Tub2 Gene Research Articles

<strong>A new species of <em>Sarcopodium</em> (Hypocreales, Nectriaceae) from China</strong>

A new species, Sarcopodium tibetense, is described on rotten stem base of maize straw from the Tibet Autonomous Region. The anatomy of perithecia, which covered with septate, curved hairs on the surface, striate ascospores, and penicillate conidiophores indicate that the fungus belongs to Sarcopodium. It is characterized by solitary to gregarious perithecia that are globose to subglobose or pyriform, not becoming cupulate upon drying, and with hyaline hairs on surface; cylindrical to clavate asci with eight ascospores that are fusiform to ellipsoidal and striate. The asexual state is penicillium-like and with subglobose, ellipsoidal, oblong to rod-shaped, aseptate, smooth, hyaline conidia aggregated in a slimy mass. Sarcopodium tibetense is distinguished from its morphologically similar species, S. circinosetiferum, by narrower conidiophores and the presence of 50 bp and 9 bp sequence divergences in the ITS and LSU regions. Sequence analysis of the combined act, ITS, LSU and tub genes confirm its taxonomic position in Sarcopodium as a species new to science.

Three New Derivatives of Zopfinol from Pseudorhypophila Mangenotii gen. et comb. nov.

Triangularia mangenotti was analyzed for the production of secondary metabolites, resulting in the isolation of known zopfinol (1) and its new derivatives zopfinol B–C (2–4), the 10-membered lactones 7-O-acetylmultiplolide A (5) and 8-O-acetylmultiplolide A (6), together with sordarin (7), sordarin B (8), and hypoxysordarin (9). The absolute configuration of 1 was elucidated by the synthesis of MPTA-esters. Compound 1 showed antimicrobial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and the fungus Mucor hiemalis. While 4 was weakly antibacterial, 3 showed stronger antibiotic activity against the Gram-positive bacteria and weak antifungal activity against M. hiemalis and Rhodotorula glutinis. We furthermore observed the cytotoxicity of 1, 3 and 4 against the mammalian cell lines KB3.1 and L929. Moreover, the new genus Pseudorhypophila is introduced herein to accommodate Triangularia mangenotii together with several species of Zopfiella—Z. marina, Z. pilifera, and Z. submersa. These taxa formed a well-supported monophyletic clade in the recently introduced family Navicularisporaceae, located far from the type species of the respective original genera, in a phylogram based on the combined dataset sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and fragments of the ribosomal polymerase II subunit 2 (rpb2) and β-tubulin (tub2) genes. Zopfiella submersa is synonymized with P. marina due to the phylogenetic and morphological similarity. The isolation of zopfinols 1–4 and sordarins 7–9 confirms the potential of this fungal order as producers of bioactive compounds and suggests these compounds as potential chemotaxonomic markers.

First Report of Pestalotiopsis lushanensis Causing Brown Leaf Spot on Sarcandra glabra in China.

Sarcandra glabra is a species of Chloranthaceae family and this family grow in the southern part of China, Japan, and Southeastern Asia (Li et al. 2019). It is a kind of precious Chinese herbal medicine, which occupies an important position in traditional Chinese herbal medicine. It plays an effective role in the treatment of cancer, rheumatism, pneumonia, digestive tract inflammations, traumatic injuries and fractures, anti-virus, anti-bacterial, antioxidant, etc. (Li et al. 2019; Zheng et al. 2003; Zhou et al. 2013). Since June 2020, we discovered a serious leaf disease in the S. glabra planting base of Shibing County (108.12E 27.03N), in Guizhou Province, with an incidence rate of 60% and yield losses of 40%. Initially, the symptoms developed as small specks where spots were purple with a dark brown halo margin, and round or oval. In later stages, the spots gradually expanded and became dry, whole severe leaf loss. To identify the pathogen, we collected the diseased leaves from S. glabra fields in Shibing County. Small tissue pieces from the edges of lesions were disinfected in 75% ethyl alcohol for 30 s and 1% hypochlorite for 1 min, rinsed five times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C in lighted incubator for 3 days. Fungal colonies were consistently isolated and transferred to PDA for morphological characterization (Fang et al. 2007). Pathogenicity tests of the novel isolate HGUP CSH-2 were conducted by spraying spore suspensions with a concentration of 1.6×108 conidia/ml on surface-disinfected (70% ethyl alcohol, 30 s) leaves, while sterile distilled water was used as the control. Plants with inoculated leaves (three per treatment) were placed in lighted growth chambers at 28°C for 5 days and watered as needed (Light to dark ratio 1:1, RH=90%). Symptoms on inoculated leaves were similar to those described previously in the field. The same pathogenic fungus was re-isolated from the infected leaves but not from the non-inoculated leaves. Colonies on PDA attaining 70 mm diam after 7 d at 28°C, with pale honey-colored, sparse aerial mycelia on the surface with black, gregarious conidiomata. Conidiogenous cell discrete or integrated, ampulliform, clavate or subcylindrical, hyaline, smooth-walled, wide at base. Conidia fusoid, ellipsoid, straight to slightly curved, 4-septate, slightly constricted at septa, 22.26-27.17×6.9-8.22 µm (av.±SD: 24.68±1.57×7.68±0.38 µm; n=30). According to the colony and conidia characteristics, the isolate was initially identified as Pestalotiopsis spp. (Liu et al. 2017). The pathogen was confirmed by amplification and sequencing of the internal transcribed spacer region (ITS) gene, the translation elongation factor-1 (TEF1) gene and the β-tubulin (TUB2) gene (Liu et al. 2017) using ITS1/ITS4, Bt2a/T1 and EF1-526F/1567R primers, respectively. The sequences of the PCR products were deposited in GenBank with accession numbers MT919215 (ITS), MT939300 (TUB2) and MT939299 (TEF1). BLAST results of the obtained sequences of the ITS, TUB2 and TEF1 genes revealed 97.16% (479/493 nucleotides), 99.56% (675/678 nucleotides) and 99.89% (890/891 nucleotides) homology with those of Pestalotiopsis lushanensis in GenBank (MG726538, KY464157 and KX895223). Maximum Likelihood method was used for phylogenetic analysis. The result showed that HGUP CSH-2 was together with P. lushanensis with a support rate of 100%. According to the morphological characteristics and molecular phylogenetic analysis, the pathogen was identified as P. lushanensis. So far as we know, our research is the first report of brown leaf spot of S. glabra caused by P. lushanensis in China. Thus, identification of P. lushanensis for this disease is important for the advancement of effective prevention and control practises as future perspectives.

Assessment of Cytospora Isolates From Conifer Cankers in China, With the Descriptions of Four New Cytospora Species.

Cytospora species are widely distributed and often occur as endophytes, saprobes or phytopathogens. They primarily cause canker and dieback diseases of woody host plants, leading to the growth weakness or death of host plants, thereby causing significant economic and ecological losses. In order to reveal the diversity of Cytospora species associated with canker and dieback diseases of coniferous trees in China, we assessed 11 Cytospora spp. represented by 28 fungal strains from symptomatic branches or twigs of coniferous trees, i.e., Juniperus procumbens, J. przewalskii, Picea crassifolia, Pinus armandii, P. bungeana, Platycladus orientalis in China. Through morphological observations and multilocus phylogeny of ITS, LSU, act, rpb2, tef1-α, and tub2 gene sequences, we focused on four novel Cytospora species (C. albodisca, C. discostoma, C. donglingensis, and C. verrucosa) associated with Platycladus orientalis. This study represented the first attempt to clarify the taxonomy of Cytospora species associated with canker and dieback symptoms of coniferous trees in China.

First Report of Leaf Spot on White Chrysanthemum (Chrysanthemum morifolium) Caused by Epicoccum sorghinum in Hubei province, China.

White Chrysanthemum (Chrysanthemum morifolium), a perennial herb of the Compositae family, is used for traditional medicine. The planting area of white chrysanthemum in Macheng city, Hubei Province is about 3333 ha and the annual output can reach more than 5000 tons. In 2019, leaf spot disease appeared on almost all middle and lower leaves of white chrysanthemum in most fields of Fengshumiao county, Macheng city (N31°29'57″, E115°05'49″). This county has 33 acres white chrysanthemum planting area, and most of the plants in the county were infected with the leaf spot disease. The average incidence of leaf spot disease was 65%, and incidence in some areas was 100%. In our observations, leaf spot disease can occur throughout the whole growth period of white chrysanthemum, and it will become more serious under the high temperature and humidity condition. Usually, the diseased leaves account for 30 to 80% of the total leaves on the plant. Leaf spot initially manifests as necrotic lesions on the edge and tip of the leaf, and then the lesions coalesce and gradually expand to form irregular light-brown to brown-black spots, eventually leading to necrosis and curling of the entire leaf. This disease seriously affects the growth and development of plants, resulting in the decline of yield and quality of white chrysanthemum. Ten symptomatic leaf samples were collected, the surfaces were disinfected with 0.1% mercuric chloride (HgCl2) for 3 min, and washed with sterile distilled water three times. Ten tissue samples at the junction of diseased and healthy areas (0.5 × 0.5 cm2) were cut and placed on potato dextrose agar (PDA) medium containing 100 µg/ml cefotaxime sodium and incubated in a dark chamber at 28°C. After 2 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA plates for further purification. Finally, eight isolates were obtained and these isolates were similar in morphology. The color of purified isolates was initially white to pale yellow. After six days of incubation, colonies had a diameter of 8 cm and the cultures were pale gray and starting to secrete scarlet pigment. After 15 days incubation, the colonies were grayish brown, while the backside was reddish-brown. Gray to tan chlamydospores were observed, nearly spherical, with a wart-like surface. Unicellular chlamydospores were 7.91 to 32.23 × 12.03 to 38.42 µm (n=30) and multicellular chlamydospores were 6.32 to 25.10 × 21.75 to 100.05 µm (n=30). The morphological characteristics were similar to Epicoccum sorghinum (Kang et al. 2019). The isolate FDY-5 was chosen for molecular identification. The sequence of rDNA-ITS, TUB, and LSU of the FDY-5 were amplified (GenBank MT800929, MT799852, and MT800935, respectively) (White et al. 1990; Carbone and Kohn 1999; Lumbsch et al. 2000). BLAST results showed that the rDNA-ITS sequences, the TUB gene sequences, and LSU gene sequences of strain FDY-5 shared 99% identity with the sequences of E. sorghinum (syn. Phoma sorghina) in GenBank (MN555348.1, MF987525.1, MK516207.1, respectively). Moreover, a phylogenetic tree of the LSU gene sequence of FDY-5 was constructed based on the Neighbor-Joining (NJ) method in MEGA6 software (Tamura et al. 2013) and revealed that strain FDY-5 was closest to E. sorghinum. Based on morphological and molecular characteristics, the fungus was identified as E. sorghinum. Pathogenicity tests were conducted on two-month-old white chrysanthemum plants. The upper three leaves of three plants were randomly selected for stab treatment and were inoculated with 5 × 5 mm mycelial discs produced from a fifteen-day-old colony on PDA. The inoculated and control (treated with sterile PDA disks) plants were incubated in a moist chamber (25 ± 2 °C, RH 85%). The first lesions appeared 1 day after inoculation on leaves, and the necrotic lesion area expanded outward and showed typical symptoms 3 days later. To fulfill Koch's postulates, the pathogen was reisolated from nine inoculated leaves by repeating the above isolating operation, and confirmed as E. sorghinum by morphology. To the best of our knowledge, this is the first report of E. sorghinum causing leaf spot on white chrysanthemum in China. E. sorghinum has a wide host range worldwide and often causes crop yield reduction. This report will facilitate the diagnosis of white chrysanthemum leaf spot of white chrysanthemum allowing control measures to be adopted to manage this disease in a timely manner. References Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Kang, Y., et al. 2019. Plant Dis. 103 (7):1787. Lumbsch, H., et al. 2000. Plant Biol. 2:525. Tamura, K., et al. 2013. Mol. Biol. Evol. 30:2725-2729. White, T. J., et al. 1990. Page 315 in:PCR protocols:a guide to methods and applications. Academic Press, San Diego, CA. Funding Funding was supported by Major Increase and Decrease Projects at the Central Level of China (2060302) and the National Key Research and Development Program (2017FYC1700704).

Diversity of Colletotrichum species causing onion anthracnose in Brazil

Foliar anthracnose is one of the main diseases of onion (Allium cepa L.) under tropical and subtropical conditions. Thus far, only Colletotrichum gloeosporioides has been reported as the causal agent of this disease in Brazil. However, there are no extensive studies characterizing Colletotrichum isolates associated with onion anthracnose in the country. Here, 38 Colletotrichum isolates obtained from onion plants displaying foliar anthracnose across major Brazilian onion-producing regions were characterized using morphometric and molecular information. The Bayesian and Maximum Liklihood methods were used for an initial analysis of the β-tubulin gene (tub2) sequences of all isolates, resulting in the discrimination of nine haplotypes. Three haplotypes grouped with the reference species of the C. acutatum complex and six with the C. gloeosporioides complex. Sequences of either the glyceraldehyde-3-phosphate dehydrogenase (gapdh), actin (act), and calmodulin genes or the intergenic spacer (IGS) region between DNA lyase (apn2) gene and the mating-type mat1–2-1 locus were used to characterize a subset of isolates representing these nine distinct tub2 gene haplotypes. These analyses revealed five anthracnose-inducing Colletotrichum species, including three members of the C. acutatum species complex (C. nymphaeae, C. scovillei, and C. tamarilloi) and two of the C. gloeosporioides species complex (C. fructicola and C. theobromicola). Bioassays confirmed that all these Colletotrichum species are pathogenic to onion, inducing typical anthracnose symptoms on bulbs and leaves. Twenty-six out of 38 isolates were identified as C. theobromicola, indicating this fungus as the prevalent anthracnose pathogen under Brazilian conditions. This Colletotrichum species diversity will affect anthracnose management strategies, including chemical and cultural control as well as the identification and deployment of onion cultivars with species-specific and/or wide-spectrum tolerance/resistance.

New species and records of Diaporthe from Jiangxi Province, China.

Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded, little is known about species able to infect forest trees in Jiangxi Province. Hence, extensive surveys were recently conducted in Jiangxi Province, China. A total of 24 isolates were identified and analysed using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions, as well as their morphological features. Results revealed five novel taxa, D. bauhiniae, D. ganzhouensis, D. schimae, D. verniciicola, D. xunwuensis spp. nov. and three known species, D. apiculatum, D. citri and D. multigutullata.

First report of Diaporthe eres causing branch canker on Cinnamomum camphora (camphor tree) in Jiangxi Province, China.

In September 2019, approximately 75 to 90% of camphor trees (Cinnamomum camphora) were observed with cankers and branch dieback symptoms in Anyi (N28°32'54'', E115°37'52'') and Xinyu (N27°37'38'', E114°50'25'') county (Jiangxi Province, China). The symptoms included dark brown to dark, oval-shaped canker lesions, sunken and cracked longitudinally, cracked and evenly swelling, or reddish brown (Figure 1 A-D). Samples were collected from symptomatic branches and were cut into small pieces (ca. 0.5 cm × 0.5 cm × 0.5 cm). Sections were surface sterilized as described by Zhang et al. (2020), then placed on potato dextrose agar amended with 0.01% penicillin and 0.015% streptomycin sulfate and incubated in the laboratory at 25℃ with darkness. After 3 to 5 days, mycelium growing out from tissues were transferred onto PDA medium. In total, 68 fungal isolates including 22 isolates of Diaporthe sp. were obtained from cankers and then were classified into five categories based on morphological characteristics and sequencing of the ITS for morphological representative strains. Pathogenicity tests were conducted in the greenhouse (Figure 1 E-M) and field (Figure 1 N-Q). Branches were surface sterilized and inoculated as described by Prencipe et al. (2017). In the greenhouse, a total of 13 representative isolates (including 6 isolates of Diaporthe sp., 2 isolates of Neofusicoccum sp., 2 isolates of Botryosphaeria sp. and 3 isolates of Colletotrichum sp.) were selected and evaluated using 2-year-old seedlings of camphor tree in pots with 5 replicates per isolate, in which 3 isolates of Collectotrichum sp. had no pathogenicity. Then, two isolates of Diaporthe sp. (Z4 and Z7) were selected for field experiment. In field tests, the same method was used as in the greenhouse. The inoculated and control branches were collected 40 days after inoculation and the fungi were isolated and placed on PDA plates to recover the inoculated fungi and complete Koch's postulates. Both isolates of Diaporthe sp. produced canker symptoms on the branches. Isolate Z4 caused discoloration also on the branch without wounding. Both isolates produced pycnidia scattered in PDA plates supplemented with stems of alfalfa, were dark brown to black, globose to subglobose (Figure 1 T). Alpha conidia were cylindrical, 5.72-9.98 µm (mean 7.64 µm) × 2.15-3.13 µm (mean 2.69 µm) (n = 30) (Figure 1 S, red arrow), while beta conidia were biguttulate, one-celled, hyaline, non-septate, and 16.21-25.52 µm (mean 21.60 µm) × 0.76~1.65 µm (mean 1.14 µm) (n = 30, green arrow) (Figure 1 S). Five isolates (Z4, S-Z4, P-Z4, Z7 and S-Z7) including those used for pathogenicity test were selected for multi-locus phylogenetic analyses of ITS (White et al., 1990), TEF1-α and TUB2 (Glass et al. 1995) gene sequences, which the accession number was MW036358- MW036362 for ITS, MW052267- MW052271 for TEF1- α, MW052276-MW052280 for TUB2. Based on the phylogenetic tree analysis using IQ-TREE 2, all five isolates were identified as D. eres (Figure 2). D. eres has been reported to cause canker on many different woody plants, such as almond (Holland et al. 2020), peach (Prencipe et al. 2017), hazelnut (Wiman et al. 2019), and so on. However, this is the first report worldwide of D. eres causing disease on Cinnamomum camphora in China.

Evaluating species in Botryosphaeriales.

The Botryosphaeriales (Dothideomycetes) includes numerous endophytic, saprobic, and plant pathogenic species associated with a wide range of symptoms, most commonly on woody plants. In a recent phylogenetic treatment of 499 isolates in the culture collection (CBS) of the Westerdijk Institute, we evaluated the families and genera accommodated in this order of important fungi. The present study presents multigene phylogenetic analyses for an additional 230 isolates, using ITS, tef1, tub2, LSU and rpb2 loci, in combination with morphological data. Based on these data, 58 species are reduced to synonymy, and eight novel species are described. They include Diplodia afrocarpi (Afrocarpus, South Africa), Dothiorella diospyricola (Diospyros, South Africa), Lasiodiplodia acaciae (Acacia, Indonesia), Neofusicoccum podocarpi (Podocarpus, South Africa), N. rapaneae (Rapanea, South Africa), Phaeobotryon ulmi (Ulmus, Germany), Saccharata grevilleae (Grevillea, Australia) and S. hakeiphila (Hakea, Australia). The results have clarified the identity of numerous isolates that lacked Latin binomials or had been deposited under incorrect names in the CBS collection in the past. They also provide a solid foundation for more in-depth future studies on taxa in the order. Sequences of the tef1, tub2 and rpb2 genes proved to be the most reliable markers. At the species level, results showed that the most informative genes were inconsistent, but that a combination of four candidate barcodes (ITS, tef1, tub2 and rpb2) provided reliable resolution. Furthermore, given the large number of additional isolates included in this study, and newly generated multigene DNA datasets, several species could also be reduced to synonymy. The study illustrates the value of reassessing the identity of older collections in culture collections utilising modern taxonomic frameworks and methods. Citation: Zhang W, Groenewald JZ, Lombard L, et al. 2021. Evaluating species in Botryosphaeriales. Persoonia 46: 63-115. https://doi.org/10.3767/persoonia.2021.46.03.

Morphological and Molecular Identification of Colletotrichum spp. Associated with Chili Anthracnose Disease in Yogyakarta Region

Colletotrichum sp., the causal agent of anthracnose disease, is one of the important pathogenic fungi in chili which can cause considerable yield losses, especially during the rainy season. This study aimed to identify the species of Colletotrichum isolates obtained from chili cultivation area in The Special Region of Yogyakarta Province both morphologically and molecularly. As a comparison, a Colletotrichum isolate obtained from Magelang Regency, Central Java Province was used as comparison isolate. From the isolation result, it was obtained 14 isolates of Colletotrichum that generally had conidia that were fusiform to cylindrical with two pointed or slightly blunt ends, or crescent shapes with a various size range between 9.02-19.38 µm x 2.37–8.57 µm. Based on morphological observations using UPGMA analysis, these 14 isolates could be divided into 4 groups with 7 different types. Representative isolates of each type in different groups and a comparison isolate were identified molecularly by multi-gene analysis using the ITS1-4, gapdh and tub2 genes. The result showed that B1, G1, K2 and Mg isolates were closely related to Colletotrichum scovillei, J1 with C. truncatum; S1 and S2 with C. siamense; and J2 with C. makassarii. From the pathogenicity test on wounded chili, it showed that C. scovillei and C. siamense isolates had higher virulence than C. truncatum and C. makassarii isolates.

First Report of Colletotrichum siamense Causing Anthracnose of Chili Pepper Fruit in Korea.

In October 2015, typical anthracnose symptoms were observed on approximately 15 to 20% of the chili fruits (cv. Manita) growing in Goesan County, Chungcheong Province, South Korea. Infection of fruits were characterized by the presence of circular, sunken lesions with concentric rings of orange conidial acervuli. Fresh samples were collected from the infected fruits and lesions from seven symptomatic fruits were cut into small pieces (5 mm2) and surface sterilized by soaking them in 1% sodium hypochlorite for 3 min, followed by rinsing thrice using sterilized water, and drying on sterilized filter paper. The tissue pieces were then placed on potato dextrose agar (PDA) and incubated at 25 ± 2°C with 12hrs photoperiod. After 2 to 3 days, single hyphal tips were transferred to fresh PDA and a total of seven isolates were selected from typical single hyphae. The upper surfaces of the colonies formed on PDA were white to gray in color with cottony mycelia, in which salmon-colored acervuli were clearly visible (Supplementary 1). Thirty conidia were examined; all were hyaline, smooth-walled, aseptate, straight, mainly cylindrical with round ends, 12 to 17 µm long, and 3 to 4.5 µm wide. Appressoria were oval to irregular inshape, dark brown in color, and range from 9.5 to 11.5 µm × 6.5 to 7.5 µm in sizes. Morphological characteristics of the seven isolates were identical and resembled those of C. siamense (Weir et al. 2012). To confirm the identification of the fungal isolates, DNA from seven isolates were extracted (Cenis et al. 1992) and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal transcribed spacer (ITS) rDNA regions, and β-Tublin-2 (TUB2) were partially amplified and sequenced. Sequences from all seven isolates were identical each other. Nucleotide sequences of ITS, GAPDH, and TUB2 from representative isolates CNU180002 and CNU180012 were deposited in GenBank under accession numbers MH085103, MH085105, and MH085107 for CNU180002 and MK033503, MK033504, and MK033505 for CNU180012, respectively. The sequences for all three genes exhibited 99 to 100% identity with C. siamense, GenBank accession nos. FJ972613 (ITS), FJ972575 (GAPDH), and FJ907438 (TUB2) for both isolates. A multi-locus phylogenetic tree with closely related reference sequences downloaded from the GenBank database demonstrated that these two isolates were aligned with C. siamense. Pathogenicity of isolates CNU180002 and CNU180012 was confirmed on healthy fruits (Manita) by using a pin-pricked wound/drop (1 mm depth) and non-wound/drop inoculation method (Oo et al. 2017) and control fruits were mock-inoculated with sterilized distilled water. Three fruits were inoculated for each isolate and pathogenicity test were repeated thrice. After inoculation, the fruits were placed on a sterilized paper tissue in moistened clean boxes with a relative humidity of approximately 90% and incubated for 7 days at 25°C in the dark. Disease symptoms were appeared 5 to 7 days after inoculation on wounded fruits whereas non-wounded fruits were observed after 10 days. The two isolates showed identical symptoms and control fruits remained symptomless. Both isolates were re-isolated from infected fruits and were identical to the original isolates in morphology characteristics as well as on molecular sequences of ITS, GAPDH and TUB2 genes. To our knowledge, this is the first report of anthracnose caused by C. siamense on chili pepper fruit in Korea.

Validation of superior reference genes for qRT-PCR and Western blot analyses in marine Emiliania huxleyi-virus model system.

To search for a set of reference genes for reliable gene expression analysis in the globally important marine coccolithophore Emiliania huxleyi-virus model system. Fifteen housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1-2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) were evaluated for their stability as potential reference genes for qRT-PCR using ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software. CDKA, TUA and TUB genes were tested as loading controls for Western blot in the same sample panel. Additionally, target genes associated with cell apoptosis, that is metacaspase genes, were applied to validate the selection of reference genes. The analysis results demonstrated that putative housekeeping genes exhibited significant variations in both mRNA and protein content during virus infection. After a comprehensive analysis with all the algorithms, CDKA and GAPDH were recommended as the most stable reference genes for E huxleyi virus (EhV) infection treatments. For Western blot, significant variation was seen for TUA and TUB, whereas CDKA was stably expressed, consistent with the results of qRT-PCR. CDKA and GAPDH are the best choice for gene and protein expression analysis than the other candidate reference genes under EhV infection conditions. The stable internal control genes identified in this work will help to improve the accuracy and reliability of gene expression analysis and gain insight into complex E. huxleyi-EhV interaction regulatory networks.

First Report of Chaetomium globosum Causing Leaf Blight on Brassica oleracea L. in China.

Cabbage (Brassica oleracea var. capitata L.) is widely cultivated in China and be of important economic value. In October 2019, all the plants of cabbage inbred line '2358' cultivated in greenhouses of the Chinese Academy of Agricultural Sciences (Beijing) were showing symptoms of leaf wilt. It usually took two weeks for the leaves to get completed wilted and the plants gradually died. It was approximately 550 plants affected, and 30 plants were collected and processed as samples. Symptomatic leaves were cut into small pieces (5×5 mm), surface sterilized with 75% ethanol for 30 s, and then sterilized with 8% NaClO for 3 mins, rinsed three times in sterile distilled water, plated on complete medium (CM: 3g Casein Enzymatic Hydrolysate + 3g Casein Acid Hydrolysate + 6g Yeast Extract+10g Sucrose + 15g Agar + 1L dH2O) and incubated at 27℃ for 6 days. Subsequently, the purified culture was obtained by tissue isolation and single-spored on CM. The colony on CM was up to about 50mm and 70mm in diameter after 4 and 7 days, respectively (Supplementary Fig. 2). At the same time, the edge of the colony gradually turned brown. Microscopic observation found that the diameter of the mycelium was about 5 µm (Supplementary Fig. 3). The conidia were transparent white and ovoid-shaped, about 0.3-0.5 µm in diameter (Supplementary Fig. 4). Fungus in liquid CM were spherical with surface hairs (Supplementary Fig. 1), which was consistent with the morphological characteristics of Chaetomium globosum. The rDNA internal transcribed spacer (ITS) of isolate ZM2019 was amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3') (Hong et al. 2013). The amplified product was sequenced and deposited in GenBank. The 573-bp amplicon (GenBank accession no. MN833403) showed a 100% homology to C. globosum isolate CES5 (MN173145). According to the morphological and molecular characterization, the fungus causing leaf blight on '2358' was identified as C. globosum. In order to further distinguish the pathogen species, the β-tubulin (tub2) gene region were amplified using primers T-F (5'-CCCCTGAACTACCCCACC-3') and T-R (5'-TATTTGACCCGACTGACC-3') and sequenced. Finally, we further identified this pathogen as C. globosum CBS 148.51 (Wang et al. 2016), according to the blast result of the sequence (GenBank accession no. MW252170) in National Center for Biotechnology Information (NCBI). To confirm the pathogenicity of this fungus, the leaves of 12 healthy seedlings of '2358' were inoculated by spraying 106 conidia/ml suspension and 12 seedlings inoculated with sterile water served as controls. All plants were incubated in a growth chamber maintained at 27℃. Ten days after inoculation, the leaves of all plants became wilted, which were consistent with the symptom in the natural state, while the control plants remained disease-free (Supplementary Fig. 6b and Fig. 6c). Subsequently, C. globosum was isolated from the inoculated seedlings again. The entire process from isolation to inoculation was repeated again and the same results were obtained. Re-isolation of C. globosum and inoculation of the host fulfiled Koch's postulates. C. globosum has been reported previously to occur on many horticulture plants such as Punica granatum (Guo et al. 2015) and Cannabis sativa (Chaffin et al. 2020); but no brassica species has been reported so far as susceptible to C. globosum. In this sense, this is the first report of leaf blight caused by C. globosum on cabbage in China, in greenhouse condition.

Population genetics and gene sequence analysis of Athelia rolfsii collected from Northern Iran

Athelia rolfsii is a globally dispersed pathogenic fungus, causing white root rot disease in many crops and horticultural plants. In this study, 90 isolates were collected from three provinces of Guilan, Mazandaran and Golestan in northern Iran. Eighteen isolates were selected for sequence analyses based on their host, sampling sites and Mycelial Compatibility Groups (MCG). Translation elongation factor 1-α(tef1-α), β-tubulin (tub2) genes and rDNA large subunit (LSU) were partially amplified and sequenced in order to conduct phylogenetic analyses. Approximately, all Iranian isolates clustered together in both tef1-α and LSU phylogenetic trees. However, after deletion of ambiguous sites, no variations were observed in tub2 sequences. ISSR and SCoT were also used to investigate the genetic structure of the population. Results of the molecular variance analysis (AMOVA) showed that 70 and 30% of the observed variance corresponded to the difference between and within the populations, respectively. According to these findings, we suggest that mating between populations would be less likely and thus, gene flow is restricted.

First Report of Root Rot Caused by Dactylonectria torresensis on Bletilla striata (Baiji) in Yunnan, China.

Bletilla striata (Thunb.) Rchb.f. (Orchidaceae family, known as Baiji in Chinese) is an endangered plant species with important medicinal value in China. Bletilla striata plants with symptoms of wilting, leaf yellowing and rotting on underground parts were found in Shizong (24.82822 N; 103.99084 E), Yunnan Province, China in July 2016. In the following years, this disease occurred and became prevalent when high temperature and high humidity prevailed in the fields from May to August. The incidence of the disease varied from 45 to 75%, with yield losses of 40 to 65% in different B. striata fields. To identify the causal agent of the disease, symptomatic vascular tissue fragments were soaked in 2% sodium hypochlorite for 2 min, rinsed twice with sterile distilled water, and then placed on 4% (w/v) potato dextrose agar (PDA) plates. The plates were incubated at 26°C in 12h light/dark for three days. Mycelia grown from the edges of the plant fragments were transferred to PDA plates and incubated at 26°C in 12h light/dark. After three days, hyphal tips were isolated from the edge of the colonies to PDA plates. Three hyphal-tip isolates from different plants were further studied. The colonies of these three isolates were dark red, with cottony mycelia of moderate density. Hyphae were transparent and branched. Numerous hyphae anastomosed frequently and formed hyphal coils. For further morphological analysis, sporulation was induced as described by Cabral et al. (2012) and Lombard et al. (2014). Macroconidia were abundant, 37.2 to 44.0 µm × 5.2 to 8.7 µm based on the measurement of 20 conidia from each isolate. Ascospores divided into two cells of equal size, ellipsoid to oblong-ellipsoid, 12.5 to 14.8 µm × 4.8 to 5.9 µm based on the measurement of 20 spores from each isolate. Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely brached, more or less cylindrical. These morphological characteristics were consistent with the description of Dactylonectria spp. by Cabral et al. (2012) and Lombard et al. (2014). From one isolate, the internal transcribed spacer (ITS) region of ribosomal DNA and the beta-tubulin (tub2) gene were amplified by polymerase chain reaction (PCR) using the primer pairs ITS1/ITS4 (White et al. 1990) and T1/Bt-2b (Cabral et al. 2012), respectively. PCR products were sequenced and deposited in GenBank with accession numbers MH458779 (ITS) and MH626485 (tub2). BLAST search revealed that both sequences showed 99 to 100% homology with the corresponding sequences of previously identified D. torresensis isolates. Specially, MH458779 shares 100% identity with the entire 463-base pair (bp) sequence of KP411806, the ITS sequence of a D. torresensis isolate identified from olive trees (Nigro et al. 2019); MH626485 shares 99% identity with the entire 320-bp sequence of KP411801, the tub2 sequence of the same olive tree isolate. In addition, the entire 609-bp sequence of MH626485 shares 99% identity with JF735478, the tub2 sequence of a D. torresensis isolate identified from grapevines (Cabral et al. 2012). To test the pathogenicity of the fungus, plants of B. striata in plastic pots filled with sterilized nursery soil were inoculated with each of the three isolates by placing a fungal-colonized wheat kernel adjacent to each health plant. Plants inoculated with noncolonized wheat kernels were used as controls. Plants in three pots (replicates), with one plant per pot, were inoculated by each isolate. The pots were maintained in a greenhouse with a 12h photoperiod at 25°C. Ten days after inoculation, black necrotic lesions identical to those observed in the field were evident on the roots of all inoculated plants. Using the same methods described above, fungi with identical morphologies as described above were isolated from lesions caused by each of the three isolates. The control plants remained healthy, and no fungus was re-isolated. This completed Koch's postulates. Based on the morphological characteristics and molecular identification, the pathogen was determined to be D. torresensis. To our knowledge, this is the first report of D. torresensis causing root rot of B. striata in Yunnan, China. It is important to further study the impacts of this new disease on B. striata production in China.

First Report of Neofusicoccum parvum Causing Leaf Spot on Geodorum eulophioides in China.

Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13'32″，N24°44'19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 μm (avg. 13.8 μm) × 4.1 to 6.7 μm (avg. 5.1 μm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R（Alves et al. 2008；Carbone & Kohn, 1999）, and T1/T2 (O'Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.

Re-Evaluation of the Order Sordariales: Delimitation of Lasiosphaeriaceae s. str., and Introduction of the New Families Diplogelasinosporaceae, Naviculisporaceae, and Schizotheciaceae.

The order Sordariales includes the polyphyletic family Lasiosphaeriaceae, which comprises approximately 30 genera characterized by its paraphysate ascomata, asci with apical apparati, and mostly two-celled ascospores, which have a dark apical cell and a hyaline lower cell, frequently ornamented with mucilaginous appendages. To produce a more natural classification of this family, we carried out a phylogenetic analysis based on sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and fragments of ribosomal polymerase II subunit 2 (rpb2) and β-tubulin (tub2) genes of several isolates from soil and of reference strains of the Sordariales. As a result, Lasiosphaeriaceae s. str. has been circumscribed for the clade including the type species of the genus Lasiosphaeria and, consequently, its description emended. In addition, the new families Diplogelasinosporaceae, Naviculisporaceae, and Schizotheciaceae are introduced to accommodate those taxa located far from the Lasiosphaeriaceae s. str. Moreover, we propose the erection of the new genera Areotheca, Lundqvistomyces, Naviculispora, Pseudoechria, Pseudoschizothecium, and Rhypophila based on morphological and sequence data. New combinations for several species of the genera Cladorrhinum, Jugulospora, Podospora, Schizothecium, and Triangularia are proposed, their descriptions are emended, and dichotomous keys are provided to discriminate among their species.

First Report of Corynspora cassiicola Causing Leaf Spot on Jasminum sambac in China.

Jasminum sambac (Linnaeus) Aiton belonging to Oleaceae family, has been cultivated in China over 1500 years. It is an important tea crop in South China, and popular ornamental plant in North China. In June 2015, Corynespora leaf spot on Jasminum sambac was first observed in Caozhuang in Tianjin(E: 117°20', N: 39°13'). In a series of investigations, the disease incidence of Corynespora leaf spot on Jasminum sambac in Tianjin was found to be as high as 20%. The disease was also found in flower planting greenhouses in Beijing and Hebei region. The initial symptoms appeared as small circular spot with gray white in the center and dark brown in the edge. The lesions expanded rapidly to 5-10 mm diameter, surrounded by a yellow halo. The affected leaves became covered with the circular spots and defoliated eventually. Fifty-six leaf samples were collected from 20 greenhouses, then directly examined and disease tissues set aside for isolation. Conidiophores were either solitary or in clusters, straight or curved and un-branched, with 3-10 septate, pale to dark brown in color. Those were measured 80.6 to 234.8 μm long and 4.5 to 8.0 μm wide in size and were formed with 0-8 successive cylindrical proliferation (n = 50). Conidia borne from conidiophore, were single or in chains, obclavate to cylindrical, smooth. The average size of 50 conidia was 43.6 to 130.7 μm long×6.4 to 13.5 μm wide, with 4 to 8 pseudosepta (n = 50). The infected leaves were cut into small pieces, sterilized with 75% ethanol, rinsed in sterilized distilled water and then plated on potato dextrose agar (PDA). The isolated plates were incubated at 25℃ for 5 days. Thrity-five pure culture isolates were obtained from diseased leaves. The colonies were pale brown on the front and dark brown on the underneath, with thickly aerial mycelia. Morphological characteristics of the pathogen from the infected leaves and purified strains matched descriptions of Corynesora cassicola (Ellis and Holliday 1971). To confirm the pathogenicity, conidial suspensions (106 conidia mL-1 ) of 35 isolated strains were sprayed on the healthy plants at the seedling stage of Jasminum sambac respectively, 5 plants per strains. 10 healthy plants were sprayed only with sterilized water as control. Conidial suspensions were prepared by washing conidia from the pure C. cassiicola cultures. Conidial suspensions were adjusted to a concentration of 1 × 106 conidia/mL with the hemocytometer. All plants were incubated in artificial climate-room at 90% relative humidity, 25±2℃. After 7 days, small circular spots, similar to the field samples were observed on all plants inoculated with the pathogen. By comparison, control plants displayed no symptoms. The same pathogens were re-isolated from the symptomatic leaves fulfilling Koch's postulates. For molecular identification, rDNA-ITS and two housekeeping genes (TUB2 and EF-1α) gene were amplified and sequenced (Zhu et al. 2020). The sequences of three high pathogenicity isolats showed 99% homology to C.cassiicola (MK571399) with464 bp for rDNA-ITS, 100% homology to C.cassiicola (MN512639) with469 bp for TUB2 gene, 98.61% homology to C.cassiicola (MH263735) with287 bp for EF-1α gene. And all 9 sequences were submitted to GenBank (GenBank accession No. MT334575-77 for rDNA-ITS, MT886387-89 for TUB2, and MT886390-92 for EF-1α).The molecular identification and morphological characteristics showed that the isolated strains from Jasminum sambac were identified as C.cassiicola. C. cassiicola has an extensive host range, covering 397 host plants (Farr and Rossman 2020). Recently, C.cassiicola has been reported on Jasminum mesnyi of Oleaceaein China (Zhang et al. 2018). To our knowledge, this is the first report of leaf spot caused by C.cassiicola on Jasminum sambac in China and also worlwide.

Reconsideration of species boundaries and proposed DNA barcodes for Calonectria.

Calonectria represents a genus of phytopathogenic ascomycetous fungi with a worldwide distribution. In recent years, there has been an increase in the number of taxonomic studies on these fungi. Currently, there are 169 described species of Calonectria based on comparisons of DNA sequence data, combined with morphological characteristics. However, for some of these species, the sequence data utilised at the time of their description were relatively limited. This has justified an urgent need to reconsider the species boundaries for Calonectria based on robust genus-wide phylogenetic analyses. In this study, we utilised 240 available isolates including the ex-types of 128 Calonectria species, and re-sequenced eight gene regions (act, cmdA, his3, ITS, LSU, rpb2, tef1 and tub2) for them. Sequences for 44 Calonectria species, for which cultures could not be obtained, were downloaded from GenBank. DNA sequence data of all the 169 Calonectria species were then used to determine their phylogenetic relationships. As a consequence, 51 species were reduced to synonymy, two new species were identified, and the name Ca. lauri was validated. This resulted in the acceptance of 120 clearly defined Calonectria spp. The overall data revealed that the genus includes 11 species complexes, distributed across the Prolate and Sphaero-Naviculate Groups known to divide Calonectria. The results also made it possible to develop a robust set of DNA barcodes for Calonectria spp. To accomplish this goal, we evaluated the outcomes of each of the eight candidate DNA barcodes for the genus, as well as for each of the 11 species complexes. No single gene region provided a clear identity for all Calonectria species. Sequences of the tef1 and tub2 genes were the most reliable markers; those for the cmdA, his3, rpb2 and act gene regions also provided a relatively effective resolution for Calonectria spp., while the ITS and LSU failed to produce useful barcodes for species discrimination. At the species complex level, results showed that the most informative barcodes were inconsistent, but that a combination of six candidate barcodes (tef1, tub2, cmdA, his3, rpb2 and act) provided stable and reliable resolution for all 11 species complexes. A six-gene combined phylogeny resolved all 120 Calonectria species, and revealed that tef1, tub2, cmdA, his3, rpb2 and act gene regions are effective DNA barcodes for Calonectria.

First report of Leaf Spot Associated with Boeremia exigua on White Clover in China.

White clover (Trifolium repens L.) is an important perennial legume forage widely cultivated in China (Zhang et al. 2016). In April 2018, severe necrotic lesions on leaves were observed in a cultivated white clover field in Chongqing, China. Approximately 90% of plants in the field were affected. Leaf spots were amphigenous, dark-brown, elliptic to subcircular, with diameter ranging between 1 to 12 mm, well defined by brown margins and yellow halos. Severely infected leaves became withered and abscised. Stems and flowers were not affected by the disease. Symptomatic leaves were surface sterilized with 70% ethanol for 30 s followed by 0.1% HgCl2 treatment for 3 min, and rinsed in sterile water three times. Thereafter, tissue samples from margins of individual lesions were placed on potato dextrose agar (PDA) amended with 50 mg/L of chloramphenicol and incubated at 25℃ in the dark. An olivaceous gray fungal colony was consistently isolated (90.5% isolation frequency). After 15 days of incubation, subglobose, black pycnidia developed in the cultural medium. Conidia were hyaline, ellipsoid to oblong, nonseptate(n = 50), ranging from 4.0 to 7.5 μm long (5.6 ± 2.3µm) × 2.0 to 3.8 μm wide(2.8 ± 1.0 µm). On the basis of its morphological characteristics, the fungus was identified as Boeremia sp. (Aveskamp et al. 2010). To confirm the identity, the internal transcribed spacer region (ITS), large subunit ribosomal RNA (LSU), partial actin (ACT), RNA polymerase II second largest subunit (rpb2) and beta-tubulin (tub2) genes were amplified with primers ITS1/ITS4, LR0R/LR7, ACT-512F/ACT-783R, RPB2-5F2/fRPB2-7cR, and Btub2Fd/Btub4Rd, respectively, in eight representative isolates and sequenced(Aveskamp et al. 2009; Chen et al. 2015). BLAST results showed 100% identity of the ITS (506/506 nucleotides), LSU (966/966 nucleotides), ACT (244/244 nucleotides) and tub2 (297/297 nucleotides) sequences with those of B. exigua (KY419536, MK398746, EU880878, and MK514090) and 99.83% identity of those of the rpb2 (593/594 nucleotides) sequence with B. exigua (KT389572).Based on morphology and DNA sequence analysis, the associated fungus was identified as B. exigua. Representative sequences of one isolate (BT2-1) were deposited in GenBank (MN826339, MN836592, MT265217, MT265218 and MT265219). In a pathogenicity test, ten 2-month-old potted white clover plants were spray-inoculated with a spore and mycelial suspension (approximately 105 CFU/mL) and the control plants were inoculated with sterile distilled water. Plants were incubated in a greenhouse at 20 to 24°C under natural light and enclosed in plastic bags for the first 3 days to maintain high humidity. After 10 days, typical dark-brown lesions similar to those seen in naturally infected leaves developed on the inoculated leaves and not on the control plants. B. exigua was reisolated from the lesions, thus completing Koch's postulates. There is some evidence that B exigua is capable of invading seedling root tissue of white clover and causing necrotic lesions on roots (Skipp and Christensen,1982). However, to our knowledge, this is the first report of leaf spot on T. repens caused by B. exigua. This disease severely reduces forage quality and yield. Proper identification of the causal organism is essential in formulating management strategies.