The Comparative Genomics of Botryosphaeriaceae Suggests Gene Families of Botryosphaeria dothidea Related to Pathogenicity on Chinese Hickory Tree.

Trunk canker poses a major threat to the production of Chinese hickory tree (Carya cathayensis Sarg.), which is primarily determined by Botryosphaeriaceae. In our previous work, we identified Botryosphaeria dothidea as the predominant pathogen of this disease. However, it is still unclear about corresponding gene families and mechanisms associated with B. dothidea's pathogenicity on Chinese hickory tree. Here, we present a comparative analysis of high-quality genome assemblies of Botryosphaeria dothidea and other isolated pathogens, showing highly syntenic relationships between B. dothidea and its closely related species and the conservative evolution of the Botryosphaeriaceae family. Higher GC contents were found in the genomes of B. dothidea and three other isolated pathogens (Botryshaeria cortices, Botryshaeria fabicerciana, and Botryshaeria qingyuanensis) compared to Macrophomina phaseolina, Neofusicoccum parvum, Diplodia corticola, and Lasiodiplodia theobromae. An investigation of genes specific to or expanded in B. dothidea revealed that one secreted glucanase, one orsellinic acid biosynthesis enzyme, and two MFS transporters positively regulated B. dothidea's pathogenicity. We also observed an overrepresentation of viral integrase like gene and heterokaryon incompatibility proteins in the B. dothidea's genome. In addition, we observed one LRR-domain-containing protein and two Sec-domain-containing proteins (Sec_1 and Sec_7) that underwent positive selection. This study will help to understand B. dothidea's pathogenicity and potential influence on the infection of Chinese hickory, which will help in the development of disease control and ensure the security of Chinese hickory production.

High-quality genome assembly and genetic transformation system of Lasiodiplodia theobromae strain LTTK16-3, a fungal pathogen of Chinese hickory.

Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea, Botryosphaeria fabicerciana, Botryosphaeria qingyuanensis, and Botryosphaeria corticis. Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control.IMPORTANCEFungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

Development of Recombinase Polymerase Amplification-Lateral Flow Dipstick (RPA-LFD) as a Rapid On-Site Detection Technique for Fusarium oxysporum.

Fusarium oxysporum can cause many important plant diseases worldwide, such as crown rot, wilt, and root rot. During the development of strawberry crown rot, this pathogenic fungus spreads from the mother plant to the strawberry seedling through the stolon, with obvious characteristics of latent infection. Therefore, the rapid and timely detection of F. oxysporum can significantly help achieve effective disease management. Here, we present a protocol for the recombinase polymerase amplification- lateral flow dipstick (RPA-LFD) detection technique for the rapid detection of F. oxysporum on strawberry, which only takes half an hour. A significant advantage of our RPA-LFD technique is the elimination of the involvement of professional teams and laboratories, which qualifies it for field detection. We test this protocol directly on plant samples with suspected infection by F. oxysporum in the field and greenhouse. It is worth noting that this protocol can quickly, sensitively, and specifically detect F. oxysporum in soils and plants including strawberry. Key features • This protocol is used to detect whether plants such as strawberry are infected with F. oxysporum. • This protocol has potential for application in portable nucleic acid detection. • It can complete the detection of samples in the field within 30 min.

The influence of gender on the epidemiology of and outcome from sepsis associated acute kidney injury in ICU: a retrospective propensity-matched cohort study.

PURPOSES: The influence of gender on the epidemiology of and outcome from SA-AKI in ICU has not been fully clarified. Our aim is to elucidate these differences. METHODS: This study included adult patients with sepsis in MIMIC IV (V 2.2), and propensity matching analysis, cox regression and logistic regression were used to analyze gender differences in incidence, mortality and organ support rate. RESULTS: Of the 24,467 patients included in the cohort, 18,128 were retained after propensity score matching. In the matched cohort, the incidence of SA-AKI in males is higher than that in females (58.6% vs. 56.2%; P = 0.001).males were associated with a higher risk of SA-AKI (OR:1.07(1.01-1.14), P = 0.026;adjusted OR:1.07(1.01-1.14), P < 0.033).In SA-AKI patients, males were associated with a lower risk of ICU mortality(HR:0.803(0.721-0.893), P < 0.001;adjusted HR:0.836(0.746-0.937), P = 0.002) and in-hospital mortality(HR: 0.820(0.748-0.899), P < 0.001;adjusted HR:0.853(0.775-0.938), P = 0.003).there were no statistically significant differences between male and female patients in 1-year all-cause mortality (36.9% vs. 35.8%, P = 0.12), kidney replacement therapy rate (7.8% vs.7.4%, P = 0.547), mechanical ventilation rate 64.8% vs.63.9%, P = 0.369), and usage of vasoactive drugs (55.4% vs. 54.6%, P = 0.418). CONCLUSIONS: Gender may affect the incidence and outcomes of SA-AKI, further research is needed to fully understand the impact of gender on SA-AKI patients.

Low HDL-C can be a biomarker to predict persistent severe AKI in septic patients? A retrospective cohort study.

PURPOSES: Low HDL-C is associated with an increased risk of sepsis-associated AKI and subsequent decline in eGFR. HDL-C possesses anti-inflammatory, antioxidant, and endothelial repair-promoting properties. The use of Apo A-I mimetic peptides, which are the main structural components of HDL-C, has been shown to improve renal function in animal models of sepsis. However, the diagnostic value of low HDL-C in persistent sepsis-associated AKI remains unclear. METHODS: This is a retrospective cohort study based on MIMIC IV (V 2.2). The study population consisted of all adult septic patients admitted to the Beth Israel Deaconess Medical Center Intensive Care Unit from 2008 to 2019, with plasma HDL-C measured within 24 h of ICU admission. The primary endpoint was persistent severe sepsis-associated acute kidney injury (SA-AKI) and the secondary endpoint is kidney replacement therapy (KRT). Logistic regression was used to assess the correlation between HDL-C and persistent severe SA-AKI and KRT, and receiver operating characteristic (ROC) curve analysis was performed to evaluate predictive ability. RESULTS: A total of 604 cases of SA-AKI patients were included in the analysis, among which 88 cases (14.5%) experienced persistent severe SA-AKI. The median (IQR) HDL-C level in the group with persistent severe SA-AKI was lower (33.0 [24.0-45.5]) compared to the non-persistent severe SA-AKI group (42.0 [31.0-53.0]). However, HDL-C showed poor discriminatory ability with an AUROC [95%CI] of 0.62 [0.56-0.69]. Clinical prediction models based on serum creatinine concentration, 24-h creatinine change, APSIIIscore, lactate levels, APTT, and heart rate performed well in predicting persistent severe SA-AKI with an AUROC [95%CI] of 0.876 [0.84-0.91]. However, adding HDL-C to this model did not improve predictive performance. CONCLUSIONS: The plasma HDL-C measured within 24 h after admission to the ICU does not provide a good prediction for persistent severe SA-AKI, and it does not improve the clinical predictive ability compared to conventional variables.

Research on the Prediction Model of Engine Output Torque and Real-Time Estimation of the Road Rolling Resistance Coefficient in Tracked Vehicles.

Road parameter identification is of great significance for the active safety control of tracked vehicles and the improvement of vehicle driving safety. In this study, a method for establishing a prediction model of the engine output torques in tracked vehicles based on vehicle driving data was proposed, and the road rolling resistance coefficient f was further estimated using the model. First, the driving data from the tracked vehicle were collected and then screened by setting the driving conditions of the tracked vehicle. Then, the mapping relationship between the engine torque Te, the engine speed ne, and the accelerator pedal position ß was obtained by a genetic algorithm-backpropagation (GA-BP) neural network algorithm, and an engine output torque prediction model was established. Finally, based on the vehicle longitudinal dynamics model, the recursive least squares (RLS) algorithm was used to estimate the f. The experimental results showed that when the driving state of the tracked vehicle satisfied the set driving conditions, the engine output torque prediction model could predict the engine output torque T^e in real time based on the changes in the ne and ß, and then the RLS algorithm was used to estimate the road rolling resistance coefficient f^. The average coefficient of determination R of the T^e was 0.91, and the estimation accuracy of the f^ was 98.421%. This method could adequately meet the requirements for engine output torque prediction and real-time estimation of the road rolling resistance coefficient during tracked vehicle driving.

Sensitivity and resistance risk of Botryosphaeria dothidea causing Chinese hickory trunk canker to fludioxonil.

Hickory trunk canker (HTC), primarily caused by Botryosphaeria dothidea, is an aggravating disease that threatens an important regional economic tree species of Chinese hickory and few information is available in the control of this disease. Here, the sensitivity of 93 isolates to fludioxonil and the resistance risk were investigated. All the isolates tested were sensitive to fludioxonil and the EC50 ranged from 0.0028 to 0.0569 µg/mL. The tamed fludioxonil-resistant mutants remained highly resistant to fludioxonil even after 10 consecutive transfers to fludioxonil-free PDA plates. As for fitness penalty, the fludioxonil-resistant mutants demonstrated a reduction in conidia production and virulence as well as increased sensitivity to high osmotic stress. While, variations in mycelial growth and responses to SDS and H2O2 were not detected in all the resistant mutants. In addition, the resistant mutants demonstrated positive cross-resistance to iprodione but not to fungicides of other modes of action. Sequential analysis of BdNik1 showed that premature stop codon occurred in all the resistant mutants despite of point mutation (BD16-22R9 and BD16-22R20) or frameshift mutation (BD16-22R8, BD16-22R11 and BD16-22R18). Our study suggested that fludioxonil exhibited excellent inhibition activity on mycelial growth of B. dothidea in vitro, the resistance risk of B. dothidea to fludioxonil should be low to moderate and fludioxonil would be a nice candidate in controlling HTC caused by B. dothidea.

Quantitative Loop-Mediated Isothermal Amplification Detection of Ustilaginoidea virens Causing Rice False Smut.

Rice false smut caused by Ustilaginoidea virens is one of the most devastating diseases in rice worldwide, which results in serious reductions in rice quality and yield. As an airborne fungal disease, early diagnosis of rice false smut and monitoring its epidemics and distribution of its pathogens is particularly important to manage the infection. In this study, a quantitative loop-mediated isothermal amplification (q-LAMP) method for U. virens detection and quantification was developed. This method has higher sensitivity and efficiency compared to the quantitative real-time PCR (q-PCR) method. The species-specific primer that the UV-2 set used was designed based on the unique sequence of the U. virens ustiloxins biosynthetic gene (NCBI accession number: BR001221.1). The q-LAMP assay was able to detect a concentration of 6.4 spores/mL at an optimal reaction temperature of 63.4 °C within 60 min. Moreover, the q-LAMP assay could even achieve accurate quantitative detection when there were only nine spores on the tape. A linearized equation for the standard curve, y = -0.2866x + 13.829 (x is the amplification time, the spore number = 100.65y), was established for the detection and quantification of U. virens. In field detection applications, this q-LAMP method is more accurate and sensitive than traditional observation methods. Collectively, this study has established a powerful and simple monitoring tool for U. virens, which provides valuable technical support for the forecast and management of rice false smut, and a theoretical basis for precise fungicide application.

Identification and characterization of pathogens causing saffron corm rot in China.

Corm rot is the most important disease of saffron, for which fungi from several genus such as Fusarium spp. Penicillium spp. and Botrytis spp., have been previously reported to be the pathogens. In this research, we used a combination of amplicon sequencing and traditional isolation methods to identify the causal agents, main infection source. The diversity of microbial communities in diseased saffron corms and soil decreased significantly compared with healthy corms and soil. The contents of Penicillium and Botrytis in healthy and diseased corms were similarly high, indicating that them were not directly related to the occurrence of corm rot. But the relative abundance of Fusarium, Cadophora and Fusicolla were significantly higher in the diseased corms than healthy ones. The abundance of Fusarium increased, while the abundance of Oidiodendron, Paraphaeosphaeria and the endophytic beneficial bacteria Pseudomonas decreased, which may relate to the occurrence of the disease. The co-occurrence network diagram showed that the correlation between fungal and bacterial communities was mainly positive. Plant pathogens were relatively abundant in the diseased soil, according to functional gene prediction. At the same time, we also collected 100 diseased corms from the fields in Jiande, where is known as the "hometown of saffron." All isolated pathogenic strains were identified as Fusarium oxysporum through morphological observation and phylogenetic tree analysis of ITS, Tef-1α and ß-tubulin. To better clarify the biological characteristics of F. oxysporum, we cultured the isolates at different temperatures and pH values. The optimum temperature for mycelial growth and sporulation was 25°C, pH 6，carbon sources sorbitol and nitrogen sources, peptone. In short, our results suggests that F. oxysporum was the pathogen causing corm rot in Jiande and corms other than soils are the main primary infection source. These new understanding of saffron corm rot will provide the theoretical basis for its better and efficiently management.

Development and validation of a nomogram for predicting in-hospital mortality of elderly patients with persistent sepsis-associated acute kidney injury in intensive care units: a retrospective cohort study using the MIMIC-IV database.

OBJECTIVES: To identify the clinical risk factors that influence in-hospital mortality in elderly patients with persistent sepsis-associated acute kidney injury (S-AKI) and to establish and validate a nomogram to predict in-hospital mortality. DESIGN: Retrospective cohort analysis. SETTING: Data from critically ill patients at a US centre between 2008 and 2021 were extracted from the Medical Information Mart for Intensive Care (MIMIC)-IV database (V.1.0). PARTICIPANTS: Data from 1519 patients with persistent S-AKI were extracted from the MIMIC-IV database. PRIMARY OUTCOME: All-cause in-hospital death from persistent S-AKI. RESULTS: Multiple logistic regression revealed that gender (OR 0.63, 95% CI 0.45-0.88), cancer (2.5, 1.69-3.71), respiratory rate (1.06, 1.01-1.12), AKI stage (2.01, 1.24-3.24), blood urea nitrogen (1.01, 1.01-1.02), Glasgow Coma Scale score (0.75, 0.70-0.81), mechanical ventilation (1.57, 1.01-2.46) and continuous renal replacement therapy within 48 hours (9.97, 3.39-33.9) were independent risk factors for mortality from persistent S-AKI. The consistency indices of the prediction and the validation cohorts were 0.780 (95% CI: 0.75-0.82) and 0.80 (95% CI: 0.75-0.85), respectively. The model's calibration plot suggested excellent consistency between the predicted and actual probabilities. CONCLUSIONS: This study's prediction model demonstrated good discrimination and calibration abilities to predict in-hospital mortality of elderly patients with persistent S-AKI, although it warrants further external validation to verify its accuracy and applicability.

Establishment of the Recombinase Polymerase Amplification-Lateral Flow Dipstick Detection Technique for Fusarium oxysporum.

Fusarium oxysporum causes crown rot, wilt, root rot, and many other major plant diseases worldwide. During the progression of strawberry crown rot disease, the pathogen is transmitted from the mother plant to the seedling through the stolon, with obvious characteristics of latent infection. Therefore, rapid and timely detection of F. oxysporum is important for efficient disease management. In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection technique was developed for the rapid detection of F. oxysporum on strawberry plants by targeting the CYP51C gene, which is unique to Fusarium spp. Because this RPA-LFD detection technique was highly specific to F. oxysporum, other Fusarium and non-Fusarium fungi were not detected. The optimal reaction temperature and time for this technique were 39°C and 8 min, respectively. The detection limit was 1 pg of F. oxysporum genomic DNA in a 50-µl reaction system. A total of 46 strawberry plants with or without crown rot symptoms collected from Jiande, Changxing, and Haining in Zhejiang Province were further assessed for F. oxysporum infection using both RPA-LFD and traditional tissue isolation techniques. The RPA-LFD test showed that 32 of the 46 strawberry plants tested were positive for F. oxysporum, while in the traditional isolation technique, F. oxysporum was isolated from 30 of the 46 strawberry plants. These results suggest that our established RPA-LFD method is rapid, sensitive, and highly specific in detecting F. oxysporum infection in strawberry plants.

Phenotypic and Genomic Difference among Four Botryosphaeria Pathogens in Chinese Hickory Trunk Canker.

Botryosphaeria species are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea as one of the most common Botryosphaeria species. However, the information related to the widespread incidence and aggressiveness of B. dothidea among various Botryosphaeria species causing trunk cankers is still poorly investigated. In this study, the metabolic phenotypic diversity and genomic differences of four Chinese hickory canker-related Botryosphaeria pathogens, including B. dothidea, B. qingyuanensis, B. fabicerciana, and B. corticis, were systematically studied to address the competitive fitness of B. dothidea. Large-scale screening of physiologic traits using a phenotypic MicroArray/OmniLog system (PMs) found B. dothidea has a broader spectrum of nitrogen source and greater tolerance toward osmotic pressure (sodium benzoate) and alkali stress among Botryosphaeria species. Moreover, the annotation of B. dothidea species-specific genomic information via a comparative genomics analysis found 143 B. dothidea species-specific genes that not only provides crucial cues in the prediction of B. dothidea species-specific function but also give a basis for the development of a B. dothidea molecular identification method. A species-specific primer set Bd_11F/Bd_11R has been designed based on the sequence of B. dothidea species-specific gene jg11 for the accurate identification of B. dothidea in disease diagnoses. Overall, this study deepens the understanding in the widespread incidence and aggressiveness of B. dothidea among various Botryosphaeria species, providing valuable clues to assist in trunk cankers management.

Targeted deletion of three CYP51s in Fusarium fujikuroi and their different roles in determining sensitivity to 14α-demethylase inhibitor fungicides.

BACKGROUND: Fusarium fujikuroi is the pathogenic agent of rice bakanae disease and has developed serious resistance to prochloraz, a 14α-demethylase inhibitor (DMI). Prochloraz resistance in F. fujikuroi is caused by cooperation between FfCyp51B with Cyp51A and shows cross-resistance only to prothioconazole but not to tebuconazole, difenoconazole, propiconazole, metconazole, hexaconazole, and triadimefon. This study aimed to analyze the functions of the three Cyp51s in F. fujikuroi, especially their role in determining sensitivity to DMIs. RESULTS: The respective deletion of FfCyp51A, Cyp51B, and Cyp51C had no obvious effect on morphology, conidium germination, or pathogenicity. The involvement of growth, growth and ergosterol biosynthesis, and conidium production and ergosterol biosynthesis was observed for FfCyp51A, Cyp51B, and Cyp51C, respectively. Compared with the sensitive isolate of F. fujikuroi, the effect on sensitivity to the tested DMIs was divided into four groups: (i) both of Cyp51A and Cyp51B positively regulate the sensitivity to prochloraz and prothioconazole; (ii) Cyp51B positively regulate the sensitivity to tebuconazole and metconazole, but negatively regulate the sensitivity to difenoconazole; (iii) Cyp51A and Cyp51B play opposite roles in the sensitivity to triadimefon. Therefore, deletion of Cyp51A in F. fujikuroi confers a higher sensitivity to triadimefon, while deletion of Cyp51B results in a triadimefon-resistant mutant isolate; (iv) deletion of Cyp51B yielded a mutant isolate that was more resistant to propiconazole and hexaconazole. CONCLUSION: Sophisticated interactions exist within the three Cyp51 genes to DMIs fungicides sensitivity in F. fujikuroi, and Cyp51B probably plays a more critical role than Cyp51A and Cyp51C. © 2022 Society of Chemical Industry.

Fungal Pathogens Associated with Strawberry Crown Rot Disease in China.

Strawberry crown rot (SCR) is a serious disease that is generally referred to as seedling anthracnose due to its association with Colletotrichum spp. Presently, SCR is the main cause of death of strawberry seedlings. However, management strategies, including fungicides targeting Colletotrichum spp., have failed to obtain satisfactory results. Therefore, identifying the exact pathogen species causing SCR could guide its management. A total of 287 isolates were obtained from SCR-diseased plants. Based on the culture, morphology, and phylogenetic characteristics, the above 287 fungal isolates of SCR pathogens were identified as 12 different species, including Colletotrichum siamense, C. fructicola, Fusarium oxysporum, F. commune, F. equiseti, F. solani, F. tricinctum, Epicoccum sorghinum, Stemphylium lycopersici, Clonostachys rosea, Phoma herbarum, and Curvularia trifolii. Pathogenicity results showed that most isolates were pathogenic to strawberry seedlings and exhibited different degrees of virulence. In severe cases, poor growth on the ground, yellowing of the leaves, and even death of seedlings occurred. In mild cases, only black disease spots appeared on the stems of the strawberry seedlings, and a few withered leaves became necrotic. The inoculation experiments showed that the most virulent species were C. siamense and F. oxysporum, followed by F. equiseti, P. herbarum, Cl. rosea, S. lycopersici, and C. fructicola, which had disease incidences above 50%. E. sorghinum, S. lycopersici, Cl. rosea, P. herbarum and Cu. trifolii were reported to cause SCR for the first time herein. In conclusion, SCR is a sophisticated disease caused by a diversity of pathogenic fungi. This work provides new valuable data about the diversity and pathogenicity of SCR pathogens, which will help in formulating effective strategies to better control of the SCR disease.

Bioactivity of mefentrifluconazole against different Fusarium spp.

Emergence and development of resistance to 14α-demethylase inhibitors (DMIs) have become a critical issue in both agriculture and medical fields. Mefentrifluconazole, the first isopropanol triazole fungicide belonging to a new subclass of DMIs, has been proposed to show high activity, minimal adverse side effects, and inconsistent cross resistance with other DMIs due to its high structural flexibility. In this study, mefentrifluconazole showed disparate inhibitory activity against the mycelium growth of seven tested Fusarium species. The most sensitive species included F. oxysporum, F. proliferatum, F. commuae, and F. fujikuroi, followed by F. equiseti and F. graminearum, while F. solani was most insensitive. Consistently, mefentrifluconazole presented the strongest inhibiting effects on conidium germination, cell membrane integrity, and ergosterol biosynthesis in F. fujikuroi, followed by F. graminearum, while F. solani ranked last. Further results indicated that all F. fujikuroi isolates causing rice bakanae disease (RBD) were sensitive to mefentrifluconazole regardless of their resistance to prochloraz, tebuconazole, carbendazim, and phenamacril. Additionally, the inoculation tests found that mefentrifluconazole presented a better protective efficacy on rice seedlings when applied 12 h before the F. fujikuroi inoculation, compared to applied 12 h post the inoculation. Overall, this study demonstrated the various bioactivity of mefentrifluconazole combating Fusarium spp. and put new insights into RBD management as well as the applications of DMIs.

Colletotrichum Spp. Diversity Between Leaf Anthracnose and Crown Rot From the Same Strawberry Plant.

Leaf anthracnose (LA) and anthracnose crown rot (ACR) represent serious fungal diseases that pose significant threats to strawberry production. To characterize the pathogen diversity associated with above diseases, 100 strawberry plants, including varieties of "Hongjia," "Zhangji," and "Tianxianzui," were sampled from Jiande and Zhoushan, the primary plantation regions of Zhejiang province, China. A total of 309 Colletotrichum isolates were isolated from crown (150 isolates) and leaves (159 isolates) of affected samples. Among these, 100 isolates obtained from the plants showing both LA and CR symptoms were selected randomly for further characterization. Based on the morphological observations combined with phylogenetic analysis of multiple genes (ACT, ITS, CAL, GAPDH, and CHS), all the 100 tested isolates were identified as C. gloeosporioides species complex, including 91 isolates of C. siamense, 8 isolates of C. fructicola causing both LA and ACR, and one isolate of C. aenigma causing ACR. The phenotypic characteristics of these isolated species were investigated using the BIOLOG phenotype MicroArray (PM) and a total of 950 different metabolic phenotype were tested, showing the characteristics among these isolates and providing the theoretical basis for pathogenic biochemistry and metabolism. The pathogenicity tests showed that even the same Colletotrichum species isolated from different diseased tissues (leaves or crowns) had significantly different pathogenicity toward strawberry leaves and crown. C. siamense isolated from diseased leaves (CSLA) was more aggressive than C. siamense isolated from rotted crown (CSCR) during the infection on "Zhangji" leaves. Additionally, C. fructicola isolated from affected leaf (CFLA) caused more severe symptoms on the leaves of four strawberry varieties compared to C. fructicola isolated from diseased crown (CFCR). For crown rot, the pathogenicity of CSCR was higher than that of CSLA.

Effects of a Single Sub-Anesthetic Dose of Ketamine on Postoperative Emotional Responses and Inflammatory Factors in Colorectal Cancer Patients.

Objective: To investigate the effect of a single sub-anesthetic dose of ketamine on postoperative anxiety, depression, and inflammatory factors in patients with colorectal cancer. Methods: A total of 104 patients undergoing selective colorectal surgery in our hospital from Jan 2015 to Oct 2017 were included and randomly assigned (1:1:1:1) into a 0.1 mg kg-1 ketamine group (K1 group), 0.2 mg kg-1 ketamine group (K2 group), 0.3 mg kg-1 ketamine group (K3 group), or control group (C group). Corresponding doses of ketamine were given intravenously in the K groups (K1, K2, and K3 groups) 5 min before operation, and the same amount of normal saline was given in the C group. The intravenous analgesia program was identical in the four groups. The patients' emotional reactions (anxiety and depression) were assessed by the Hospital Anxiety and Depression Scale (HAD), the quality of postoperative recovery was evaluated by the Quality of Recovery-40 (QoR-40) questionnaire, and the levels of IL-6, IL-8, and TNF-α in peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA) on the day before operation and within 24, 48, and 72 h post-operation respectively. Pain was estimated by the visual analog scale (VAS), and sedation was assessed with Ramsay score 30 min after extubation. The time points of anesthetic end and extubation were recorded. The complications during anesthesia and recovery such as cough and agitation 30 min after extubation were recorded. Results: The anxiety score (HAD-A) and depression score (HAD-D) of the K3 group were significantly lower than those of the C group post-operation (p < 0.05). The QoR-40 score of the K3 group was significantly higher than that of the C group (p < 0.05). The serum levels of IL-6, IL-8, and TNF-α in the K3 group were significantly lower than those in the C group (p < 0.05 and p < 0.01). There were no significant differences in HAD-A, HAD-D, and QoR-40 scores or serum levels of IL-6, IL-8, and TNF-α between the K1 and K2 groups and the C group. There were no significant differences in VAS pain score or Ramsay sedation score among the four groups 30 min after extubation. There were no significant differences in extubation time, postoperative cough, emergence agitation, or delirium among the four groups. Dizziness, nausea, vomiting, diplopia, or other adverse reactions were not found 30 min after extubation. Conclusion: A single sub-anesthetic dose (0.3 mg kg-1) of ketamine can significantly improve the postoperative anxiety and depression of colorectal cancer patients and reduce the levels of IL-6, IL-8, and TNF-α.

First Report of Seedling Stem Rot on Jinxianlian (Anoectochilus roxburghii) Caused by Fusarium oxysporum in China.

Anoectochilus roxburghii is an important Chinese herbal medicine plant belonging to Orchidaceae and known as Jinxianlian. This orchid is cultivated and mostly adopted to treat diabetes and hepatitis. About 2 billion artificially cultivated seedlings of Jinxianlian are required each year and approximately $600 million in fresh A. roxburghii seedlings is produced in China. From 2011, sporadic occurrence of stem rot on Jinxianlian have been observed in greenhouses in Jinhua City (N29°05', E119°38'), Zhejiang Province. In 2018, nearly 30% of seedlings of Jinxianlian grown in greenhouse conditions were affected by stem rot in Jinhua City. Symptoms initially occurred in the stem at the soil line causing dark discoloration lesions, rotted tissues, wilting, and eventually leading to the death of the plants. A total of 23 diseased seedlings collected from seven different greenhouses were surface sterilized with 1.5% sodium hypochlorite for 3 min, then rinsed in water. Pieces of tissues disinfected from each sample were plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days (Kirk et al. 2008). A total of 19 isolates were recovered. They developed colonies with purple mycelia and beige or orange colors after 7 days of incubation under 25°C on PDA and carnation leaf agar (CLA) media (Kirk et al. 2008; Zhang et al. 2016). Colonies on PDA had an average radial growth rate of 3.1 to 4.0 mm /d at 25°C. Colony surface was pale vinaceous, floccose with abundant aerial mycelium. On CLA, aerial mycelium was sparse with abundant bright orange sporodochia forming on the carnation leaves. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.7 to 9.3 × 1.3 to 2.9 µm) (n=19). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (27.4 to 35.6 × 3.2 to 4.2 µm) (n=19). These morphological features were consistent with Fusarium oxysporum (Sun et al. 2008; Lombard et al., 2019). To confirm the identification based on these morphological features, the internal transcribed spacer region (ITS) and translation elongation factor1 (TEF) were amplified from the DNA of 3 out of 19 isolates chosen at random respectively using the set primer ITS1/ITS4 and EF1/ EF2 (Sun, S., et al. 2018; Lombard et al., 2019). BLAST analysis revealed that the ITS sequences (OK147619, OK147620, OK147621) had 99% identity to that of F. oxysporum isolate JJF2 (GenBank MN626452) and TEF sequence (OK155999, OK156000, OK156001) had 100% identity to that of F. oxysporum isolate gss100 (GenBank MH341210). A multilocus phylogenetic analysis by Bayesian inference (BI) and maximum likelihood (ML) trees based on ITS and TEF indicated that the pathogen grouped consistently with F. oxysporum. Three out of 19 isolates chosen at random were selected to evaluate pathogenicity. Uninfected healthy A. roxburghii seedlings about 40 day-old planted in sterilized substrates were sprayed with distilled water containing 2 x 106 conidia per ml suspensions as inoculums, and plants sprayed with distilled water alone served as controls. Plants were then incubated at 25°C and 85% relative humidity. Ten plants were inoculated for each isolate. After 10 days, all plants inoculated developed stem rot symptoms, while control plants remained healthy. Cultures of Fusarium spp. were re-isolated only from inoculated plants with the frequency of 100% and re-identified by morphological characteristics as F. oxysporum, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F. oxysporum causing stem rot on A. roxburghii seedlings. As F. oxysporum is a devastating pathogenic fungus with a broad host range, measures should be taken in advance to manage stem rot of A. roxburghii.