Genetic variability in the E6 and E7 oncogenes of HPV52 and its prevalence in the Taizhou area, China.

BACKGROUND: Human papillomavirus (HPV) 52 is one of the prevalent oncogenic HPV genotypes in East Asia. Chinese women have the highest susceptibility to the HPV52 type, but research data on HPV52 genetic variability and its carcinogenicity in China is lacking. METHODS: The present study aimed to investigate the genetic variability of HPV52 currently circulating among Chinese women by PCR sequencing the entire E6 and E7 oncogenes. HPV52 sequence alignment, genetic heterogeneity analyses and maximum-likelihood phylogenetic tree construction were performed by BioEdit software and MEGA X software. RESULTS: Between 2016 and 2018, the overall HPV infection rate was 21.3%, of which HPV52 was the most prevalent high-risk type (17.2%) in the Taizhou area, China. A total of 339 single HPV52-positive samples were included in this study. We obtained 27 distinct variation patterns of HPV52 with the accession GenBank numbers ON529577-ON529603. Phylogenetic analysis showed that 96.6% of HPV52 variants belonged to lineage B, which seemed to be uniquely defined by G350T, A379G (K93R) in the E6 gene and C751T, A801G in the E7 gene. Due to the dominance of lineage B in our study population, the results could not be used to assess the association of the HPV52 (sub)lineage with the risk of cervical lesions. In addition, no significant trends were observed between the nucleotide substitutions of HPV52 variants and the risk of cervical carcinogenesis. CONCLUSION: Our data showed that HPV52 variants were strongly biased towards lineage B. These results confirmed that cervical lesions in the Taizhou area are highly attributable to HPV52, which may be due to the high infection rate of lineage B in the population.

First Report of Post-Harvest Anthracnose of Kiwifruit Caused by Colleotrichum fioriniae in Liaoning and Sichuan Province, China.

Kiwifruit (Actinidia spp.) is one of the most important fruit crops in China. Post-harvest anthracnose symptoms were observed on kiwifruit in October 2021. Kiwifruits 'Longcheng 2' (n=200) were obtained from an orchard in Kuandian city of Liaoning province, China (124°32'E, 40°20'N). And cultivar 'Cuiyu' (n=100) were harvested from orchards in Mianzhu city, Sichuan Province, China (104°03'E, 31°15'N). After storage at 24 °C and 80% relative humidity (RH) for 8 days, the disease incidence of 'Longcheng 2' and 'Cuiyu' was 30% and 15%, respectively. Symptoms of diseased fruits appeared as water-soaked, irregular and light brown lesions. Orangish conidial masses were observed on some fruits. Ten lesion margins (5×5 mm) from 'Longcheng2' or 'Cuiyu' were respectively excised, surface sterilized by 70% ethanol (1 min), 1% NaOCl (5 min), washed, dried, plated on potato dextrose agar (PDA), and incubated at 25 °C for 5 days. Eight isolates were obtained from 'Longcheng 2' (LC1-3 to LC1-10) and nine strains from 'Cuiyu' (CY1-2 to CY1-10). The representative isolates LC1-3 and CY1-2 were put on PDA, and appeared white to pale gray on the upper side. However, isolate LC1-3 secreted red pigments after 7 days of culture. Conidia of LC1-3 were hyaline, smooth-walled, single-celled, cylindrical (3.0 to 4.9×7.2 to 14.7 µm, n=50). Ellipsoidal single cell conidia of CY1-2 were hyaline, and ranged in size from 3.2 to 5.0×8.5 to 13.9 µm (n=50) born on conidiophores. Appressorium of isolates LC1-3 and CY1-2 were globose to ellipsoid with 4.2 to 7.4×7.3 to 10.8 µm and 3.0 to 4.9×6.3 to 10.3 µm in size, respectively (n=50) (Fu et al. 2019 ). Four genes (ACT, CHS, GAPDH, TUB2) and the ITS region were successfully amplified and sequenced from all isolates (Weir et al. 2012). Based on sequence alignment, the isolates from 'Longcheng 2' or 'Cuiyu' were identical. BLAST analysis of the ACT, CHS, GAPDH, ITS and TUB2 sequences of LC1-3 (ON018724, ON018722, ON018720, OM980324, ON018718) or CY1-2 (ON018725, ON018723, ON018721, OM980325, ON018719) showed high similarity with C. fioriniae (CBS 128517; JQ949613, JQ948953, JQ948622, MH865005, JQ949943) were 97.1% to 99.7% or 98.1% to 99.7%, respectively. Phylogenetic analysis using concatenated sequences (maximum likelihood method) with MEGA 11 showed LC1-3 and CY1-2 were located within the same clade with C. fioriniae. Previous studies showed that C. fioriniae was classified into three subclades (Damm et al. 2012; Fu et al. 2019). However, LC1-3 and CY1-2 were located within a new subclade, namely the subclade IV. To test pathogenicity, healthy and mature kiwifruits 'Donghong', 'Cuiyu', 'Xuxiang', 'Hayward' and 'Jinyan' were surface sterilized. Each un-wounded fruit was dropped with 10 µl conidial suspension (105 conidia/ml) on the fruit surface. All fruits were placed into a plastic box and stored at 24 °C under 80% RH. Each treatment consisted of 10 fruits and were repeated three times. After 8 days, typical anthracnose lesions were observed on all inoculated fruits. Whereas, the controls treated with sterile distilled water remained asymptomatic. The pathogens re-isolated from diseased fruits were similar morphological and identical to the original isolates, fulfilling Koch's postulates. Anthracnose caused by C. fioriniae has been reported on many fruits (Ling et al. 2020; Waller et al. 2021), but to our knowledge, this is the first report of anthracnose on kiwifruit caused by C. fioriniae. The results will provide valuable information for avoiding post-harvest anthracnose on kiwifruit.

First Report of Leaf Spot Caused by Colletotrichum fructicola on Kiwifruit in China.

Kiwifruit (Actinidia spp.) is an important fruit with high nutritional and economic value, which is widely cultivated in China. In April 2021, leaf spots were observed on the leaves of 'Xuxiang' (A. deliciosa) in a kiwifruit plantation of Hefei city, Anhui province, China (117°26'E, 31°85'N). Disease incidence was about 10% of the observed plants. Small yellow spots initially developed on the leaves and gradually expanded into irregular dark brown spots, and eventually the diseased leaves curled and withered. Leaf tissues (n=10, 5×5 mm) were collected from five infected plants, sterilized in 75% ethanol solution for 30 s and 1% NaOCl for 5 min, washed, dried and plated on PDA at 25°C. In total, ten isolates were obtained, including two previously reported Botryosphaeria dothidea (Zhou et al. 2015) and Diaporthe actinidiae strains (Bai et al. 2017) and eight unknown isolates with similar morphology. All unknown isolates initially appeared white with many aerial hyphae, and at the later stage, the center of all colonies turned gray. Colonies were transferred to new PDA with 0.1% yeast extract for three days. Then, aerial hyphae were scraped with sterile cotton swabs, and continued to grow for four days. Orange conidial masses were produced. Conidia were hyaline, smooth-walled, single-celled, cylindrical with broadly rounded ends, with average size around 4.1-5.5×13.2-18.2 µm (n=100). Appressoria (n=50) were ovoid in shape with average size around 4.9-6.7×8.6-11.8 µm. Morphological features were similar to Colletotrichum. gloeosporioides species complex (Weir et al. 2012). To confirm their species identification, internal transcribed spacers (ITS), ß-tubulin (TUB2), glyceraldehydec-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS) were amplified by PCR using the primer pairs ITS1/ITS4, Bt2a/Bt2b, GDF/GDR, ACT-512F/ACT-783R CHS-79F/CHS-234R, respectively (Weir et al. 2012). Based on alignment analysis, sequences of the eight unknown isolates were 100% homologous. The representative isolate LSD3-1 was selected for further study. BLAST analysis showed that the ITS (OM033371), TUB2 (OM044376), GAPDH (OM044377), ACT (OM044379) and CHS (OM044378) sequences of isolate LSD3-1 were 98.7%-100% identical with the collected sequences of C. fructicola strain ICMP:18581 (NR_144783, JX010405, JX010033, JX009866, JX009501). Phylogenetic analysis of multiple genes was conducted with the Maximum likelihood method using MEGA 7. Based on morphological and molecular characteristics, the LSD3-1 was identified as Colletotrichum fructicola (Prihastuti et al., 2009). Koch's postulates were performed on six one-year-old 'Xuxiang' plants, which were used to test pathogenicity in the greenhouse (at 28℃, relative humidity 80%, 16/8 h light/dark). Surface-sterilized leaves were sprayed with a conidial suspension (107 conidia/mL). Yellow and brown lesions were formed 14 to 21 days after inoculation, whereas the mock-inoculated controls remained asymptomatic. The experiment was performed three times. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Although C. fructicola has been reported as a leaf spot disease on many plants (Shi et al. 2018), this is the first report of leaf spot caused by C. fructicola on kiwifruit in China. This result is helpful to better understanding the pathogen of kiwifruit leaf spot diseases in China and formulate effective control strategies.

First Report of root rot Caused by Fusarium solani and F. breve on kiwifruit in China.

China is considered as the main producer of kiwifruit (Actinidia spp.) in the world. During 2020-2021, root rot (~8000 plants, ~5% disease incidence) of 3-year-old kiwifruit (cv. Xuxiang) was observed in Lujiang County (117°24'E, 31°15'N), Anhui, China. This disease usually occurred in fields with poor drainage in hot and humid summers. Symptoms started on leaves showing dehydration and curling, the last root of diseased plant turned black and died. Dig out the skin on rotten root was cracking and flaking and white mycelium covered on surface. Twenty rotten tissues from ten plants were cut and surface disinfected with 1% NaOCl for 5 min, rinsed in sterile water, and cultured on potato dextrose agar (PDA) at 25 ± 2°C in the dark. Fifteen fungal isolates were obtained. The first type (KWRR1, 3-10) was cotton-like, reverse with white outer margin, and light brown inner region on PDA. The second type (KWRR2, 11-15) was cotton-like on PDA but appeared pale yellow in reverse. On oatmeal agar, the KWRR1 colony was flat with little aerial hyphae and was red, while KWRR2 was hyaline. On carnation leaf agar (CLA), microconidia of the KWRR1 and KWRR2 isolates were reniform, fusiform or oblong, 0-1 septate, and measuring 1.9-4.3×8.4-15.7 µm and 3.0-3.8×8.2-16.7 µm, respectively (n=50). The macroconidia of KWRR1 were straight or moderately curved, 3-5 septa (2.7-4.6×21.5-52.6 µm in size, n=50). For KWRR2, the macroconidia were straight or slightly curved and with 3-4 septate, 4.1-4.8×26.1-30.8 µm (n=50). Chlamydospores of the KWRR1 and KWRR2 isolates were 1-2 celled, irregular globose, measuring 4.5-8.5 µm and 7.6-9.0 µm diam, respectively (n=50). To identify the isolates, four DNA fragments (RPB1, RPB2, ITS and TEF-1α) were amplified and sequenced from all isolates (O'Donnell, et al. 2012; White et al. 1990; O'Donnell et al. 2022). BLAST analysis of the RPB1, RPB2, ITS and TEF-1α sequences of the KWRR1 isolates (OL474057, OL474055, OL468550, OP382187) showed highest identity with F. solani (NRRL66304; MW218134, KT313623, KT313633, KT313611) at 98.2%-99.8%, while KWRR2 (OL505579, OL474056, OL468551, OP382188) showed that their homology with F. breve (NRRL28009; HM347149, EF470136, DQ094351, DQ246869) at 98.2%-99.4%. F. solani and F. breve belong to clade 3 of the F. solani species complex (FSSC) (Geiser et al. 2021). Phylogenetic analysis based on RPB2, ITS and TEF-1α sequences with MEGA7 software (Sisic et al. 2018), placed the KWRR1 sequences with F. solani (FSSC5), while there of KWRR2 nested with F. breve (FSSC15). One-year-old seedlings (n=6) of 'Xu Xiang', growing in a greenhouse (at 28℃, relative humidity 80%), were inoculated by drenching the soil with a conidial suspension with one of the two isolates (30 ml, 106 conidia/ml). Control plants (n=6) were inoculated with sterilized water and the pathogenicity assay was repeated three times. One month post-inoculation, the leaves of inoculated plants became chlorotic, wilted and died, whereas the controls were disease-free. F. solani and F. breve were successfully reisolated from diseased samples (n=6) and verified based on morphology and sequencing as described above, fulfilling Koch's postulates. Members of the FSSC cause root rot on many hosts (Coleman. 2016; Schroers et al. 2016), but this is the first report of F. solani and F. breve causing root rot disease on kiwifruit in China. The result will serve as the foundation for management of root rot of kiwifruit.

Anti-Proliferation Activity of a Decapeptide from Perinereies aibuhitensis toward Human Lung Cancer H1299 Cells.

Perinereis aibuhitensis peptide (PAP) is a decapeptide (Ile-Glu-Pro-Gly-Thr-Val-Gly-Met-Met-Phe, IEPGTVGMMF) with anticancer activity that was purified from an enzymatic hydrolysate of Perinereis aibuhitensis. In the present study, the anticancer effect of PAP on H1299 cell proliferation was investigated. Our results showed that PAP promoted apoptosis and inhibited the proliferation of H1299 cells in a time- and dose-dependent manner. When the PAP concentration reached 0.92 mM, more than 95% of treated cells died after 72 h of treatment. Changes in cell morphology were further analyzed using an inverted microscope and AO/EB staining and flow cytometry was adopted for detecting apoptosis and cell cycle phase. The results showed that the early and late apoptosis rates of H1299 cells increased significantly after treatment with PAP and the total apoptosis rate was significantly higher than that of the control group. Moreover, after treatment with PAP, the number of cells in the S phase of cells was significantly reduced and the ability for the cells to proliferate was also reduced. H1299 cells were arrested in the G2/M phase and cell cycle progression was inhibited. Furthermore, the results of western blotting showed that nm23-H1 and vascular endothelial growth factor (VEGF) protein levels decreased in a dose-dependent manner, while the pro-apoptotic protein and anti-apoptotic protein ratios and the level of apoptosis-related caspase protein increased in a dose-dependent manner. In conclusion, our results indicated that PAP, as a natural marine bioactive substance, inhibited proliferation and induced apoptosis of human lung cancer H1299 cells. PAP is likely to be exploited as the functional food or adjuvant that may be used for prevention or treatment of human non-small cell lung cancer in the future.