Single-spore Isolates Research Articles

First report of tomato wilt caused by Fusarium brachygibbosum in China.

Tomato (Solanum lycopersicum) is a common vegetable in the Solanaceae family, which as a nutritious vegetable, is widely planted in China. In July 2022, typical wilt symptoms were observed in tomato fields located in the regions of Shiyan (31.343781°N, 110.901005°E), Hubei. Surveys were performed on tomato plants showing symptoms of leaf chlorosis, dry wilt, and vascular wilts on stem and root. The disease incidence ranged from 40% to 70% in 12 surveyed fields, 11.2 ha in total.. After using a sterilized scalpel to cut a small piece of diseased tomato stem and root tissue, diseased tissue was surface disinfested in 75% ethanol for 30s, placed onto potato dextrose agar (PDA) medium,and incubated at 25℃ for 3 days. Then, the emerging single fungal hypha tip was cut and transferred into PDA plates to obtain single spore isolates. Sixteen fungi growing on PDA plates were initially white colonies with abundant aerial mycelium. After seven days of growth, the center of the plate was yellow to orange and red pigmentation was produced. The five-day-old cultures grown on mung bean medium produced macroconidia that were scarce and scattered, three to four septa, wide central cells, slightly sharp apexes, and ranged in size from 12.6-23.6 μm×2.8-4.1 μm (n=30). Microconidia were slightly curved, ovoid with zero to two septa, measuring 5.2-11.8 μm×1.8-2.7μm (n=30). Spherical chlamydospores were terminal or intercalary, and measured a diameter of 8.1-11.6 μm (n =30). Therefore, sixteen isolates were morphologically identified as Fusarium spp. Moreover, the genomic DNA of the isolates (HBSY-1,HBSY-2, and HBSY-3) was extracted for amplification and sequencing of the regions of internal transcribed spacer (ITS) (White et al., 1990), nuclear large subunit rRNA (nLSU) (O'Donnell, 1992; Vilgalys and Hester, 1990), and the translation elongation factor 1-alpha (EF1-α) (O'Donnell et al. 1998) with primers ITS1/ITS4, NL1/LR3, and EF1/2, respectively. Sequences were submitted to GenBank under accession numbers OP959509, OQ568650, OQ568651 (ITS), OQ186731, OQ568652, OQ568653 (nLSU), and OP957576, OQ572485, OQ572486 (EF1-α). BLASTn showed that the sequences ITS, nLSU, and EF1-α were matched 99.61% (508/510 bp; KU528864.1), 99.90% (993/994 bp; GQ505450.1), and 99.85% (651/652; ON032449.1) to Fusarium brachygibbosum, respectively. Multilocus phylogenetic analysis showed that the isolate was of the same clade as F. brachygibbosum. Therefore, morphological characterization and molecular data identified the fungus as F. brachygibbosum. Pathogenicity of the isolate (HBSY-1) was tested on ten seedlings of tomato (cv. Hezuo908). Tomatoes were inoculated by spraying with conidial suspensions (1×107 spores/mL) at the rootstock region of each plant. In addition, ten negative control plants were treated with sterile water. All plants were incubated in an artificial climate box (LongYue, ShangHai) at 25℃ for 12 days. The experiment was repeated three times. Twelve days later, inoculated tomatoes developed typical wilting symptoms of leaves and vascular wilts of stem and root, while the control plants remained healthy. Thus, pathogens were reisolated from the stems of inoculated plants and not from control plants. To our knowledge, this is the first report of F. brachygibbosum causing leaf wilt and vascular wilts of stem and root on tomatoes in China.

First report of Rice panicle blight caused by Fusarium sporotrichioides in China.

Rice (Oryza sativa L.) panicle blight, caused by various fungal and bacterial pathogens, is an emerging threat to rice production, due to the impact on rice yield and quality. In the autumn of 2020, a survey was conducted to understand the etiology of the disease in Liaoning province, an important rice growing area in northeastern China. Rice seeds with typical reddish or brown spots on the glumes were collected from various rice fields. Symptomatic seeds were sterilized with 5% sodium hypochlorite and 75% ethanol, rinsed in sterile distilled water, and placed on potato dextrose agar (PDA) plates. After 3 to 5 days of incubation at 25°C, suspected Fusarium strains showing cream to salmon colonies on PDA were purified by the single-spore isolation method. The identification of these strains were performed based on morphological and molecular characteristics. Fusarium incarnatum-equiseti species complex was the most frequently, followed by the members of Fusarium graminearum species complex and Fusarium fujikuroi species complex. However, one strain was identified as F. sporotrichioides Sherb. based on the following results: (I) Colonies on PDA produced dense mycelia and typical red pigment on the undersurface. Macroconidia were usually 3- to 5-septate, moderately curved to straight, and 27.46 ± 7.16 × 3.78 ± 0.8 μm (n = 50). Microconidia were ellipsoid to fusoid and 9.77 ± 2.29 × 2.99 ± 0.65 μm (n = 50). (II) Genomic DNA was extracted by AxyPrep Multisource Genomic DNA Miniprep Kit according to the manufacturer's instructions; the sequence analysis of partial translation elongation factor-1α (TEF-1α) and RNA polymerase II second largest subunit (RPB2) were accomplished with the primers EF1/2 and RPB5F/7CR, respectively. BLAST searches of the obtained sequences had 99-100% homology with several F. sporotrichioides strains from GenBank. DNA sequences of partial TEF-1α and RPB2 were deposited in GenBank as OQ068267 and OQ068269, respectively. (III) This strain can synthesis T-2, HT-2, diacetoxyscirpenol (DAS), and Neosolaniol (NEO) toxin at the concentration of about 5000, 600, 700 and 8000 μg/kg in rice culture, respectively, according to the previous culture and analysis methods (1,2). (IV) Pathogenicity tests were conducted with the rice variety Nanjing 9108 by spraying spore suspension (106 conidia/ml) on rice spikes (5 mL per spike) at the flowering stage. Control spikes were inoculated with sterile distilled water. Three weeks after inoculation, the inoculated rice glumes showed similar symptoms with the original samples in the field. No symptoms were observed on controls. Besides, F. sporotrichioides was successfully recovered from the inoculated rice spikes but not from controls. To our knowledge, this is the first report of F. sporotrichioides causing rice panicle blight in China and this disease appears to be a serious risk to food safety and human health. Funding: This work was supported by Jiangsu Agriculture Science and Technology (CX(21)1005).

First Report of Leaf Spot on Soybean Caused by Epicoccum sorghinum in Heilongjiang Province, China.

Soybean (Glycine max (Linn.) Merr.) is one of the important oil crops in China. In September 2022, a new soybean leaf spot disease was found in Zhaoyuan County, Suihua City, Heilongjiang Province, China. Symptoms of the initial formation of irregular brown lesions on the leaves, dark brown inside, the periphery is yellow, vein chlorotic yellow, severe leaf spots connected into pieces, late fall off, not the same as previously reported soybean leaf spot (Fig. 1A). The leaf samples of infected plants were collected, and the leaf tissue (5 × 5 mm) was cut from the edge of the lesion, and then surface sterilized with 3% sodium hypochlorite for 5 min, rinsed with sterile distilled water for 3 times, and inoculated on potato dextrose agar (PDA) at 28°C. Isolates growing around the tissues from samples were subcultured on PDA, and 3 isolates were obtained using the single-spore isolation method. The fungal hyphae were white or grayish white in early stage, and the hyphae with light green concentric ring appeared on the front of the colony after 3 days, appeared orange, pink or white convex, irregular shape, reddish brown on the front of the colony for 10 days and black spherical pycnidium can be produced in the hyphae layer for 15 days (Fig.1D, E). Conidia were oval, hyaline, unicellular, aseptate, and 2.3 to 3.7 × 4.1 to 6.8 μm (n=30, Fig. 1F). Chlamydospores were subglobose, light brown, unicellular or multicellular, and 7.2 to 14.7 × 12.2 to 43.9 μm (n=30, Fig. 1H, I). Pycnidia mostly spheroid, brown, and 47.1 to 114.4 × 72.6 to 167.4 μm (n=30, Fig. 1G). A cetyl trimethyl ammonium bromide method was used to extract DNA from 7-day-old. Internal transcribed spacer (ITS), RNA polymerase II (RPB2) and β-tubulin (TUB) gene were amplified using ITS1/ITS4 (White et al. 1990), RPB2-5F/RPB2-7cR (Liu et al. 1999) and BT2a/Bt2b (O'Donnell et al. 1997) primers respectively. The sequences obtained by polymerase chain reaction (PCR) were sequenced and the results showed that the DNA sequences of the 3 isolates were identical. Therefore, the sequence of isolate DNES22-01, DNES22-02 and DNES22-03 was submitted to GenBank. According to BLAST search, the ITS (OP884646), RPB2 (OP910000) and TUB (OP909999) sequences showed 99.81% similarity to Epicoccum sorghinum strain LC12103 (MN215621.1), 99.07% to strain P-XW-9A (MW446946.1), and 98.85% with the strain UMS (OM048108.1), respectively. Phylogenetic analysis by maximum likelihood method (MEGA7.0) generated based on the ITS, RPB2 and TUB sequences indicated that the isolates formed a supported clade to the related E. sorghinum type sequences. Isolates was found to be most closely related to E. sorghinum and far from other species. Based on morphological and phylogenetic characteristics, isolates DNES22-01, DNES22-02 and DNES22-03 was identified as E. sorghinum (Bao et al. 2019; Chen et al. 2021; Zhang et al. 2022). At the 4-leaf-stage, 10 soybean plants were inoculated by spraying with a conidial suspension (1 × 106 spores·ml-1). Sterile water served as a control. The test was repeated 3 times. All samples were incubated in a growth chamber at 27°C. Symptoms typical developed on the leaves after 7 days, but control samples remained healthy (Fig.1B, C). The fungus was reisolated from symptomatic tissues and identified as E. sorghinum by morphology characteristics and molecular characterization. To our knowledge, this is the first report of E. sorghinum causing leaf spot on soybean in Heilongjiang, China. The results can provide the basis for future studies on the occurrence, prevention, and management of this disease.

First Report of Fusarium asiaticum Causing Sheath Rot of Zizania latifolia in China.

Zizania latifolia is perennial plant, belonging to the rice tribe (Oryzeae) of the grass family Poaceae (Xu et al. 2020), which is also called jiaobai in China and commonly consumed as a vegetable crop. In 2022, a sheath rot occurred on Z. latifolia plants in Lishui, the Zhejiang Province of China. Symptoms occurred on the leaf sheath and initially showed as water-soaked chlorotic spots, later enlarging to irregular, elliptic, and elongated dark brown necrotic lesions. Later, lesions fused and extended to most of the leaf sheath leading to wilting. Almost 60% of the surveyed Z. latifolia plants in 100 hectare were affected. Diseased samples were collected for pathogen isolation. Symptomatic tissues were taken from the edge of lesions, sterilized for 10 s in 70% ethanol, then 2 min in 1% NaClO, washed three times with sterile distilled water, and placed on potato dextrose agar (PDA) at 26 °C in the dark. Fungal colonies displaying similar morphology were picked and purified by single spore isolation. In total, 8 isolates were obtained from 8 plant samples. When cultured on PDA, fungal colonies were white, gradually turning pale yellow with time. Macroconidia only were produced on Carnation leaf agar (CLA) and were hyaline, slender, falcate with single foot cells, 3 to 5 septate, and measured 29 to 50 μm × 3.75 to 5.0 μm. Chlamydospores were globose to subglobose and measured 6.8 to 16.5 μm. These morphological features were consistent with the description of Fusarium asiaticum (Leslie and Summerell 2006). For molecular identification, the partial translation elongation factor 1 alpha (TEF1-α) gene and RNA polymerase II second largest subunit (RPB2) gene of three representative isolates were amplified and sequenced (O'Donnell et al. 1998). These sequences were identical to each other, and one representative, Z-3-1, was deposited in GenBank (Accession No. OQ129437 and OQ858619, respectively). Analysis of the TEF1-α and RPB2 sequences of Z-3-1 showed that they were 99.85% (688/689) and 100% (945/945) identical to F. asiaticum strain Daya350-3 (KT380124) and MRC 1976 (MH582121), respectively, in NCBI, and had 99.38% and 100% identity to F. asiaticum strain CBS 110257 (AF212451 and JX171573) in Fusarium-ID. A combined phylogenetic tree based on the TEF1-α and RPB2 sequences showed that Z-3-1 was clustered with F. asiaticum using the neighbor-joining algorithm. Pathogenicity testing was conducted by inoculating potted Z. latifolia plants with a 1×105 conidial suspension of isolate Z-3-1, which was prepared by culturing the fungal strain in PDB at 26°C for 4 days in a shaker incubator. Conidial suspensions (1 mL) were dropped onto sheaths of potted Z. latifolia plants with sterile water serving as controls. All inoculated plants were covered with plastic bags and maintained in a humid growth chamber at 26°C with a photoperiod of 16 h. The inoculation experiment was repeated twice with 5 replicates per test. Four days later, the sheaths of potted inoculated plants displayed symptoms similar to those observed in the field. No symptoms were observed on control plants. Fusarium asiaticum was re-isolated specifically from the symptomatic inoculated Z. latifolia plants and confirmed by morphological and molecular methods, thus fulfilling Koch's postulates. Fusarium asiaticum has been reported to be a pathogen of other plants in China, such as Ligusticum (Zhu et al. 2022) and Setaria italica (Kong et al. 2022). To our knowledge, this is the first report of F. asiaticum causing sheath rot of Z. latifolia in China. The identification of the pathogen is the first step in developing appropriate field management strategies for this new disease.

First report of Fusarium equiseti causing bulb rot on lily (Lilium 'White planet' ) in China.

Lily (Lilium spp.) is one of the main ornamental plants grown in the world. In addition, bulbs of lily have been extensively used as edible and medicinal herbs in northern and eastern Asia, especially in China (Yu et al. 2015; China Pharmacopoeia Committee 2020; Tang et al. 2021). In August of 2021, a disease of stem and leaf rot was observed on lily cultivar 'White planet' with approximately 25% disease incidence in the greenhouse and fields at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China). The bulbs of symptomatic plants were brown and rotten, with sunken lesions. Symptomatic plants showed short, discolored leaves, and eventually lead to stem wilt and death of the whole plants. Infected bulbs were surface sterilized in 75% ethanol for 30 s, then in 2% sodium hypochlorite for 5 min, and rinsed three times with sterile distilled water. A 0.5×0.5 cm2 tissue piece was then placed on potato dextrose agar (PDA) medium and incubated at 25±1℃. After 5 days, the isolate was purified by single spore isolation technique. The singled-spored fungal colony was characterized by fluffy white aerial mycelia, and produced orange pigments with age. After seven days on Spezieller Nahrstoffarmer agar (SNA), conidia produced from simple lateral phialides. Macroconidia have pronounced dorsiventral curvature typical, significantly enlarged in the middle, a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 3 to 6 septate, measuring 18.71 to 43.01×2.89 to 5.56 μm with an average size of 26.98×3.90 μm (n=30). Microconidia were not observed. Typical verrucose thick chlamydospore with rough walls were profuse in chains or clumps, ellipsoidal to subglobose. These morphological characteristics were consistent with Fusarium spp. (Leslie et al. 2006). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymeraseⅡsubunit 2 (RPB2) genes were amplified using primers ITS1/ITS4, EF1/EF2 and 5F2/7cR respectively and sequenced (White et al. 1990; Jiang et al. 2018; O'Donnell et al. 2007). Sequences were submitted to GenBank under accession numbers OM078499 (ITS), Accession OM638086 (TEF1-α) and OM638085 (RPB2). BLAST analysis showed that ITS, TEF1-α and RPB2 sequences shared 100%, 99.8%, 99.2% identity to F. equiseti (OM956073, KY081599, MW364892) in GenBank, respectively. In addition, ITS, TEF1-α and RPB2 sequences shared 100%, 99.53%, 100% identity with Fusarium lacertarum (LC7927, Fusarium incarnatum-equiseti species complex) in the Fusarium-ID database. Based on the morphological characteristics and molecular sequences, the isolates were identified as Fusarium equiseti. A pathogenicity test was performed on potted lily ('White planet') under greenhouse conditions (25±1℃ with a 16 h light and 8 h dark cycle). Three healthy lily bulbs were selected and one bulb was planted in each pot filled with sterilized soil. Each pot was inoculated with 5 mL of conidia suspension (1×107 conidia/mL) in te soil around bulbs with a stem length of 3 cm, with an equal amount of sterilized water as a control. This test had three replicates. After 15 days of inoculation, typical symptoms of bulb rotten, like those observed in the greenhouse and fields, developed on the inoculated plants but not on the controls. The same fungus was consistently reisolated from the diseased plants. To our knowledge, this is the first report that F. equiseti caused bulb rot on Lilium in China. Our result should help with future monitoring and control of lily wilt disease.

First Report of black spot of Shine Muscat fruit caused by Cladosporium allicinum in China.

Shine muscat is a Vitis vinifera hybrid (Akitsu-21 × Hakunan), that has emerged as a popular table grape cultivar in China. In recent years, shine muscat has been widely cultivated with 66,667 ha being cultivated in 2021. In November 2021, symptoms of fruit spot were observed on shine muscat during the storage at 0~3℃ and 85%~90% RH, while stored at National Agricultural Product Preservation Engineering Technology Research Center, in Tianjin (N 116°20', E 39°09'), China. The incidence of this disease was about 35%. Affected grape berries initially had small brown spots. The spots on the fruit expanded to an ellipse or circular sunken area with a black center. The central peel of the diseased spots were ruptured and collapsed. The diseased fruits eventually fell off the vine. To isolate the pathogen, grape peels with typical symptoms were cut into small pieces, sterilized with 75% ethanol for 45-sec, rinsed with sterilized distilled water three times, and then transferred onto potato dextrose agar (PDA) medium.The plates were incubated at 25°C in the dark. After 10 days, 26 single spore isolates with similar morphology were obtained from 30 symptomatic grape berries. Fungal colonies were grayish brown, with abundant conidia on the obverse-side on PDA. Conidiophores were cylindrical, straight with unbranched, solitary or clustered, elongation at the tip and ranged in size from 3.2-6.8 × 35.6-150.9 µm (n=50). Conidia were grew in chains, ovoid, aseptate, and 2.2-6.0 × 8.3-16.8 µm (n=50). The morphological characteristics were consistent with Cladosporium allicinum (Bensch et al. 2012). Molecular data were also used to support the microscopic identification by extracting genomic DNA from 26 isolates using a Plant Genomic DNA kit (Tiangen, China). Amplicons were generated for the internal transcribed spacer (ITS), translation elongation factor 1-alpha(tef1-α), and actin (act) using the following primers ITS1/ITS4, EF1-728F/ EF1-986R and ACT-512F/ ACT-783R, respectively (Bensch et al. 2012). Blast analysis showed that three amplified fragments of 26 isolates were highly similar to C. allicinum, with 98.96~100% sequence identity with Cladosporium allicinum accessions in GenBank (ITS, OK661041; tef1-α, MF473332; act, LN834537). Three amplified fragments of representative isolate YG03 were deposited in GenBank with accession nos. OP799670 for ITS, OP888001 for tef1-α and OP887999 for act, respectively. Neighbor-joining trees based on concatenated sequences of three genes were constructed using MEGA5.2. The results showed that the strain YG03 from shine muscat was closely related to C. allicinum. Pathogenicity tests of 26 isolates were performed on healthy shine muscat berries using pin pricks and a humidor. In each wound, 5 μL of conidial suspension (1×106 conidia/mL) and sterile distilled water were inoculated on 30 berries, and maintained in a dark incubator at 25°C, 90% relative humidity. Each treatment was repeated twice. After 10 days, the wounded berries inoculated with the spore suspension showed dark brown spots, similar to the original diseased fruits, while no symptoms were observed on the control treament. Pathogen re-isolated from inoculated fruits were identical to the original strains on colony and microscopic morphology, and identified to Cladosporium allicinum based on act gene by molecular method, thereby fulfilling Koch's postulates. C.allicinum has been reported causing leaf spot on 11 host plants around the world (Bensch et al. 2012, 2015; Quaedvlieg et al. 2014; Jurisoo et al. 2019). To our knowledge, this is the first report of C. allicinum causing black spot on fruit of Vitis vinifera worldwide. The identification of this disease could establish a foundation for developing management strategies to reduce losses in storage period.

First report of leaf spot caused by Botrytis cinerea on Bletilla striata in Anhui province, China.

Bletilla striata (Thunb.) is a perennial herb plant of the orchidaceous family and is used as an ornamental plant in Europe and the United States. Furthermore, it is important as traditional Chinese medicine (TCM) in East Asian countries, such as China, Japan, Korea, Mongolia, and Myanmar (Gou et al. 2022). In April 2023, a severe disease similar to gray mold occurred in a B. striata plantation in Anqing, Anhui province, China (N30°27'15″, E116°18'32″), causing disease on about 20% of the plants in the field. Early symptoms were characterized by brown spots or stripes on the leaves of B. striata, and as the disease progressed, large brown irregular spots appeared. Eventually disease spots coalesced, covering the entire leaf surface and causing leaf death. A gray mildew layer was observed on the senescent leaves. To investigate the causal agent, 10 plants with typical symptoms were collected from the field. Leaf pieces (5 × 5 mm) from the border of infected areas were soaked in 75% ethanol for 10 seconds, and then transferred into 0.1% mercury bichloride for three min, rinsed three times with sterile water, and transferred to PDA at 25 °C for three days. Pure cultures were obtained by single spore isolation, and the resulting colonies were morphologically similar, indicating a single pathogen, of which the representative BSFC-7 was selected for further study. BSFC-7 colonies were initially white to gray-brown, and cottony aerial hyphae grew over the entire petri dish after five days of incubation. Grayish, branched conidiophores and their terminal unicellular conidia were observed under a microscope after additional two days at 25 °C. Conidia were colorless or gray, elliptical or oval, and 7.06-12.54 × 8.33-13.55 μm (n=30). Sclerotia appeared in BSFC-7 culture up to about two weeks and were black, hard, and round or irregularly shaped (0.81-4.32 × 0.97-5.68 mm, n=20). The morphological characteristics fit the description of Botrytis cinerea (Li et al. 2016). To further identify the species, genomic DNA of BSFC-7 was extracted. PCR analysis was performed with species-specific primer pairs C729+/C729- and two nuclear genes G3PDH and RPB2 with their corresponding primer pairs G3PDH-F/G3PDH-R and RPB2-F/RPB2-R (Rigotti et al. 2002; Aktaruzzaman et al. 2018). The sequences for all three PCR products of C729, G3PDH, and RPB2 (GenBank accession nos. OR287069, OR255923, and OR255924 respectively) exhibited 99 to 100% similarity with other B. cinerea isolates. In the pathogenicity test, detached leaves of B. striata were inoculated with the BSFC-7 isolate. The leaves were soaked in sodium hypochlorite (1%) for two min, washed with sterile distilled water, and then inoculated with 10 µl of conidial suspension (106 conidia/ml). Sterile water was used as control and samples were incubated at 25 °C. After three days, all leaves inoculated with conidia showed dark brown water-soaked lesions similar to those observed in the field, while the control leaves remained healthy. The pathogen was re-isolated from the affected leaves, fulfilling Koch's postulates. B. cinerea is a common pathogen on a wide range of host plant species worldwide and has been reported to infect B. striata in Yunnan province, China (Romanazzi and Feliziani 2014; Zhang et al. 2020). To our knowledge, this is the first report of B. cinerea causing leaf spots on B. striata in Anhui province, China. This study will provide a basis for controlling the prevalence and economic losses of gray mold on B. striata.

First report of leaf anthracnose caused by Colletotrichum camelliae on tea plants (Camellia sinensis) in South Korea.

The tea plant (Camellia sinensis (L.) O. Kuntze) is a popular non-alcoholic beverage crop worldwide. The tea market in South Korea is projected to increase annually by 4.59% (Statista, 2022). Boseong, Hadong, and Jeju Island are the main tea-growing regions in South Korea. Anthracnose is one of the major diseases of tea plants and is responsible for substantial yield loss and poor tea quality. In 2021, anthracnose of tea (disease incidence of 30%) was observed in a garden (33°28'45.5"N 126°42'02.2"E) at Jeju Island, where the Yabukita cultivar has been cultivated. The typical symptoms consisted of round or irregularly shaped lesions with gray-white centers and purple-brown borders. Twelve morphologically similar isolates were recovered from 12 infected leaves using the single spore isolation method on solid potato dextrose agar (PDA) (Cai et al. 2009). Four representative isolates (GT6, GT7, GT8, and GT11) were identified based on morphology, molecular analysis, and pathogenicity tests. The upper side of seven-day-old colonies on PDA (incubated at 25 °C in the dark) was off-white with white aerial mycelia and gray-white with black zonation on their reverse side. Conidia were hyaline, aseptate, cylindrical, with both obtuse ends, and measuring 12.3 - 25.8 µm × 4.4 - 9.3 µm (n = 50). Appressoria were dark brown, irregularly shaped with a smooth edge, and measuring 7.3 -18.8 µm × 6.9 - 11.3 µm (n = 50). According to morphological characteristics, the fungal isolates were tentatively identified as the Colletotrichum gloeosporioides complex, including C. caelliae (Wang et al. 2016; Weir et al. 2012). The genomic DNA was extracted, and the ribosomal internal transcribed spacer (ITS), β-tubulin-2 (TUB2) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, actin (ACT), calmodulin (CAL), and the Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat) genes were amplified and subsequently sequenced using primer sets ITS1/ITS4, BT2a/BT2b, GDF1/GDR1, ACT-512F/ACT-783R1, CL1C/CL2C, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting sequences were deposited in GenBank accession numbers (LC738932-LC738959). All the representative isolates were identified as C. camelliae by constructing the 50% majority rule consensus and maximum likelihood phylogenetic treebased on the combined ITS, TUB2, GAPDH, ACT, CAL, and ApMat sequences using MrBayes v. 3.2.2 and Mega X, respectively (Kumar et al., 2018; Ronquist et al. 2012). The pathogenicity of these isolates was tested on healthy leaves of 2- years-old tea seedlings (the Yabukita cultivar). Onside of unwounded or wounded leaves of seedlings were inoculated with 20 µL of conidial suspension (1 × 106 conidia or spores/ml) per spot (3-4 wounded or unwounded spots per side per leaf). Another side of the leaves received sterile distilled water and served as a control. Each treatment was replicated three times (three seedlings/isolate and four leaves per seedling) and this experiment was repeated twice. All plants were covered with plastic bags, placed in a growth chamber, and incubated at 25 °C with a 12-h photoperiod and 90% relative humidity. Typical anthracnose symptoms appeared on wounded leaves after two days of inoculation. Unwounded and controlled leaves remain asymptotic. To confirm Koch's postulates, fungal isolates were re-isolated from inoculated leaf lesions and identified as C. camelliae based on morphology and ITS sequences. Colletotrichum camelliae is a very common pathogen associated with tea anthracnose worldwide, including China (Liu et al. 2015; Wang et al. 2016).To the best of our knowledge, this is the first report of anthracnose in tea trees caused by C. camelliae in South Korea. The results of this study could help come up with better ways to keep an eye on and deal with this devastating on tea plants. Key words: Tea anthracnose, Colletotrichum camelliae, pathogenicity References Cai, L., et al. 2009. Fungal Divers. 39:183. Kumar, S., et al. 2018. Mol. Biol. Evol. 35:1547. Liu, F. et al. 2015. Persoonia. 35: 63-86. Ronquist, F. et al. 2012. Syst. Biol. 61:539-542. Silva, D. N. et al. 2012. Mycologia. 104:396-409. Statista 2022. Statista Digital Market out Look. Available at www.statista.com. Wang, Y.-C. et al. 2016. Sci. Rep. 6: 35287. Weir, B. S., et al. 2012. Stud. Mycol. 73:115.

First report of peanut root rot caused by Fusarium acuminatum in Shandong Province, China.

Peanut (Arachis hypogaea L.) is one of the most economically important crops as a major source of edible oil and protein. In July 2021, a root rot disease was observed on peanut in Laiwu (36º22' N, 117º67' E), Shandong Province, China. Disease incidence was approximately 35%. Disease symptoms included root rot, vessels with a brown to dark brown discoloration, plus progressive yellowing and wilting of leaves from the base leading to whole plant death. To determine the causal agent, symptomatic roots with typical lesions were cut into small pieces, surface sterilized in 75% ethanol for 30 s, and 2% NaClO for 5 min, rinsed three times in sterile water and placed on potato dextrose agar (PDA) at 25℃ (Leslie and Summerell 2006). After 3 days of incubation, whitish-pink to red colonies growing from the roots were observed. Eight single-spore isolates had identical morphological traits that were similar to those of Fusarium spp. A representative isolate (LW-5) was used for morphological characterization, molecular analysis, and pathogenicity test. On PDA, the isolate formed dense aerial mycelia, which were initially white, then became deep pink with age and formed red pigments in the medium. On carnation leaf agar (CLA), macroconidia with 3 to 5 septa were abundant, relatively slender, curved to lunate, that measured 23.7 to 52.2 × 3.6 to 5.4 μm (n=50). Microconidia were oval, 0 to 1 septa. Chlamydospores were globose with a smooth outer wall in chains or single. Following DNA extraction of isolate LW-5, primers EF1-728F/EF1-986R (Carbone et al., 1999), RPB1U/RPB1R, and RPB2U/RPB2R (Ponts et al., 2020) were used to amplify the partial translation elongation factor 1 alpha (TEF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) regions for DNA sequencing, respectively. BLASTn analysis of TEF1-α (GenBank accession No. OP838084), RPB1 (OP838085), and RPB2 (OP838086) sequences, revealed 99.66, 99.87, and 99.09% identity with those of F. acuminatum (OL772800, OL772952 and OL773104), respectively. Isolate LW-5 was identified as F. acuminatum based on morphology and molecular analysis. Twenty Huayu36 peanut seeds were each planted in a 500-ml sterile pot containing 300 g of autoclaved potting medium (nutritive soil: vermiculite=2:1 in volume). Two weeks after seedling emergence, 1 cm depth of the potting medium was dug around the plants to expose the taproot. Two 5-mm wounds per taproot were scratched with a sterile syringe needle. Potting medium in each pot of 10 inoculated plants was mixed with 5 ml of conidial suspension (106 conidia per ml). The other 10 plants were used as non-inoculated controls and treated with sterile water in the same manner. The seedlings were placed in a plant growth chamber maintained at 25°C, RH >70%, 16-h light per day, and irrigated with sterile water. After 4 weeks, inoculated plants exhibited yellowing and wilting symptoms that were similar to those observed in the field, while non-inoculated control plants had no symptoms. F. acuminatum was re-isolated from diseased roots and confirmed using morphological features and DNA sequence analysis of TEF1-α, RPB1 and RPB2. F. acuminatum was reported to cause root rot on Ophiopogon japonicus (Linn. f.) (Tang et al., 2020), Polygonatum odoratum (Li et al., 2021), and Schisandra chinensis (Shen et al., 2022) in China. To our knowledge, this is the first report of root rot on peanut caused by F. acuminatum in Shandong Province, China. Our report will provide crucial information for studying the epidemiology and management of this disease.

First Record of Neofusicoccum luteum and Colletotrichum gloeosporioides Causing Anthracnose and Stem End Rot on Avocado Fruits in Greece.

In May 2022, rot symptoms were observed 5 days after storage on fresh avocado fruits cv "Lamb Hass" harvested from a 3.4 ha organic orchard in Chania, Crete exhibiting 30% symptom incidence. Brownish-green sunken lesions and soft rot with dark brown lesions covering up to 50% of the mesocarp on fruits and blackish soft lesions on fruit stem ends were observed. To isolate the pathogens, fruits were surface sterilized using 1% NaOCl for 1 min, placed in 70% ethanol for 30 s and washed twice with sterile distilled water. Then, small pieces were excised from the fruit rot margins and transferred on PDA amended with 0.015% streptomycin-sulfate. Single-spore isolates were incubated on PDA for 10 days and subjected to morphological examination. Two distinct pathogenic fungal isolates were obtained, and their symptoms were re-examined on avocado fruits. The first isolate (A1) obtained from the fruit stem end, initially produced hyaline dense aerial mycelia, being gray and black on the upper and lower surface of the Petri dishes, respectively. The second isolate (A2) obtained from the main body of the fruit, formed round, grayish colonies, with orange conidial aggregates. Based on morphological characteristics (Phillips et al.,2013; Weir et al., 2012), isolates were preliminary identified as Neofusicoccum sp. (A1) and Colletotrichum sp. (A2). Isolates were molecularly identified by sequencing of the ITS-5.8S rRNA, translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2) genes. PCRs were conducted using primer pairs ITS4/ITS5, EF1-728F/EF1986R and Bt2a/Bt2b as well as ITS4/ITS5 and 5'-tef1/3'-tef1 and Bt2a/Bt2b for isolates A1 and A2, respectively (Carbone & Kohn, 1999; Glass & Donaldson, 1995; Rojas et al., 2010; Weir et al., 2012; White et al., 1990). The sequences were deposited into GenBank under the accession numbers OQ852465, OQ867962, OQ867965 for N. luteum and, OQ852466, OQ867963 and OQ867964 for C. gloeosporioides. Based on Multilocus sequence analysis (MLSA), a phylogenetic tree was constructed using concatenated sequences, following Kimura's two parameter model (1980), which confirmed their identity as N luteum and C. gloeosporioides strains. Mature avocado fruits (cv. Hass) were surface sterilized and dried. Consequently, incised fruits were inoculated with mycelial agar plugs (5 mm in diameter) cut from the edge of rapidly growing colonies of N. luteum and C. gloeosporioides strains. Fruits incubated in moist chambers and at 25°C for 5 days in the dark. Fruit bodies and stems were inoculated with the respective isolates and sterile agar plugs in the case of the control. Five fruits were used for each pathogenic trial per fungal isolate, which was repeated twice. After symptom occurrence, these pathogenic isolates were re-isolated successfully and molecularly identified, while exhibiting similar to original symptoms confirming Koch's postulates. While other reports exist on the presence of these pathogens in different countries worldwide, this is the first report of C. gloeosporioides and N. luteum as post-harvest pathogens of avocado, which is an economically important crop of Crete, in Greece (Akgül et al., 2016). This study provides the means for the accurate identification of these fungal pathogens causing avocado fruit rots and taking into consideration the available treatment options can contribute to establishing effective management strategies.

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

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

First Report of Alternaria Leaf Blight Caused by Alternaria alternata on Convallaria majalis in China.

Convallaria majalis is native to temperate zones in the northern hemisphere, Europe, Asia, North America and China, and has excellent ornamental properties and medicinal value. In July 2021, leaf blight was observed on nearly 70~90% of C. majalis plants growing in Heilongjiang University of Chinese Medicine campus (45.72°N, 126.68°E) from Harbin City, China. The typical symptom on the leaves is irregular dark brown lesions. As the brown lesions expanded and eventually coalesce, they form large necrotic areas, with chlorosis, curling, and wilting at the apical edge of the diseased leaf. Ten symptomatic leaves were randomly collected from twenty different plants at the site. Several fragments of diseased tissues (5×5mm) were disinfected in 75% ethyl alcohol for 30 s and 7% NaOCl for 60 s, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25 °C in the dark for 7 days. Twenty purified fungal isolates were obtained by single spore isolation. The morphology of all the twenty isolates was similar, and two isolates were randomly selected (LL, LL01) for further study. Colonies of these isolates on PDA were off-white to black with abundant cotton-like aerial hyphae, and the diameter of the colony is 72 to 85 mm. On potato carrot agar (PCA) medium, these isolates produced light brown and solitary conidiophore with septum. Conidia were ovate to pear-shaped, brown to black in color, with 1-4 transverse septa and 0-2 longitudinal septa, and measured 20.5 to 38.5 × 7 to 13.5 µm (n=100). The isolates were identified as Alternaria alternata according to their morphological characteristics (Simmons 2007). Two representative isolates LL and LL01 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(White et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nawashima 2020) and Alt-for/Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS, OM319508, OP799847; RPB2, OM649830, OP830846; GAPDH, OM296234, OP830845; TEF1, OM393717, OP830844; Alta1, OM171258, OP830847). Phylogenetic analyses showed 100% identity between LL and LL01 and the type strain CBS 118815. 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/ml of A. alternata isolates LL and LL01 on leaves of six healthy C. majalis plants, separately. Another six plants were sprayed with sterile distilled water as control 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. After 15 days inoculation, the similar symptoms were observed on the inoculated plants, whereas control plants remained healthy. 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 C. majalis in China and worldwide. The result will serve as the foundation for management leaf blight of C. majalis.

First Report of Neck Rot Caused by Botrytis aclada on Onion (Allium cepa L.) in Gansu Province, China.

Onion (Allium cepa L.) is one of the most important cultivated vegetable crops in many countries, including China (Hanci 2018; Steentjes et al. 2021). Gansu, in the northwest region of China, is a major area of onion production (Zhang et al. 2022). In October 2021, typical symptoms of neck rot were observed on stored onion bulbs (cv. Honghe) in Jiuquan, Gansu Province, China. Further surveys of 20 bags of onion bulbs randomly selected in a storage facility with bulbs harvested from 73 ha indicated that approximately 5% of the bulbs had typical neck rot symptoms. The neck of infected bulbs developed a water-soaked decay, with softened and discolored inner scales with white to gray mycelium in the neck. Infection usually began in the neck and sometimes spread through the entire bulb. In severely affected bulbs, the fleshy scales were span style="font-family:'Times New Roman'">decayed and the bulbs had shrunk, with black sclerotia between the rotting scales in the neck and shoulders of the bulb. Small pieces cut from the margins of lesions were surface-disinfested with 75% ethanol for 10 s, and 1% NaClO for 1 min, rinsed three times in sterile distilled water, dried on sterile filter paper, placed onto potato dextrose agar (PDA), and incubated at 20 ± 1℃ for 5 days in the dark. Ten pure cultures were obtained by single-spore isolation (one from each of 10 bulbs). All 10 isolates produced colonies that initially were white, and became gray to dark in color with gray mycelium that was covered with abundant conidia resembling Botrytis species when cultured on PDA at 20 ± 1℃ for 10 days in the dark. Conidia (n = 100 per isolate) were one-celled, ellipsoid or ovoid, beige to dark brown, and 5.6 to 14.8 × 3.9 to 8.5 μm. Sclerotia were not produced by any of the isolates on PDA after incubation at 20 ± 1°C for 30 days. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene, heat shock protein 60 (HSP60) gene, and DNA-dependent RNA polymerase II core subunit (RPB2) gene of two representative isolates, JQ21AC03 and JQ21AC09, were amplified and sequenced with primers ITS1/ITS4, G3PDH-F/G3PDH-R, HSP60-F/HSP60-R, and RPB2-F/RPB2-R, respectively (Staats et al. 2005), and deposited in GenBank (ITS: OP604277 and OP604283; G3PDH: OP627512 and OP627515; HSP60: OP627513 and OP627516; and RPB2: OP627514 and OP627517). BLASTn analysis of the resulting sequences showed 98 to 100% similarity with those of Botrytis aclada. A maximum likelihood phylogenetic tree generated by combining the sequenced loci using MEGA11.0, clustered isolates JQ21AC03 and JQ21AC09 with B. aclada with 99% bootstrap support. Based on these results, isolates JQ21AC03 and JQ21AC09 were identified as B. aclada. Pathogenicity of the two isolates on the onion cv. Honghe was confirmed by inoculating 30 heathy onion bulbs per isolate with 100 μL of conidial suspension (1×105 conidia/ml), while control bulbs were inoculated with 100 μL sterile distilled water. All 30 bulbs inoculated with each isolates developed neck rot symptoms after 30 days incubation at 15 ± 1ºC in the dark, with symptoms identical to those observed in the original storage facility, whereas control bulbs remained symptomless. The pathogen was re-isolated from the symptomatic tissue of inoculated bulbs, fulfilling Koch's postulates. hang et al (2008) documented this pathogen causing bulb rot of onions in Wuhan, Hubei Province, 1,700 km from Gansu. To the best of our knowledge, this is the first report of B. aclada causing neck rot on onion bulbs in storage in Gansu Province, which is the main onion production region in China. Considering that onion is the main source of income for growers in Gansu, further studies will be required to understand the epidemiology of this disease and foster appropriate disease management measures to avert disease outbreaks in the future.

First Report of Neofusicoccum australe Causing Branch Dieback of English Walnut cv. Chandler in Central Chile.

The English walnut (Juglans regia L.) is the second most important fruit crop of importance in Chile, with 43,700 hectares mainly in the Central Valley (www.odepa.cl, 2022). For several seasons symptoms of a branch dieback have been observed in walnut orchards with 3 to 50% of trees incidence levels. During the 2020 winter season (July) a total of 150 symptomatic spurs of 15 trees were sampled from an 8-year-old walnut cv. Chandler orchard located in Buin (33°42' S, 70° 42' W). The collected spurs showed external and internal brown necroses, starting from the tip with well-defined margins. The symptomatic tissue was cut in to small pieces (5 x 4 x 2 mm), surface disinfected by dipping in a 10% solution made from a commercial bleach solution (4,9% NaOHCl), rinsed twice in sterile water and plated on APDA (PDA Difco laboratories acidified with lactic acid (2,5 ml of 25% (vol/vol) per liter of medium). After five days at 20 °C in darkness, fast-growing, white-grey turning to black colonies were obtained, tentatively classified as a member of the Botryosphaeraceae family and two single-spore isolates (SS1, SS2) were selected for identification. Colony mycelia were first white and turned to light grey, dark grey or black, with tufts of mouse gray aerial mycelia. The pycnidia and conidia production was induced by inoculating autoclaved pine needles placed on APDA an incubation for 25 to 30 days at 20 °C in darkness. Black pycnidia solitary and globose were obtained producing hyaline, aseptate, fusiform to obovoid conidia with truncated ends with dimensions of (22.6-) 19.1 ± 1.4 (-13.3) x (6.7-) 5.5 ± 0.5 (-3.7) µm and 3.5 length/width ratio (n=100). Both isolates were identified using dichotomous keys confirming the description of Crous et al, 2006 as Neofusicoccum australe. The identification was molecularly confirmed by amplifying the nuclear ribosomal gene 5,8S (ITS1-5.8S-ITS2) using the ITS1/ITS4 primers, a partial region of β-tubulin gene (Bt2a/Bt2b), and the translation elongation factor 1-α gene (TEF1) with TEF1-728F/TEF1-986R primers. The BLASTn search revealed 100% of identity for ITS and TEF according to sequences of N. australe reference strains MT587467.1 and MK759852.1, respectively; and over 99% for β-tubulin compared to N. australe strain KX464929.1. The DNA sequences were submitted to the GenBank (ITS, OP142414, OP142416; BT, OP209981, OP209978; and TEF OP209979, OP209980) for SS1 and SS2 isolates, respectively, and deposited in the fungal collection of CChRGM - INIA, Chillán, Chile (RGM 3409 and 3410). Pathogenicity of both isolates was tested in 8-year-old asymtomatic English walnut cv. Chandler in the field during 2020 spring season, by cutting transversally 15 twigs of different tress and inoculating with a 5 day-old PDA plug. An equal number of wounded twigs were inoculated with a sterile PDA plug and served as control. After six months, all inoculated twigs developed the same necrotic lesions observed in field of 2.0 to 10.1 cm (SS1) and 1.9 to 10.8 cm (SS2) in length while control twigs showed only a scar without any dieback tissues. The inoculated pathogens of N. australe were recovered from the diseased tissues, thus fulfilling Koch's postulates. A similar dieback of walnut was reported in Chile, which caused Diplodia mutila (Díaz et al, 2018), and N. parvum (Luna et al, 2022) while N. australe has been reported in other hosts (Auger et al, 2013, Besoain et al, 2013). To the best of our knowledge, this is the first report of N. australe associated with walnut branch dieback in Chile.

First report of rot disease in Prunus salicina var. taoxingli fruit caused by Fusarium lateritium in China.

Shengzhou nane (Prunus salicina var. taoxingli) as main cultivated plum in Zhejiang province, China, ripened in summer and was susceptible to microbial infection during the maturation stages because of high ambient temperature. In July 2022, Shengzhou nane fruits were found with symptoms of dark skin and grey-white lesions on surface, and an average incidence of approximate 16% (data obtained from investigation of ten trees in each orchard, and fifty fruits in each tree) in four orchards in Shengzhou city, Zhejiang province, China. To isolate the pathogen, four symptomatic partially rotted fruits (about 25%) from four orchards were collected. The plum tissue from the margin of lesions was excised into small blocks (4×4×3 mm). Eight blocks were disinfested with 70% ethanol for 30 seconds and 2% NaClO for 3 minutes, rinsed at least twice with distilled water, and incubated on PDA medium supplemented with 30.0 μg/mL Chloramphenicol at 25°C for 5 days in darkness until sporulation. Using single-spore isolation, four uniform isolates were obtained. The purified mycelium of isolates on PDA were milky white, the reverse on agar was dark pink. Colonies grew with a mean mycelium growth rate of 4.62 ± 0.38 mm per day on PDA. Macroconidia was shaped like a sickle and the size was 27.27 ± 3.43×4.54 ± 0.39 μm (n=50) with 2 to 3 septate. No microconidia and chlamydospores were observed. These morphological characteristics were consistent with previous descriptions of Fusarium lateritium (F. lateritium) species (Yun et al. 2013). Molecular identification was performed by sequencing nuclear ribosomal internal transcribed spacer region (ITS) gene with ITS1/ITS4 (White et al. 1990) and translation elongation factor-1α (TEF) with EF1-728F/EF1-986R (Carbone and Kohn. 1999). The representative sequences were submitted to Genbank (Accession Nos. OP315081 for the ITS and OP394153 for the TEF-1α). Blastn result indicated that ITS and TEF-1a sequences shared 99.28% and 100% similarity with two strains of F. lateritium (GenBank Accession Nos. MK311296.1 and JF740854.1). To fulfill Koch's postulates, the isolated F. lateritium (TXL-IT5) with a conidial suspension (1×106 spores per mL) was inoculated on ten fresh harvested healthy mature Shengzhou nane fruits (20 μL per fruits) after surface sterilization. The control fruits were inoculated with 20 μL distilled water. The experiment was repeated three times.Fruits were kept in a sealed plastic boxes at 25 °C with 90% relative humidity in dark. Four days after inoculation, all inoculated fruits were infected and appeared the same symptoms which were found in naturally infected fruits while those in the control group remained symptomless. The pathogen was re-isolated from symptomatic rotten fruits using above-mentioned method and was identified asF. lateritiumbased on morphological characteristics as well asITS and TEFgene sequences. In addition, this pathogen was reported to infect Dalbergia tonkinensisin north Vietnam (Nhung et al. 2018), causing shoot dieback of Acer negundo in northeast Poland (Patejuk et al. 2022), and rot of Chinese cherry during postharvest storage (Wang et al. 2020). However, to the best of our knowledge, this is the first report of F. lateritium causing rot on Shenshou nane fruits. Our findings serve as a warning for scientists and growers who should pay attention to this disease and take effective control strategies to reduce epidemics of this disease in China and other regions.

First Report of Leaf Spot Caused by Clonostachys rosea on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis), which originated in southwest of China 60 - 70 million years ago, is widely consumed as a beverage for its potential enhancing effect on human health with rich polyphenol content (Pan et al. 2022).From October to December in 2021, a disease with symptoms similar to leaf spot affected the quality and yield of teaPuer (102°73 'E, 25°07' N), Yunnan province, China. Based on the survey, leaf spot symptoms were observed on approximately 60% of tea plants in a 5,700 m2 field. The symptoms initially appeared as shrinking, yellowing, and later became circular or irregular brown spots. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees, and portions of the diseased tissue (0.5×0.5 cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol and 2 min with 3% NaOCl, washed three times with sterilized distilled water), the disinfected pieces were dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Four single-spore isolates, FH-1, FH-5, FH-6 and FH-7, were obtained, these isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS] and translation elongation factor 1-alpha [TEF] genes. Therefore, the representative isolate FH-5 was used for further study. Fungal colonies were white or light yellow on PDA after 7 days incubated at 28ºC. Conidia were hyaline, round or oval, aseptate, occur singly or in clusters on hyphae or conidia stalks, and measured as 2.94 ± 1.79 × 1.82 ± 0.2 μm (n = 50). Primary conidiophores is Verticillium-like (Fig1.K,L), which generally formed first, 1-3-level verticillate, mostly with divergent branches and phialides, and measured as 16.67 ± 4.39 µm (n = 50). Secondary conidiophores is penicillate (Fig1.I,J), which generally appearing after one week, sometimes even more often branched, and with a length of 16.02 ± 3.83 µm (n = 50). The morphological features were consistent with the descriptions of Clonostachys rosea Schroers H.J. (Schroers et al. 1999). The pathogen was confirmed to be C. rosea by amplification and sequencing of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4 and EF1-728F/EF1-986R, respectively (Fu Rongtao 2019). The sequences of PCR products were deposited in GenBank with accession numbers ON332533 (ITS) and OP080234 (TEF). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides) and 98.37% (241/245 nucleotides) homology with those of C. rosea HQ-9-1 form GenBank (MZ433177 and MZ451399, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate FH-5 in a well-supported cluster with C. rosea. The pathogenicity of FH-5 was tested through a pot assay. Ten healthy tea plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (105 spores·mL-1) of FH-5 onto leaves until runoff, and the control leaves sprayed with sterile water. Inoculated plants were put in an artificial climate box at 25℃, 70% relative humidity. The pathogenicity test was replicated three times. Symptoms developed on all inoculated leaves but not on the control leaves. Lesions around the wound edge became pale yellow, and brown spots were first observed at 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after two weeks. The same fungus was reisolated and identifiedbased on themorphological characterization and molecular analyses (ITSand TEF)from the infected leaves but not from the noninoculated leaves. In addition, C. rosea has also been reported to cause diseases to broad bean (N. Afshari et al. 2017 ), garlic (Diaz et al. 2022), beet (Haque M.E et al. 2020) and other plants. To our knowledge, this is the first report of leaf spot on tea caused by C. rosea in China. This study provides valuable information for the identification and control of the leaf spot on tea.

Leaf spot disease on Juglans regia caused by Ragnhildiana diffusa in China.

Juglans regia L. is commercially important for its edible nuts, which is a major species of walnut trees in Sichuan Province (Luo et al. 2020). In September 2021, brown leaf spot symptoms were observed on roughly 75% of 60 J. regia trees surveyed in an orchard of Chongzhou city (30°40'6''N, 103°40'18''E). Initially, the lesions measuring 2-10 mm were reddish to brown with a yellowish halo, then increased in size and coalesced to cover the whole leaf, eventually resulting in severe defoliation. Six symptomatic leaves from different trees were collected, and a single fungal isolate was obtained from each of the sampled leaves using single-spore isolation (Chomnunti et al. 2014). The isolates were incubated on potato dextrose agar (PDA) with a 12h photoperiod at 25 ℃, and deposited at the Culture Collection of Sichuan Agricultural University. Colonies were identical with black center and reddish-brown periphery, and the diameter reached 2 cm after 7 days. On the host, conidiophores were mostly reduced to conidiogenous cells, with prominent and thickened conidiogenous loci. Conidia were light green to light brown, and curved with a thickened and darked hilum at the base, 0-17 septate, tapering toward the distal end, and measuring 20-120 × 3-5 μm ((x ) ̅= 56 × 4, n = 30). Morphological characteristics fit the description of Ragnhildiana diffusa (Heald & F.A. Wolf) Videira & Crous (Synonym: Sirosporium diffusum (Heald & F. A. Wolf) Deighton) (Poletto et al. 2017). The internal transcribed spacer (ITS) region, the large subunit of the nrDNA (LSU), and RNA polymerase II second largest subunit (rpb2) were amplified by polymerase chain reaction and sequenced with primers ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys & Hester 1990), fRPB2-5F/Rpb2-R3 (Liu et al. 1999, Videira et al. 2017), respectively. The nucleotide blast of the two isolates (SICAUCC 22-0077, SICAUCC 22-0078) showed 99.7% and 99.5% (ITS, 472/473 bp, 471/473 bp), 100% (LSU, 725/725 bp, 725/725 bp), 99.8% (rpb2, 866/867 bp, 866/867 bp) identities with the ex-type strain of Ragnhildiana diffusa (CBS 106.14). The phylogenetic tree combined with ITS, LSU, and rpb2 genes and morphological characteristics confirmed the identification as R. diffusa. These sequences of the three gene regions of two isolates were deposited in GenBank with accession numbers ON409525 and ON409526 (ITS), ON409559 and ON409560 (LSU), ON417473 and ON417474 (rpb2), respectively. The isolate SICAUCC 22-0077 was used for pathogenicity test to fulfill Koch's postulates. Three leaves of each walnut seedlings (2-year-old seedlings) were inoculated by placing a mycelium plug onto fresh wounds on the upper leaf surface punctured via a fine needle (0.7 mm in diameter), and three replicate seedlings were inoculated. For the control, a sterile PDA plug was placed on the same number of replicate leaves on the plants. The inoculated and control plants were placed in a growth chamber at 25°C with relative humidity >80% and a 12-h photoperiod. Irregular light to dark brown spots developed on inoculated leaves after twenty days, and no symptoms were observed on controls. The re-isolation and examination of the fungus showed it to be morphologically and phylogenetically identical to the originally isolated pathogen. R. diffusa has been described on J. regia in Mexico (Farr & Rossman 2022). To our knowledge, this is the first report of R. diffusa causing brown leaf spot on J. regia in China. The identification of the pathogen will provide a basis for disease management in walnut planting areas.

First report of Alternaria alternata causing leaf blight on Trollius chinensis in China.

Trollius chinensis is widely distributed in east Asian countries that include China, Siberia, and Japan, with antibacterial, antiviral, anti-inflammatory and analgesic activity for medical applications. In August 2021, leaf blight was observed on nearly 80~95% of T. chinensis plants growing in Daxinganling (51.43°N, 126.39°E) from Heilongjiang Province, China. Initial symptoms were gray-black necrosis, wilting progressing from the leaf margin, and eventual defoliation. Six T. chinensis plants with typical symptoms were randomly collected, and three fresh leaf samples were collected from each plant. Diseased leaf pieces that measured 5 mm square were disinfected in 75% ethyl alcohol for 30 s and 7% NaClO for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Twelve fungal isolates, obtained by single-spore isolations, were selected for further. These isolates produced colonies that measured 63 to 73 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces, neat edges, olive green on the back. 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 12.5 to 37.5 × 5.0 to 12.5 μm(n=150). Conidiophores were dark, erect or curved, branched, with pronounced spore marks, and measured 35.0 to 50.0 × 4.0 to 5.0 μm(n=150). All twelve fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates jlh01 and jlh02 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, OM095427, OM108099; RPB2, OM131213, OM131214; GAPDH, OM201165, OM201166; TEF1, OM131211, OM131212; Alta1, OM201167, OM201168). Phylogenetic tree results showed 100% similarity between jlh01, jlh02 and the type strain CBS 118812. Morphological and molecular analysis results confirmed the identity of the fungus as A. alternata. Pathogenicity tests were done by spraying water-spore suspensions containing 106 spores per ml of A. alternata isolates jlh01 and jlh02 on leaves of six healthy T. chinensis plants, separately. Six 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 all plants after 48 h. Black brown lesions and concentric rings developed on spore-inoculated plants after 15 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 T. chinensis in China. Based on the plant's medicinal value, this report provides the basis for further research and control of T. chinensis leaf blight.

First Report of Black Spot Disease Caused by Alternaria alternata on Persimmon Fruit in China.

Sweet persimmon is native to Japan and valued for its fruit, which are high in sugar and vitamins. In October 2021, symptoms were observed on persimmon (Diospyros kaki L. cv. Yangfeng) fruits in cold storage room in Suiping county, Henan Province (32.59 °N, 15 113.37 °E). Initially, small circular dark-brown spots were visible on the fruit rind, turning into irregular sunken dark areas, and eventually rotting 15% of 200 fruits after four weeks of cold storage (10°C, 95% relative humidity). To isolate the causal agent, 10 fruits of symptomatic tissues (4 mm2) were surface-sterilized in 2% sodium hypochlorite (NaOCl) for 1 minute, washed three times in sterile distilled water, then aseptically transferred to potato dextrose agar (PDA) and incubated for 7 days at 25°C. Fungal colonies were isolated from plant tissue, and on three colonies of similar morphology, single-spore isolation was performed. On PDA, the isolates produced circular colonies of fluffy aerial mycelia, gray-brown in the center with gray-white margins. Conidia were dark brown, obclavate or pyriform, with 0 to 3 longitudinal septa and 1 to 5 transverse septa, and a size range of 19.2 - 35.1 × 7.9 - 14.6 μm (n=100). Conidiophores were olivaceous, septate, straight, or bent, with a length of 18 - 60 × 1 - 3 μm (n=100). These morphological characteristics identify the isolates as Alternaria alternata (Simmons. 2007). Genomic DNA was extracted from a representative isolate YX and re-isolated strain Re-YX by cetyltrimethylammonium bromide (CTAB). The primers of ITS1/4, Alt-F/R, GPD-F/R, EF1/2, EPG-F/R (Chen et al. 2022), RPB2-5F/7cR (Liu et al. 1999), and H3-1a/1b (Lousie et al. 1995) were used to amplify the partial internal transcribed spacer (ITS) region, Alternaria major allergen (Alt a1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF), endo-polygalacturonase (endoPG), RNA polymerase second largest subunit (RPB2) and Histone 3 (His3), respectively. GenBank accession No of ITS, Alt a1, GAPDH, TEF, endoPG, RPB2, His3 were ON182066, ON160008 to ON160013 for YX and OP559163, OP575313 to OP575318 for Re-YX respectively. Sequence data of Alternaria spp. were downloaded from GenBank and the BLAST analysis showed 99%-100% homology between various A. alternata strains (ITS: MT498268; Alt a1: MF381763; GAPDH: KY814638; TEF: MW981281; endoPG: KJ146866; RPB2: MN649031; His3: MH824346). A phylogenetic analysis based on ITS, Alt a1, GAPDH, TEF, and RPB2 sequences using MEGA7 (Molecular Evolutionary Genetics Analysis) revealed that the isolate YX and Re-YX were clustered in A. alternata clade (Demers M. 2022). For the pathogenicity test, seven-day-old cultures were used to create spores suspensions (5.0 × 105 spores/mL) of each of the three isolates. Ten µL aliquots from each isolate were inoculated onto ten needle-wounded persimmon fruits; ten additional fruits were inoculated with water only to serve as controls. The pathogenicity test was three replications. Fruits were deposited in a climate box at 25°C, 95% relative humidity. Seven days post-inoculation, the wounded fruit treated with spore suspensions displayed black spot symptoms similar to the symptoms on the original fruit. There were no symptoms on the control fruits. The strain Re-YX was re-isolated from the symptomatic tissue of inoculated fruits and its identity confirmed using the morphological and molecular methods previously mentioned, fulfilling Koch's postulates. The persimmon fruit rot caused by A. alternata had been reported in Turkey and Spain (Kurt et al., 2010, Palou et al., 2012). According to our knowledge, this is the first report of black spot disease on persimmon fruits caused by A. alternata in China. The disease could infect persimmon fruits during cold storage, so more control methods should be developed to prevent postharvest disease of persimmon in the future.

First Report ofMucor irregularisCausing Flower Rot onSelenicereus undatusin China.

Dragon fruit (Selenicereus undatus (Haw.) D.R.Hunt is a famous tropical fruit (Korotkova et al. 2017). In May 2021, a flower rot disease was found on Dragon fruit in a field (21˚19'42''N, 110˚28'32''E), Zhanjiang, Guangdong Province, China. The incidencerate was approximately 30% (n=500 investigated plants from about 30 hectares). Flower rot was evident, and was light brown, watery, soft, and covered with white mycelia. The pathogen could continue to infect the fruit during the fruit ripening stage with about 20% rot rate. Ten samples of symptomatic flowers were collected in the field. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Three representative isolates (HUM-1,HUM-2, and HUM-3) by single-spore isolation were randomly selected for further study. Colonies on PDA were circular with massive aerial hyphae, white to ochraceous in color. Nonseptate hyphae were hyaline. Sporangiophores arose from hyphae. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 3.15 to 6.55 µm × 1.35 to 2.85 µm (n =50). Morphological characteristics of isolates were consistent with the description of Mucor irregularis (Lima et al. 2018). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and LR0R1/LR5 (Vilgalys et al. 1990). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376751-OL376753, and LSU, OM672239-OM672241. BLAST analysis of these sequences revealed a 100% identity with M. irregularis in GenBank. The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered withM. irregularis (the type strain CBS 103.93).The pathogenicity was tested through in vivo experiments. Nine healthy flowers of Dragon fruit were inoculated with 3-day-old mycelial plugs (5 × 5 mm) of isolates, while another five healthy flowers were treated with PDA plugs (controls). Those plugs were embedded inside the calyxes, and each flower was inoculated with one plug in one calyx. Besides, the inoculated and control flowers (n = 5) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The plants were grown in pots in a greenhouse at 28°C, with relativehumility approximately80%. The test was repeated three times. After 3 days of incubation, rot symptoms developed on the inoculated flowers, which were similar to those observed on the naturally samples in the field. The control flowers remained healthy. The fungus was reisolated from the inoculated flowers and confirmed as M. irregularis by morphology and ITS analysis. M. irregulariswas reported as a pathogen causing human skin diseases and post-harvest diseases of crop (Álvarez et al. 2011; Lima et al. 2018; Wang et al. 2022). This is the first report ofM. irregulariscausing flower rot of Dragon fruit and reduce yield in China. This research can provide a theoretical basis for the fruit industry to maintain yield.