First Report of Anthracnose on Cinnamomum camphora (Camphor tree) Caused by Colletotrichum fioriniae and Colletotrichum siamense in China

Camphor tree (Cinnamomum camphora) is native to east Asia, which could produce pharmaceutical metabolites, such as camphor, linalool, and so on (Chen, Tang et al. 2020). In September 2020, severe anthracnose symptoms were observed on the leaves of camphor trees in Nanchang, and estimated incidences ranged from 30% to 80%, which could inhibit leaf growth and reduce their biomass. The lesions were appeared on the leaves of annual branchlets, which the irregular dead areas appeared on leaf tips or margins (Figure 1 A and B), sometimes moving onto the shoots and small twigs. For pathogen isolation, fifteen leaves with typical symptom were randomly collected in Jiangxi Agricultural University (N28°45'38", E115°50'0.006") and the fungi were isolated from the symptomatic-asymptomatic junction and cultured on potato dextrose agar (PDA) at 25℃ in darkness. A total of 40 isolates were obtained from tissue samples, in which 32 isolates were identified as belonging to Colletotrichum spp. following the published works (Damm, Cannon et al. 2012, Damm, Cannon et al. 2012, Weir, Johnston et al. 2012). Based on the morphologies of conidia, all the 32 isolates were classified into two categories. For further precise identification, the represented isolate YK1 and YK18 were selected to analyzed using morphological characters after 7 days of incubation, and multiple genes including ITS (White, Bruns et al. 1990), ACT, GAPDH, TUB (Damm, Woudenberg et al. 2009) and RPB2 (Réblová, Gams et al. 2011). Sequences were deposited in GenBank with accession numbers from MZ229311 to MZ229326. Conidia of isolate YK1 were aseptate, primarily fusiform and measured 14.07-21.21 µm × 4.99-6.79 µm (n = 51) (Figure 1 L) and acervulus were 60.24 to 113.56 µm × 44.24 to 102.63 µm (n = 6) (Figure 1 K), while that of YK18 were one-celled, cylindric with obtuse ends (Figure 1 N) and measured 13.28-16.51 µm × 4.10-5.82 µm (n = 52) and acervulus were 73.85 to 131.70 µm × 63.93 to 105.66 µm (n = 6) (Figure 1 M). Acervulus of isolate YK1 and YK18 were produced on alfalfa stems 40 days after inoculation and dark brown to black in color. For all the genes showed greater than 99% similarity to multiple C. fioriniae and C. siamense accessions, respectively. The phylogram reconstructed from the combined dataset using W-IQ-TREE (Trifinopoulos, Nguyen et al. 2016) showed that isolate YK1 and YK18 clustered with C. fioriniae and C. siamense, respectively. Pathogenicity of both species was tested in the field by ten inoculating surface-sterilized mature leaves with puncture wound (Figure 1 C and D) and ten non-wounded young leaves with 20 µL of a conidial suspension (105 conidia ml-1) (Figure F and G). Leaves treated with sterilized water under the same conditions served as controls. After 4 to 7 days, the inoculated leaves of camphor tree developed typical dark brown to black lesions, similar to symptoms observed in the field, whereas controls remained symptomless. To fill the Koch’s postulates, C. fioriniae and C. siamense were consistently re-isolated, and confirmed morphologically and molecularly. C. siamense have been found to cause anthracnose on Cinnamomum camphora in China (Xu, 2017). To our knowledge, this is the first report of anthracnose on Cinnamomum camphora with C. fioriniae in China. In addition, this is an indication to the complexes about pathogens to anthracnose on camphor tree, which can pose serious threat to the production of Cinnamomum camphora in China.

Electron Microscope Characterization of Platelet Activation State Reveals That Wound Closure and P2Y 12 Receptors Are Major Early Determinants of Thrombus Structure in a Venous Puncture Model

Vascular damage presents in many forms and varying geometries. Nevertheless, the platelet response to endothelial damage to the blood vessel wall, be it through a prick or a full puncture wound, is thought to be staged by a qualitatively similar temporal variance in signaling agonists. For example, endothelial damage in the microvasculature is thought to be initially dominated by thrombin and later by platelet released ADP and thromboxane. The same temporal sequence in signaling has been proposed to exist in a profusely bleeding puncture wound 1. If so, platelet morphology, a gold standard of platelet activation state, could provide a strong readout of temporally distinct signaling effects. Platelet morphology has long been considered to be a reliable indicator of a strong agonist such as thrombin acting through PAR receptors that produces a rounded, pseudopod extending, degranulated, highly adhesive platelet versus weaker agonists such as ADP or thromboxane acting through P2Y 12 receptors to produce a less adhesive, somewhat rounded platelet. A testable prediction of existing hemostasis models is that temporal staging of signaling leads to temporal differences in platelet morphology within the forming/remodeling thrombus. Such hypothesized temporal differences in signaling are clinically significant as they form the basis for hypothesizing phenotypically distinct outcomes for direct acting anti-coagulants (DOACs) affecting thrombin versus anti-platelet drugs affecting P2Y 12, ADP receptors. Advances in imaging, e.g., wide area transmission electron microscopy (WA-TEM), make possible the local determination of platelet activation state with high precision 2. Taking a mouse jugular vein puncture wound model 1,2, we found that all morphologically recognized platelet activation states were present early, 1 min post puncture, with loosely bound discoid shaped platelets being the most peripherally located. For bleeding, early-stage puncture wound, these loosely adherent, low activation state platelets were located on both intravascular and extravascular thrombus aggregates. Once the puncture wound is closed, loosely adherent platelets were only found on the intravascular surfaces of the thrombus. We propose that this result is most consistent with a platelet conversion model in which new loosely adherent platelets rapidly convert to tightly packed platelets. As the thrombus remodels, 5 and 20 min post-puncture, the thrombus continued to accumulate platelets both intravascularly and extravascularly. Peripheral, discoid shaped platelets provided a source for intravascular thrombus growth. However, any subsequent extravascular thrombus growth must be due to platelet migration. Significantly, we found that cangrelor, a direct acting P2Y 12 inhibitor, stalled thrombus formation/remodeling at an early stage (Figure 1A,C,E see also ref 1,2). By WA-TEM, the accumulation of discoid-shaped, loosely adherent platelets appeared to be enhanced in a cangrelor treated 5 min thrombus (Figure 1E,F). We suggest that P2Y 12 receptors must act early in thrombus formation with the conversion of discoid to more activated platelets being most affected. In contrast, a 5-min post puncture dabigatran (DOAC) treated showed deformed architecture with inhibition of the accumulation of discoid shaped platelets/rounded loosely adherent platelets being most affected (Figure 1D,F, see also ref 2). Accumulation of degranulated platelets appeared to be lessened in both cangrelor and dabigatran treated thrombi. We propose that the simplest explanation of these results is that multiple signaling pathways act in parallel with select activation states being more dependent on one pathway than another. Clinically, our results suggest that P2Y 12 inhibitors can affect thrombus formation at early time points in addition to the late time points projected by current models. 1. Tomaiuolo M., Matzko C.N., Posentud-Fuentes I., Weisel J.W., Brass L.F. & Stalker T.J. Interrelationships between structure and function during the hemostatic response to injury. Proc Natl Acad Sci USA. 116. 2243-2252 (2019). 2. Rhee, Pokrovskaya I.D.,BallK., LingK., VedanapartiY., CohenJ., CruzD., ZhaoO.S., AronovaM.A., ZhangG., Kamykowski J.A., LeapmanR.D., & StorrieB. Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates. Commun. Biol., accepted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

First Report of Leaf Spot Caused by Corynespora cassiicola on Viburnum odoratissimum Ker-Gawl. var. awabuki (K. Koch) Zabel ex Rumpl. (sweet viburnum) in China

Sweet viburnum [Viburnum odoratissimum Ker-Gawl. var. awabuki (K. Koch) Zabel ex Rumpl.] belonging to the family Adoxaceae, is a medical and landscape plant, native to Korea (Jeju Island), Taiwan, and Japan (Edita 1988). In June and September 2019, leaf spots were observed on approximately 65% to 80% of sweet viburnum plants in a hedgerow located in Fenghe Xincheng District (28°41'52.9"N 115°52'14.3"E) in Nanchang, China. Initial symptoms of disease appeared as dark brown spots surrounded by red halos (Figure 1 A), which expanded irregularly. Finally, the center of the lesions desiccated and became light-brown, surrounded by a deep-red halos (Figure 1 B). Ten leaf samples with typical symptoms were collected and washed with tap water for about 15 min. The tissue between the healthy and necrotic area (ca. 4 mm × 4 mm) was cut with a sterile scalpel and surface sterilized with 70% alcohol for 45 s, 2% NaClO for 2 min, washed in sterile deionized water three times, dried on sterilized filter paper, then placed in Petri dishes and incubated at 25℃ in the dark. After 3 to 5 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA dishes. Eventually, 54 fungal isolates were obtained and, of these, 39 isolates were identical in their morphological characteristics. Morphological analysis was performed according with Ellis (1971). The isolate S18, chosen as representative, formed a gray to grayish brown colony with concentric circleson PDA, and a diameter of 8.5 to 9 cm after 7 days incubation at 25℃ (Figure 1 G). Conidia were hyaline, straight or slightly curved, needle shaped, truncate at the base, and acuminate at the tip, with 2 to 6 pseudosepta, 18.90 to 38.38 µm (avg. = 27.51 µm) × 1.64 to 4.50 µm (avg. = 2.60 µm) (n = 36) (Figure 1 H). The genes of fungal isolates (i.e., ITS, tub2 and ACT) were amplified with ITS4/ITS5 for ITS (White, Bruns et al. 1990), Bt2a/Bt2b for tub2 (Glass and Donaldson 1995) and ACT783R/ACT512F for ACT (Carbone and Kohn 1999) and sequenced. The sequences were deposited in GenBank (MW165772 for ITS, MW175900 for ACT and MW168659 for tub2), which showing greater than 99.1% similarity to multiple C. cassiicola accessions, respectively. Pathogenicity tests were performed on healthy leaves in field by inoculating surface-sterilized mature leaves with puncture wound (Figure C) and non-wounded young leaves with 20 µL of a conidial suspension (105 conidia ml-1) (Figure F and G) at 26℃. After 4 to 7 days, all inoculated leaves reproduced similar symptoms as observed initially in the field (Figure 1 C, E and F). To fulfill Koch’s postulates, the fungus was isolated on PDA from the margins of leaf spots on inoculated leaves and confirmed as C. cassiicola by morphological characters and ITS gene sequencing. Previously, C. cassiicola was reported as an endophyte on Viburnum spp. and Viburnum odoratissimum (Alfieri et al. 1994). More recently, C. cassiicola has been reported as a pathogen of many plant species in China, such as kiwifruit (Cui, Gong et al. 2015), American sweetgum (Mao, Zheng et al. 2021), castor bean (Tang, Liu et al. 2020), and holly mangrove (Xie, He et al. 2020). To our knowledge, this is the first report of leaf spot disease on sweet viburnum caused by C. cassiicola in China and the precise identification of the causal agent will be useful for its management.

Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates

Primary hemostasis results in a platelet-rich thrombus that has long been assumed to form a solid plug. Unexpectedly, our 3-dimensional (3D) electron microscopy of mouse jugular vein puncture wounds revealed that the resulting thrombi were structured about localized, nucleated platelet aggregates, pedestals and columns, that produced a vaulted thrombus capped by extravascular platelet adherence. Pedestal and column surfaces were lined by procoagulant platelets. Furthermore, early steps in thrombus assembly were sensitive to P2Y12 inhibition and late steps to thrombin inhibition. Based on these results, we propose a Cap and Build, puncture wound paradigm that should have translational implications for bleeding control and hemostasis.

Misdiagnosis of Systemic Metastatic Melanoma for fungal infection: A case report

BackgroundMost of malignant melanomas originate from skin and often metastasize to the lungs, rarely metastasizes to the liver and bone. However, imageology characters of lung metastasis tumor are commonly similar to those of fungal infections. Case presentationA patient was admitted with unhealed plantar puncture wound for 3 years, and cough and expectoration for 2 years. The chest computed tomography (CT) revealed multiple nodules with cavities, and the patient was diagnosed of pulmonary fungal infection in another hospital and received antifungal therapy for more than 8 months, but the clinical symptoms and chest imaging findings continue to progress. After admission, the pathological results of both lung biopsy and biopsy of the plantar wound 3 years ago indicated malignant melanoma. ConclusionsThe diagnosis of lung lesions cannot rely solely on imaging diagnosis, lung biopsy should be performed if necessary.

Apple Fruit (Malus domestica Borkh.) Metabolic Response to Infestation by Invasive Brown Marmorated Stink Bug (Halyomorpha halys Stal.)

The brown marmorated stink bug (BMSB; Halyomorpha halys Stal.) has become a significant pest in Slovenia, especially in apple, pear, peach, and cherry orchards. In our study, apple fruit of the apple cultivar ‘Red Pinova’ were evaluated for visual injury and sugar, organic acid, and phenolic contents. The chemical composition of the area around the puncture wound, the uninjured part of the infested apple, was compared to, as a control, only uninjured apples. There was a significant response of the apple around the puncture wound, resulting in an 11.9 g/kg FW higher total sugar content, a 1.4 g/kg FW lower total organic acid content, and an 11.9 g/kg FW higher total phenolic content compared with control apples. A strong phenolic response in the puncture wound area, with high flavanol and hydroxycinnamic acid contents, with increases of 118% and 237%, respectively, compared with control apples, was detected. The brown marmorated stink bug induces a strong phenolic response in the injured area of the apple. The results of this study illustrated how apple fruit responds to the BMSB injury, not only sensorily (visual injury, odor), but also chemically in the form of metabolic responses.

Do High-Risk Patients with Infected Puncture Wounds Get Appropriate Tetanus Prophylaxis?

Background: To evaluate clinicians' compliance to follow national guidelines for tetanus vaccination prophylaxis in high-risk foot patients. Methods: We retrospectively evaluated 114 consecutive patients between June 2011 and March 2019 who presented with a foot infection resulting from a puncture injury through the emergency department. Eighty-three patients had diabetes mellitus and 31 patients did not have diabetes mellitus. Electronic medical records were used to collect a broad range of study data on patient demographics, previous medical history, previous tetanus immunization history and tetanus status upon presentation to the emergency department (ED), peripheral arterial disease, sensory neuropathy, laboratory values, and clinical / surgical outcomes. Results: 46.5% of the patients who presented to the ED with a puncture wound did not have up-to-date tetanus immunization. Of those patients, 79.2% received a tetanus-containing vaccine booster, 3.8% received intramuscular tetanus immunoglobulins (TIG), 3.8% received both tetanus-containing vaccine booster and TIG, and 20.8% received no form of tetanus prophylaxis. When comparing data between patients with and without diabetes, there were no statistical significant differences in tetanus prophylaxis. Conclusion: Guidelines for tetanus prophylaxis amongst high-risk foot patients in this study center are not followed in all patients. Patients with DM are at high risks of exposure to tetanus, we recommend physicians to take a detailed tetanus immunization history and vaccinate them if tetanus history is unclear.