Identity, pathogenicity and genetic diversity of Lasiodiplodia associated with stem-end rot of avocado in China.

Stem-end rot (SER) causes brown necrotic lesions in the pulp near the base of the fruit pedicel and is one of the most devastating postharvest diseases of avocados in all avocado growing regions of the world. China's avocado industry is growing very rapidly, and the planting area is expanding, but little is known about the pathogens and genetic diversity of avocado SER. To determine the causal agents of SER, avocado fruits were sampled from the main avocado-producing areas in China during 2020 and 2021. Fungal isolates were obtained from SER symptomatic avocado fruits and identified by morphology combined with phylogenetic analysis of internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and ß-tubulin (TUB2) gene sequences. All 101 isolates belonged to Lasiodiplodia spp., and four Lasiodiplodia species were identified, namely L. pseudotheobromae (59.41%), L. theobromae (24.75%), L. mahajangana (7.92%), L. euphorbiaceicola (1.98%), and six others are classified as Lasiodiplodia sp. (5.94%). There were only slight morphological differences in colonies and conidia of these four species of Lasiodiplodia. The pathogenicity tests showed symptoms of SER, and the 92.08% of the isolates exhibited a high level of virulence on avocado (disease index > 70), related to the disease severity on avocado fruit. All tested isolates grew well under conditions from 23 to 33℃. There was a significant difference in mycelial growth between the four species of Lasiodiplodia after treatment with high temperature or low temperature. L. pseudotheobromae growth was the fastest at 13 to 18℃, but was the lowest at 38℃ (P < 0.05). Red pigment could be produced by all tested isolates after culturing for 7 days at 38℃. The mycelial growth rate was the fastest on PDA medium, and the slowest on OMA medium but promoted spore formation (P < 0.05). In addition, was determined the genetic diversity of Lasiodiplodia pathogenic species associated with SER collected from avocado, mango, guava and soursop fruits was determined. A total of 74 isolates were clustered into 4 main ISSR groups by unweighted pair-group method with arithmetic mean (UPGMA) analysis, and the classification of this group was related to the host. Extensive diversity was detected in the Lasiodiplodia populations. The diverse geographical origins and host species significantly influenced the population differentiation, and most of the genetic variation occurred within populations (P < 0.001). This is the first study to identify the major pathogens of avocado SER in China and to survey their occurrence, pathogenicity and include a comparative analysis of genetic diversity with Lasiodiplodia spp. causing SER on other fruit hosts. Collectively, the Lasiodiplodia species complex affecting avocado showed high pathogenicity and diversity, while L. pseudotheobromae was the most frequently isolated species in China. The results of this study provide insights into the aspects of epidemic of SER disease caused by Lasiodiplodia species, which will help in developing strategies for the management and control of stem end-rot in avocado.

Biochemical and Molecular Characterization of Prochloraz Resistance in Lasiodiplodia theobromae Field Isolates.

Stem-end rot (SER), caused by Lasiodiplodia theobromae, is one of the most critical diseases of mango in China. The demethylation inhibitor fungicide prochloraz has been widely used in China to control mango diseases. Isolates (n = 139) of L. theobromae were collected in 2019 from six mango-producing regions in Hainan Province, China. The fungicide sensitivity of L. theobromae isolates to prochloraz revealed that the EC50 (50% effective concentration) values ranged from 0.0006 to 16.4131 µg/ml. In total, 21 of the 139 isolates were categorized as resistant to prochloraz. The resistant isolates sprayed with prochloraz could not be effectively controlled in detached fruit. The mycelial growth, conidia germination, and ability to grow at temperatures ranging from 12 to 35°C of resistant isolates decreased, suggesting fitness penalties. The experiment showed that, after treatment with prochloraz at 10 µg/ml, the content of ergosterol in the mycelia of the sensitive isolate decreased by 80.23%, whereas the resistant strain decreased by only 57.52%. The damage to membranes in the sensitive isolates was more serious than for resistant isolates. The target gene CYP51 and the ATP-binding cassette (ABC) subfamily ABCG gene were cloned but no mutation was found. When treated with prochloraz, the expression of CYP51 and ABCG in resistant isolates was significantly higher than in the sensitive isolates. Thus, induced expression of its target gene combined with the induction of expression drug efflux transporters appeared to mediate the prochloraz resistance of L. theobromae.

Analysis of the Effect of Fast Recovery Surgery Concept on Perioperative Nursing Care of Patients with Radical Resection of Cervical Cancer and Its Influence on Psychological Status.

Objective: To explore the applied effect of fast-track surgery concept in the perioperative nursing of patients undergoing radical cervical cancer surgery and its influence on mental state. Methods: The clinical data of 110 patients undergoing radical cervical cancer surgery in our hospital from May 2015 to May 2017 were retrospectively analyzed, and they were randomly divided into a research group (n = 55) and a reference group (n = 55). The reference group received routine clinical nursing, and the research group received fast-track surgical nursing. Then, we compared their nursing effect and influence on the mental state of patients in the two groups. Results: The Karnofsky performance status (KPS) scores of patients in the two groups after intervention were significantly lower than those before the intervention (P < 0.001), and after intervention, patients in the research group had significantly lower KPS scores as compared to patients in the reference group (P < 0.001). There was a significant decrease in the self-rating anxiety scale (SAS) scores and self-rating depression scale (SDS) scores of patients in the two groups after intervention in comparison with before intervention (P < 0.001), and patients in the research group after intervention had significantly lower SAS and SDS scores compared to the reference group (P < 0.001). Patients in the research group spent short time on expelling gas, eating, and getting out of bed as compared to the reference group (P < 0.001); after intervention, compared to patients in the reference group, patients in the research group had significantly higher scores in cognitive function, emotional function, social function, and physical function (P < 0.05). There was no significant difference in IgA, IgM, and IgG levels before intervention (P > 0.05). After intervention, the IgA, IgM, and IgG levels of patients in the two groups were all lower than before intervention, and patients in the research group had significantly higher IgA, IgM, and IgG levels as compared to the reference group (P < 0.001); the complication rate of patients in the research group was significantly lower than that in the reference group (P < 0.05). Conclusion: The fast-track surgery concept effectively helps improve the negative emotion of patients, shorten recovery time, improve quality of life, and reduce the impact on immune function in the radical cervical cancer surgery, and it is worthy of promotion and application, with a high safety.

Association between short-term exposure to ambient air pollution and dementia mortality in Chinese adults.

BACKGROUND: Short-term exposure to ambient air pollution has been linked to an increased risk of mortality from a variety of causes, but its effects on mortality from dementia remain largely unknown. OBJECTIVES: To investigate the association between short-term exposure to ambient air pollution and dementia mortality, and quantitatively assess the excess mortality. METHODS: In this time-stratified case-crossover study, 47,108 dementia deaths were identified in Jiangsu province, China during 2015-2019. Exposure to particulate matter with an aerodynamic diameter ≤ 2.5 µm (PM2.5), PM10, sulfur dioxide (SO2), nitrogen dioxide (NO2), carbon monoxide (CO), and ozone (O3) was assessed by extracting daily concentrations from a validated grid dataset based on each subject's residential address. Conditional logistic regression models were applied for exposure-response analyses. RESULTS: There were 47,108 case days and 159,852 control days during the study period. Each 10 µg/m3 increase of lag 04-day exposure to PM2.5, PM10, and NO2 was significantly associated with a 1.43 % (95 % CI: 0.77, 2.09 %), 1.06 % (0.59, 1.54 %), and 2.80 % (1.51, 4.10 %) increase in odds of dementia mortality, corresponding to an excess mortality of 4.87 %, 5.50 %, and 6.43 %, respectively. We estimated that reducing ambient air pollutant exposures to the WHO air quality guidelines would avoid up to 4.17 % of the dementia deaths, while the ambient air quality standards in China would only help avoid up to 0.39 %. CONCLUSIONS: This study provides consistent evidence that short-term exposure to PM2.5, PM10, and NO2 is associated with increased odds of dementia mortality, which can be translated to a considerable excess mortality. Our findings highlight a potential approach to prevent deaths from dementia by reducing individual exposures to ambient air pollution, especially in areas with high levels of ambient air pollution.

Nomograms for primary mucinous ovarian cancer: A SEER population-based study.

BACKGROUND: To develop predictive nomograms of overall survival (OS) and cancer-specific survival (CSS) in patients with primary mucinous ovarian cancer (PMOC). METHODS: Patients diagnosed with PMOC from 2010 to 2015 were obtained from the Surveillance, Epidemiology, and End Results (SEER) database, and randomly divided into a training cohort and a validation cohort. Univariate and multivariate Cox regression analyses were conducted to identify the independent risk factors. Nomograms were constructed and then verified by calibration plots, the concordance index (C-index), and the area under the receiver operating characteristic curve (AUC). RESULTS: A total of 991 patients with PMOC were enrolled and randomly divided into a training cohort (n=695) and a validation cohort (n=296) at a ratio of 7:3. Multivariate Cox regression analyses demonstrated that independent risk factors for OS included age, laterality, and American Joint Committee on Cancer (AJCC) stage. Independent risk factors for CSS included age, laterality, grade, and AJCC stage. Predictive nomograms for OS and CSS were developed with respective independent risk variables. In the training cohort, the C-index of the CSS and OS nomograms were 0.88 [95% confidence interval (CI): 0.84-0.92] and 0.85 (95% CI: 0.81-0.89), respectively. In the validation cohort, the C-index of the predictive CSS and OS nomograms were 0.86 (95% CI: 0.80-0.92) and 0.80 (95% CI: 0.74-0.87), respectively. The AUCs were higher in both cohorts. Furthermore, the calibration curves in both cohorts showed good consistency between the predicted results and the actual results. CONCLUSION: The nomograms demonstrated good predictability for the survival of patients with PMOC, and could serve as an applicable tool to help clinicians improve treatment plans.

Association of short-term exposure to ambient air pollution with mortality from ischemic and hemorrhagic stroke.

BACKGROUND: Short-term exposure to ambient air pollution has been linked to increased risk of stroke mortality, but its adverse effects on mortality from specific types of stroke including ischemic stroke and hemorrhagic stroke remain poorly understood. METHODS: Using the China National Mortality Surveillance System, we conducted a time-stratified case-crossover study among 412,567 stroke deaths in Jiangsu province, China during 2015-2019. Residential daily PM2.5 , PM10 , SO2 , NO2 , CO, and O3 exposure concentrations were extracted from the ChinaHighAirPollutants dataset for each subject. Conditional logistic regression models were performed to conduct exposure-response analyses. RESULTS: Each 10 µg/m3 increase of PM2.5 , PM10 , SO2 , NO2 , CO, and O3 was respectively associated with a 1.44%, 0.93%, 5.55%, 2.90%, 0.148%, and 0.54% increase in odds of mortality from ischemic stroke, which was significantly stronger than that from hemorrhagic stroke (percent change in odds: 0.74%, 0.51%, 3.11%, 1.15%, 0.090%, and 0.10%). The excess fraction of ischemic stroke mortality associated with PM2.5 , PM10 , SO2 , NO2 , CO, and O3 exposure was 6.90%, 6.48%, 8.21%, 8.61%, 9.67%, and 4.76%, respectively, which was also significantly higher than that of hemorrhagic stroke mortality (excess fraction: 3.49%, 3.48%, 4.69%, 3.48%, 5.86%, and 0.88%). These differences in adverse effects generally remained across sex, age, and season. CONCLUSIONS: Short-term exposure to ambient air pollution was significantly associated with increased risk of both ischemic and hemorrhagic stroke mortality and posed considerable excess mortality. Our results suggest that air pollution exposure may lead to substantially greater adverse effects on mortality from ischemic stroke than that from hemorrhagic stroke.

Molecular Characterization of Mg-Chelatase CHLI Subunit in Pea (Pisum sativum L.).

As a rate-limiting enzyme for chlorophyll biosynthesis, Mg-chelatase is a promising target for improving photosynthetic efficiency. It consists of CHLH, CHLD, and CHLI subunits. In pea (Pisum sativum L.), two putative CHLI genes (PsCHLI1 and PsCHLI2) were revealed recently by the whole genome sequencing, but their molecular features are not fully characterized. In this study, PsCHLI1 and PsCHLI2 cDNAs were identified by PCR-based cloning and sequencing. Phylogenetic analysis showed that PsCHLIs were derived from an ancient duplication in legumes. Both PsCHLIs were more highly expressed in leaves than in other organs and downregulated by abscisic acid and heat treatments, while PsCHLI1 was more highly expressed than PsCHLI2. PsCHLI1 and PsCHLI2 encode 422- and 417-amino acid proteins, respectively, which shared 82% amino acid identity and were located in chloroplasts. Plants with a silenced PsCHLI1 closely resembled PsCHLI1 and PsCHLI2 double-silenced plants, as both exhibited yellow leaves with barely detectable Mg-chelatase activity and chlorophyll content. Furthermore, plants with a silenced PsCHLI2 showed no obvious phenotype. In addition, the N-terminal fragment of PsCHLI1 (PsCHLI1N, Val63-Cys191) and the middle fragment of PsCHLI1 (PsCHLI1M, Gly192-Ser336) mediated the formation of homodimers and the interaction with CHLD, respectively, while active PsCHLI1 was only achieved by combining PsCHLI1N, PsCHLI1M, and the C-terminal fragment of PsCHLI1 (Ser337-Ser422). Taken together, PsCHLI1 is the key CHLI subunit, and its peptide fragments are essential for maintaining Mg-chelatase activity, which can be used to improve photosynthetic efficiency by manipulating Mg-chelatase in pea.

Association of short-term exposure to air pollution with recurrent ischemic cerebrovascular events in older adults.

The acute effects of ambient air pollution on recurrence of ischemic cerebrovascular events (ICEs) remains largely unknown. We therefore conducted a time-stratified case-crossover study of 43,896 patients who were 60 years or older and were admitted to hospital for recurrent ICEs including ischemic stroke and transient ischemic attack in Guangzhou, China during 2016-2019. Based on each patient's home address and pollutant data from its neighboring air quality monitoring stations, we used an inverse distance weighting method to assess exposures to particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5), particulate matter with an aerodynamic diameter ≤10 µm (PM10), sulfur dioxide (SO2), nitrogen dioxide (NO2), carbon monoxide (CO) and ozone (O3). Conditional logistic regression models were used to quantify exposure-response associations. During the study period, there were 43,896 case days and 149,131 control days. In single-pollutant models, each 10 µg/m3 increase in exposure to PM10, NO2 and CO (mean exposure on date of admission and 1 day prior) was significantly associated with a 0.74% (95% confidence interval [CI]: 0.13-1.36%), 2.15% (1.38-2.93%) and 0.14% (0.07-0.21%) increase in odds of hospital admissions for recurrent ICEs, respectively, and no significant departures from linearity were detected. The association for NO2 exposure remained consistent in 2-pollutant models, while the associations for PM10 and CO disappeared or changed materially with adjustment for other pollutants. Stronger association for NO2 exposure was observed in cool season than that in warm season. We found that short-term exposure to ambient air pollutants, especially NO2, was associated with increased risk of hospital admissions for recurrent ICEs in older adults.

Long-term fertilization and tillage regimes have limited effects on structuring bacterial and denitrifier communities in a sandy loam UK soil.

Denitrification causes loss of available nitrogen from soil systems, thereby reducing crop productivity and increasing reliance on agrochemicals. The dynamics of denitrification and denitrifying communities are thought to be altered by land management practices, which affect the physicochemical properties of the soil. In this study, we look at the effects of long-term tillage and fertilization regimes on arable soils following 16 years of treatment in a factorial field trial. By studying the bacterial community composition based on 16S rRNA amplicons, absolute bacterial abundance and diversity of denitrification functional genes (nirK, nirS and nosZ), under conditions of minimum/conventional tillage and organic/synthetic mineral fertilizer, we tested how specific land management histories affect the diversity and distribution of both bacteria and denitrification genes. Bacterial and denitrifier communities were largely unaffected by land management history and clustered predominantly by spatial location, indicating that the variability in bacterial community composition in these arable soils is governed by innate environmental differences and Euclidean distance rather than agricultural management intervention.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

First report of Lasiodiplodia theobromae causing dieback in custard apple (Annona squamosa) tree in China.

Custard apple (Annona squamosa Linn) is popular for its sweet taste and rich aroma. Hainan province is the major production area of custard apple in China. In September 2020, wilting of leaves and branches, discoloration of the vascular system and dieback of trees were observed in plantings in Lingao County of Hainnan Province, China (Fig. 1a-c). The incidence of dieback in three orchards was at least 19%, and affected samples were brought to the laboratory. Fragments of approximately 5 mm in length were obtained from five diseased branches, which were collected from different plants and orchards. Fragment surface were sterilized in 75% ethanol for 1 min, and 1% mercury chloride for 1 min, then rinsed three times with sterile distilled water. These tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated at 28°C for 3 days. Fungal colonies were transferred to fresh PDA plates, and single-spore cultures were obtained. We isolated twenty-six fungal strains, of which twenty-three isolates were morphologically identified as Lasiodiplodia species (Phillips et al. 2013). The colony morphology was initially round and white, then turned to grey or black after 5-7 days at 28°C, and formed pycnidia for 20 days (Fig. 1d). The immature conidia were ellipsoid, colorless, hyaline and unicellular, becoming brown, bicellular with longitudinal striations at maturity (Fig. 1e). Mature conidial size was: 26.61±1.57×14.87±1.14 µm (n=60). For molecular identification, genomic DNA of three isolates (HSYF01, HSYF02 and HSYF03) was extracted using E.Z.N.A.® HP Plant DNA kit (Omega Bio-Tek). The internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α) and ß-tubulin (TUB) regions were amplified using the primers ITS1/4, EF1-728F/986R and Bt2a/Bt2b, respectively (Carbone and Kohn 1999; Glass and Donaldson 1995; White et al. 1990). A BLAST search of ITS, EF1-α and TUB gene sequences (Accession nos. MW625913-MW625918, MW876481-MW876483) had in 99.8% (493bp out of 494 bp), 99.31% (286bp out of 288 bp) and 100% (372bp out of 372 bp) identity to CBS164.96 of the L. theobromae (Accession nos. AY640255, AY640258 and KU887532). The identity of the putative pathogen isolates was further confirmed by phylogenetic analysis (Fig. 2). Ten healthy 1-2-year-old custard apple trees were used to confirm pathogenicity. Custard apple plants were wounded approximately 15-20 cm below the tips with a sterilized scalpel. Each cut was inoculated with a 5 mm agar plugs with mycelium from the PDA cultures. The wound site was moisturized with wet cotton wool and wrapped with laboratory film (Silveira et al. 2018). Two seedlings treated with sterile agar plugs served as a controls. The pathogenicity test was repeated twice. After 14 days of incubation in a glasshouse, all inoculated seedlings had characteristic discoloration and necrotic lesions starting from the apical branches, (Figs. 1f and 1g). The stems exhibited browning and vascular streaking of the wood from the inoculation point (Figs. 1h), while the control seedlings remained symptomless. Typical colonies of L. theobromae were isolated and identified from all inoculated seedings, fulfilling Koch´s postulates. Although, postharvest fruit stem-end rot on custard apple caused by L. theobromae was previously described (Hu et al. 2003; Meng et al. 2017), this is the first report of L. theobromae causing dieback in mature custard apple trees in China.

Association of exposure to residential greenness with semen quality: A retrospective longitudinal study of sperm donation volunteers in Guangdong province, China.

BACKGROUND: Exposure to residential greenness has been associated with benefits on certain reproductive health outcomes. However, its potential benefits on semen quality remain unknown. OBJECTIVES: To quantitatively explore the association between exposure to residential greenness and semen quality. METHODS: We investigated 9142 sperm donation volunteers who underwent 38,682 semen examinations at Guangdong provincial human sperm bank in China during 2016-2019. Exposure to residential greenness was assessed using mean daily Normalized Difference Vegetation Index (NDVI) at each subject's residential address with a 400 m buffer during 0-90 days before each semen collection. Multivariate linear mixed models and linear regression models were used to assess the association between exposure to residential greenness and semen quality. RESULTS: An interquartile range increase in exposure to residential greenness was significantly associated with a 0.034 (95% confidence interval [CI]: 0.005, 0.063) ml, 4.06 (95% CI: 0.76, 7.37) × 106, and 0.32% (95% CI: 0.22%, 0.41%) increase in semen volume, total sperm number, and normal forms, respectively; similar trends were observed across quartiles of exposure to residential greenness (all p-values for liner trend <0.05 except for semen volume). The association of greenness exposure with semen volume and total sperm number was stronger in subjects 18-25 years, while the association with normal forms was stronger in subjects 26 years or older. The association for sperm concentration, total sperm number, and normal forms were stronger in cool season, while the association for semen volume was stronger in warm season. CONCLUSION: We found that exposure to residential greenness was significantly associated with higher semen quality. Further studies are warranted to determine the causality of the association and its underlying mechanisms.

Knockdown of ribosomal protein S6 suppresses proliferation, migration, and invasion in epithelial ovarian cancer.

BACKGROUND: Ovarian cancer typically is diagnosed late because insensitivity and lack of specificity of current biomarkers prior to its clinical detection. Ribosomal protein S6 (RPS6) is a ribosomal protein involved in the ribosomal 40S subunit, but its biological role in epithelial ovarian cancer (EOC) is still unknown. RESULTS: RPS6 was elevated in EOC compared to normal ovarian tissues and adenomas. Higher expression of RPS6 predicted worse prognosis in EOC. The level of RPS6 was correlated with clinical stage, histological type and pathological grade. Knockdown of RPS6 reduced the proliferation of ovarian cancer cell lines SKOV-3 and HO8910, and inhibit the migration and invasion ability. It revealed that cells arrested at G0G1 phase after knockdown of RPS6, and the expressions of CyclinD1, Cyclin E, CDK2, CDK4, CDK6 and pRb were also reduced. CONCLUSIONS: RPS6 is involved in EOC and knockdown of RPS6 could inhibit the proliferation, invasion and migration ability of EOC in vitro by inducing G0/G1 phase arrest. RPS6 is expected to be a novel biomarker and molecular target to the EOC.