Internal Transcribed Spacer Sequence Analysis Research Articles

Genetic Diversity and Population Structure of Actinidia eriantha by ITS Sequences

Background: Actinidia eriantha Benth is a widely used natural product from Traditional Chinese Medicine in the Actinidiaceae family. However, its wild resources have been declining due to over-exploitation. It has become urgent to investigate the genetic diversity for the conservation of A. eriantha, to evaluate the current species and discover strategies for preservation. Methods: The internal transcribed spacer (ITS) was used to evaluate the genetic diversity among and within populations of this species. Dnasp, PERMUT and Arlequin 3.0 software were used to calculate the genetic diversity index, and MEGA 5.0 software was used to construct the neighbor-joining (NJ) tree. Results: A total of 27 haplotypes were obtained by ITS sequence analysis of 12 populations, and the most frequently haplotype observed was H1. AMOVA analysis revealed that the genetic variation rates were 10.91% (FST = 0.22290) and 77.71% (FSC = 0.12306) among and within populations, respectively, with high genetic diversity at the species level (Hd = 0.692). The genetic distance among populations ranged from 0 to 0.004. The results of Permut analysis showed that there was no significant correlation between genetic distance and geographic distance (NST > GST). The NJ tree was divided into two Clades (Clade A and Clade B), Clade B has obvious geographical specificity, and haplotypes of this clade are all specific to the GX-ZY population. Four types were found according to ITS sequences of A. eriantha, haplotypes H1 and H5 were ancient haplotypes. Conclusion: Our findings indicated that genetic diversity within populations was higher than observed among populations. This study is significant for further research endeavors focused on the efficient collection and preservation of wild resources of A. eriantha.

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 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 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.

Identification and reproduction of dagger nematode, Xiphinema americanum, in potato

Summary Plant-parasitic nematodes limit potato production by feeding on roots, reducing tuber yield and quality, and resulting in poor growth. Dagger nematodes (Xiphinema spp.) can pose a significant threat to crop production even in low numbers. Dagger nematodes have been reported in North Dakota, USA, potato fields. In this study, a dagger nematode species was identified and its reproduction potential in 21 potato cultivars was evaluated. The dagger nematode was identified as X. americanum via morphometric measurements and DNA sequence analysis. At the molecular level, D2-D3 of 28S ribosomal RNA (rRNA) and internal transcribed spacer (ITS)-regions of rDNA were targeted. Sequencing data of D2-D3 (749 bp) showed 98.18% similarity with three isolates of X. americanum. ITS sequence (860 bp) analysis showed 97.68% similarity with an isolate of X. americanum but lower similarity with other species of Xiphinema. The phylogenetic analysis of ITS region further confirmed the species of dagger nematode as X. americanum. Using naturally infested soil, two glasshouse experiments were conducted to assess the reproduction rate of X. americanum on 21 potato cultivars belonging to four distinct market potato classes (yellow, red, white and russet). The reproduction rates were found to vary among the potato classes and cultivars. Numerically, X. americanum population density was higher in ‘Soraya’, whereas ‘Ranger Russet’, ‘Manistee’, ‘Kennebec’, ‘Russet Norkotah 278’, ‘Modoc’, ‘Pomerelle Russet’ and ‘Dakota Rose’ reduced nematode reproduction in both trials. This is the first study demonstrating the reproduction of X. americanum in potato cultivars and could be helpful to manage dagger nematodes.

First Report of Fusarium acuminatum Causing Leaf Spot on Schisandra chinensis in China.

Schisandra chinensis (Turcz.) Baill. is a popular and widely cultivated medicinal herb in China, which has rich nutritional value and medicinal effect. In August 2022, leaves with oval and irregularly circular light brown spots from 2 to 10 mm wide with white centers were found on Schisandra chinensis growing in Fusong district (127°28'E, 42°33'N) of Jilin, China. The symptoms were observed in 20% of the plants of a 2 ha-1 field of Schisandra chinensis. About 50% of the leaf areas were affected. As the disease developed, the lesions grew larger and developed necrotic centers. Leaves with light brown spot symptoms from five plants were collected from the field. Five leaf pieces (3 to 5 mm2) were excised from lesion margins, surface sterilized based on Ju et al. (Ju et al. 2021), and incubated on potato dextrose agar (PDA) at 25°C. Six single spores were isolated from five independently infected isolates for pure culture using the single spore isolation technique (Zhang. 2003). Representative single spore isolate (ZWWZH) was selected from pure cultures for further culture. After 5 days, fluffy white aerial mycelium with pink pigmentation on the underside of the colony were observed on PDA. Mycelia became pinkish-brown as the culture aged. Microscopic observations showed the presence of elongated or pointed, and thick-walled macroconidia (n = 50), predominantly three septate, 3.40 to 7.50 × 40.34 to 61.29 μm were observed. Chlamydospores formed in chains within or on top of the mycelium. The primers ITS1/ITS4 (White et al. 1990) and Bt-2a/Bt-2b (Robideau et al., 2011) were used to amplify the internal transcribed spacer (ITS) rDNA and β-tubulin (TUB2) region, respectively. The obtained sequences were submitted to GenBank under accession numbers for OQ629789 (ITS) and OQ803521(TUB2). BLASTn analysis of both ITS sequence and TUB2 sequence, revealed 100% and 99.92% sequence identity with F. acuminatum MT566456, MT560377 and KJ396328, respectively. The pathogen was identified as F. acuminatum based on morphological and molecular data. Pathogenicity tests were carried out in the greenhouse. Select five healthy Schisandra chinensis seedlings, each with each healthy leaf surfaces inoculated a 1 × 106 spores/mL solution, 3 wells on one side, 10 µL per well. Sterile ddH2O was used in the control experiment. The inoculated seedlings were incubated at 25°C with a relative humidity of 65 to 70% in a greenhouse. Four days after inoculation, all inoculated leaves exhibited the same symptoms as observed in the field, while the controls showed no symptoms. The experiment was repeated three more times with similar results. The re-isolated fungi from the inoculated plants had the same morphology and DNA sequences as the original isolate (ZWWZH) obtained from the field samples, completing Koch's postulates. To our knowledge, this is the first report of F. acuminatum causing leaf spot on Schisandra chinensis in China. F. acuminatum has seriously affected the quality of Schisandra chinensis production. The identification of leaf spot caused by F. acuminatum will enable farmers to identify practices to minimize disease on this important crop.

First Report of Penicillium oxalicum Causing Leaf Blight on Maize in China.

In August 2022, two-month-old maize plants (Zea mays cv. 'Zihei'; "Chinese purple corn") exhibited irregular lesions on leaves and leaf blight symptoms (Figure 1). Although the lesions were yellow at the early infection stages, they turned brown during the pathogen advancement and culminated in leaf blight. Nearly 60% of plants from a non-commercial maize field (0.2 ha) in south-eastern Jiangsu (Nantong municipality, China; 120.54º E, 31.58º N) exhibited brown lesions, and about 4% of the diseased plants showed advanced leaf blight symptoms. The disease resulted in approximately a 9% yield loss compared to previous years when no disease symptoms were observed. Thirty small leaf pieces, approximately 0.3 cm2 in size and showing disease symptoms, were surface sterilized in 1.5% NaOCl for 1 min and washed twice with sterile ddH2O. The pathogen was cultured on PDA medium in the dark at 25 ºC, with grayish colonies observed after 5 days. Morphological analysis showed the presence of round/oval conidia (8.81 ± 0.50 μm diameter; n = 86) and branched conidiophores, which was consistent with the morphology of Penicillium spp. (Visagie et al. 2014). Nine representative isolates were obtained from different leaf pieces via single spore isolation, and the internal transcribed spacer (ITS), β-tubulin (TUB2) and calmodulin (CMD) genes were amplified using ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively. The obtained ITS (OP954496-OP954497 and OP942428-OP942434), TUB2 (OP966781-OP966784 and OQ025045-OQ025049) and CMD (OQ078664-OQ078672) sequences were submitted in GenBank. Two isolates belonged to the P. citrinum species, while seven of the isolates belonged to the P. oxalicum species. A blast search revealed that the obtained P. citrinum ITS and CMD sequences had 99.39% and 100% homology to the ex-type strain P. citrinum NRRL 1841; GenBank numbers: AF033422 and GU944638 (Peterson & Horn 2009). Additionally, the obtained P. oxalicum ITS and CMD sequences had 99.82-100% and 94.64-95.49% homology to the ex-type strain P. oxalicum NRRL 787; GenBank numbers: AF033438 and KF296367 (Visagie et al. 2015). A molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen (Figure 2). To confirm pathogenicity, 3-week-old healthy 'Zihei' plants were used. The leaves were sprayed with aqueous solutions (sterilized ddH2O) that contained 1 × 106 spores/mL of each isolate. For the control experiment, sterilized ddH2O was used. After 5 days in a growth chamber at 25 ºC and 70% relative humidity, yellow lesions were observed. The number of lesions was higher when inoculating with P. oxalicum than when inoculating with P. citrinum. This result, together with the higher occurrence of P. oxalicum isolates, suggests that P. oxalicum is the main species causing the disease symptoms. The pathogen was recovered from the infected plants, and its identity was confirmed by ITS sequencing and morphological analysis. As far as we know, this is the first report of P. citrinum and P. oxalicum causing maize leaf blight worldwide. These species have previously been associated with maize kernels, as a source of mycotoxins posing relevant hazards to human health (Keller et al. 2013; Yang et al. 2020). P. citrinum was recently identified as the causal agent of green mold on Dictyophora rubrovalvata in China (Qin et al. 2022), while P. oxalicum was reported to cause citrus rot, pineapple leaf spot, and blue mold on Gastrodia elata, Astralagus membranaceus and muskmelon (Tang et al. 2020; Wu et al. 2022; Zheng et al. 2022). China is one of the world's largest producers of maize, harvesting more than 171 million tons in 2021. This report will help to better understand the pathogens that affect China's maize production.

Fruit Rot on Persimmon Caused by Neopestalotiopsis saprophytica and Neopestalotiopsis ellipsospora in Guangxi, China.

Persimmon (Diospyros kaki Thunb.) is widely cultivated in China. On October 15, 2019, about 10% of persimmon fruits showed fruit rot in the orchards of Guilin, Guangxi, China (24°45' N, 110°24' E), which could cause more than 15% of yield losses. The initial symptoms of fruit rot exhibited irregular brown to black spots (range from 2 to 4 cm in diameter), the areas surrounding the blackened spots would be soft and rotten, and three diseased fruit samples were collected from three orchards, respectively. Tissues (5×5 mm) were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on potato dextrose agar (PDA) at 25°C under 12/12 h light/darkness for a week. Forty-one tissues yielded morphologically similar cultures, and three representative isolates LPG1-1, LPG1-2, and YSG-1 were selected from three samples for further study, respectively. Their colonies showed wavy edges, white surfaces, and dense aerial hyphae on PDA after two weeks. Conidia were fusiform, straight to slightly curved, and 4-septate; basal cells were conical, hyaline, thin, and verruculose with two or three long and hyaline apical appendages and one short apical appendage; three median cells of LPG1-1 with length 14.06 to 17.69 μm (n=100), and LPG1-2 with length 14.03 to 17.61 μm (n=100) were dark brown to olivaceous, while three median cells of YSG-1 with length 12.54 to 15.58 μm (n=100) were dark brown. The conidial sizes of LPG1-1, LPG1-2, and YSG-1 were 17.41 to 27.68 × 4.63 to 8.55 μm (n=100), 18.06 to 27.41 × 4.33 to 8.21 μm (n=100), and 16.58 to 27.73 × 4.99 to 8.39 μm (n=100), respectively. The morphological characteristics were consistent with Neopestalotiopsis spp. (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014). Primer pairs ITS4/ITS5, BT2a/BT2b, and EF1-526F/EF-1567R were used to amplify internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1 alpha (TEF1-α), respectively (Shu et al., 2020). All DNA fragments were sequenced bySangonBiotechCo., Ltd. (Shanghai, China). Sequences have been deposited in GenBank (ITS: OM349120 to OM349122, TUB2: OM688188 to OM688190, TEF1-α: OM688191 to OM688193). Based on BLASTn analysis of ITS, TUB2, and TEF1-α sequences, the LPG1-1 and LPG1-2 showed over 99% similarity to N. saprophytica, and YSG-1 showed over 99% similarity to N. ellipsospora. Phylogenetic analysis of the three isolates was performed with MEGA10 (version 10.0) based on sequences of ITS, TUB2, and TEF1-α using maximum parsimony analysis. The results revealed that LPG1-1 and LPG1-2 were clustered with N. saprophytica, and YSG-1 was clustered with N. ellipsospora. Pathogenicity tests of three isolates were conducted on 72 healthy persimmon fruits with and without wounds, and 9 fruits are for each treatment. The wound was made by a sterilized needle. Fruits were pre-processed with 75% ethanol for 10 s, 1% NaClO for 2 min and rinsed three times in sterile water. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site (4 sites/fruit). Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton. The inoculated fruits were placed in a plastic box to maintain humidity at 28℃. After 5 days, all wounded fruits showed fruit rot, whereas unwounded and control fruits remained asymptomatic, there were significant differences (P<0.05) in aggressiveness between N. saprophytica (average lesion diameter 13.1 mm) and N. ellipsospora (average lesion diameter 14.9 mm). Koch's postulates were fulfilled by re-isolating the causal agents from inoculated fruits. N. ellipsospora was previously reported as an endophyte in D. montana in southern India (Reddy et al. 2016). N. saprophytica could cause leaf spot of Erythropalum scandens and Magnolia sp., and fruit rot of Litsea rotundifolia in China and leaf spot of Elaeis guineensis in Malaysia (Yang et al. 2021, Ismail et al. 2017). To our knowledge, this is the first report of N. ellipsospora and N. saprophytica causing fruit rot on persimmon in the world. The results will provide a foundation for controlling fruit rot caused by pestalotioid fungi on persimmon.

Isolation of Zygosaccharomyces siamensis kiy1 as a novel arabitol-producing yeast and its arabitol production

Arabitol is gaining attention in the food industry as an alternative sweetener owing to its low-caloric and non-cariogenic characteristics. The yeast strain kiy1 was newly isolated from unpasteurized honey for arabitol production. Based on internal transcribed spacer sequence analysis, the isolated strain was identified as Zygosaccharomyces siamensis. In this study, the effects of different substrates and sugar concentrations on arabitol production were investigated. When three types of carbon sources (glycerol, fructose, and glucose) were used, glucose was the most suitable substrate for arabitol production (68.7 g/L). Maximum arabitol production (101.4 g/L) was observed at a glucose concentration of 30%, and the highest arabitol production yield was 0.34 g/g of initial glucose. In the time-course production of sugar alcohols by strain kiy1, glucose was completely consumed for 8 days. The concentration of arabitol exceeded that of glycerol after 3 days, and the final arabitol concentration reached 83.6 g/L after 10 days. The maximum production rate was 16.7 g/L/day. The yeast produced glycerol as an intracellular sugar alcohol in the early stage of culture and switched its metabolism to arabitol production after the middle stage. Z. siamensis kiy1 possessed an NADP+-dependent arabitol dehydrogenase, which indicated that it probably produces arabitol via ribulose from glucose. These results suggest that the novel yeast strain, Z. siamensis kiy1, is promising for arabitol production. The proposed arabitol production approach can contribute toward its production at the industrial scale.Graphical

Phylogenetic analysis and morphology of Ganoderma multipileum, a Ganoderma species associated with dieback of the metropolitan woody plant Delonix regia (Boj. ex Hook.) Raf. in Vietnam.

Ganoderma multipileum, a wood decay mushroom, was initially discovered and classified in Taiwan through the analysis of its morphology and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. In this study, we identified a mushroom associated with the dieback of Delonix regia (Boj. ex Hook.) Raf., a woody ornamental street tree in Vietnam, as Ganoderma multipileum. This classification was based on phylogenetic analysis of ITS, RPB2, and TEF1 sequences, as well as morphology assessment and scanning electron microscope observation of basidiospores. The phylogenetic analysis revealed that the specimens collected in Vietnam formed a monophyletic group of Ganoderma multipileum with a high bootstrap value and posterior probability (100%/1.00). Furthermore, the morphological features consistent with laccate Ganoderma, including a thin pileipellis composed of enlarged and bulbous hyphae, and the basidiomes exhibited two different phenotypes. Notably, scanning electron microscopy of the basidiospores revealed ovoid spores with numerous echinules, providing the first documented evidence of this characteristic for Ganoderma multipileum. This research represents the first recorded instance of Ganoderma multipileum in Vietnam associated with the dieback of Delonix regia.

First Report of Nalanthamala psidii causing wilt disease of guava in Guangdong, China.

Guava (Psidium guajava L.) is a tropical fruit with great economic value. Guangdong is one of the most important guava production areas. In November 2019, guava wilt disease (GWD) was observed in a 10.6 HA commercial orchard in NanSha district, Guangzhou, Guangdong (22°37'37.626" N, 113°35'56.089" E). Disease incidence was up to 35%. Initially, leaves on the top of some branches became purple or yellow interveinal chlorosis, later dry. Infection severely became systemic developing vascular discoloration of stem, black root rot, eventually entire trees wilted and died. The root tissues were cut into 5-mm2 pieces and surface disinfected with 70% ethanol for 30 sec, 3 % sodium hypochlorite for 4 min, rinsed by the sterile water, then plated onto potato dextrose agar and incubated for 5 days at 25°C. A total of 8 monoconidial isolates with identical colony morphology were obtained. All formed cottony, whitish to pale yellow colonies. Conidiophores were dimorphic, penicillate and acremonium-like. Penicillate conidiophores gave rise to ovoidal, one-celled conidia (4.15 to 6.55×2.28 to 4.61 μm) (n=100) with truncated ends. Cylindrical or fusiform conidia (7.02 to 15.57×2.01 to 5.30 μm) (n=100) arose in long chains on acremonium-like conidiophores. Morphological characteristics of the isolates were consistent with those of Nalanthamala psidii (syn. Myxosporium psidii) reported by Schroers (2005). The rDNA internal transcribed spacer (ITS) and partial nuclear large-subunit ribosomal DNA (LSU) of two representative isolates (GDNS02 and GDNS08) were amplified using the primers pairs ITS4/ITS5 (White et al. 1990) and V9G/LR5 (de Hoog and Gerrits van den. 1998), respectively. The obtained sequences were deposited in GenBank under the accession nos. OM278372 to 73 (ITS) and OM278377 to 78 (LSU). BLASTn analysis showed 99.81% and 100% identities with the reported sequences of N. psidii CBS 116952 (AY864836) and CBS 110507 (AY554243). Maximum likelihood analyses of combined ITS and LSU sequences indicated that these two isolates being clustered with N. psidii strains. Pathogenicity tests were performed twice using healthy seedlings (60-70 cm height, cv. pearl). Each stem of five seedlings was wounded using a 5-mm sterile cork borer, and 5-day-old mycelium plugs of isolate GDNS08 were inoculated into the holes (25-cm above the soil line) and covered with Parafilm, sterile PDA plugs were placed into the wounds of additional 5 control seedlings. All plants were kept in a greenhouse (25℃, 80% relative humidity, 16/8-h day/night). After 3 months, all inoculated plants developed purple leaf, defoliation and wilt symptoms resembling those observed in the orchards, while the controls remained asymptomatic. Nalanthamala psidii was reisolated from the roots tissue of the inoculated plants, identity was confirmed by morphological characteristics and ITS sequence analyses as described above, but not from the controls, fulfilling Koch's postulates. Nalanthamala psidii has been previously reported as the causal agent of guava wilt in Taiwan, Philippines, South Africa and Bangladesh (Hsieh et al. 1976; Opina 1995; Schoeman et al. 1997; Alam et al. 2019). To our knowledge, this is the first report of N. psidii causing guava wilt in Guangdong, China. The outbreak of GWD in South Africa in the 1980s resulted in devastating losses to guava industry (Schoeman et al. 1997). Further research is needed to develop the integrated management to constrain this disease from spreading.

Gray Blight Disease on Euonymus japonicas Caused by Pestalotiopsis disseminata in China.

Euonymus japonicas is widely planted as an important landscape species throughout China. In June 2021, a serious gray blight disease was detected on E. japonicas in Henan Province (32°30'58" N, 112°19'44" E), causing severe defoliation of infected trees with a foliar disease incidence of 52 to 70% (n = 100). Gray spots initially appeared on leaves, gradually expanded into irregular white blotches with dark brown borders, eventually leading to wilting and death of the leaves. The junctions between the lesion and healthy tissue of infected leaves were cut into 3 × 3-mm pieces, surface sterilized with 1% NaClO solution for 1 min, rinsed in sterile water, and placed on PDA plates with 50 μg/ml of streptomycin. Three isolates (HY94, HY95, and HY98) were selected for subsequent experiments. The colonies reached 80-85 mm diam after 7 days at 25°C, with undulated margins, white to pale in color, with moderate aerial mycelium on the surface. Conidiomata were globose, solitary, and dark black. Conidia were ellipsoid, straight to slightly curved, 4-septate, 19 to 26.4 × 5 to 7.5 μm (n=100). The apical cell was cylindrical and hyaline, with 2 to 3 tubular apical appendages, unbranched, filiform, 2.5 to 3.5 μm in length. The basal appendage was single, unbranched, centric, 1.5 to 3 μm long. The characteristics were close to those of Pestalotiopsis spp. (Maharachchikumbura et al. 2013). The genomic DNA was extracted, and the rDNA internal transcribed spacer (ITS), the β-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified by primers ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Carbone and Kohn, 1999). Sequences were submitted to GenBank with accession numbers OL840327-OL840329(ITS), OL961454-OL961456(TUB), and OL961448-OL961450 (TEF1). BLASTn analyses of ITS, TUB, and TEF1 sequences exhibited 99.46, 99.05, and 96.53% similarity to the sequences of Pestalotiopsis disseminata strain MEAN1166 (ITS, 548/551 bp; MT374688) (Silva et al. 2020), PSH2000I-066 (TUB, 418/422 bp; DQ333575), and TAP29O082 (TEF1, 250/259 bp; AB453850), respectively in GenBank. The three isolates formed a clade with the type strains, MEAN 1166 and MAFF238347 of P. disseminata in phylogenetic trees, being clearly seperated from other Pestalotiopsis spp. Based on morphological and molecular evidence, the pathogen was identified as P. disseminata (Maharachchikumbura et al. 2011). To fulfill Koch's postulates, pathogenicity was tested with three isolates. Ten healthy leaves of 5-year-old intact plants were used per isolate and inoculated with mycelial plugs on both nonwounded and wounded leaves. Control leaves were inoculated with agar plugs. The inoculated plants were placed at 28°C in a greenhouse (90% relative humidity). Distinct lesions were observed after 10 days. The pathogen reisolated was identical to that of the original cultures according to phenotype and ITS sequences. The control leaves showed no obvious symptoms. P. disseminata is known to cause disease on several important plants in China, such as Camellia japonica (Zhang et al. 2012), Pinus armandii (Hu et al. 2007), and Tripterygium wilfordii (Kumar et al. 2004). This is the first report of gray blight disease caused by P. disseminata on E. japonicas in China and worldwide. The fungal pathogen identification will provide valuable information for prevention and management of gray blight disease associated with E. japonicas.

First Report of Colletotrichum fructicola Causing Anthracnose on Rosa chinensis in China.

China rose (Rosa chinensis Jacq.) is a popular ornamental plant grown widely in China. In September 2021, a serious leaf spot disease was observed on R. chinensis in Rose plantation of Nanyang Academy of Agricultural Sciences in Nanyang (112°25'41″N, 32°54'28″E), Henan Province, causing severe defoliation of infected plants with a foliar disease incidence of 50 to 70% (n = 100). The early symptoms were irregular brown specks on the leaves, mostly at the tip and margin of the leaves. Then the specks gradually expanded into round amorphous and became dark brown, eventually leading to large irregular or circular lesions. Twenty symptomatic samples were collected from several individual plants, and the junction areas between infected and healthy tissues were cut into 3×3 mm pieces. These tissues were sterilized in 75% ethanol for 30 seconds and 1% HgCl solution for 3 min, rinsed thrice in sterile water, and placed on potato dextrose agar (PDA) plates, incubated at 25°C for 3 days. The edges of the colony were cut and transferred to new PDA plates for purification. These isolates were isolated from the original diseased leaves and showed similar phenotypes in morphological characters. Three representative purified strains (YJY20, YJY21, and YJY30) were used for further study. Colonies were villiform, initially white, later turning gray and greyish-green. Conidia were unitunicate, clavate, and averaged 17.36 (11.61 to 22.12) - 5.29 (3.92 to 7.04) µm in diameter (n=100). The characteristics were close to those of Colletotrichum spp. (Weir et al. 2012). The genomic DNA was extracted, and the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), calmodulin genes (CAL), actin genes (ACT), chitin synthase 1 genes (CHS-1), manganesesuperoxide dismutase (SOD2), and β-tubulin 2 genes (TUB2) were amplified from genomic DNA by primers ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-345R, SODglo2-F/SODglo2-R, and Bt2a/Bt2b, respectively (Weir et al. 2012). Sequences were submitted to GenBank with accession numbers OP535983, OP535993, OP535994(ITS), OP554748, OP546349, OP546350(GAPDH), OP546351-OP546353(CAL), OP546354-OP546356(ACT), OP554742-OP554744(CHS-1), OP554745-OP554747(SOD2), and OP554749-OP554751(TUB2). BLASTn analyses of ITS, GAPDH, CAL, ACT, CHS-1, SDO2 and TUB2 sequences exhibited 99.62%, 98.40%, 99.72%-99.86%, 96.85%-96.86%, 99.26%-100%, 100% and 99.33% similarity to the sequences of Colletotrichum fructicola strain ICMP 18581, respectively in GenBank. These morphological features and molecular identification indicated that the pathogen possessed identical characteristics as C. fructicola (Weir et al. 2012). Pathogenicity was tested through in vivo experiments. Six 1-year-old intact plants were used per isolate. The test leaves of plants were gently scratched with a sterilized needle. Conidial suspension of the pathogen strains were inoculated on the wounded leaves at a concentration of 107 conidial/mL. The control leaves were inoculated with distilled water. The inoculated plants were placed in greenhouse at 28℃ and 90% humidity. After 3-6 days，anthracnose-like symptoms were observed on inoculated leaves of five plants while the control plants remained healthy. The strains of C. fructicola were reisolated from the symptomatic inoculated leaves, confirming Koch's postulates. To our knowledge, this is the first report of C. fructicola causing anthracnose on Rosa chinensis in China. C. fructicola has been reported to affect numerous plants worldwide, including grape, citrus, apple, cassava, mango （Qili Li et al. 2019）, and tea-oil tree (X. G. Chen et al. 2022), among others (Oliveira et al. 2018). This identification research will facilitate subsequent assistance with disease control and field management of plants.

First Report of Phytophthora infestans Causing Late Blight on Pepino (Solanum muricatum) in China.

Solanum muricatum, known as pepino or melon pear, is a species of evergreen shrub grown for its sweet edible fruits, which was introduced in Yunnan about 20 years ago. Since 2019 to now, serious blight disease was observed on foliages, haulms and fruits of pepino plants in Shilin (25°N, 103°E), which is the biggest pepino production area in China. The symptomatic blighted plants showed water-soaked and brown foliar lesions, haulm brown necrosis, black-brown and rotting fruits, and overall plant decline. The samples with typical disease symptoms were collected for pathogen isolation. After surface sterilization, disease samples were cut into small pieces and placed on rye sucrose agar medium amended with both 25 mg/liter rifampin and 50 mg/liter ampicillin, then incubated in the dark at 25℃ for 3 to 5 days. White fluffy colonies of mycelia that grew from the edge of diseased tissues were further purified and subcultured on rye agar plates. All purified isolates were identified as Phytophthora spp. based on morphological characteristics (Fry 2008). Sporangiophores were nodular and sympodial branches with swellings at the points where sporangia were attached. Sporangia that were hyaline and the average size were 22×40 μm were formed on the tip of sporangiophores and appeared as subspherical, ovoid, ellipsoid or lemon shaped with half-papillate on the spire. Mature sporangia were easilly detached from sporangiophores. For pathogenicity tests, healthy leaves, haulms and fruits of pepino were inoculated with 1×104 cfu/ml zoospore suspension of Phytophthora islolate (RSG2101), and controls were treated with sterile distilled water, respectively. After 5 to 7 days postinoculation, all Phytophthora-inoculated leaves and haulms showed water-soaked and brown lesions with white mold layer, fruits showed dark-brown firm lesions which got expanded and rotted the entire fruit. The symptoms were same as those occurred in natural fields. In contrast, no disease symptoms appeared in the control tissues. Phytophthora isolates could be reisolated and showed same morphological characteristics from the infected tissues of leaves, haulms, and fruits, sufficing Koch's postulates. Two common molecular targets of the internal transcribed spacer (ITS) region of ribosomal DNA and partial cytochrome c oxidase subunit II (CoxII) of the Phytophthora isolate (RSG2101) were amplified and sequenced with primers ITS1/ITS4 and FM75F/FM78R (Kroon et al. 2004). The ITS and CoxII sequence data were deposited in GenBank under accession numbers OM671258 and OM687527, respectively. Blastn analysis of both ITS and CoxII sequences showed 100% identity with isolates of P. infestans (MG865512, MG845685, AY770731 and DQ365743, respectively). Phylogenetic analysis also indicated that RSG2101 isolate and known P. infestans isolates localized in the same evolutionary branch based on sequences of ITS and CoxII, respectively. Based on these results, the pathogen was identified as P. infestans. It is known that P. infestans infection of pepino occurrs in Latin America and then it was recorded in other parts of the world such as New Zeeland and India (Hill, 1982; Abad and Abad,1997; Mohan et al. 2000).To our knowledge, this is the first report of late blight on pepino caused by P. infestans in China, which will be helpful to develop efficient blight management strategies on pepino.

Biodiversity and Functional Distribution of Macrofungi from Plant Genetic Conservation Area, Chanthaburi Province, Thailand

The aims of this study were to investigate the biodiversity and functional distribution of macrofungi within the Plant Genetic Conservation Area of Rambhai Barni Rajabhat University, Chanthaburi Province, Thailand, and to identify the macrofungi by sequence analysis of their internal transcribed spacer (ITS) regions. One hundred and eighty-five macrofungi samples were collected from the survey routes in 2021. The macrofungi with different morphologies were selected to perform molecular identification by sequence analysis of ITS. A total of 41 samples of representative macrofungi were classified into 2 phyla, 5 classes, 11 orders, 21 families, and 34 genera. The macrofungi were found to be in the phylum Basidiomycota (35 taxa, 85.4%), the family Polyporaceae (10 taxa, 24.4%), and Microporus xanthopus was the most frequently found species in every month of samples collection. Their role in the ecosystem was saprotroph (40 taxa, 97.6%), symbiotroph (7 taxa, 17.0%), and pathotroph (4 taxa, 9.8%). In addition, there was no published information about the edibility of many of the macrofungi (29 taxa, 70.7%); however, some edible (8 taxa, 19.5%) and poisonous macrofungi (4 taxa, 9.8%) had previously been reported. Interestingly, some macrofungi samples need more investigation for further identification, and additional genes may be required for the study.

Efficient control of the fungal pathogens Colletotrichum gloeosporioides and Penicillium digitatum infecting citrus fruits by native soilborne Bacillus velezensis strains

Destruction of citrus fruits by fungal pathogens during preharvest and postharvest stages can result in severe losses for the citrus industry. Antagonistic microorganisms used as biological agents to control citrus pathogens are considered alternatives to synthetic fungicides. In this study, we aimed to identify fungal pathogens causing dominant diseases on citrus fruits in a specialized citrus cultivation region of Vietnam and inspect soilborne Bacillus isolates with antifungal activity against these pathogens. Two fungal pathogens were characterized as Colletotrichum gloeosporioides and Penicillium digitatum based on morphological characteristics and ribosomal DNA internal transcribed spacer sequence analyses. Reinfection assays of orange fruits confirmed that C. gloeosporioides causes stem-end rot, and P. digitatum triggers green mold disease. By the heterologous expression of the green fluorescent protein (GFP) in C. gloeosporioides using Agrobacterium tumefaciens-mediated transformation, we could observe the fungal infection process of the citrus fruit stem-end rot caused by C. gloeosporioides for the first time. Furthermore, we isolated and selected two soilborne Bacillus strains with strong antagonistic activity for preventing the decay of citrus fruits by these pathogens. Molecular analyses of 16 S rRNA and gyrB genes showed that both isolates belong to B. velezensis. Antifungal activity assays indicated that bacterial culture suspensions could strongly inhibit C. gloeosporioides and P. digitatum, and shield orange fruits from the invasion of the pathogens. Our work provides a highly effective Bacillus-based preservative solution for combating the fungal pathogens C. gloeosporioides and P. digitatum to protect citrus fruits at the postharvest stages.

First Report of Corynespora cassiicola Causing Leaf spot of Strobilanthes cusia in China.

Strobilanthes cusia (Nees) Kuntze is a vital medicinal and industrial herb, planted extensively in southern China (Hu, et al. 2011.). In July and August of 2021, leaf spot incidence on >60% plants and reduced yields >20% for fresh leaves were observed in S. cusia cultivar 'Malan No.1' across the Shufeng whole Township, Xianyou County, Fujian province. Initial symptoms on leaves were observed as small, dark-brown, spots surrounded by a yellow halo, expanding irregularly or into semicircular spots. As symptoms developed, the spots became dark brown, thin and fragile, forming small holes. In severe cases plants were defoliated. The pathogen was isolated from the margin of 60 symptomatic leaf lesions， surfacesterilized with 75% ethanol for 45 s, rinsed three times with sterile water, air dried, and cultured on PDA at 25°C in the dark. Pure cultures were obtained by single-spore isolation after subculture. Ten representative single-spore isolates (MY-1 to MY-10) from 154 pathogens in 10 sampling points were selected for morphological characterization and identification. After 7 days, mycelial colonies were gray to dark gray with few aerial hyphae. Conidia (32.3 to 132.8 × 5.8 to 8.4 μm, average 81.4 × 6.3 μm, n=50) were pale to brown, erect or curved, solitary or in chains, with 0 to 15 pseudosepta. Based on morphological characteristics, the isolates were preliminarily identified as Corynespora cassiicola. Genomic DNA of isolate MY-2 (randomly selected from 10 isolates as representative) was extracted from mycelia using the Ezup DNA extraction kit (Sangon Biotech Co., Ltd. Shanghai, China). The ITS (internal transcribed spacer) region of rDNA, TEF1-α (translation elongation factor 1 alpha) and TUB2 (beta-tubulin) genes were amplified and sequenced with primers ITS4/ITS5, EF1-728F/EF-986R (Wang et al. 2021) and Bt2a/Bt2b (Glass et al. 1995), respectively. BLASTN sequence analyses of ITS (538 bp), TEF1-α (302 bp) and TUB2 (436 bp) of isolate MY-2 (GenBank accessions OK355515, OM339443, OM339442) showed 100%, 97.6%, 100% identity with C. cassiicola in GenBank (Accession numbers JX908713, MW961421, AB539228). A neighbor-joining phylogenetic analysis based on ITS and TEF1-α sequences using MEGA7 showed that MY-2 clustered in the same clade with C. cassiicola. For pathogenicity tests, five S. cusia plants were inoculated onto the adaxial surface of leaves with mycelial plugs from ten isolates of 8-day-oldcultures on PDA. Five leaves per plant were inoculated, covered with wet cotton, and kept in a controlled greenhouse (26~33 °C, RH 80% ~ 90%). Leaves inoculated with sterile PDA plugs served as a negative control. At 3-5 days post inoculation, all 25 inoculated leaves of each isolate showed leaf spot lesions similar to those observed in the field, and control leaves were symptomless. C. cassiicola was successfully reisolated from the diseased leaves. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. In view of morphology, pathogenicity and sequence results, the isolates were identified as C. cassiicola, a pathogen reported from many important crops (Lu et al. 2021). This is the first report of C. cassiicola as a pathogen in China which poses a potential threat to leaf production and S. cusia processing.

First Report of Tobacco Fusarium Root Rot Caused by Fusarium redolens in China

First Report of Tobacco Fusarium Root Rot Caused by <i>Fusarium redolens</i> in China