATTACAAATTTGGGCT-3') /ITS 5u (5'-CAAGGTTTCTGTAGGTG-3') were used to amplify internal transcribed spacer (ITS) and Rust2inv (5'-GATGAAGAACACAGTGAAA-3') /LR6 (5'-CGCCAGTTCTGCTTACC-3') to amplify large subunit (LSU) rDNA regions (Zhao et al. 2021). The sequences were submitted to NCBI (ITS: OQ991182 OQ991183, LSU: OQ979612 OQ979613), and blastn analyses of ITS and LSU showed 99.81% and 99.84% identity with the sequences from N. vitis (ITS: OQ304336, LSU: OM420266), respectively, found in GenBank. ITS combined with LSU sequences from the current study and reference sequences from Zhao et al. (2021) were used to construct Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees using CIPRES website. ML and BI phylogenetic trees showed that the sequences from the current study grouped with N. vitis, with bootstrap support and posterior probability of 98% and 1.0, respectively (Fig. 2). Morphological and molecular analyses confirmed that the rust fungus was N. vitis. In the pathogenicity test, urediniospores were picked from fresh diseased leaves with sterile tweezers, prepared into spore suspensions (1.0 × 106 urediniospores/ml), and sprayed on a one-week-old leaf from three healthy of potted plants of P. semicordata, while three other healthy leaves were sprayed with sterile water as control. Each treated leaf was wrapped in a plastic bag and incubated in the dark at 25℃ for 48 h after which plastic bags were removed and the plant were moved to a greenhouse at 25℃. At 15 days after inoculation, symptoms and the morphology of urediniospores were confirmed to be the same as those observed in the field collection and no symptoms or uredinia were observed in the control group (Fig.1 g, h). Yoshitaka first discovered leaf rust on P. semicordata caused by N. vitis in Nepal(Yoshitaka 2000). To our knowledge, this is the first report of N. vitis causing leaf rust on P. semicordata in China. P. semicordata is often used as an ornamental plant for exterior wall decoration in urban landscaping and court. The occurrence of this disease can lead to the decline of the P. semicordata leaves and the impairment of the plants aesthetically.

First Report of Diplodia intermedia Causing Canker and Dieback Diseases on Apple Trees in Canada.

A dieback of apple trees (Malus domestica (Suckow) Borkh.) associated with cankers was observed in commercial orchards in southwestern Ontario, Canada, in 2019. Fifteen 2 to 10-year-old symptomatic trees were collected from three orchards exhibiting up to 37% disease incidence. Small sections of diseased wood (1 cm long) were surface sterilized with 70% ethanol for 30 sec and 1% NaClO for 20 min, rinsed thrice in sterile water, placed on 2% PDA (Difco) amended with kanamycin (50 mg liter-1), and incubated at 22°C for 5 days in the dark (Ilyukhin et al. 2023). Fungal colonies that were consistently isolated were hyphal-tipped, transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified isolates with same characteristics were classed into morphotypes. One morphotype was initially white and turned dark olivaceous with dense aerial mycelium. Pycnidia were produced on pine needles on PDA (Fig. S2) after incubation at 22°C for 17 days in the dark. Conidia were brown, aseptate, ovoid, and measured 27.9 to 31.3 μm x 12.1 to 14.2 μm (mean ± S.D. of 15 conidia = 29.9 ± 0.9 μm × 13.2 ± 0.6 μm), the typical morphology of a Diplodia sp. (Phillips et al. 2012). Genomic DNA was extracted from a 7-day-old culture of a representative isolate M45-28, using the Plant/Fungi DNA Isolation Kit (Norgen Biotech, Canada). The internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin gene regions were amplified and sequenced with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b and deposited in GenBank with accession numbers MZ970605, MZ995430 and MZ995431, respectively. Based on the sequence, the fungus was identified as Diplodia intermedia A.J.L. Phillips et al. and matched isolates from different hosts and countries (ITS: 100%, MG220378; EF1-α: 100%, MG220385; β-tubulin: 99.24%, MT592502). The maximum likelihood-based phylogenetic analysis of ITS, EF1-α and β-tubulin concatenated sequences was performed using IQ-Tree 2.2.2.7 (Minh et al. 2020). M45-28 was clustered with high bootstrap support values with D. intermedia isolates from the Westerdijk Fungal Biodiversity Institute collection, including the ex-holotype (CBS 124462) (Fig. S1). A living culture of M45-28 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252253). Pathogenicity assay was conducted by inoculating mycelial plugs from a 7-day-old culture of M45-28 into wounds made on the trunk of 5 eight-month-old potted healthy 'Royal Gala' apple seedlings. Five control seedlings were inoculated with sterile plugs. Canker symptoms appeared 15 days after inoculation, spread around, up and down the main stem from the inoculation point, and by 7 weeks the upper portion of the seedling was dead (Fig. S2). Diplodia intermedia was re-isolated from all inoculated seedlings and species identity was confirmed by sequencing as described above, fulfilling Koch's postulates. Control seedlings remained symptomless and the fungus was not isolated from the wood. Diplodia intermedia was reported to cause cankers on apple in Uruguay (Delgado-Cerrone et al. 2016), wild apple (Malus sylvestris) in Portugal (Phillips et al. 2012), grapevines in France (Comont et al. 2016) and forest trees in Iran (Kazemzadeh Chakusary et al. 2019). To the best of our knowledge, this is the first report of D. intermedia causing canker and dieback diseases on apple trees in Canada. Further studies are required to better understand the epidemiology involved in the dynamic spread of the disease in order to recommend an adequate phytosanitary program for its control.

First Report of Root Rot on Hydroponically Grown Romaine Lettuce Caused by Pythium dissotocum in Korea.

The romaine lettuce (Lactuca sativa L.) is one of the most frequently consumed vegetables in Korea. In January 2023, the romaine lettuce cultured within an indoor hydroponic farm in South Korea displayed severe disease, with an incidence of approximately 13.7% of 300 plants. The diseased plants showed symptoms of stunted growth, lower leaf yellowing, and brown or black-colored soft and mushy root rot, in which the outer layer of root was sloughed off, leaving a thread-like appearance. These symptoms were similar to those of Pythium root rot previously reported to occur on lettuce (McGehee et al., 2018; Stanghellini and Kronland, 1986). Samples of romaine lettuce with rot symptoms were collected from the hydroponic farm. The infected roots were rinsed three times with sterilized distilled water (SDW), dried on sterilized filter paper, and sliced into segments (about 0.5 cm in length), which were placed into Petri dishes (9 cm in diameter) containing V8 juice agar (V8A: 8% V8 juice and 1.5% agar powder) and cultured at 25°C for 2 days. The emerging hyphae were transferred to new Petri dishes containing V8A. After four rounds of sub-culturing, a total of 11 strains were isolated and all of them exhibited the same morphology. Strain KNU2301TP was purified via isolation of a single zoospore and stored at -80°C. The mycelia were non-pigmented. The hyphae obtained from a three-day-old culture grown in V8A were aseptate and the diameter of major hyphae was up to 7 µm. The filamentous sporangia were not to slightly inflated and formed dendroid-like branches. Vesicles containing zoospores were formed on the filamentous sporangia. The encysted zoospores were spherical with a diameter of 8.1 to10 µm (average 8.9 ± 0.6 µm; n = 50). These morphological characteristics of KNU2301TP were similar to those of a previously reported oomycete, Pythium dissotocum (Van der Plaats-Niterink 1981). The genomic DNA was extracted from mycelia cultured on V8A by a previously described extraction method (Chi et al. 2009). Sequences of internal transcribed spacer (ITS) region and cytochrome c oxidase subunit II (cox2) gene were amplified with paired primers ITS1/ITS4 and cox2_F/R, respectively (Callaghan et al., 2022; Hudspeth et al. 2000; White et al. 1990). The resulting sequences were deposited in GenBank (GenBank Accession Nos.; ITS: OQ683867; cox2: OQ700848). The sequences of ITS and cox2 gene of strain KNU2301TP were 99.61% and 100% identical to the sequences of ITS (MG719858) and cox2 gene (MG719859) of P. dissotocum strain YNP-3, respectively. Based on the result of the morphological characterization and sequence analysis, the strain KNU2301TP was identified as P. dissotocum. For Koch's postulates, 15 lettuce seedlings (eighteen-day-old) were inoculated by immersing the roots in a spore suspension (1 × 105 zoospores/ mL) and incubated at 25°C under 16/8 h light/dark cycles for 8 days. Fifteen plants of same age were treated with SDW in the same manner as a control The symptoms resembling those originally found at the farm were developed on the inoculated plants, but not on controls. The strain reisolated from the inoculated plants by same method mentioned above was confirmed as strain KNU2301TP by analysis of morphology and ITS and cox2 sequences. To our knowledge, this is the first report of root rot on hydroponically grown lettuce caused by P. dissotocum in Korea. Root rot on lettuce caused by P. dissotocum has been previously reported in USA, Canada, and Finland (McGehee et al. 2018; Stanghellini and Rasmussen 1994). Since lettuce is an important and popular leafy vegetable around the world, further work would focus on developing efficient strategies to manage this Pythium root rot disease.

Xenoacremonium palmarum sp. nov., a novel species associated with Phoenix dactylifera in Iran

The date palm (Phoenix dactylifera L.) is one of the most important fruit crops in Iran, where it has been cultivated in the south, southeast, and southwest of Iran. Root fragments of date palm showing root rot symptoms were collected from different areas in Khuzestan and Bushehr provinces to isolate their fungal associations. Accordingly, six isolates of a new species, Xenoacremonium palmarum, were obtained, herein described and illustrated. This new species is identified based on both morphology and phylogenetic analyses of combined ITS (the internal transcribed spacer region and intervening 5.8S nrRNA), tub2 (β-tubulin) and tef1α (translation elongation factor 1-alpha) sequence data. Our isolates grouped in a highly-supported clade, distinct from previously known species of Xenoacremonium. The new species and X. allantoideum differ from both X. falcatum and X. minutisporum in not having lateral phialidic pegs on their somatic hyphae. In addition, conidia in X. falcatum are more curved than those in X. allantoideum, X. palmarum and X. minutisporum. New species, X. palmarum, differs from X. allantoideum in having longer conidia and lacking mycelial ropes.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Xanthium orientale in Korea.

Xanthium orientale L. (syn. Xanthium canadense Mill., Asteraceae), known as cocklebur, is an annual weed native to North America, which is now a neophyte distributed throughout the world. This plant was accidentally introduced to Korea in the late 1970s ( So et al. 2008) and is considered a problematic exotic weed in orchards, for which many herbicides are ineffective (Kim et al. 2020). In September 2018, powdery mildew was observed on X. orientale in Jeju, Korea. The disease incidence ranged from 40 to 60%. Voucher specimens were deposited in the Korea University Herbarium (Accession No. KUS-F30795) and Kunsan National University Herbarium (KSNUH1988). Symptoms appeared as round to irregular white patches with abundant hyphal growth on the leaf surface. Hyphal appressoria were nipple-shaped, and 3 to 6 μm diam. Conidiophores (n = 30) were 145 to 206 × 9 to 11.6 µm and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot-cells of the conidiophores were straight, cylindrical, and 43 to 100.9 μm long. Conidia (n = 30) were ellipsoid-ovoid, doliiform to somewhat limoniform, 25.2 to 31.8 × 13.6 to 16.8 μm (l/w 1.6 to 2.1), and devoid of distinct fibrosin bodies. The morphological characteristics corresponded to those of Golovinomyces ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012, under Golovinomyces spadiceus (Beck. & M.A. Curtis) U. Braun; Qiu et al. 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS), large subunit (LSU) (Bradshaw and Tobin 2020), the intergenic spacer (IGS) of rDNA, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Bradshaw et al. 2022) were amplified for a herbarium specimen (KUS-F30795). A BLASTn search of these sequences revealed 100% identity with reference sequences of G. ambrosiae on diverse Asteraceae plants (AB077644 for ITS, AB077643 for LSU, ON361171 for IGS, and ON075648 for GAPDH). However, there was a single nucleotide difference on both the IGS and GAPDH sequences when compared to the closely related species Golovinomyces latisporus. The sequences were deposited in GenBank (Accession No. OQ165157 (ITS), OQ165164 (LSU), OR050524 (IGS), and OR086076 (GAPDH)). Phylogenetic analyses of ITS, LSU, IGS, and GAPDH sequences revealed the Korean sample formed a well-supported group with other G. ambrosiae sequences, confirming its identity. A pathogenicity test was performed through inoculation by gently pressing diseased leaves onto the leaves of five healthy plants. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25±2°C. Powdery mildew colonies developed on the inoculated plants after ten days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the initially diseased leaves, fulfilling Koch's postulates. Powdery mildew on X. orientale has previously been reported as Golovinomyces cichoracearum (≡ Erysiphe cichoracearum) sensu lato in the USA, G. ambrosiae (= G. spadiceus) throughout all continents, and Podosphaera fusca sensu lato (now P. xanthii) in Korea (Braun and Cook 2012; Farr and Rossman 2023). To date, powdery mildew in Korea has been reported only on Xanthium strumarium as G. cichoracearum s. lat. and Podosphaera xanthii (KSPP 2022). To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on X. orientale in Korea.

First Report of Root Rot Caused by Macrophomina phaseolina in Peanut Shandong Province, China.

Peanut (Arachis hypogaea L.) is a widely grown oilseed crop of great agricultural importance worldwide. In July 2022, disease symptoms were observed on peanut roots in Laixi (36º85' N, 120º54' E), Shandong Province, China. About 25% of the plants showed various symptoms, including stem and root rot and blackening, microsclerotia on the stem, yellowing and wilting of leaves, and even death. Twenty diseased plants were collected to confirm the pathogen. Symptomatic roots were cut into small pieces, disinfested with 75% ethanol for 1 min and 0.5% NaClO for 2 min, rinsed three times with sterile water, dried on sterile filter paper, and then spread on potato dextrose agar (PDA) supplemented with 100 μg/mL chloramphenicol and incubated at 25°C in the dark. At the beginning of growth, the fungus formed sparse, white mycelia, which white, then darkened with age and microsclerotia were formed in the medium after 5 days. The mycelium aggregated into black, round to oblong or irregularly shaped microsclerotia 84 to 163 μm long and 54 to 125 μm wide (n=40). These morphological characteristics were consistent with the description of Macrophomina phaseolina (Holliday and Punithalingam, 1970). Molecular identification was performed by sequencing the internal transcribed spacer (ITS) region with ITS1 and ITS4 and translation elongation factor 1-alpha (TEF) with EF1-728F/EF1-986R (Glass and Donaldson 1995) of a representative isolate SXY183. ITS (OR056369) and TEF (OR098356) of SXY183 showed 100% and 97.74% similarity with M. phaseolina (KF951622, KF951997), respectively. Phylogenetic analysis was performed using Neighbor-Joining (NJ) analysis based on the gene sequences of ITS and TEF. The fungus was identified as M. phaseolina based on molecular analysis and morphological characteristics. The pathogenicity of a representative isolate (SXY183) was tested on peanuts under greenhouse conditions. Two-week-old peanut (Huayu No. 9115) seedlings were inoculated with a mycelial plug (8 mm diameter) at the root base of each plant and cultured in a greenhouse (30°C during the day and 25°C at night, a 12-h photoperiod, and 80% RH). Ten plants were inoculated with a plug of non-colonized PDA as a control. Brown lesions were observed on the stem and root of all inoculated seedlings 7 days after inoculation, but not on the control plants. The experiment was repeated three times. M. phaseolina was re-isolated from the symptomatic root and confirmed based on morphological characteristics and DNA sequence analysis of ITS and TEF. M. phaseolina is a soil-borne fungus that is distributed worldwide and has a broad host range. Disease agent has previously been reported on several host plants such as adzuki bean, faba bean, watermelon, Plukenetia volubilis, Atractylodes lancea and Curcuma longa in China (Cai et al., 2020; Sun et al. 2016; Sun et al., 2019; Sun et al., 2020; Wang et al., 2020; Wu et al., 2022). However, this is the first report in which M. phaseolina was found to cause peanut root rot in Shandong Province, China. Our report will provide important information for studying the epidemiology and management of this disease.

Identification of four autochthonous yeasts from the intestines of goldfish, Carassius auratus with potential probiotic properties and their effects on the most common fish bacterial pathogens

In aquaculture, probiotic yeasts have gained particular interest because of their numerous health benefits for farmed fish. Many autochthonous yeasts have been isolated and identified from fish species with potential probiotic characteristics. In the present study, four autochthonous yeast strains were identified and characterized from the intestinal tracts of 16 healthy goldfish, Carassius auratus. Their in vitro probiotic properties were examined in terms of cell surface hydrophobicity, co-aggregation, and tolerability to different pH values and bile salt concentrations. These strains were identified by culture characters and sequence analysis of ITS (Internal Transcribed Spacer) gene regions. Four strains, namely Cutaneotrichosporon jirovecii isolate jpn01, Debaryomyces nepalensis isolate jpn02, Blastobotrys proliferans isolate jpn05, and Diutina catenulata isolate jpn06, were identified and added to the NCBI GenBank with accession numbers defined as MT584874.1, MT584873.1, MT649918.1, and MT501155.1, respectively. Results demonstrated the capability of these strains to co-aggregate with several fish-associated bacterial pathogens such as Lactococcus garvieae, Vagococcus salmoninarum, Vibrio anguillarum, Yersinia ruckeri, and Aeromonas hydrophila. Only the jpn05 strain did not co-aggregate with A. hydrophila. All identified yeast isolates could grow and tolerate low pH conditions (pH 2.0) and bile salt concentrations (up to 1.5%). Of interest, the hydrophobicity (%) of the yeast isolates was 80%, 94.0%, 80.6%, and 66.4% for jpn01, jpn02, jpn05, and jpn06 isolates, respectively. In this context, our data provide important in vitro evidence for the potential probiotic features of the yeast isolates. These strains could be considered candidate probiotic yeasts; however, their application in aquaculture nutrition necessitates further in vivo assays.