First report of leaf blight on pumpkin caused by Nigrospora sphaerica in China.

Pumpkin (Cucurbita moschata), which belongs to the gourd family (Cucurbitaceae), is widely planted throughout the world. In June 2023, many pumpkin plants (cv. Miben) displayed leaf blight and chlorosis in fields located in Suizhou (31.99°N, 113.02°E), Hubei Province, China. The disease incidence ranged from 30 to 40% in nine fields, 6.3 ha in total. The symptoms were irregularly shaped lesions that expanded along the mid-vein until the leaf turned brown and wilted. Fungal isolations were performed as described previously (Liu et al. 2023). Twenty pumpkin leaf samples with typical symptoms were collected and cut into 1 cm×1 cm pieces. The diseased tissue was surface-sterilized in 75% ethanol for 30 sec, plated on potato dextrose agar (PDA) medium and incubated at 25℃ for 3 days. Then, the emerging single fungal hyphal tip was transferred onto PDA plates to obtain purified isolates. A total of eighteen isolates on PDA plates were initially white and then developed to dark gray. The 5-day-old cultures growing on mung bean medium produced conidia that were black, single-celled, smooth, spherical or oblate, and ranged in size from 14.5 to 20.8 µm×13.3 to 20.5 µm (n=50). Therefore, the isolates were morphologically identified as Nigrospora sphaerica. Moreover, the genomic DNA of the isolates (HB-P1,HB-P2, and HB-P3) 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; Rehner and Samuels 1994), and ß-tubulin (TUB2) (Glass and Donaldson 1995), with primers ITS1/ITS4, LROR/LR3, and Bt2a/Bt2b, respectively. Sequences were submitted to GenBank under accession numbers PP348112, PP348113, PP348114 (ITS), PP411414, PP411415, PP411416 (nLSU), and PP357438, PP357439, PP357440 (TUB2). BLASTn showed that the sequences ITS, nLSU, and TUB2 of HB-P1, HB-P2, and HB-P3 had >99% nucleotide identities ((ITS: 100%, 508/508 bp, MF996488.1; 99.8%, 506/507, ON326588.1; 100%, 500/500 ,MK748317.1), (nLSU: 99.83%, 573/574, KT462720.1; 99.83% , 574/575 bp, KT462720.1; 99.65%, 575/577, KT462720.1), and (TUB2: 100%, 388/388, MN719407.1; 99.74%, 387/388, MN719407.1; 100%, 387/387, MN719407.1)) with Nigrospora sphaerica, respectively. A multilocus (ITS, nLSU and TUB2) phylogenetic analysis indicated that the isolates were Nigrospora sphaerica. Pathogenicity of three isolates were tested on pumpkin plants (cv. Miben). Fifteen pumpkin plants were inoculated by spraying the leaves (1×106 spores/ml), respectively, and 10 pumpkin plants were treated with sterile water as a negative control. 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, the inoculated pumpkin plants developed symptoms of leaf blight, while the control plants remained healthy. Then, pathogens were re-isolated from the each leaf of inoculated pumpkin plants and not from the control plants. Nigrospora sphaerica has been previously reported to cause leaf spot on watermelon in Malaysia (Ismail and Abd Razak 2021). To our knowledge, this is the first report of N. sphaerica causing leaf blight on pumpkin in China. This new disease can cause leaf blight, which may affect pumpkin productivity.

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.