Tissue Infections Research Articles

First Report of Leaf Blight of Syringa oblata Caused by Botryosphaeria dothidea in China.

As an important landscaping plant, Syringa oblata is widely planted in northern China with high ornamental, medicinal, and edible value (Men et al. 2023). In September 2023, a new leaf blight disease on S. oblata was observed in Tianjin (39.0916°N, 117.1019°E), China. The onset of foliar symptoms was marked by the appearance of yellow-brown spots that originated from the tip and margin, subsequently evolving into irregularly shaped brown lesions. Finally, the lesions are distributed throughout the leaf surface, causing the leaf to wilt and seriously affecting photosynthesis. To identify the pathogen responsible for leaf blight of S. oblata, symptomatic leaves were collected and cut into square leaf blocks with a size of 0.3 cm², which were sterilized by immersion in 75% ethanol for 60 s and 5% NaClO for 30 s, and rinsed three times with sterile distilled water. The sterilized leaf pieces were then placed on potato dextrose agar (PDA) and incubated at 25 °C for 3-5 days. The peripheral hyphae of the fungal colony which developed from the infected tissues were isolated onto PDA plates. The fungal cultures on PDA plates were used for morphological observation and identification of the fungus. Colonies of B. dothidea on PDA medium were initially off-white, batting-shaped and gradually grayish-black. Aerial hyphae were well developed and could reach the tip of the petri dish. To induce sporulation, the hyphae were picked into medium containing sterilized pine needles. Conidia were found on pine needles after 30 d of incubation at 25°C. Conidia were hyaline, unicellular, oblong to spindle-shaped, and 17.5-23.0 µm in size × 6.1-8.5 µm(n=50). Based on these characteristics, the isolates were preliminarily identified as B. dothidea (Phillips et al. 2005). To provide additional evidence for the classification of the isolate, genomic DNA was extracted from the isolates of B. dothidea and used for Polymerase Chain Reaction (PCR). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), and actin (ACT) were amplified with the primer pairs ITS1/ITS4, GDP1/GDP2, and ACT-512F/ACT-783R, respectively (Jiang et al. 2022). A BLAST search of sequences showed the ITS, GPDH, and ACT sequences had >99% identity with homologous sequences from B. dothidea isolates Bb158-4(HQ392696.1), PPO-46523(MG761771.1), and CMW7779 (AY972117.1), respectively. Phylogenetic analysis determined that the isolate was in the same clade position as B. dothidea, which confirmed the above morphological identification. To assess pathogenicity, the fungal cakes (6 mm diameter) were obtained from the edge of a fresh colony (cultured on a PDA plate for 7 days) using a sterile perforator. Ten surface-sterilized leaves of healthy S. oblata with uniform growth condition were collected and inoculated with fungal cakes after wounding. Ten leaves were inoculated with sterile PDA medium blocks as control. The test was repeated three times. All leaves were kept at 25°C and sterile H2O was sprayed daily to keep leaves surface moist. After five days, all vaccination sites showed lesions similar to those of the S. oblata diseased leaves in the field, while the controls were asymptomatic. B. dothidea was reisolated from symptomatic tissues, thus fulfilling Koch's postulates. B. dothidea is a member of Botryosphaeriaceae. Currently, B. dothidea infection of Syringa spp. plants has not been reported. However, in China, it has been reported to cause stem rot on Forsythia suspense (He et al. 2022) and leaf dieback on sweet osmanthus (Ling et al. 2010), demonstrating that B. dothidea can infect Oleaceae species. This study found that S. oblata could be infected by B. dothidea. To the best of our knowledge, this is the first report of B. dothidea causing leaf blight on S. oblata in China. Identifying the pathogen of S. oblata leaf blight is essential for the prevention and management of disease associated with S. oblata.

An inhalable nanoparticle enabling virulence factor elimination and antibiotics delivery for pneumococcal pneumonia therapy

Streptococcus pneumoniae (S. pneumoniae) is a major cause of community-acquired pneumonia. Current standard clinical therapies mainly focus on combating S. pneumoniae through antibiotics. However, the limited delivery of antibiotics and the undetoxified hydrogen peroxide (H2O2) virulence factor secreted by S. pneumoniae impede the therapeutic outcomes. Here we report an inhalable catalase (CAT)-tannic acid (TA) nanoassembly for local antibiotic (levofloxacin) delivery and simultaneously neutralizing the secreted H2O2 virulence factors to treat pneumococcal pneumonia. After aerosol inhalation, the inhalable formulation (denoted as CT@LVX) effectively accumulates in lung tissues through TA-mediated mucoadhesion. CAT can reduce alveolar epithelial cells apoptosis by catalyzing the decomposition of accumulated H2O2 in the infected lung tissues. In synergy with antibiotic LVX-mediated S. pneumoniae elimination, CT@LVX significantly decreases lung injury companied with reduced inflammatory, resulting in 100 % survival of mice with pneumonia. In a clinically isolated S. pneumoniae strain-induced pneumonia mouse model, CT@LVX also shows superior outcomes compared to the traditional antibiotic treatment, highlighting its potential clinical application prospects.

First Report of Xanthomonas campestris pv. raphani Causing Leaf Spot Disease on Broccoli in China.

Broccoli (Brassica oleracea L. var. italica) is one of the important cruciferous vegetables in China, known for its considerable economic and nutritional value (Li et al. 2021). In September 2023, leaf spot disease was observed on broccoli seedlings in the commercial fields in Weifang City, Shandong Province, China (119°15'E, 36°70'N). Further investigation revealed that the disease incidence was approximately 60% in 4.5 square hectometers (hm2) broccoli field, resulting in substantial economic losses. This disease primarily affects the leaves, manifesting distinct symptoms such as circular, dark necrotic spots that gradually lighten and are encircled by a chlorotic halo. Some lesions further develop a black or purplish border, exhibit concentric zonation, and eventually fall off, leaving behind holes, as shown in Figure S1B and C. In severe cases, decay originates from the central perforation and spreads outwards, as shown in Figure S1A. To identify the causal agent of this disease, infected leaf tissues were collected and surface disinfected by immersing in 75% ethanol for 30 seconds, followed by three rinses with sterile water. The samples were grinded in sterile deionized water, and the extract was plated on NA. After incubation at 28°C for 48 h, individual colonies were transferred to fresh NA plates. A total of 12 strains with the similar morphological characteristics were isolated from diseased samples collected from the three plots. After 48 hours of growth on NA medium at 28℃, each colony attained a diameter ranging from 3 to 5 mm. These colonies appeared yellow, slightly elevated, nearly circular in shape, with a smooth and moist edge. Three representative strains were selected for further investigation. All strains were Gram-negative, aerobic, and rod-shaped. The analysis of BIOLOG GENIII microplate system revealed the capability of three isolates using cellobiose, trehalose, glucose, mannose, galactose, and sucrose. Furthermore, the isolates were unable to hydrolyze arginine and utilize rhamnose and inositol. The 16S rRNA gene was amplified and sequenced to confirm that three isolated bacteria belong to the genus Xanthomonas (OR772321-OR772323), followed by PCR amplification for 4 housekeeping genes atpD, dnaK, gyrB, and rpoD (Young et al. 2008; Saux et al. 2015). The obtained sequences were submitted to the GenBank under accessions OR789628-OR789630, OR785471-OR785473, OR789631-OR789633, and OR785468-OR785470. Gene sequences were aligned, concatenated, and used to generate a phylogenetic tree using the neighbor-joining method in MEGA11 (Tamura et al. 2021). The phylogenetic analysis revealed that all the three isolates were clustered with Xanthomonas campestris pv. raphani strains 5055 and 576, respectively (Figure S2). (Dubrow et al. 2022). These results were consistent with those of the reported X. campestris pv. raphanin (Cruz et al. 2015). To verify the pathogenicity of these strains, we used a spray inoculation method. In detail, bacterial suspensions (30 mL per treatment) containing approximately 108 CFU/ml were sprayed onto healthy, four-week-old broccoli plants and incubated in a phytotron at 28°C and above 90% RH. Negative controls were performed using sterile distilled water. Each isolate underwent three trials and each treatment included 12 broccoli seedlings. Leaf spot symptoms were observed on 5 days post inoculation, as shown in Figure S1E, F and G. Negative control plants showed no symptoms (Figure S1D). We further re-isolated the bacterium from the symptomatic plants and verifying the bacteria as X. campestris pv. raphanin with the aforementioned sequence analysis, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of X. campestris pv. raphani causing leaf spot disease on broccoli in China. This study enhances our understanding of the pathogenic bacterium on broccoli and lays the groundwork for developing targeted management strategies.

Clinical application of parasacral artery perforator flap in the treatment of Pilonidal Sinus Diseases

Objective: To investigate clinical efficacy of parasacral perforator flap (PPF) on postoperative wound healing in pilonidal sinus diseases (PSDs). Methods: The surgery steps were as follows: (1) To preoperatively detect parasacral perforator arteries with the handhold Doppler probe and mark them; (2) To remove the infected and necrotic tissues of PSDs completely; (3) To design the PPF according to the wound size and the parasacral perforator arteries' localization; (4) To harvest the flap from the gluteus maximus muscle surface and transfer it to the wound without tension. Several data were documented, including surgical duration, flap length, flap width, drainage tube placement duration, hospital stay, duration from operation to stitch removal, postsurgical complications and recurrence. Results: There were six patients with PSDs whose postoperative wound healing was repaired by PPF, admitted in our department from March 2021 to March 2023. Of them, five were male and one was female. Their median age was 24 (range: 18-33) years old. Their median surgical duration was 165 (range: 134-207) minutes, median length of PPF was 8 (range: 7-11) cm, median width of PPF was 3 (range: 3-4) cm, mean duration of drainage tube placement was 8 (range: 4-17) days, mean hospital stay was 13 (range: 6-23) days, mean duration from operation to stitch removal was 14 (range: 14-17) days, median follow-up time was 6-16 months. Incisions of all six cases achieved first-intention healing without early- or late-stage complications. No recurrence occurred during follow-up. All patients involved were satisfied with their clinical efficacy. Conclusion: The utility of PPF in postoperative wound healing of PPDs was effective, safe and reliable.

Pseudorabies Virus UL4 protein promotes the ASC-dependent inflammasome activation and pyroptosis to exacerbate inflammation.

Pseudorabies virus (PRV) infection causes systemic inflammatory responses and inflammatory damages in infected animals, which are associated with the activation of inflammasome and pyroptosis in infected tissues. Here, we identified a critical function of PRV non-structural protein UL4 that enhanced ASC-dependent inflammasome activation to promote pyroptosis. Whereas, the deficiency of viral UL4 was able to reduce ASC-dependent inflammasome activation and the occurrences of pyroptosis. Mechanistically, the 132-145 aa of UL4 permitted its translocation from the nucleus to the cytoplasm to interact with cytoplasmic ASC to promote the activation of NLRP3 and AIM2 inflammasome. Further research showed that UL4 promoted the phosphorylation levels of SYK and JNK to enhance the ASC phosphorylation, which led to the increase of ASC oligomerization, thus promoting the activation of NLRP3 and AIM2 inflammasome and enhanced GSDMD-mediated pyroptosis. In vivo experiments further showed that PRV UL4 (132DVAADAAAEAAAAE145) mutated strain (PRV-UL4mut) infection did not lead to a significant decrease in viral titers at 12 h. p. i, but it induced lower levels of IL-1β, IL-18, and GSDMD-NT, which led to an alleviated inflammatory infiltration and pathological damage in the lungs and brains, and a lower death rate compared with wild-type PRV strain infection. Taken together, our findings unravel that UL4 is an important viral regulator to manipulate the inflammasome signaling and pyroptosis of host cells to promote the pathogenicity of PRV, which might be further exploited as a new target for live attenuated vaccines or therapeutic strategies against pseudorabies in the future.

Encapsulation of Mentha aquatica methanol extract in alginate hydrogel promotes wound healing in a murine model of Pseudomonas aeruginosa burn infection

Burn injuries are the fourth most prevalent devastating form of trauma worldwide. Among the most extensively explored materials, composite dressings with alginate loaded with herbal extract can be mentioned. This research aimed to develop a sodium alginate (SA)-based hydrogel encapsulated with Mentha aquatica (MA) methanol extract and investigate its therapeutic efficacy in the infected burn mouse model. SEM, FTIR, in vitro extract release, HPLC, mechanical test, contact angle, swelling, degradability, and temperature response properties were used to analyze the hydrogel scaffold's physicochemical structure. Additionally, the antibacterial activity and MIC level of the extract, cell cytotoxicity, and macroscopic and microscopic analysis of the wound healing process were done using Masson's trichrome and hematoxylin-eosin staining. The physicochemical properties of the SA hydrogel encapsulated with MA extract were verified. The lowest inhibitory dose of the extract was determined to be 12.5 mg/ml. Application of SA/MA hydrogel to localized wounds of deep third-degree burns demonstrated faster tissue regeneration, collagen recovery, and eradication of bacterial infection. This research focused on the design and the preparation of a novel and effective biomaterials-based medical product, which has the potential to rehabilitate infected and injured skin tissue; therefore, it can be a promising candidate for wound dressing applications.

Insights into the molecular basis of beet curly top resistance in sugar beet through a transcriptomic approach at the early stage of symptom development

Curly top disease caused by Beet curly top virus (BCTV) is a limiting factor for sugar beet production. The most economical and sustainable control of BCTV in sugar beet would be via the growth of resistant cultivars, although most commercial cultivars possess only low-to-moderate quantitative resistance. A double haploid line (KDH13) showed a high level of resistance to BCTV infection. However, the mechanism of resistance and response of this line to BCTV infection is unknown. Here, we tested the response of this line to both local and systemic BCTV infections. The virus replicated at a high level in locally infected tissue but lower than in susceptible KDH19 plants. Resistant KDH13 plants systemically infected with BCTV showed only mild enation without leaf curling after 30 days. In contrast, severe leaf curling appeared after 12 days in susceptible plants with higher virus accumulation. Transcriptome analysis of the BCTV-infected KDH13 plants at the early stage of symptom development showed only 132 genes that were exclusively deregulated compared to the regulation of a large number of genes (1018 genes) in KDH19 plants. Pathway enrichment analysis showed that differentially expressed genes were predominantly involved in hormone metabolism, DNA methylation, immune response, cell cycle, biotic stress and oxidative stress. The auxin level in both resistant and susceptible plants increased in response to BCTV infection. Remarkably, exogenous application of auxin caused leaf curling phenotype in the absence of the virus. This study demonstrates the response of resistant and susceptible plants to BCTV infection at both local and systemic infections and highlights the defence-related genes and metabolic pathways including auxin for their contribution towards BCTV symptom development and resistance in sugar beet.

Emerging severe acute respiratory syndrome coronavirus 2 variants and their impact on immune evasion and vaccine-induced immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants harboring mutations in the structural protein, especially in the receptor binding domain (RBD) of spike protein, have raised concern about potential immune escape. The spike protein of SARS-CoV-2 plays a vital role in infection and is an important target for neutralizing antibodies. The mutations that occur in the structural proteins, especially in the spike protein, lead to changes in the virus attributes of transmissibility, an increase in disease severity, a notable reduction in neutralizing antibodies generated and thus a decreased response to vaccines and therapy. The observed multiple mutations in the RBD of the spike protein showed immune escape because it increases the affinity of spike protein binding with the ACE-2 receptor of host cells and increases resistance to neutralizing antibodies. Cytotoxic T-cell responses are crucial in controlling SARS-CoV-2 infections from the infected tissues and clearing them from circulation. Cytotoxic Tcells efficiently recognized the infected cells and killed them by releasing soluble mediator's perforin and granzymes. However, the overwhelming response of Tcells and, subsequently, the overproduction of inflammatory mediators during severe infections with SARS-CoV-2 may lead to poor outcomes. This review article summarizes the impact of mutations in the spike protein of SARS-CoV-2, especially mutations of RBD, on immunogenicity, immune escape and vaccine-induced immunity, which could contribute to future studies focusing on vaccine design and immunotherapy.

Morphological and molecular identification of Euclinostomum heterostomum (Trematoda: Clinostomidae) from spotted snakehead Channa punctata in Bangladesh.

Parasites belonging to the trematode family Clinostomidae have a global distribution. Freshwater fish in Bangladesh frequently serve as hosts for different clinostomid species. During the metacercaria stage, the digenetic trematode Euclinostomum heterostomum exhibits a specific preference for Channidae species, where it undergoes encystment within the liver, kidneys, and muscles of these hosts. The study aimed to identify E. heterostomum in Channa punctata through combined morphological and molecular analysis. Light and scanning electron microscopy were conducted to investigate the morphological characteristics of the species. The histological examination of the infected liver tissue revealed the presence of encysted flukes, inflammatory leukocyte infiltrates, and degeneration and loosening of hepatic tissue. In molecular analysis, the generated internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA and ITS2 regions sequence (GenBank accession no: OR591452) of 830 bp showed 100% identity with Euclinostomum heterostomum identified in India (MT785786). The phylogenetic reconstruction provided substantial evidence of genetic similarities (0-1% genetic distance) among different isolates of the genus Euclinostomum, suggesting that there is a shared heritage.

Chestnut quality classification by THz Time-Domain Hyperspectral Imaging combined with unsupervised learning analysis

Chestnut crops are threatened by fungal pathogens such as Gnomoniopsis castaneae, which cause significant degradation of quality. Early detection of such infections is crucial to maintain the quality of chestnuts in the food industry. This study explores the application of Terahertz Time-Domain Hyperspectral Imaging (THz-TDHIS) combined with unsupervised learning techniques to identify fungal infections in chestnuts. Unlike conventional methods that rely on light attenuation, this approach leverages the unique spectral signatures of infected tissues. By employing Principal Component Analysis, K-Means Clustering, and Agglomerative Clustering, we effectively differentiate between healthy and infected portions of chestnuts. Our findings indicate that spectral features, rather than just intensity variations, provide more reliable markers for infection. In addition, we demonstrate that these methods enable the quantification of the degree of infection in chestnuts. The robustness of these unsupervised learning methods in handling large and heterogeneous data sets further underscores their potential in agricultural applications. This integrated THz-TDHIS and machine learning approach presents a promising solution to ensure chestnut quality and safety.

Ultrasound-assisted minimally invasive technique for tissue collection from bovine mammary glands.

The objective of this case report was to describe an ultrasound-guided, minimally invasive method for longitudinal mammary gland tissue collection from the bovine species. Biopsies were performed on 14 8-week-old calves and 113 10-week-old calves. A subset of 36 animals had repeated mammary gland biopsies through the first lactation. Mammary gland biopsies were performed using a disposable biopsy punch. The technique was also performed on multiparous cows on other independent research trials. One-hundred and thirteen animals healed from the 10-week biopsies with no complications. Of the 36 animals that received repeated biopsies, one developed mastitis due to premature suture removal and one had recurring mastitis in all quarters. Thirty-three animals underwent all biopsies during gestation. Thirty of the original 36 are currently in lactation and still undergoing repeated biopsies. The method has also been successfully replicated on multiparous cows in separate studies. The described technique is a safe, reliable method for cattle mammary gland biopsies beginning at eight weeks of age and can be utilized to obtain repeated tissue collection from individual animals. The technique is also straightforward to perform and utilizes simple tools while providing acceptable amounts of tissue for most applications, with low risk for infection and long-term tissue damage.

Cyclic stretch enhances neutrophil extracellular trap formation

BackgroundNeutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries.ResultsUsing this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization.ConclusionsIn summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target.

Rabies, The Administration of Vaccines and Public Health Concern, Neglected Challenges and Historical Perspective in China

Rabies virus is transmitted via contact of saliva of a rabid animal with a person ,s mucosa or a skin lesion. It is a fatal encephalomyelitis caused by members of expanding Lyssavirus genus, and the genus included 17 species. The aim of this literature review is to survey on rabies and vaccination in China, threats and challenges to eliminate it, especially in China. The information provided is obtained from randomized control experiments, review articles, and analytical observations and studies which were gathered from various literature sources such as Scopus, Google Scholar, PubMed, and Science Direct. The keywords were dogs, glycoprotein, lyssavirus, post-exposure prophylaxis, and rabies virus. Rabies virus is a bullet shaped enveloped virion, and the classical rabies virus and its field strains are discovered worldwide and induces to rabies in animals and humans. In recent years, China has made wonderful and significant achievements in rabies prevention and control, and currently, the mortality and incidence rate of human infection with rabies have decreased to the minimum level historically, which has caused a notable foundation for the ultimate elimination of human rabies. Generally, five proteins are available in mature rabies virus particles, phosphoprotein, nucleoprotein, glycoprotein, matrix protein, and RNA-dependent RNA polymerase. The main carriers of rabies in China are dogs which are accountable for most of the human rabies deaths in China. Most common way of entry of rabies virus into the body is both via infected neural tissue through open cuts in the skin and salvia. The most notable parameters which limit rabies vaccinations are limited laboratory-based surveillance, diminished community outreach and education, underestimation of the population at risk, irregular vaccine applications in space and time, inaccessible naïve free-ranging dogs, exclusion of puppies, cold chain failure, translocation of cases, effect of other species, including cats, wildlife, and livestock, accessibility to remote hotspots, competing priorities, lack of adequate resources, impact of poverty on responsible social ownership and routine animal health care, cultural disparities, Sisyphean fatigue, frustration and staff burn-out. Rabies remain a public health problem and continue to present health risks for both animals and human; It is important to improve access to PEP in endemic countries where human rabies deaths still happen, but the big problem is its costs that should be curtailed and surveillance strengthened for controlling and eliminating it.

ADP-heptose attenuates Helicobacter pylori-induced dendritic cell activation

ABSTRACT Sophisticated immune evasion strategies enable Helicobacter pylori (H. pylori) to colonize the gastric mucosa of approximately half of the world’s population. Persistent infection and the resulting chronic inflammation are a major cause of gastric cancer. To understand the intricate interplay between H. pylori and host immunity, spatial profiling was used to monitor immune cells in H. pylori infected gastric tissue. Dendritic cell (DC) and T cell phenotypes were further investigated in gastric organoid/immune cell co-cultures and mechanistic insights were acquired by proteomics of human DCs. Here, we show that ADP-heptose, a bacterial metabolite originally reported to act as a bona fide PAMP, reduces H. pylori-induced DC maturation and subsequent T cell responses. Mechanistically, we report that H. pylori uptake and subsequent DC activation by an ADP-heptose deficient H. pylori strain depends on TLR2. Moreover, ADP-heptose attenuates full-fledged activation of primary human DCs in the context of H. pylori infection by impairing type I IFN signaling. This study reveals that ADP-heptose mitigates host immunity during H. pylori infection.

First Report of Klebsiella variicola Causing Tomato Pith Necrosis in Yunnan Province, China.

Tomato (Solanum lycopersicum L.) is a key vegetable crop in China. In August 2023, an outbreak of bacterial pith necrosis in tomato occurred in Lufeng County, Yunnan Province, China, affecting over 40% of the tomato plants in a greenhouse. The stems of infected plants developed a waterlogged soft rot and the disease progressed, the lower leaves and lateral branches of infected plants gradually wilted and died. A longitudinal cut of the stem revealed hollow pith with brown vascular tissue. To isolate the pathogen, the plant surface was disinfested with 75% ethanol. Then, a piece of infected tissue from the base of the stem was excised and immersed in sterile water for 2 min. A small amount of liquid was streaked onto TTC (2,3,5-triphenyltetrazolium chloride) agar medium using an inoculation loop, and plates were incubated at 28℃ for 24 h. Colonies on the TTC plate were white, indicating that the pathogen was not Ralstonia solanacearum. Colonies grown on LB (Luria-Bertani) agar medium were randomly selected and subjected to preliminary pathogenicity tests. Based on the results, a colony named Kv4 was selected and purified through six subcultures in LB agar medium. Biochemical tests showed the strain utilized D-sorbitol, raffinose and citrate but not adonitol, and was positive for methyl red, D-glucose (acid), urea hydrolysis, lysine decarboxylase, and motility, and negative for phenylalanine deaminase, H2S production, indole production, and ornithine decarboxylase. These characteristics align with Klebsiella species (Garrity et al. 2007). To determine the species of strain Kv4, partial sequences of the 16S rDNA, phoE, leuS, and rpoB genes were amplified (Barrios-Camacho et al. 2019) and sequenced. Through BLASTn analysis, strain Kv4 sequences of 16S rDNA (OR888750) had 99.47% identity (1488/1496 bp), phoE (OR899599) had 98.69% (605/613 bp) identity, leuS (OR899598) had 99.07% identity (959/968 bp), and rpoB (OR899597) had 97.69% (633/648 bp) identity with Klebsiella variicola strain FF0907. Using the ClustalW algorithm in MEGA11 for nucleotide sequence alignment, phylogenetic trees were constructed with 16S and phoE, leuS, and rpoB via the neighbor-joining method, confirming strain Kv4 as K. variicola. To test pathogenicity, the roots of 25 'Moneymaker' tomato plants with four to five true leaves were wounded, then each plant inoculated with a 15 mL bacterial suspension (OD600=0.6) of strain Kv4, while the control plants received sterile water. Plants were incubated at 28℃ with a 16 h photoperiod. Experiments were done twice. At 15 days after inoculation (DAI), all plants inoculated with Kv4 showed yellowing, unevenly distributed small black necrotic spots on the leaf surface, and purple-brown soft rot at the stem base. By 18 DAI, there was a gradual transformation of the stem bases from green to purplish brown. At 21 DAI, 60% of the inoculated plants displayed brownish soft rot at the stem base. In contrast, the control plants remained symptom-free. The pathogen was re-isolated from the stem and identified as K. variicola via sequence analysis of 16S, phoE, leuS, and rpoB. In recent years, several new bacterial pith necrosis diseases were reported in tomato (Guo et al. 2023; Ivić et al. 2023). This is the first study documenting K. variicola causing bacterial pith necrosis in tomato. Once considered a benign plant endophyte, Sun et al. (2023) reported K. variicola causing banana sheath rot in Guangdong and Guangxi Provinces, China. Malik et al. (2023) reported that K. variicola caused leaf streak in sorghum in India. This report of bacterial pith necrosis in tomato caused by K. variicola strain Kv4 underscores the escalating threat posed by emerging pathogens to agricultural production. The emergence of K. variicola as a tomato pathogen complicates plant disease management strategies.

In vivo Antifungal Efficacy and Safety of Natural Compounds for Topical Treatment of Cutaneous Candidiasis

In vivo toxicity and antifungal therapeutic evaluation of selected natural compounds for topical applications was analyzed using standard methods. The natural compounds, blue vitriol, brimstone, trona, alum, palm ash and black stone were hygienically selected and purchased at a local market in southeastern Nigeria. The test organisms were isolated from high vaginal swab specimens obtained from cases of cutaneous candidiasis. The isolates were identified based on their morphological, physiological and molecular characteristics. The toxicological screening of the test samples was done using Draize’ skin irritancy test, Murine local lymph node assay (LLNA) and necropsy. The in vivo evaluation of antifungal activity of the solutions was done using eight-week-old albino rats weighing 120g – 150g. The animals were infected with the Candida isolates percutaneously; and treated using various formulations of the natural compounds. The skin sections were carefully observed for histopathological changes, before and after treatment. The total yeast counts of the infected skin tissues were also determined. The isolates include Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. Draize’ skin test and Murine local lymph node assay showed no observable skin irritancy reaction (P>0.05). The normal skin architecture of the infected animals was restored after 5 days of treatment with the test samples; while the total yeast count decreased from 108 CFU/mL to 102 CFU/mL. The findings have revealed that natural compounds are effective as well as non-toxic topically, in treatment of cutaneous candidiasis.

Transcriptome Mining, Identification and In silico Characterization of Thaumatin-Like Protein Sequences from Mentha Longifolia

Background: Thaumatin-like proteins (TLPs) constitute the PR-5 family of plant pathogenesis- related (PR) proteins and are responsible for host defense and various growth and development- related processes. Mints (Mentha sp.) are essential oil-bearing plants, mainly affected by water availability and fungal pathogens. Objective: Identification of candidate TLPs in Mentha longifolia that may help to counter pathogen infection Methods: Several bioinformatics procedures were used to identify TLP gene sequences from M. longifolia and to characterize them for nucleotide length and composition, their translated proteins, subcellular location, signal peptides, post translational modifications, allergenicity potential, evolutionary relatedness, and miRNA targeting. Result: 19 TLP candidates were identified in M. longifolia (Mlo_TLP001 - Mlo_TLP0019) annotating the available transcriptome data. The molecular mass of Mlo_TLPs ranged between 35.43 kDa - 12.79 kDa. Expression analysis of the Mlo_TLPs revealed that Mlo_TLP003, 006, 008, 010, 012, 013, 014, and 018 were upregulated in M. longifolia stem as compared to the root under control conditions. Further, the analysis of control and V. dahliae infected M. longifolia plants has shown Mlo_TLP006 to be upregulated in both the infected stem and root tissues as compared to the control. Conclusion: Mlo_TLP006 is a probable candidate that imparts resistance to fungal pathogens in Mentha longifolia

Multi-gene phylogeny and phenotypic analyses revealed an association of different Colletotrichum species with inflorescence dieback and leaf spot of arecanut in India

Arecanut is one of the economically important plantation crops. Among the various diseases, inflorescence dieback and leaf spot/blight diseases caused by Colletotrichum spp. have re-emerged as major factors impeding arecanut production in India in the last few years. To understand the Colletotrichum species complex in the arecanut system, we have characterized Colletotrichum spp. associated with inflorescence dieback and leaf spot/blight of arecanut based on a combination of phenotypic, multi-locus (ITS, ACT, CHS-1, GAPDH, HIS3 and TUB2) phylogeny and pathogenicity studies. A total of 26 Colletotrichum isolates were obtained from arecanut inflorescence dieback (13 isolates) and leaf spot/blight (13 isolates) infected tissues collected from disease-endemic regions of Karnataka, Kerala and Tripura states of India. The combined multi-locus sequence analyses and a critical examination of phenotypic characters revealed the association of Colletotrichum aoteaora as a dominant species with arecanut inflorescence dieback disease. On the other hand, two Colletotrichum species, viz., C. siamense and C. fructicola, were distinctly associated with leaf spot/blight disease. All the Colletotrichum isolates studied were pathogenic to arecanut. Significant variations in lesion size were observed among the different species with cross-infection ability on inflorescence and leaves. This study represents the first comprehensive study of Colletotrichum spp. associated with arecanut inflorescence dieback and leaf spot/blight diseases. In addition, the study is the first record of C. aoteaora, C. siamense, and C. fructicola associated with the arecanut crop in this country. It provides valuable information for implementing effective IDM strategies against these diseases in India.

Mapping Staphylococcus aureus at Early and Late Stages of Infection in a Clinically Representative Hip Prosthetic Joint Infection Rat Model.

Prosthetic joint infection (PJI) continues to be a devastating complication following total joint replacement surgeries where Staphylococcus aureus is the main offending organism. To improve our understanding of the disease pathogenesis, a histological analysis of infected peri-implant tissue in a hip PJI rat model was utilized to assess S. aureus spread and tissue reaction at early and late stages of infection. Sprague-Dawley rats were used and received a left cemented hip hemiarthroplasty using a 3D-printed titanium femoral stem. The rats received an intra-articular injection of S. aureus Xen36. These infected rats were sacrificed either at 3 days post-infection (early-stage infection) or at 13-days post-infection (late-stage infection). The femoral and acetabular tissues of all animals were harvested at euthanasia. Histological analysis for the harvested tissue was performed using immunohistochemistry, hematoxylin and eosin, as well as Masson's trichrome stains. Histological examination revealed significant quantitative and qualitative differences in peri-implant tissue response to infection at early and late stages. This hip PJI rat model identified clear histologic differences between early and late stages of S. aureus infection and how quickly bacterial infiltration could occur. These findings can provide insight into why certain surgical strategies like debridement and antibiotics may be associated with high failure rates.

Visualizing of engineered five-helix bundle protein for blocking SARS-CoV-2 variants in live animals

Variants of Concern (VOCs), and variant under monitoring (VUM) of SARS-CoV-2 have emerged with heightened infectivity, antibody resistance, and vaccine breakthrough. In light of this, it is imperative to establish efficient and streamlined testing models for the prompt detection of newly emerging mutant strains. The 5-Helix Bundle (5-HB) protein was engineered as potent inhibitors predicated on the S2 subunit of the SARS-CoV-2 spike protein, exerting a broad-spectrum inhibitory influence against SARS-CoV-2 infections. Positron emission tomography (PET) quantifications revealed significantly reduced radioactive uptake in infected tissue of 5-HB protein-treated mice. The reduction was much lower than that of untreated infected mice imaged at 4 h (48.6 % reduction), 12 h (45.2 % reduction), and 24 h (36.9 % reduction) (p < 0.01). The newly engineered 5-HB protein features with the capability of binding to conservative pre-fusion spike of SARS-CoV-2 in live animals, highlighting its entry-inhibition capacity for a wide spectrum of SARS-CoV-2 variants.