Aerial Hyphae Research Articles

First Report of Clonostachys rosea Causing rot of Morchella sextelata in Anhui Province, China.

Morel mushroom (Morchella spp.) is a valuable mushroom, which has extremely high nutritional and economic value. In the early March of 2022, a serious rot disease was observed on approximately 30% fruiting bodies at an M. esculenta farm of Suzhou City, Anhui province, China. A white mold-like hyphae was initially present on the pileus, which then gradually spread to the whole fruiting body, eventually resulting in softening of the fruiting body and death. This disease developed rapidly at relatively high temperature (>20°C) and humidity (>85%), resulting in approximately 80% loss of yield. Twenty infected tissues were cut into small pieces (5 × 5 mm) and placed on potato dextrose agar (PDA) and incubated at 25°C for 5 days. Fifteen morphologically similar isolates were obtained and purified using the single spore isolation technique. Colonies of these isolates were yellowish-white, and tomentose with thick aerial hyphae after 7 days at 25°C on PDA plates. Conidiophores were dimorphic: primary conidiophores were Verticillium-like, secondary conidiophores were penicillate. Primary conidiophore stipe length ranged from 68.6 to 180.5 µm, and the base width was 3.2-5.6 µm. Phialides were solitary, straight, generally slightly tapering towards the tip, each producing a small, hyaline drop of conidia. Secondary conidiophores stipe length ranged from 68.4 to 120.5 µm, the base measured 3.3-6.1µm. Phialides were straight to slightly curved, slightly flask-shaped, with widest point below the middle, slightly tapering in the upper part, without visible collarette. Conidia were colorless, smooth, slightly curved, and distally broadly rounded with an average size of 6.3 to 8.2 × 2.4 to 3.7 μm (n=30). These isolates were initially identified as Clonostachys rosea based on morphological features (Schroers et al. 1999). To confirm the identity of C. rosea, primers ITS1/ITS4 (White et al. 1990) and EF1-728F/ EF1-986R (Carbone and Kohn 1999), were used to amplify the internal transcribed spacer (ITS), and translation elongation factor 1-alpha (EF-1α) genes of the representative isolate 5-3-2. These sequences were deposited in GenBank (GenBank accession nos. ON614093 and ON630916) and had 100% and 99.45% nucleotide identity with Clonostachys rosea E5R(17) and Clonostachys rosea KGSJ26 (GenBank accession nos. MK752437 and MT462122), respectively. Single conidium were isolated and multiplied on PDA for pathogenicity testing. To fulfill Koch's postulates, pathogenicity tests were performed using the fruiting bodies. Spores of C. rosea isolates 5-3-2 were collected and diluted with sterile distilled water at a concentration of 1 × 106 conidia/ml. Five healthy fruiting bodies were inoculated with 1 ml of the spore suspension, which were maintained in an artificial climate chamber at 22°C and 85% humidity. Sterile water inoculated on other 10 healthy fruiting bodies served as control. Mycelia grew rapidly and overgrew more than half of the fruiting bodies within 3 days. The fruiting bodies died five days after inoculation. Clonostachys rosea isolates were re-isolated from symptomatic fruiting bodies and identified by the methods described above. The control group showed no symptoms. The experiment was conducted twice. To our knowledge, this is the first identification of Clonostachys rosea as the causal agent of the Morchella sextelata rot.

Streptomyces beihaiensis sp. nov., a chitin-degrading actinobacterium, isolated from shrimp pond soil.

A Gram-stain-positive actinobacterium, designated strain GXMU-J5T, was isolated from a sample of shrimp pond soil collected in Tieshangang Saltern, Beihai, PR China. The morphological, chemotaxonomic and phylogenetic characteristics were consistent with its classification in the genus Streptomyces. The organism formed an extensively branched substrate mycelium, with abundant aerial hyphae that differentiated into spores. Phylogenetic analysis of 16S rRNA gene sequences showed that strain GXMU-J5T was most related to Streptomyces kunmingensis DSM 41681T (similarity 97.74 %) and Streptomyces endophyticus YIM 65594T (similarity 96.80 %). However, the values of digital DNA-DNA hybridization, average nucleotide identity and evolutionary distance of multilocus sequence analysis between strain GXMU-J5T and its closest relatives indicated that it represented a distinct species. Strain GXMU-J5T contained ll-diaminopimelic acid and the major whole-cell hydrolysates were xylose and galactose. The predominant menaquinones of strain GXMU-J5T were revealed as MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids consisted of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannosides and phospholipids of unknown structure containing glucosamine. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and anteiso-C17 : 0. The whole genome size of strain GXMU-J5T was 6.79 Mbp with a 71.39 mol% G+C content. Genomic analysis indicated that strain GXMU-J5T had the potential to degrade chitin. On the basis of these genotypic and phenotypic data, it is supported that strain GXMU-J5T represents a novel species of the genus Streptomyces, for which the name Streptomyces beihaiensis sp. nov. is proposed. The type strain is strain GXMU-J5T (=MCCC 1K08064T=JCM 35629T).

Sphaerisporangium perillae sp. nov., isolated from the root of Perilla frutescens (Linn.) Britt.

A novel protease-producing actinomycete, designated strain NEAU-ZS1T, was isolated from the root of Perilla frutescens (Linn.) Britt collected from Jiamusi, Heilongjiang, PR China. Comparative 16S rRNA gene sequencing showed that strain NEAU-ZS1T belonged to the genus Sphaerisporangium and was most closely related to 'Sphaerisporangium corydalis' NEAU-YHS15T (99.2%) and Sphaerisporangium cinnabarinum JCM 3291T (99.0%). Phylogenetic tree analysis revealed that strain NEAU-ZS1T formed a monophyletic clade with 'S. corydalis' NEAU-YHS15T. The genome size was 9.3 Mbp with a DNA G+C content of 70.3 mol%. Digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values between the genome sequence of strain NEAU-ZS1T and those of 'S. corydalis' NEAU-YHS15T (28.6, 83.9 and 79.1 %) and S. cinnabarinum JCM 3291T (18.5, 70.6 and 50.2 %) were below the recommended thresholds for species delineation. The strain formed spherical spore vesicles produced on the aerial hyphae. The cell wall contained meso-diaminopimelic acid and the whole-cell sugars were glucose and madurose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, an unidentified phospholipid and an unidentified glycolipid. The menaquinones were MK-9(H4), MK-9(H6) and MK-9(H2). The major fatty acids were iso-C16 : 0, C16 : 1 ω5c, 10-methy C17 : 0 and C17 : 1 ω7c. On the basis of the results of a polyphasic taxonomic study, it is concluded that strain NEAU-ZS1T represents a novel species of the genus Sphaerisporangium, for which the name Sphaerisporangium perillae sp. nov. is proposed. The type strain is NEAU-ZS1T (=CCTCC AA 2021019T= JCM 35655T).

Integrated enzymes activity and transcriptome reveal the effect of exogenous melatonin on the strain degeneration of Cordyceps militaris.

As a valuable medicinal and edible fungus, Cordyceps militaris has been industrialized with broad development prospects. It contains a lot of bioactive compounds that are beneficial to our health. However, during artificial cultivation, strain degeneration is a challenge that inhibits the industrialization utility of C. militaris. Exogenous melatonin (MT) can scavenge for reactive oxygen species (ROS) in fungus and can alleviate strain degeneration. To establish the significance and molecular mechanisms of MT on strain degeneration, we investigated the third-generation strain (W5-3) of C. militaris via morphological, biochemical, and transcriptomic approaches under MT treatment. Morphological analyses revealed that colony angulation of C. militaris was significantly weakened, and the aerial hypha was reduced by 60 μmol L-1 MT treatment. Biochemical analyses showed low levels of ROS and malondialdehyde (MDA), as well as increasing endogenous MT levels as exogenous MT increased. RNA-Seq revealed that compared with the control, several antioxidant enzyme-related genes were up-regulated under 60 μmol L-1 MT treatment. Among them, glutathione s-transferase genes were up-regulated by a factor of 11.04. In addition, genes that are potentially involved in cordycepin, adenosine and active compound biosynthesis for the growth and development of mycelium were up-regulated. Collectively, these findings provide the basis for further elucidation of the molecular mechanisms involved in C. militaris strain degeneration.

Cellular differentiation into hyphae and spores in halophilic archaea

Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea.

First Report of Leaf Spot Disease Caused by Alternaria brassicae on Camptotheca acuminata in Sichuan, China.

Camptotheca acuminata (C. acuminata), is belongs to a monotypic genus endemic to southwestern China and listed as the first class national protected plant in China in 1999 (Wen, et al. 2020). Camptothecin, isolated from the wood and bark of C. acuminata Decne, which exhibits clinical effects in various cancer treatments (Pommier, et al. 2006; Kang, et al. 2021). In October 2021, we investigated leaf spot disease occurrence on C. acuminata (FigS1.A) with 80% incidence in Beichuan County, Sichuan Province of China. Leaf symptoms were randomly distributed on the adaxial surfaces and consisted of punctate spots of alternating light gray and dark brown in the early stage of onset (FigS1. B, C). As the disease progressed, these spots expanded irregularly shaped regions of necrotic tissue, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. Infected tissues from symptomatic leaves disinfected in 75% ethanol for 45 s, and with 0.1% HgCl2 for 1 min, rinsed then plated on potato dextrose agar (PDA) medium supplemented with ampicillin and carbenicillin (50 μg/ml each). Plates were incubated for 3 days at 25°C. Then prepared by transferring hyphal tips from the edges of these colonies onto fresh PDA medium for subculture. Aerial hyphae had a cotton-like appearance with white to pale gray color (FigS1.D). Conidia were present in long chains, with conidiophores being present in clusters or in isolation (FigS1E), with 1-6 transverse septa, 0-3 oblique and longitudinal septa and an ellipsoidal to obpyriform structure, measuring 10.0-50.9 μm in length and 5.6-11.8 μm in width (n = 20) (FigS1E, G). On the basis of conidial and cultural characteristics, the fungus was consistent with those of members of the Alternaria genus (Simmons, 2008). To confirm this tentative identification, DNA was extracted from isolate XS9, the internal transcribed spacer rDNA regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), partial RNA polymerase II largest subunit (RPB2) genes were amplified with primers pairs ITS1/ITS4 (White et al.1990), GDF/GDR (Templeton et al.), TEF-728F/TEF-986R (Carbone & Kohn 1999) and RPB2-5F2/RPB2-7cR (Sung et al. 1990; Liu et al. 1999), Bt-2a/Bt-2b (Glass and Donaldson 1995) respectively. The resulting sequences were deposited in GenBank (ITS, OP113690; GAPDH, OP120953; TEF, OP120952; RPB2, OP120954). Further phylogenetic analyses of isolate XS9 revealed it to cluster in the A. brassicae clade with 97% bootstrap support. Pathogenicity identification of isolate XS9 was carried out on the detached leaves. The pure agar plugs (as control) or spraying water on the leaf surface were inoculated on detached leaves, the controls remained healthy after 8 days (FigS1.H-J). but the leaves inoculated with other the mycelium plugs (Fig S1K, L) or the conidia suspension (2×105 conidia/mL) of isolate XS9 was sprayed on the detached leaves (Fig S1M, N), both showed brown necrotic lesions that are similar to the symptoms observed in the field. The pathogen was reisolated and confirmed to be A. brassicae. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on C. acuminata in China. Leaf spot disease causes the branches and leaves of camptotheca acuminata to wither and even the whole plant to die. To ensure the protection of the irreplaceable species, effective measures should be taken to prevent the spread of the leaf spot disease.

First report of leaf spot on passion fruit in China, caused by Alternaria alternata.

Passion fruit (Passiflora edulis Sims), which is native to South America, is an important fruit crop in tropical and subtropical countries. Passion fruit growing areas have increased rapidly in southern China. In 2018 to 2019, circular spots on passion fruit were observed in Shangsi, Guangxi, China (21°15'N, 107°98'E). The disease occurred from June to April of the following year. The disease incidence was generally between 10% to 30%, but could reach up to 50% in purple passion fruit 'Tainong No.1'. The initial lesions on the fruits were small, with a brown center and a greasy margin, and then became sunken and lighter brown with a diameter of about 1 cm in later stages. The spots on the leaves were often surrounded by a yellow halo and turned into larger lesions after coalescence.. Five typical symptomatic fruit and leaves were collected from Shangsi county for the presumed pathogen isolation. Section of the samples were surface sterilized to isolate the fungus on potato dextrose agar (PDA) at 28°C. Five fungal isolates with similar morphology on PDA were obtained by single spore isolation. Colonies at the age of 7 days accompany with flourishing aerial hyphae, showed surface color varying from white to grey. Conidia were ovate or elliptic, light brown to brown, with 2 to 5 diaphragms, 0 to 4 longitudinal-oblique diaphragms, and mostly 8.2 to 36.7 μm × 5.4 to 15.8 μm. The morphology of the fungus resembled Alternaria alternata (Fr.) Keissl (Simmons, 2007). Each of the five isolates (SF-001, SF-002, SF-003, SF-004 and SF-005) was molecularly identified using genomic regions of 18S nrDNA (SSU), 28S nrDNA (LSU), RNA polymerase second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) (Jayawardena et al., 2019). Nucleotide sequences of SSU (MZ275254, ON055696, ON055697, ON055698 and ON055699), LSU (MZ275253, ON062947, ON062948, ON062949, ON062950), RPB2 (MZ275251, ON055377, ON055378, ON055379 and ON055380), ITS (MW866522, MW866523, ON053451, ON053452 and ON053453), GAPDH (MZ286628, ON055381, ON055382, ON055383 and ON055384) and TEF1 (MZ275255, ON055373, ON055374, ON055375 and ON055376) were deposited in GenBank database. The LSU, GAPDH and TEF1 sequences showed 100% identity with A. alternata in NCBI (KX609773, MK683852 and MK637432, respectively). The SSU, RPB2 and ITS sequences showed 99% identity to A. alternata (U05194, MK605898 and MN856409, respectively). In pathogenicity test (Zhang et al., 2020), 3-month-old grafted 'Tainong No.1' seedlings and mature fruit were used. Five-mm-diameter mycelial plugs taken from 7-day-old PDA colonies of each of 5 isolates were placed on the leaves and fruit that were wounded with a sterilized needle to form 3 pinpricks. Sterile PDA plugs were used as control. Three plants and three fruits were used in each treatment, and the test was repeated twice. The inoculated plants and fruit were kept in plastic bags and grown in a chamber at 28℃. Typical lesions were observed on inoculated plants and fruit after 3 days, but the controls remained healthy. A. alternata was consistently reisolated from these typical lesions. Previously, leaf spot on passion fruit caused by A. alternata has only been recorded in New Zealand (Rheinländer, 2010). To our knowledge, this is the first report of A. alternata (Fr.) Keissl. causing leaf spot on passion fruit in China. The identification of the pathogen may help to take effective management strategies of controlling this disease.

First Report of Crown and Root Rot Caused by Fusarium solani on Photinia × fraseriDress in China.

Photinia × fraseriDress was introduced to China at the end of the 20th century. It is a kind of colorful ornamental tree species with great ornamental value.From 2020 to 2021, adiseasewasfoundinRailway Gymnasium, Xuanwudistrict of Nanjing which approximately 40% P. × fraserishowed symptoms of blight with discoloration and dieback of crown and root . Symptomatic root tissues collected from three 2-year-old plants were rinsed with water, cut into 2-mm pieces which were surface-sterilized in 70 % ethanol for 60 s, and plated onto Potato Dextrose Agar (PDA), and incubated in the dark at 26 °C for 3 days. Mycelium emerged from 75 % of the samples. Two representative isolates (SG31, SG32) were obtained and deposited in China's Forestry Culture Collection Center. The colony growed in a circular shape, and the early aerial hyphae were white. Later a floccose, white, colony which was dull yellow on the underside was observed . The two isolates had identical morphological features. The macroconidia were sickle-shaped with two to three septa, 22.8 - 43.7 µm × 4.1 - 5.8 µm in size (n=50). The microconidia were numerous, oval, fusiform, renal, or oblong, with zero or one septum, 10.96 - 14.63 µm × 3.89 - 5.76 µm in size (n=50). The hyphae begin to germinate from one or both ends of the spore . Thus, the two isolates were identified as Fusarium solani. For molecular identification, the DNA of the two isolates was extracted. The internal transcribed spacer (ITS) region, β-tubulin (TUB2), and actin gene (ACT) region were amplified using the primer pairs ITS5 / ITS4, T1 / T2 , and ACT-512F / ACT-783R), respectively. The sequences were deposited in GenBank under accession numbers ON329814, ON366356, and ON366358 for SG31 and ON329813, ON366357, and ON366359 for SG32. The ITS, TUB2, and ACT sequences of isolate SG31 were 99.83% (574 / 575 nt), 99.81% (517 / 518 nt), and 100% (248 / 248 nt) identical to those of SF_450 (MT529726.1), CH64 (KU938961.1), and Co.Karbala-IQ1 (MW080737.1), respectively (The comparison results of SG32 were shown in Appendix.). Based on morphological and molecular analysis, the two isolates were identified as F. solani. The pathogenicity of SG31 and SG32 were tested on potted 1-yr-old (30-cm tall) P. × fraseri. Nine plants were dug up to expose root balls, which were wounded before inoculations with a sterile needle, and then inoculated with conidial suspension (106 conidia / mL). Controls were treated with ddH2O. Three seedlings/isolate were used for each treatment. All plants were repotted using the original sterilized potting mix and pots. After inoculation, the plants were covered with plastic bags, and sterilized H2O was sprayed into the bags twice per day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2 / 16 ± 2 ℃. Within 30 days, all the inoculated plants showed lesions similar to those observed in the field, whereas controls were asymptomatic. The isolates were reisolated from the lesions (whereas not from controls) and sequenced as F. solani. Globally, this is the first report of F. solani causing crown blight and root rot of P. × fraseri. Additional surveys are being conducted for mapping the distribution of F. solani in Jiangsu Province of China.

First Report of Alternaria Leaf Blight Caused by Alternaria alternata on Phedimus aizoon in China.

Phedimus aizoon is native to east Asian countries that including China, Siberia, Korea, Mongolia, and Japan. In China, the plant is highly valued for use in folk medicine, for detoxification and analgesia, blood pressure, hemostasis, and used as an ornamental. In August 2021, a leaf spot and blight disease were observed on P. aizoon in a 120-ha field in Pizhou, Jiangsu Province, China where disease incidence reached 90%, and almost every leaf was withered. Early symptoms appeared as dark brown lesions on leaf margins that enlarged and coalesced to form large necrotic areas. In efforts to determine the cause of the disease, ten symptomatic leaves were randomly collected from ten different plants at the site. Diseased leaf pieces that measured 5 mm2 were disinfected in 75% ethyl alcohol for 30 s and 7% NaOCl for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Ten fungal isolates obtained by single-spore isolations were selected for further study. These isolates produced colonies that measured 70 to 82 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces. The isolates produced conidia that were ovate to pear-shaped, brown to black in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, smooth surfaced, parietal cells extending into the beak, and measured 10 to 35.5 × 5.0 to 12.5 μm. Conidiophores were brown, erect or curved, branched, with pronounced spore marks, and measured 7.5 to 37.5 × 2.5 to 5.0 μm. All ten fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates FC01 and FC02 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5, RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nakashima 2020) and Alt-for/Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS, ON584560, ON564492; RPB2, ON729984, ON703241; GAPDH, ON652866, ON652867; TEF1, ON652868, ON652869; Alta1, ON652870, ON652871). Phylogenetic analyses showed 100% identity between FC01 and FC02 and the type strain CBS 916.96. Thus, the fungus was identified as A. alternata based on morphology and molecular analysis. Pathogenicity tests were done by spraying conidial suspensions containing 106 conidia per ml of A. alternata isolates FC01 and FC02 on leaves of five healthy P. aizoon plants, separately. Five control plants were sprayed with distilled water and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25⁰ C. Plastic bags were removed from plants after 48 h. Dark brown lesions developed on inoculated plants after 16 days and control plants remained symptomless. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on P. aizoon in China and worldwide. Based on the plant's medicinal value, further studies should be directed toward control of this disease.

A contractile injection system is required for developmentally regulated cell death in Streptomyces coelicolor

Diverse bacterial species produce extracellular contractile injection systems (eCISs). Although closely related to contractile phage tails, eCISs can inject toxic proteins into eukaryotic cells. Thus, these systems are commonly viewed as cytotoxic defense mechanisms that are not central to other aspects of bacterial biology. Here, we provide evidence that eCISs appear to participate in the complex developmental process of the bacterium Streptomyces coelicolor. In particular, we show that S. coelicolor produces eCIS particles during its normal growth cycle, and that strains lacking functional eCIS particles exhibit pronounced alterations in their developmental program. Furthermore, eCIS-deficient mutants display reduced levels of cell death and altered morphology during growth in liquid media. Our results suggest that the main role of eCISs in S. coelicolor is to modulate the developmental switch that leads to aerial hyphae formation and sporulation, rather than to attack other species.

Leaf Spot Disease on Euonymus japonicas Caused by Nigrospora oryzae in China.

Euonymus japonicas belong to the family Celastraceae and is native to Japan. This ornamental plant has been widely introduced for cultivation as a hedge plant in China. From August to October 2021, severe leaf lesions were observed on E. japonicas in Meicheng garden in Nanyang (32°59'42"N, 112°33'13"E), Henan Province, China. The disease had very wide coverage in the surveyed areas, with foliar diseases reaching 50%-69% (n=200). The early symptoms were yellow or 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 and even branch necrosis. Twenty symptomatic samples were collected from several individual plants, and the junction areas between infected and healthy tissues were cut into 5×5 mm pieces. The tissues were sterilized in 75% ethanol for 30 seconds and 1% NaClO solution for 1 min, rinsed thrice in sterile water and placed on potato dextrose agar (PDA) plates supplemented with 50 µg/ml of streptomycin, incubated at 25°C for 3 days. The edges of the colony were cut and transferred to new PDA plates for purification. These strains showed similar phenotypes in morphological characters. Three representative purified strains (HY12, HY16, and HY17) were used for further study. Colonies were fast-growing, massive sparse aerial hyphae, initially white, later turning gray and black. Hyphae were branched, septa, and transparent. Conidia were single-celled, dark black, oblate, or nearly spherical, and measured 10.7 to 15.4 μm × 9.8 to 15.5 μm in diameter (n=100). For molecular identification, the rDNA internal transcribed spacer (ITS), the β-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified from genomic DNA by primers ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Carbone and Kohn, 1999). Sequences were submitted to GenBank with accession numbers OL840319, OL840320, OL840321 for the ITS sequences, OL961451, OL961452, OL961453 for the TUB sequences, and OL961445, OL961446, OL961447 for the TEF1 sequences of the strains HY12, HY16, and HY17, respectively. BLASTn analyses of these sequences exhibited 99 to 100% identity to Nigrospora oryzae strains 62L1, LC6923, and DP-J2 (MZ151384 of ITS, KY019581 of TUB, and MW562242 of TEF1). These morphological features and molecular identification indicated that the pathogen possessed identical characteristics as N. oryzae (Berk. &Broome) Petch. Pathogenicity was tested through in vivo experiments. Mycelial plugs of the pathogen strains were inoculated on the wounded leaflets, meanwhile, agar plugs served as blank controls. Five 2-year-old plants were grown in pots in a climate incubator maintained at a temperature of 28°C and relative humidity of approximately 90%. Symptoms consistent with those previously described were observed on the inoculated leaves of four plants after 3 to 7 days while the control plants remained healthy. The strains of N. oryzae were reisolated from the symptomatic inoculated leaves, fulfilling Koch's postulates. N. oryzae is known to cause disease on a variety of ornamental plants in China, such as purple blow maple (Sun et al. 2011), cleyera (Wang et al. 2017), cotton rose (Han et al. 2021), and Costus speciosus (Sun et al. 2021). To our knowledge, this is the first report of N. oryzae leaf spot on E. japonicas in China. This identification research will be helpful for subsequent disease control and field management of hedge plants.

First report of Fusarium commune causing root rot of blueberry in Guizhou Province, China.

In February 2017, an unknown root disease was observed on blueberry (Vaccinium uliginosum L.) plants in Fenggang county, Guizhou Province, China. Root symptoms began with small necrotic lesions; subsequently, dark brown rot of the tap root and stem base was observed; finally, the whole plant withered and died. Root tissue was obtained from the junction of diseased and healthy tissue, and the samples were rinsed 3 times with sterile water. Then the surface was disinfected in 75% alcohol for 30 s, washed three times with sterile water, washed with 3% (v/v) sodium hypochloride for 3 min, rinsed three times in sterile water and cultured on PDA at 28 ℃ for 3-5 days. Fungal colonies were subcultured on PDA 3-4 times until purified colonies were obtained. Eight isolates exhibiting morphological characteristics of Fusarium were recovered from symptomatic roots. Six isolates showed morphological characteristics consistent with Fusarium commune. The colonies of LMGF6, a representative, on PDA were white and pink, and there was abundant aerial hyphae. Microconidia were cylindrical, usually without septa, with a dimension of 5.0 to 12.0 μm × 2.0 to 3.5 μm; macroconidia were usually 3-septate and 10.5 µm to 36.5 µm × 3.0 to 5.0 μm in size. Chlamydospores were smooth and spherical. DNA fragments were amplified from LMGF1-6 from the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al. 1990), DNA-directed RNA polymerase II second largest subunit (RPB2) gene using 5f2/7cr primers and translation elongation factor 1-alpha (TEF1) gene using EF1/EF2 primers (Zhong et al. 2021; O'Donnell et al. 2022). The sequences were submitted to GenBank (GenBank accession Nos. OP035965 to OP035970 for ITS, OP106349 to OP106354 for RPB2, and OP106355 to OP106360 for TEF1). BLAST analysis showed 100% identity to those of F. commune. Ten plants were used for the experimental and control. The roots of experimental bushes were inoculated with 50 ml of a 1ⅹ106 conidial suspension. The roots of control plants were irrigated with sterile water. The plants were grown under the ambient conditions (monthly average maximum temperature of 30℃, monthly average minimum temperature of 20℃). After 25 days, all inoculated plants showed symptoms similar to those of the natural infection observed, but no lesions were observed on the control plants. The same fungus was successfully reisolated from all of the inoculated plants and was identified as F. commune based on colony morphology, phylogenetic analysis of concatenated ITS-RPB2-TEF1 sequences was performed using MEGA7.0 and pathogenicity tests. To our knowledge, this is the first report of F. commune causing root rot on blueberry plants in Guizhou Province, China.

First Report of Postharvest Fruit Rot on Citrus reticulata Blanco Caused by Fusarium concentricum in China.

Nanfeng tangerine (Citrus reticulata Blanco) is highly regarded for its nutritional and economic value. In January 2022, an unknown fruit rot was observed on Nanfeng tangerine fruits harvested from Nanfeng County (27.22 °N, 116.53 °E), Fuzhou City, Jiangxi Province after 70 days in storage (25 °C, 90% relative humidity). The disease mostly started from the pedicel or a wound. Symptoms initiated with dark brown lesions that rapidly expanded between the fruit center and pulp capsule causing total fruit rot. The surface of symptomatic fruit was sterilized with 75% ethanol for 30 s and 2% NaClO for 30 s. Small diseased tissue pieces (2 mm2) between diseased and healthy tissues were placed on potato dextrose agar (PDA) and put in an incubator (25 ± 1 °C) for 3 days. The representative isolate NFMJ-1 was subcultured onto PDA using single-spore purification. Colonies on PDA were light yellow to white, with abundant flocculent aerial hyphae. Microconidia were oval, obovoid to allantoid, 0 septate, occasionally 1 septate, 4.07 to 17.53 × 1.69 to 3.56 μm (average=7.40 μm × 2.55 μm, n=50). Macroconidia were slender, with a beaked apical cell and a foot-shaped basal cell, 3 to 5 septate, 22.99 to 81.12 × 2.34 to 3.81 μm (average=45.04 μm × 3.12 μm, n=50). According to morphological characteristics, the isolate was tentatively identified as Fusarium sp. (Leslie and Summerell 2006). To confirm the identification, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), RNA polymerase II second largest subunit (RPB2), beta-tubulin gene (TUB2), and calmodulin gene (CaM) sequences were amplified with primers ITS1/ITS4 (Gardes et al. 1993), TEF1/TEF2 (O'Donnell et al. 2010), RPB2-5f2/RPB2-7cr (Liu et al. 1999), Bt2a/Bt2b (Glass and Donaldson 1995), and CL1C/CL2C (Weir et al. 2012), respectively. The obtained sequences (ON184033, ON212051, ON212052, ON212053, ON212054) showed homology with F. concentricum ITS (MW016417.1; 514/514 bp), TEF (MK609902.1; 667/667 bp), RPB2 (LC631461.1; 941/972 bp), TUB2 (MT942588.1; 331/337 bp), and CaM (MK609916.1; 558/597 bp). A phylogenetic analysis of concatenated ITS-RPB2-TEF sequences was performed by MEGA7.0 with the maximum likelihood and Kimura 2-parameter model, revealing that the isolate was placed in the F. concentricum clade. To confirm pathogenicity, 36 healthy tangerine fruits were surface sterilized with 75% alcohol, then 18 disinfected fruits were wounded with sterile needles and 18 remained unwounded. Half of the wounded and un-wounded fruits were inoculated with 10 μL of a conidial suspension (1.0 × 106 conidia/ml) of isolate NFMJ-1 cultured for 7 days on PDA. Half of the wounded and un-wounded fruits were mock-inoculated with sterile water as controls. After incubation in an incubator (25 ± 1°C, 90% relative humidity) for 7 days, the wounded fruits inoculated with F. concentricum showed similar symptoms to the original diseased fruits, while the mock-inoculated fruits were asymptomatic. The pathogenicity test was repeated three times. The pathogen was re-isolated from the wound-inoculated fruits and identified as F. concentricum by morphological and molecular analysis, completing Koch's postulates. F. concentricum has been reported as a pathogen of Podocarpus macrophyllus (Dong et al. 2022), Capsicum annuum (Wang et al. 2013) and Zea mays (Du et al. 2020) in China. This is the first report of fruit rot caused by F. concentricum on Citrus reticulata in China. Appropriate prevention and control measure of the pathogen need to be developed to preserve marketability of this economically important citrus fruit.

First Report of Leaf Spot Disease Caused by Boeremia linicola on Trifolium repens in China.

Trifolium repens L. (White clover) a multipurpose legume crop, primarily utilized as a green manure in China. In March 2018, field investigations showed that a leaf spot disease occurred on T. repens in three fields with 50% to 80% incidence (50 plants in each field were investigated) in Nanchong City, Sichuan Province of China. Infected leaves showed symptoms of irregular dark brown spots in the center of leaves or at the leaf margins. Symptomatic leaves were surface sterilized with 3% NaClO for 3 min followed by 75% ethanol for 30 s and then rinsed in sterile water three times. Thereafter, tissue samples from margins of individual lesions were placed on potato dextrose agar and incubated at 25°C in the dark. Four pure cultures (Y3-1; Y3-2; Y3-3; Y3-4) were obtained by single spore isolation. On oatmeal agar medium, a colony reached 57 mm diameter after 9 d in alternating light and dark at 25℃. Fimbriate aerial hypha were flat and compact with pale brown to dark brown color. Conidiogenous cells were hyaline, smooth, ampulliform to doliiform (n = 30), ranging from 4.1 to 10.5 μm long (6.6 ± 1.8 µm) × 2.2 to 5.7 μm wide (4.1 ± 0.8 µm). Conidia were ellipsoidal to cylindrical, hyaline, thin walled, smooth, aseptate, with 2 to 4 polar guttules (n = 50), ranging from 3.2 to 11.3 μm long (6.3 ± 1.1 µm) × 2.1 to 4.1 μm wide (2.8 ± 0.4 µm). Conidial matrix was whitish. Morphologically these isolates resembled species in Boeremia (Chen et al. 2015). Genomic DNA of each culture was extracted from mycelia using the quick and safe method (Chi et al. 2009). The 28S large subunit of nuclear ribosomal RNA (LSU) region, internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α (TEF 1-α), β-Tubulin (TUB2) were amplified with corresponding primers (Carbone and Kohn 1999; Liu et al. 1999; Rehner and Samuels 1994; Sung et al. 2007; Vilgalys and Hester 1990; White et al. 1990; Woudenberg et al.2009). Sequences were deposited in GenBank with accession numbers: ON705759 to ON705766, ON734043 to ON734046 and ON841585 to ON841592. Phylogenetic analysis was conducted with combined sequences of the five loci using the maximum likelihood (ML) and the maximum parsimony (MP) methods. The four isolates and the extype strain of B. linicola (CBS 116.76) clustered together with high bootstrap support (BS) values (MLBS = 100; MPBS = 98). All sequences showed 100% identity to those of CBS 116.76, except the ITS region of our isolates (ON705759 to ON705762), which show 99.6% identity to that of CBS 116.76. Based on morphological characteristics and phylogenetic results, our isolates were identified as B. linicola, although the morphological characteristics of CBS 116.76 had not been characterized. To assess pathogenicity, a conidial suspension (approximately 105 CFU/mL) of isolate Y3-1 was sprayed on 1-month-old healthy plants in a greenhouse at 22℃ to 28℃. Plants sprayed with sterilized water were used as negative controls. The test was conducted three times, each with 3 plants. After 10 days, the leaves of the plants showed irregular brown lesions that were similar to the symptoms observed in the field, control plants remained healthy. The pathogen was reisolated and confirmed to be B. linicola, thus completing the verification of Koch's Postulates. Compared to B. exigua, a causal pathogen associated with leaf spot on white clover reported by Wang et al (Wang et al. 2021), B. linicola produced larger conidia, and the two species did not cluster together in the phylogenetic tree. To our knowledge, this is the first report of leaf spot disease caused by B. linicola on Trifolium repens in China.

First Report of Brown Leaf Spot Caused by Neofusicoccum parvum on Photinia × fraseri Dress (Christmas berry) in China.

Christmas berry (Photinia × fraseri Dress), known as red tip photinia, is an important landscape color plant in South China (Zhu et al., 2021). In summer 2021, brown leaf spots of Christmas berry were observed with an incidence of 45-70% on the campus of Nanchang Institute of Technology, Jiangxi Province (28°41'32.61"N, 116°1'53.75"E), China. These spots have a negative influence on the photosynthetic activity and severely reduce the ornamental value of these plants. Lesions originally occurred on the margins or tips of the leaves as red patches (Figure 1 A) and then developed into irregular reddish-brown necrotic spots with yellow halos (Figure 1 B) that finally were grey lesions with dark brown ring circles surround with yellow halos (Figure 1 C). Ten leaf samples with typical symptoms from five plants were collected. The tissue between the healthy and necrotic area (ca. 5 mm × 5 mm) was cut with a sterile scalpel and surface sterilized in 70% alcohol for 45 s, followed by 2% NaClO for 2 min and washed in sterile deionized water three times, The sterilized leaf parts were placed on potato dextrose agar (PDA) and incubated at 25℃ for 3 to 5 days. A total of 28 fungal isolates were obtained from ten symptomatic leaves, which were classified into five species by morphological characteristics. Neofusicoccum was the genus with the highest isolation frequency (50%). For pathogenicity tests, healthy leaves of Christmas berry were surface-sterilized, wounded using a sterile needle and inoculated with mycelial plugs (6 mm in diameter) on the left site of the leaves. Five representative isolates were chosen and ten leaves were used for every isolate. The controls on the same leaves on the right side of the leaves were treated with sterile PDA plugs. All samples were placed in an artificial climate box (RH 90±3%, 27±1℃, 12 h light) for 3 days. Only the leaves inoculated with isolate NH7 showed dark brown leaf spots (Figure 1 D). The control leaves and those inoculated with the other four isolates remained symptomless. To confirm pathogenicity, mycelial plugs of isolate NH7 were applied on punctured leaves of Christmas berry in the field as the artificial climate box (Figure 1 E-H). Inoculation with a sterile PDA plug served as control. All the leaves were covered with plastic bags for 48 h to maintain high relative humidity. Seven days later, symptoms similar to those observed in the field developed on all leaves inoculated with isolated NH7, while the controls remained symptomless. To fulfill Koch's postulates, fungal isolates were reisolated from symptomatic leaves and identified by morphological and molecular characteristics. The colony of isolate NH7 developed aerial hyphae, which had a grey center surrounded by grey-white hyphae (Figure 1 I). Conidia were aseptate, primarily fusiform and measured 11.1-19.8 × 3.2-7.1 µm (n = 50) (Figure 1 J). For further confirmation of the identity, five genes, including ITS (White et al., 1990), RPB2 (Pavlic et al., 2009), tub2 (Glass & Donaldson, 1995), and tef1 (Carbone & Kohn, 1999) were sequenced. The sequences were deposited in GenBank (OL584411 for ITS, OL606622 for ACT, OL694623 for tub2, OM141481 for RPB2 and OM141482 for tef1), Based on the phylogenetic tree analysis using IQ-TREE, isolate NH7 clustered with Neofusicoccum parvum (Figure 2). N. parvum has been reported to cause leaf spot disease on different plants including Myristica fragrans (Jayakumar et al., 2011), Ginkgo biloba (Mirhosseini et al., 2014), Vitis heyneana (Wu et al., 2015), rubber tree (Liu et al., 2017) and Geodorum eulophioides (Du et al., 2021). To our knowledge, this is the first report of N. parvum causing leaf spot disease on Christmas berry in China. As one of the most widely planted ornamental shrubs in China, the detailed knowledge of the pathogens targeting Christmas berry is critical.

First report of leaf blight on Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa caused by Alternaria alternata in China.

Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa is one of the sources of Chinese herb "Gao-Ben". It is widely distributed in the Northeastern China. L. jeholense has antipyretic, antibacterial and anti-inflammatory effects (Zhang et al. 2021). In September 2021, a serious leaf blight was found in a 1.2 ha plantation of L. jeholense in Harbin, Heilongjiang Province, and the incidence was about 85%. The foliar symptoms were grayish-brown lesions, surrounded by a yellow margin at the edge of the leaf. In serious cases, the lesions extended into the middle of the leaf, and finally the whole leaf withered. A total of 12 samples (5×5mm) from symptomatic and healthy junction of 12 infected leaves from 6 different plants of L. jeholense with typical symptoms were cut and surface disinfected in 75% ethanol, and with 7% NaClO for 1 min, then rinsed three times with sterilized water. These tissues were placed onto Potato dextrose agar (PDA) plates at 28℃ in the dark. The colonies cultured for 7 days were obtained and transferred onto new PDA and potato carrot agar (PCA) plates by single spore method to further purify. After 7 days, the colonies on PDA were 63 to 75 mm in diameter, circular, grayish, with white aerial hyphae on the edge, the back of the colonies were grayish green. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear, 2 to 6 transverse septa, 0 to 3 longitudinal or oblique septa, 16.5 to 67.5 × 8.5 to 20.5 μm. The beaks were conical or cylindrical, 2.5 to 25.3 × 2.0 to 3.0 μm. Conidiophores were grayish brown, erect or bent, separated, 57.0 to 137.0 × 5.1 to 13.7 μm. Morphological characteristic showed the 12 isolates were the same fungus and similar to Alternaria sp. (Simmons 2007). Two typical strains (LGB and LGB2) from twelve isolates were randomly selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates on PDA by modified CTAB method, and internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit (RPB2) and Alternaria major allergen (Alt a 1), translation elongation factor 1-alpha (TEF) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified and sequenced with the primers ITS1/ITS4, RPB2-5F2/RPB2-7CR, Alt-F /Alt-R, TEF-F/TEF-R and gpd-F/gpd-R (Woudenberg et al. 2015). The obtained sequences were deposited in GenBank (ITS: OM319506, OM943431; RPB2: OM393721, OM984854; Alt a 1: OM649816, OM984853; TEF: OM238108, OM984852; gpd: OM296228, OM984851). The phylogenetic analysis of maximum-likelihood tree by MEGA7 showed the LGB and LGB2 had 100% identity with A. alternata CBS 916.96. For pathogenicity test, conidial suspension (1 × 106 spores/mL) of the strain LGB and LGB2 was sprayed on 10 healthy 40-day-old L. jenholense plants and five plants with sterile water as control. The plants were incubated at 25℃. After 28 days, grayish withering appeared on the leaves. The test was repeated three times. The same fungi were re-isolated from the inoculated leaves and with the same morphological and molecular characteristics as A. alternata, fulfill the Koch's postulates. No symptoms and fungi were found in the control group. This is the first report of leaf blight on L. jenholense caused by A. alternata. Leaf blight could reduce the yields of L. jenholense. This study provides a reference for the prevention and treatment to the leaf blight of L. jenholense.

First report of leaf spot on Clematis brevicaudata DC. caused by Alternaria alternata in China.

Clematis brevicaudata DC. is distributed in China, Korea, Mongolia, Russia and Japan. This plant is both ornamental and medical, used in the treatment of nervous disease, dyskinesia and other diseases. In September, 2019, a leaf spot on C. brevicaudata was first found in a 5 ha C. brevicaudata plantation in Harbin, Heilongjiang Province, China. The incidence was about 80%. The symptoms were elliptical, circular, or irregular brown to black necrotic lesions in leaf apex and leaf margin. Ten fresh sample leaves with typical symptoms were collected from ten C. brevicaudata plants. The tissues (5mm×5mm) between symptomatic and healthy junction were cut and surface disinfected in 75% ethanol, and with 7% NaClO for 1 min, then rinsed three times with sterilized water, 30s each time. The sterilized tissues were inoculated on potato dextrose agar (PDA) plates for 7 days at 25℃. The colonies were obtained and transferred onto new PDA and potato carrot agar (PCA) plates by single spore method to further purify. After 7 days, the colonies on PDA were 50 to 63 mm in diameter, circular, grayish brown, with white aerial hyphae. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear, 2 to 7 transverse septa, 0 to 3 longitudinal or oblique septa, 17.5 to 57.5 × 7.5 to 17.5 μm. Beaks and supposititious beaks were mostly columnar, rarely conical, 2.5 to 6.0 × 2.0 to 3.0 μm. Conidiophores were solitary or clustered, pale brown, erect or bent, branched or unbranched, separated, 112.0 to 151.0 × 5.1 to 14.7 μm. Ten isolates purified on PDA were obtained. Morphological identification showed the ten isolates were similar and appeared to be Alternaria alternata (Simmons, 2007). Two strains from ten isolates were selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates (LD2020520 and LD2020521) on PDA using a modified CTAB method. Internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit gene (RPB2), Alternaria major allergen (Alt a 1), endopolygalacturonase (endoPG) and glyceraldehyde 3-phosphate dehydrogenase (gpd) were amplified and sequenced using two directional sequencing with the primers ITS1/ITS4, RPB2-F/RPB2-R, Alt-F/Alt-R, end-F/end-R and gpd-F/gpd-R (Woudenberg et al. 2015). The sequences obtained were deposited in GenBank (ITS: MT501762, OK571395; RPB2: MT506027, OK631891; Alt a 1: MT506026, OK631890; endoPG: ON054189, ON054188; gpd: ON054191, ON054190). The phylogenetic analysis of maximum-likelihood tree by MEGA 7 software showed that the two isolates had 99% identity with the A. alternata CBS 916.96. For pathogenicity testing, eighteen leaves of six 5-week-old plants were sprayed with spore suspensions (1×106 spores /mL) of the 7 days-old isolates LD2020521 and LD2020520 (Each isolate infected three plants and each infected three leaves). Three plants were sprayed with sterile distilled water as a control group. The plants were incubated at 25℃. After 15 days, taupe irregular spots appeared on the leaves. The pathogenicity test was repeated three times. The same fungi were re-isolated from the inoculated leaves and with the same morphological and molecular characteristics as LD2020520 and LD 2020521, fulfilling Koch's postulates. No fungi were isolated from the control group. This is the first report of leaf spot on C. brevicaudata caused by A. alternata. Leaf spot can reduce the yields of C. brevicaudata. This study provides a reference for the prevention and treatment to the leaf spot of C. brevicaudata.

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 Verticillium dahliae causing Verticillium Wilt on Radish in South Korea.

Radish (Raphanus sativus L.) is an important crop grown in Korea primarily for its use in kimchi, and is cultivated on an area of about 23,000ha. In September 2016, radish leaves were observed with yellowing and wilting symptoms in Gangneung (37.55406°N, 128.84871°E) and Jeongseon (37.42895°N, 128.85882°E), Gangwon province, South Korea. Disease incidence was estimated at approximately 10% in two fields, respectively. About 30% of radish plants (cv. Gwangdongyeoreum) with foliar symptoms exhibited vascular discoloration in the roots. Small pieces of discolored root tissue were surface sterilized in 1% NaOCl for 30s, and then rinsed in sterile water. The tissue pieces were placed on water agar and incubated at 25°C for 10 days. Eight isolates were obtained through single spore isolation, and a representative isolate NC16557 (from Gangneung) was selected for identification. After 14 days, colonies on potato dextrose agar (PDA) were 3.7 cm in diameter and becoming dark due to the formation of microsclerotia. Aerial hyphae were smooth-walled, 3 to 4 μm wide. Conidiophores were erect or slanted, verticillately branched or unbranched, and hyaline. Conidia were hyaline, smooth-walled, non-septate, cylindrical to oval, 4.8 to 6.6 × 2.6 to 3.4 μm (mean 5.7 × 2.9 μm, l/w =2.0, n =80). Microsclerotia were immersed in the agar, composed of rounded, brown-pigmented cells and elongate or irregular in shape, 42 to 60 μm diam. Based on morphology, NC16557 isolate was tentatively identified as Verticillium dahliae (Hawksworth and Talboys, 1970; Isaac, 1967). To confirm taxonomic placement, DNA extracted from mycelia of the same isolate was PCR amplified and sequenced targeting internal transcribed spacer (ITS) regions of rDNA, translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes (Inderbitzin et al., 2011). The sequences were deposited in GenBank with accession numbers MZ723402, and MZ735720-MZ735723, respectively. The sequencing results showed 100% (ITS=479/479, TEF=500/500, ACT=185/185, and TS=295/295) and 99.61% (GPD=257/258) similarity with the sequences of V. dahlia type strain PD322 (LR026889, HQ414624, HQ206921, HQ414909, and HQ414719) by BLAST. Based on the morphology and multigene sequence analysis, the isolate was identified as V. dahliae. Pathogenicity of two isolates (NC16557 and NC16547) was carried out in the greenhouse using ten 10-day-old seedlings (cv. Gwangdongyeoreum) by root-tip cutting and then soaking the roots in a fungal spore suspension of 106 conidia mL-1 for 1 hour. Ten seedlings were treated with sterile distilled water as a control. All plants were placed in the greenhouse at 15°C (night)/25°C (day) with natural light. After 6 weeks, all inoculated plants showed vascular discoloration in the roots while control plants remained asymptomatic. However, the above-ground symptoms of inoculated plants, such as yellowing and wilting, were indistinguishable from control plants. V. dahliae was consistently re-isolated from the symptomatic root tissues and the pathogen identity was confirmed by observing morphological characteristics. Verticillium wilt of radish has been reported from China(Yan et al., 2019). In Korea, this is the first report that V. dahliae causes Verticillium wilt of radish, although Dumin et al. (2020) already reported on Verticillium wilt of Chinese cabbage in Gangwon province, the main production area of Chinese cabbage and radish in summer. With these findings in Chinese cabbage and now radish, it will be critical to identify or develop Verticillium-resistant varieties and other management strategies for the stable production of these crops in Korea.

First report of Fusarium tricinctum causing fruit rot on navel orange (Citrus sinensis (L.) Osbeck) in China.

Citrus sinensis (L.) Osbeck is popular with consumers for its delicious taste. In December 2020, a rot symptom causing about 15% losses of a total of 450 fruits was observed on 'Newhall' navel oranges after 70 d storage (20℃, 85%-90% RH) at Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables (28.68° N, 115.85° E). The fruits were harvested from an orchard in Ganzhou City, Jiangxi Province, China (25.53° N, 114.79° E). Incipiently, the pedicles of infected fruits were brown, the peels became softened and showed yellowish-brown lesions which, gradually expanded and had white hyphae (Fig. S1A). To isolate the pathogen, the surface of diseased fruits was disinfected with NaClO (2%) for 2 min and ethanol (75%) for 0.5 min, then washed with sterile water three times. Tissues (5 × 5 mm) around the lesion were incubated on potato dextrose agar (PDA) at 28 ± 1℃ (L: D=12: 12) for 5 days. Five cultures with similar morphology were obtained and colonies initially produced white aerial hyphae and became khaki then turned pink on PDA (Fig. S1F, G, H). Abundant microconidia, macroconidia and rare chlamydospores were observed after 10 days on PDA and no glucose PDA media (Zhang et al. 2020). Macroconidia were falciform and curved to lunate, 2-4 septa, 29.38 × 3.75 µm in size (n=50) (Fig. S1K, Fig. S3). Microconidia were oval, napiform or pyriform, 0-1 septa, 12.00 × 3.43 µm in size (n=50) (Fig. S1L1 to L4, Fig. S3). Chlamydospores were found in hyphae, ellipsoidal or orbicular (Fig. S1I-1 to I-2, J-1 to J-2). The morphological features of five isolates were similar to Fusarium (Leslie and Summerell 2006). Genomic DNA of five isolates was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), ITS1/ITS4, EF1Ha/EF2Tb and fRPB2-5F/fRPB2-7cR primers were used to amplify the internal transcribed spacer region (ITS), and the transcriptional elongation factor-1 alpha (TEF-1α), and RNA polymerase II (RPB2) gene sequences (White et al. 1990; Carbone and Kohn 1999; Liu et al. 1999). The ITS, TEF-1α and RPB2 sequences of five isolates were deposited in GenBank and showed 99-100% identity with corresponding sequences from F. tricinctum (Table S1). A phylogenetic tree was constructed with ITS-TEF-1α-RPB2 concatenated sequences in MEGA7.0 (Li et al. 2021) and all five isolates were placed in F. tricinctum clade with 100% bootstrap support (Fig. S2). To confirm pathogenicity, ten healthy C. sinensis fruits were surface-sterilized with 75% ethanol and inoculated with 10 µL spore suspension (1.0 × 106 spore/mL) including five wounded (with sterilized needle) and five unwounded (Fig. S1B to E). Control fruits were inoculated with 10 µL sterile water. All fruits were incubated at 28 ± 1℃, 90% RH for 7 days. The experiment was conducted three times. The lesion diameter of inoculated wounded fruits was 21.01 ± 2.52 mm and showed similar symptoms to original rotten fruits. However, the control and unwounded fruits remained healthy. To fulfill Koch's postulates, F. tricinctum was re-isolated from the inoculated fruits and deposited in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. To our knowledge, F. tricinctum has been reported on apple tree and kiwi plant in China (Zhang et al. 2021; Ma et al. 2022), but this is the first report of F. tricinctum causing fruit rot on navel orange in China. This finding provides important information for preventing postharvest disease of citrus.