First Report of Monosporascus cannonballus causing Root Rot and Vine Decline in Watermelon (Citrullus lanatus) in China.

In June 2023, a sudden outbreak root rot and vine decline symptoms was observed during a watermelon (Citrullus lanatus T.) variety demonstration trial located in Taizhou City, Zhejiang Province, China, with an incidence rate ranging from 75% to 100% and an affected area of nearly 2,000 square meters. The disease initially appeared with a rapid and alarming invasion of root rot and vine decline symptoms within watermelon plants. Affected plants exhibited rapid deterioration, showing symptoms of wilting, yellowing and eventual demise, predominantly during the pre-harvest stage. Notably, numerous black, spherical, erumpent perithecia were clearly visible on the watermelon's root epidermis, a characteristic trait of the disease. Symptomatic plant samples were rigorously disinfected with 75% ethanol, and plated on potato dextrose agar medium for incubation at 25°C, successfully isolate two potential strains. These isolates were inoculated in oatmeal agar and incubated in a 25℃ light incubator. After 30 days, mature perithecia, the same as those found on the watermelon's root epidermis, reached a diameter of 500 µm. Each perithecium contained several pear-shaped asci, 56 to 108.5 µm in length and 30.5 to 46.4 µm in width, typically holding 1, rarely 2 ascospores. These characteristics align precisely with the typical strains of Monosporascus cannonballus Pollack and Uecker (1974). Additionally, sequencing the internal transcribed spacer region of ribosomal DNA (ITS) gene (White et al., 1990), large subunit ribosomal RNA (LSU) gene (Rehner and Samuels 1995), and beta-tubulin (TUB) gene (Glass and Donaldson, 1995) were performed. BLAST analysis indicated the highest nucleotide sequence identity with M. cannonballus CBS 586.93 reference sequence (ITS: 100%, JQ771930; TUB: 98.99%, JQ907292). Representative sequences of isolate ZJUP0990-2 from these regions were deposited in GenBank (Accession No.: OR357656 for ITS; OR474500 for LSU; OR365762 for TUB). A multigene phylogenomic analysis (ITS-LSU-TUB) was undertaken to ascertain the exact phylogenetic position of M. cannonballus within the genus Monosporascus. The amalgamation of both morphological and molecular insights consistently reaffirmed the accurate classification of the causative agent as M. cannonballus. To validate the pathogenicity of M. cannonballus, a controlled greenhouse experiment was conducted using watermelon (cv. Nabite) as the subject. Mycelium fragments, harvested from the edge of the colony ZJUP0990-2, were inoculated into oat liquid medium and cultivated under dark conditions at a consistent temperature of 30°C for 7 days. After 20 days, the inoculated plants exhibited root rot and wilting, mirroring the symptoms observed during the field outbreak. In contrast, the control plants did not exhibit any signs of disease. M. cannonballus was successfully re-isolated from the symptomatic roots of the inoculated plants, satisfying Koch's postulates. This experiment was repeated three times. This pathogenic fungus has previously been documented as a menace to melons in various regions including Mexico (Chew-Madinaveitia et al., 2012) and Brazil (Sales et al., 2004), as well as watermelons in Brazil (Sales et al., 2010), northern Mexico (Gaytan-Mascorro et al., 2012), and Saudi Arabia (Karlatti et al., 1997). To our knowledge, this is the first reported presence of M. cannonballus on watermelons in China. This new disease poses a serious threat to watermelon production, potentially leading to severe economic losses and impacting food security.

First report of Trunk Canker Caused by Botryosphaeria dothidea on Cinnamomum camphora in China.

Cinnamomum camphora, an essential woody plant in China, experienced a severe outbreak of trunk canker affecting an area of 540,000 square meters in the Xiaoshan District of Zhejiang province during the early summer of 2022. The observed symptoms included stem canker, dieback, twig blight, and extensive vascular discoloration, with an incidence rate ranging from 45% to 70%. To investigate the etiology of the disease, symptomatic plant samples were subjected to rigorous surface sterilization involving washing with running tap water, followed by surface sterilization using 75% ethanol. The samples were then rinsed twice with sterile distilled water, plated on potato dextrose agar (PDA) medium, and incubated at a temperature of 28°C. The isolated fungi exhibited characteristics consistent with the species Botryosphaeria dothidea. Fungal isolates displayed abundant white aerial mycelium, which darkened to grey after five to seven days, eventually giving rise to black pycnidia. Single hyphal tip cultures of putative two isolates were stored at the Agricultural Experiment Station of Zhejiang University. Conidia formed on pycnidia were one-celled, hyaline, aseptate, and fusiform, with dimensions of 18.34-27.12 µm x 3.77-6.84 µm (average 22.90 µm x 5.20 µm) (one hundred conidia were measured). To determine the fungal species, genomic DNA was extracted from individual isolates ZJUP0868, and subjected to DNA sequence analysis of four gene regions: the internal transcribed spacer (ITS) using the primer-pair ITS4 and ITS1 (White et al., 1990), the large subunit (LSU) gene with LR0R and LR5 (Rehner and Samuels, 1995), the small subunit (SSU) gene with SR1R and SR7 (Zoller et al., 1999), and the translation elongation factor 1-alpha (tef1) with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). Furthermore, DNA sequence analysis of above four genes was performed. BLAST analysis indicated the highest nucleotide sequence identity with Botryosphaeria dothidea CBS 115476 reference sequence (ITS: 99.59%, KF766151; LSU: 99.88%, DQ377852; SSU: 100%, NG_062738; tef1: 98.93%, AY236898). Representative sequences of isolate ZJUP0868 from these regions were deposited in GenBank (ITS: Accession No. OR192838; LSU: Accession No:OR248147; SSU: Accession No:OR248174; tef1: Accession No. OR262053). A phylogenomic analysis was conducted to determine the phylogenetic position of Botryosphaeria dothidea in the family Botryosphaeriaceae. The combined morphological and molecular findings confirmed the identification of the pathogen as Botryosphaeria dothidea. Pathogenicity tests were conducted by stem inoculation of two-year-old Cinnamomum camphora baranches. Mycelial plugs (2-3 mm in diameter) from actively growing colonies of B. dothidea (PDA) were applied to bark of similar size on the middle point of the stems. Inoculated barks were wrapped with Parafilm, while control branches received sterile PDA plugs. Inoculated and control branches (7 each) were maintained in a greenhouse at 28°C. After two weeks, all inoculated plants exhibited dark vascular stem tissue, whereas the control plants remained healthy. B. dothidea was re-isolated from symptomatic tissues, thus fulfilling Koch's postulates. No symptoms were observed in the control branched, and B. dothidea was not re-isolated from their tissues. B. dothidea , the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales), is commonly associated with cankers and dieback in woody plants. Previous reports have identified Botryosphaeria dothidea as a pathogen causing stem dieback and trunk canker on Glycine max (Chen et al., 2020), Camellia oleifera (Hao et al., 2022), and Gleditsia sinensis (Huang et al., 2020). Additionally, B. dothidea has been reported to cause leave wilt on various plant species in China, including Daimyo oak (Liu et al., 2023) and Cornus officinalis (Zhang et al., 2022). To the best of our knowledge, this is the first report of B. dothidea on C. camphora in China. This findings contribute to a better understanding of the pathogens affecting Cinnamomum camphora in the China.