TUB

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.

Bitter rot is an emerging disease of apple (Malus domestica) fruit in Ontario in part due to changing weather conditions. The disease was mostly documented in warm and humid regions such as the southern USA, and Central and South America. Thirteen Ontario orchards in the fall of 2019 and 15 in 2020 were scouted for bitter rot based on their previous history of the disease. 100 fruit were collected from ten asymptomatic trees per cultivar and two susceptible cultivars, 'Empire' and 'Ambrosia' were scouted per orchard. If an orchard did not have either one of these cultivars, 'Honeycrisp' or 'Gala' were used. The fruit was stored at 4-5 ^oC for five months and then left at 22 ^oC for two weeks and assessed thereafter for bitter rot symptoms. Monoconidial cultures of Colletrotrichum spp. were established from the symptomatic fruit using potato dextrose agar media with antibiotics at 22 ^oC 14-hour light cycles. The fungal isolates were divided into two groups based on colony morphology observations seven days after culturing. The first group showed light grey, cottony, aerial mycelium on top and a pink to dark red color on the reverse of the plate. The second group showed light to dark grey, cottony mycelium on top and dark green colonies on the reverse of the plate. It is difficult to identify Colletrotricuhum species solely based on morphology, therefore the representative isolates from each group were used for multilocus gene sequencing and species identification. Genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit according to the manufacturer's protocols. The ITS region was amplified and sequenced using the primers ITS-1F (Grades & Bruns 1993) and ITS-4 (White et al. 1990). Primers T1 and T2 (O'Donnell & Cigelnik 1997) were used to amplify and sequence the 750 bp region of the TUB gene. Primers GDF1 and GDR1 (Guerber et. al. 2003) were used to amplify a 280 bp region of the GADPH gene. Following an NCBI nucleotide blast search, the isolates from group 1 were identified as C. fioriniae.The ITS sequences of group 2 isolates were matched 100% to the Colletotrichum godetiae type strain CBS133.44 and they were 99% matched to the closest species C. Johnstonii CBS128532. The TUB sequences matched 100% identity to 20 sequences belonging to C. godetiae, 99.59 % identity to C. godetiae type strain CBS133.44, and 97.75% to C. Johnstonii CBS128532. The GADPH sequences matched with 100% identity to C. godetiae ON241087.1 or MT816329.1 and 99-99.5% identity to the type strain CBS133.44 and 98.61-99% identity to CBS128532. Based on the blast results the group 2 isolates were identified as C. godetiae and their sequences were submitted to GenBank with ID OP702962 for ITS and OP972240 and OP972241 for GADPH and OP972242 for the TUB gene. Out of 50 isolates collected in this work, 94% belonged to group 1 and 6% belonged to group 2. Koch's postulates were performed on selected isolates by artificial inoculation of 5 healthy detached fruits of the cultivar, 'Empire.' Fruit surfaces were wiped with 70% ethanol, dried, wounded with a sterile needle, and then inoculated with a 10 µl of spore suspension containing 1x10^6 spores /ml. Inoculated fruits were incubated in a humid chamber at 22°C in dark. Symptoms started to appear at 5 days post-inoculation and looked like small brown circular lesions which developed orange spore masses as they grew into larger lesions. The non-inoculated control fruit did not develop lesions. Fungal colonies were established from the spores collected from the inoculated fruit and found to have identical morphological characteristics to the original isolates. C. godetiae has previously been reported to cause bitter rot in apples from various countries in Europe (Baroncelli et al. 2014; Munda, 2014; Wenneker et al. 2016). C. fioriniae has previously been reported as the dominant species causing bitter rot in apples although other species including C. chrysophilum and C. noveboracense have also been reported as causal agents from the Eastern USA (Khodadadi et al. 2020). To the best of our knowledge, this is the first report of Colletotrichum godetiae causing the bitter rot of apples in Ontario, Canada.

Polypropylene microplastics (PP-MPs) are emerging environmental contaminants that have the potential to cause adverse effects on aquatic organisms. Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) is a valuable tool for assessing the gene expression profiles under PP-MPs stress. To obtain an accurate gene expression profile of tissue inflammation and apoptosis that reflects the molecular mechanisms underlying the impact of PP-MPs on Chinese sturgeon, identifying reliable reference genes is crucial for RT-qPCR analysis. In this study, we constructed an experiment model of Chinese sturgeon exposed to PP-MPs, assessed the pathological injury, metabolic profile responses and oxidative stress in liver, evaluated the reliability of 8 reliable reference genes by 4 commonly used algorithms including GeNorm, NormFinder, BeatKeeper, Delta Ct, and then analyzed the performance of inflammatory response genes in liver, spleen and kidney with the best reference gene. HE staining revealed that the cytoplasm full small vacuoles and nucleus diameter increased were occurred in the liver cell of PP-MPs in the treatment groups. Additionally, oxidative and biochemical parameters were significantly changes in the liver of treatment groups. For the reference genes in PP-MPs exposure experiments, this study screening the optimal reference genes including: EF1α and GAPDH for liver and spleen, and GAPDH and RPS18 for kidney. Besides, 2 inflammatory response genes (NLRP3, TNF-α) were chosen to assess the optimal reference genes using the least stable reference gene (TUB) as a control, verified the practicality of the select reference genes in different tissues. We also found that the low concentration of PP-MPs could generate the liver tissue damage and inflammatory response in Chinese sturgeon. Our study initially evaluated the impact of short-time exposure with PP-MPs in Chinese sturgeon and provided 3 sets of validated optimal reference genes in Chinese sturgeon exposure to PP-MPs.