Accession Number Research Articles

Điều tra bệnh hại sau thu hoạch trên giống chuối Tiêu hồng (Musa acuminata) và chuối Nam Mỹ (Musa paradisiaca) ở Việt Nam

The survey implemented in the 2021 winter season on the Tieu hong banana cultivar (Musa acuminata) grown in Phu Tho and Hung Yen provinces showed that the most severe diseases were fruit rot and fruit spot with infection rates of 79.1% and 75.5%, respectively. Peel cracking and end rot (cigar rot) were also recorded at less than 5.0%. Fruit spot was the main disease that affected the South-American banana cultivar (Musa paradisiaca) during the 2022 spring-summer season in Gia Lai province. Of the 24 fungal and 37 bacterial strains isolated from the disease samples collected during the investigation and tested for their pathogenicity via artificial infection on healthy bananas, only 17 fungal strains showed the ability of lesion formation of which 4 strains caused typical symptoms of the fruit rot disease. There was not a single bacterial strain ascertained as phytopathogen indicating that fungi were the primary agents of post-harvest diseases on bananas. These four fungal strains were taxonomically characterized based on their morphology (colony and spore), comparative analysis of the ITS sequences of the 18S rDNA gene, and were then identified as Corynespora torulosa B2B, Microdochium colombiense B6, Fusarium circinatum B13F, and Aspergillus niger GL2.1. The ITS sequences of these strains were deposited to GenBank under the accession numbers OP855524, OP855525, OP855526, and OP855527, respectively.

Evaluation of a plant salinity-stress B2-protein gene in wild barley (Hordeum spontaneum) and cultivated barley (Hordeum vulgare L.)

Dehydration-inducing conditions are major factors affecting plants geographic distribution worldwide. Salinity is a major factor affecting crop productivity and survival. Advances in molecular studies enabled scientists to explore salinity and genetic factors that control and contribute to salinity tolerance in plants. We previously identified clones from barley substractive library “from plants growing under high salinity regime” that was similar to previously identified B2-protein gene. In the current study, the relative expression of the B2-protein gene is assessed in wild barley (Hordeum spontaneum) and cultivated barley (Hordeum vulgare L) varieties. Our results indicated that the relative expression of the B2 gene displayed 8.00, 10.34, 12.12 and 11.37 folds at 100 mM NaCl and 4.28, 3.62, 2.70 and 1.58 folds at 200 mM NaCl compared to the non-salt stressed control in wild barley, Giza 129, Giza 132 and Giza 123, respectively. The full-length sequence of the B2 gene (1449 bp) was successfully assembled “accession number MT249004” from wild barley. Comparison studies of the wild barley B2 sequence to its published homologue in cultivated barley indicated the presence of an extra 23 bp long sequence, but its deduced amino acids sequence is five a.a. residues shorter (354 vs. 359 a.a.) with 96.1 % identity. Computational analysis at the protein level indicated the presence of differences in the physicochemical properties between the two proteins. Also, the subcellular localization prediction of the protein revealed the detection of the former protein in the nucleus and the later in the chloroplast. However, the protein modelling and structure prediction do not reveal differences since around 60 % of the protein is disordered. On the other hand, B2-protein interaction predicted networks composed of proteins directly or indirectly associated with the stress responses. In conclusion, the B2- protein gene is a promising candidate for dehydration stress tolerance.

Withanolides production by the endophytic fungus Penicillium oxalicum associated with Withania somnifera (L.) Dunal.

Withanolides are steroidal lactones with diverse bioactive potential and their production from plant sources varies with genotype, age, culture conditions, and geographical region. Endophytic fungi serve as an alternative source to produce withanolides, like their host plant, Withania somnifera (L.) Dunal. The present study aimed to isolate endophytic fungi capable of producing withanolides, characterization and investigation of biological activities of these molecules. The methanolic fungal crude extract of one of the fungal isolates WSE16 showed maximum withanolide production (219mg/L). The fungal isolate WSE16 was identified as Penicillium oxalicum based on its morphological and internal transcribed spacer (ITS) sequence analysis and submitted in NCBI (accession number OR888725). The methanolic crude extract of P. oxalicum was further purified by column chromatography, and collected fractions were assessed for the presence of withanolides. Fractions F3 and F4 showed a higher content of withanolides (51.8 and 59.1mg/L, respectively) than other fractions. Fractions F3 and F4 exhibited antibacterial activity against Staphylococcus aureus with an IC50 of 23.52 and 17.39µg/ml, respectively. These fractions also showed antioxidant activity (DPPH assay with IC50 of 39.42 and 38.71µg/ml, superoxide anion scavenging assay with IC50 of 41.10 and 38.84µg/ml, and reducing power assay with IC50 of 42.61 and 41.40µg/ml, respectively) and acetylcholinesterase inhibitory activity (IC50 of 30.34 and 22.05µg/ml, respectively). The withanolides present in fraction 3 and fraction 4 were identified as (20S, 22R)-1a-Acetoxy-27-hydroxywitha-5, 24-dienolide-3b-(O-b-D-glucopyranoside) and withanamide A, respectively, using UV, FTIR, HRMS, and NMR analysis. These results suggest that P. oxalicum, an endophytic fungus isolated from W. somnifera, is a potential source for producing bioactive withanolides.

First report of Diaporthe eres causing leaf spot on Rosa roxburghii Tratt in Guizhou Province, China.

Rosa roxburghii Tratt is a plant from the Rosaceae family whose fruits are rich in vitamins, dietary fiber, flavonoids, phenolic acids, and other active components (Jiang, et al. 2024). In July 2023, about R. roxburghii 500 plants were investigated in a field of 6000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou province, China, and the results showed a leaf spot incidence of s 20 to 30%. . The affected leaves had irregular, black lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. Fifteen symptomatic leaves were collected from 10 plants washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were cut. After surface sterilization for30 s with 75% ethanol, 2 min with 3% NaOCl, three washes in sterilized distilled water, the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Three isolates (H3-Y-1-1, H3-Y-1-2, H3-Y-1-3) with identical morphology were obtained, and the isolate H3-Y-1-1was selected for further study. The colonies on PDA exhibited irregular growth patterns, with white felty aerial mycelium on the upper surface, and white mycelium on the lower surface. Conidiomata were irregularly distributed over the agar surface. The isolate H3-Y-1-1 produced darkly pigmented pycnidia on PDA after 30 days and oozed milky mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 4.74 - 5.96 × 1.52 - 2.24 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.13 - 25.74 × 0.86 - 1.29 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers PP411998 (ITS), PP502153 (TUB), PP502156 (TEF). BLAST searches of the sequences revealed (96%) (500/523nt), 97% (479/494 nt) and 99% (334/338 nt) homology with those of D. eres CBS 138594 from GenBank (OM698848, OM752196 and OM752197), respectively. Phylogenetic analysis using maximum-likelihood and Bayesian methods placed the isolate H3-Y-1-1 in a well-supported cluster with D. eres CBS 101742. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). To assess its pathogenicity, healthy R. roxburghii potted plants were inoculated with H3-Y-1-1 spore suspensions. Symptomatic leaves mirroring field symptoms were observed after XX days of incubations at XX°C, while control plants exhibited no symptoms. Diaporthe eres was consistently reisolated from the infected leaves showing brown irregular or round lesions at the initial stage of the disease, expanding and become more irregular over time ultimately causing leaf curling and plant death. To our knowledge, this is the first report of leaf spot on R. roxburghii caused by D. eres in China. The disease may become a serious threat to fruit of R. roxburghii production in China. Therefore, detection of this pathogen is very important to ensure timely disease management.

Phytochemical and biological assessment of secondary metabolites isolated from a rhizosphere strain, Sphingomonas sanguinis DM of Datura metel

BackgroundThe plant roots excrete a large number of organic compounds into the soil. The rhizosphere, a thin soil zone around the roots, is a hotspot for microbial activity, making it a crucial component of the soil ecosystem. Secondary metabolites produced by rhizospheric Sphingomonas sanguinis DM have sparked significant curiosity in investigating their possible biological impacts.MethodsA bacterial strain has been isolated from the rhizosphere of Datura metel. The bacterium’s identification, fermentation, and working up have been outlined. The ethyl acetate fraction of the propagated culture media of Sphingomonas sanguinis DM was fractioned and purified using various chromatographic techniques. The characterization of the isolated compounds was accomplished through the utilization of various spectroscopic techniques, such as UV, MS, 1D, and 2D-NMR. Furthermore, the evaluation of their antimicrobial activity was conducted using the agar well diffusion method, while cytotoxicity was assessed using the MTT test.ResultsThe extract from Sphingomonas sanguinis DM provided two distinct compounds: n-dibutyl phthalic acid (1) and Bis (2-methyl heptyl) phthalate (2) within its ethyl acetate fraction. Furthermore, the 16S rRNA gene sequence of Sphingomonas sanguinis DM has been registered under the NCBI GenBank database with the accession number PP422198. The bacterial extract exhibited its effect against gram-positive bacteria, inhibiting Streptococcus mutans (12.6 ± 0.6 mm) and Staphylococcus aureus (10.6 ± 0.6 mm) compared to standard antibiotics. Conversely, compound 1 showed a considerable effect against phytopathogenic fungi such as Alternaria alternate (56.3 ± 10.6 mm) and Fusarium oxysporum (21.3 ± 1.5 mm) with a MIC value of 17.5 µg/mL. However, it was slightly active against Klebsiella pneumonia (11.0 ± 1.0 mm). Furthermore, compound 2 was the most active metabolite, having a significant antimicrobial efficacy against Rhizoctonia solani (63.6 ± 1.1 mm), Pseudomonas aeruginosa (16.7 ± 0.6 mm), and Alternaria alternate (20.3 ± 0.6 mm) with MIC value at 15 µg/mL. In addition, compound 2 exhibited the most potency against hepatocellular (HepG-2) and skin (A-431) carcinoma cell lines with IC50 values of 107.16 µg/mL and 111.36 µg/mL, respectively.ConclusionSphingomonas sanguinis DM, a rhizosphere bacterium of Datura metel, was studied for its phytochemical and biological characteristics, resulting in the identification of two compounds with moderate antimicrobial and cytotoxic activities.

Assessment of antimicrobial activity and GC-MS using culture filtrate of local marine Bacillus strains

Secondary metabolites produced by Bacillus species from marine sources encompass a variety of compounds such as lipopeptides, isocoumarins, polyketides, macrolactones, polypeptides and fatty acids. These bioactive substances exhibit various biological activities, including antibiotic, antifungal, antiviral, and antitumor properties. This study aimed to isolate and identify a particular species of Bacillus from marine water and organisms that can produce bioactive secondary metabolites. Among the 73 Bacillus isolates collected, only 5 exhibited antagonistic activity against various viral and bacterial pathogens. The active isolates were subjected to 16S rRNA sequencing to determine their taxonomical affiliation. Among them, Bacillus tequilensis CCASU-2024-66 strain no. 42, with the accession number ON 054302 in GenBank, exhibited the highest inhibitory potential. It displayed an inhibition zone of 21 mm against Bacillus cereus while showing a minimum zone of inhibition of 9 mm against Escherichia coli and gave different inhibition against pathogenic fungi, the highest inhibition zone 15 mm against Candida albicans but the lowest inhibition zone 10 mm was against Botrytis cinerea, Fusarium oxysporum. Furthermore, it demonstrated the highest percentage of virucidal effect against the Newcastle virus and influenza virus, with rates of 98.6% and 98.1%, respectively. Furthermore, GC-MS analysis was employed to examine the bioactive substance components, specifically focusing on volatile and polysaccharide compounds. Based on these results, Bacillus tequilensis strain 42 may have the potential to be employed as an antiviral agent in poultry cultures to combat Newcastle and influenza, two extremely destructive viruses, thus reducing economic losses in the poultry production sector. Bacteria can be harnessed for the purpose of preserving food and controlling pathogenic fungi in both human and plant environments. Molecular docking for the three highly active derivatives 2,3-Butanediol, 2TMS, D-Xylopyranose, 4TMS, and Glucofuranoside, methyl 2,3,5,6-tetrakis-O-(trimethylsilyl) was carried out against the active sites of Bacillus cereus, Listeria monocytogenes, Candida albicans, Newcastle virus and influenza virus. The data obtained from molecular docking is highly correlated with that obtained from biology. Moreover, these highly active compounds exhibited excellent proposed ADMET profile.

Morphological and molecular characterization of two species of genus Ageratum.

The species of genus Ageratum (family Asteraceae) are distributed in various parts of the world. Ageratum conyzoides and A. houstonianum are the most commonly occurring species in India. These species are quite similar in their morphology thus creating a challenge in identification during the field survey and taxonomic validation. The accurate identification of the species is highly significant especially when those are of medicinal interest. To overcome the barriers in morphological based identification, DNA barcoding has been employed during the present investigation. Morphological and DNA barcodes matK and ITS genes, were employed to differentiate between Ageratum conyzoides and A. houstonianum. The obtained matK and ITS gene sequences were submitted to GenBank and BOLD system to obtain accession numbers. The DNA sequences were aligned with database sequences using BLAST and phylogenetic trees were constructed through neighbor-joining algorithm in MEGA 11 software. The distinguish features of A. conyzoides include ovate to elliptic-oblong leaves with a cuneate base and inflorescence heads forming domed to flat-topped clusters. However, A. houstonianum has triangular to ovate leaves with a cordate to truncate base, cymose clusters in the inflorescence and stipulate glandular involucre bracts. The matK gene has shown the highest identity percentages (100%) for A. houstonianum and 99.87% for A. conyzoides. The phylogenetic tree analysis has demonstrated a close association of A. conyzoides and A. houstonianum with their respective species, supported by bootstrap values in the matK and ITS trees. This study revealed that morphological and molecular data can be successfully utilized in the identification of A. conyzoides and A. houstonianum. The matK and ITS barcodes provide promising results in the identification of Ageratum species, with their phylogeny supporting classification within the family asteraceae.

#1922 Patterns and 2-year outcomes of preemptive vascular access creation in France

Abstract Background and Aims Most studies on clinical practices regarding the type and the timing of vascular access creation are limited to patients on kidney replacement therapy (KRT) from North American settings. Our aim was to describe patterns of preemptive vascular access creation in patients under nephrology care in France, and to assess the subsequent risks of hemodialysis (HD) initiation with arteriovenous (AV) access use, HD initiation with central venous catheter (CVC) use, initiation of other KRT modality, and competing death. Method From 2013 through 2016, the CKD-REIN cohort included 3033 adult patients with CKD stages 3 to 5 from 40 outpatient nephrology clinics in France, nationally representative geographically and in terms of facility legal status (public, private not for profit, and private for profit). Participants were followed for 5 years or until initiation of kidney replacement therapy (KRT) or death, whichever came first. Patients who underwent a first vascular access creation during follow-up were eligible for this analysis. We described the type, anatomical site and timing of the first vascular access created, as well as patients’ associated characteristics. The number and type of subsequent accesses were also described. Among patients who underwent AV fistula creation first, we used the cumulative incidence functions to estimate the 2-year probabilities of HD initiation with AV access use (independently of its type or order), HD initiation with CVC use, initiation of other KRT modality, and death. Results Over a median follow-up of 5.0 years (interquartile range [IQR], 4.6 to 5.2), 535 patients (18% of the total population) underwent a first vascular access creation. 408 patients (75%) had an AV fistula as their first vascular access, 7 patients (1%), an AV graft, and 127 patients (24%), a CVC. AV fistulas were predominantly located in the lower arm (76%). The median eGFR levels at access creation were 13 (IQR, 10 to 16), 12 (10 to 14) and 9 (6 to 13) ml/min/1.73 m2 for AV fistula, AV graft, and CVC, respectively. Groups defined by their first vascular access did not differ with regards to diabetes status, but patients who received AV fistula as their first vascular access stood out for being younger and having less cardiovascular comorbidities than their counterparts who received AV graft or CVC (Table 1). In contrast to the 7 patients with first AV graft who did not have any subsequent access creation, 22% of those with first AV fistula had at least one subsequent access creation before initiating KRT. The subsequent access was a fistula in 49% of cases (54% of them in the lower arm), a CVC in 48% and a graft in 2%. The 2-year cumulative incidences of HD initiation with AV access use, HD initiation with CVC use, preemptive kidney transplantation and pre-KRT death were 61%, 14%, 2% and 5%, respectively (Fig. 1); 18% of patients undergoing a first AV fistula creation did not experience any event within two years, and 25% did not start HD. No patients received peritoneal dialysis as other KRT modality. Conclusion In this nationwide description of patterns of preemptive vascular access creation, we highlight that lower arm AV fistula is largely prioritized, even after a first failed AV fistula creation. This may contribute to the higher use of AV fistula among HD patients in France compared to North American countries. Most patients who undergo a first AV fistula creation eventually start hemodialysis with an AV access within 2 years of this creation, but 1 out of 4 do not start HD at all (because of death, preemptive transplantation, or stable kidney function). Novel tools assisting clinical decision making in preemptive AV fistula creation are needed.

#2331 Distal arteriovenous fistulas for hemodialysis – too many interventions?

Abstract Background and Aims Distal arteriovenous fistula (dAVF) for haemodialysis (HD) is preferred due to lesser complications, despite greater risk of maturation failure. In HD patients, age and comorbid conditions increased the risk of access failure and number of procedures necessary for fistula patency. Preferred location for AVF poses a challenging decision. Advantages of dAVF must be taken into account although these may lead an unreasonably high number of interventions. Our aim was to determine time to functioning distal AFV in our centre and number of procedures necessary to achieve such status. Method Single centre retrospective analysis was performed on a cohort of patients submitted to distal AFV construction from 2015-2021. Success rate was defined as AFV patency in time for dialysis. Results Of 528 patients submitted to AVF construction from 2015 - 2021, 110 radiocephalic fistulas were seen, 59% male patients, mean age 68.5±12,0 yrs. Comorbidities were prevalent (80.9%, n=89), DM (54.6%) and HF (41.1%). Outcomes at 6 weeks were adequate maturation in 4.5% (n=59), 31.8% (n=35) late maturation (LM) and 14.5%(n=16) primary failure (PF). Causes of LM included inflow and outflow stenosis, 34.3% and 37.1%, respectively. LM (> 6 weeks) requiring no further intervention was seen in 28.6%. PF patients had smaller artery diameters on pre-operative US, with no statistical significance (0.23±0.07; 0.30 ± 0.2; p=0,375). Assisted patency rate was 78.2%. Number of procedures for access permeability was 1, 2 e 3 in 40%, 25.5% and 6.4%, respectively. Mean of 1,5±0,7 interventions per patient. Median time-to-adequate AFV was 2.0 (IQR 2,0 - 6,45) months. Success rate was 72,8%. Conclusion Outcomes at 6 weeks in our cohort were similar to those reported in literature. Timely referencing may increase access patency with resort to vascular intervention. Individualized management is essential, discussing risks and benefits taking into account the treatment burden associated with numerous procedures. Patency rates and outcomes after HD initiation and even cost analysis are essential to guide recommendations.

First report of postharvest pear bitter rot caused by Colletotrichum acutatum in Italy.

The fungal genus Colletotrichum includes numerous important plant pathogenic species, some of which causes fruit bitter rot as well as leaf lesions (leaf black spot) on major crops, leading to yield losses (Fu et al. 2019; Talhinhas & Baroncelli, 2023). C. acutatum was reported associated with black spot on fallen, immature fruit of pear (Pyrus pyrifolia) in New Zealand (Damm et al. 2012); however, to our knowledge, this species has not been reported in Italy or nowhere else. In 2022, a significant increase of anthracnose symptoms was observed on pears in Emilia-Romagna region, Italy. Symptoms, such as round brown lesions of 1 to 4 cm, appeared on more than 50% of refrigerated stored fruit. These symptoms were undetectable at the end of September 2022 and appeared after a five-month period of storage (February 2023) at 4°C (e-Xtra 1A and B). Fungal isolates were obtained from symptomatic pears after surfaces sterilization with 96% ethanol by culturing necrotic tissue pieces on Potato Dextrose Agar at 25°C in the dark (e-Xtra 1C and D). Cultures were shades of coral color, from opalescent to sunkist coral, with slight aerial mycelium becoming grey and darker with age. When observed from the reverse side, the color was pink and, with age, became coral orange to dark amaranth. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. (e-Xtra 1E) One reference isolate, named L51, was used for molecular characterization. Total genomic DNA was extracted, and the ITS region of rDNA amplified using the universal primers ITS1 and ITS4, then sequenced. The resulting sequences were 100% identical to those of C. acutatum (NR_144794.1: strain CBS 112996 ITS region; from TYPE material). Based on Damm et al. (2012), partial ACT, GAPDH, CHS and TUB2 gene sequences were also amplified and sequenced (GenBank Accession numbers: ITS: OR882016, ACT: OR882013, GAPDH: OR882011, CHS: OR882012, TUB2: OR882010), to characterize the isolates. Additionally, the multilocus phylogenetic analysis carried out with the obtained and reference sequences (Damm et al. 2012) revealed the species of analyzed isolates and confirmed the BLAST results, identifying the strain as C. acutatum (e-Xtra 1F). Koch's postulates were performed on 10 'Kaiser' pears. Surfaces sterilized fruits with 96% ethanol were subjected to wound inoculation with a conidial suspension (106 conidia ml-1) while 10 fruit were used as negative control and inoculated with sterile water. Following an incubation period of 8-14 days at 15-20°C, symptoms around the inoculation site resembled those initially observed, while the negative control showed no symptoms. Fungal colonies re-isolated from the lesions exhibited the same morphological characteristics as the original isolates. To our knowledge, this is the first report of pear bitter rot caused by C. acutatum in Italy and in Europe (Talhinhas & Baroncelli, 2023). Yet, bitter rot had not been recognized as a notable issue in pear cultivation. Nevertheless, given that pears rank as the 8th most cultivated fruit globally and economically very significant for the Emilia Romagna region in Italy the emergence of pear bitter rot caused by Colletotrichum species has the potential to evolve into a significant worldwide problem, warranting further investigation.

First Report of Diaporthe tectonendophytica Causing Leaf Spot on Dalbergia odorifera in Guangxi, China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is a national level II protected plant in China, with extremely high economic value and medical properties (Zhao et al. 2020). In June 2023, an unknown leaf spot was found in a garden land of Pingxiang city, Guangxi, China, and approximately 80% of the plants covered an area of 500 m2 displayed similar symptoms. The spots were grey to white, 4~6 mm in diameter (n=30) with black pycnida on the spots surface (Fig S1, A-D). Multiple disease spots were observed on a single leaf. The pycnida on the lesion were picked and mashed, to make a conidia suspension using sterile water. The conidial solution was then spread onto a potato dextrose agar (PDA) plate containing streptomycin, with 10 mg of streptomycin per 100 mL, and incubated for 3 days at 28°C with a 12 hour photoperiod. Three isolates (GXPX01, GXPX02 and GXPX03) were obtained by re-culturing the colonies on fresh PDA plates. The colony on PDA were white with aerial mycelia (Fig S1, E-F). Black conidiomata developed at 28°C with a 12 hour photoperiod in 20 days (Fig S1, G-H). Alpha conidia were 4.2~6.4 μm × 1.8~2.6 μm (average =5.1 × 2.3 μm, n = 30), mostly bi-guttulate, hyaline, ellipsoid, apex bluntly rounded, base obtuse to subtruncate, smooth (Fig S1, I). Beta conidia were 15.1~33.5 μm × 1~1.8 μm (average = 24.5 × 1.5 μm, n = 30), filiform, hyaline, curved or hamate, aseptate, base subtruncate (Fig S1, J). Morphological characteristics of the three isolates matched those of Diaporthe spp.(Gomes et al. 2013). The rDNA internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1), the calmodulin (CAL), the histone H3 (HIS) and the β-tubulin (TUB2) genes of the three isolates were amplified using the primer pairs ITS4/ITS5, EF1-728F/EF1-986R, CAL-228F/CAL2Rd, CYLH3F/H3-1B, and T1 /CYLTUB1R, respectively (Crous et al. 2004, Sun et al. 2021). The sequences were all deposited in GenBank (accession numbers OR437511 to OR437513 for ITS, OR454965 to OR454967 for TEF1, OR454968 to OR454970 for CAL, OR454971 to OR454973 for TUB2, OR454974 to OR454976 for H3). Sequences had 98.36% to 100% homology with the corresponding sequences of known Diaporthe tectonendophytica strains MFLUCC 13-0471 in the NCBI database. Phylogenetic analysis was based on combined ITS, TEF1, TUB2 and CAL sequences data using MEGA 11 software to construct phylogenetic tree with Maximum Likelihood (Doilom et al. 2017). In the phylogenetic tree, the combined sequences attributed the three isolates to the D. tectonendophytica (Fig S2). The pathogenicity was tested on leaves of 1.5-year-old D. odorifera seedlings. Three leaves were wounded with a sterile needle and individually inoculated with a 5 mm mycelial disk of PDA culture from each isolate. Sterile PDA disks inoculated leaves as a control. The test was repeated three times. The inoculated plants were placed in a greenhouse at 25℃ and 90% humidity, with a photoperiod of 12 hours. Five days after inoculation, necrotic lesions appeared on inoculated leaves and symptoms from all three isolates were the same as those form natural infections ( Fig S1, K-N), whereas all the control remained symptomless (Fig S1, P). The pathogen was reisolated from the inoculated leaves and again identified as D. tectonendophytica, with the same methodology used for the initial identification. D. tectonendophytica was reported to cause plant diseases, such as stem gray blight of red-fleshed dragon fruit (Hylocereus polyrhizus) (Rahim et al. 2021), leaf spots disease on Elaeagnus conferta and Pometia pinnata (Sun et al. 2021). To our knowledge, this is the first report of D. ctonendophytica causing leaf spot disease on D. odorifera.

Integrating 16S rRNA profiling and in-silico analysis for an epitope-based vaccine strategy against Achromobacter xylosoxidans infection

Achromobacter xylosoxidans is an aerobic, catalase-positive, non-pigment-forming, Gram-negative, and motile bacterium. It potentially causes a wide range of human infections in cystic fibrosis and non-cystic fibrosis patients. However, developing a safe preventive or therapeutic solution against A. xylosoxidans remains challenging. This study aimed to construct an epitope-based vaccine candidate using immunoinformatic techniques. A. xylosoxidans was isolated from an auto workshop in Lahore, and its identification was confirmed through 16S rRNA amplification and bioinformatic analysis. Two protein targets with GenBank accession numbers AKP90890.1 and AKP90355.1 were selected for the vaccine construct. Both proteins exhibited antigenicity, with scores of 0.757 and 0.580, respectively and the epitopes were selected based on the IC50 value using the ANN 4.0 and NN-align 2.3 epitope prediction method for MHC I and MHC II epitopes respectively and predicted epitopes were analyzed for antigenicity, allergenicity and pathogenicity. The vaccine construct demonstrated structural stability, thermostability, solubility, and hydrophilicity. The vaccine produced 250 B-memory cells per mm3 and approximately 16,000 IgM + IgG counts, indicating an effective immune response against A. xylosoxidans. Moreover, the vaccine candidate interacted stably with toll-like receptor 5, a pattern recognition receptor, with a confidence score of 0.98. These results highlight the potency of the designed vaccine candidate, suggesting its potential to withstand rigorous in vitro and in vivo clinical trials. This epitope-based vaccine could serve as the first preventive immunotherapy against A. xylosoxidans infections, addressing this bacterium’s health and financial burdens. The findings demonstrate the value of employing immunoinformatic tools in vaccine development, paving the way for more precise and tailored approaches to combating microbial threats.

Reduced Tolerogenic Program Death-Ligand 1-Expressing Conventional Type 1 Dendritic Cells Are Associated with Rapid Decline in Chronic Obstructive Pulmonary Disease

Background: Chronic obstructive pulmonary disease (COPD) is characterized, at least in part, by autoimmunity through amplified T helper 1 and 17 (Th1 and Th17) immune responses. The loss of immune tolerance controlled by programmed death-ligand 1 (PD-L1) may contribute to this. Objectives: We studied the tolerogenic role of PD-L1+ dendritic cells (DCs) and their subtypes in relation to specific T cell immunity and the clinical phenotypes of COPD. Methods: We used flow cytometry to analyze PD-L1 expression by the DCs and their subtypes in the peripheral blood mononuclear cells (PBMCs) from normal participants and those with COPD. T cell proliferation and the signature cytokines of T cell subtypes stimulated with elastin as autoantigens were measured using flow cytometry and enzyme-linked immunosorbent assays (ELISA), respectively. Measurement and main results: A total of 83 participants were enrolled (normal, n = 29; COPD, n = 54). A reduced PD-L1+ conventional dendritic cell 1 (cDC1) ratio in the PBMCs of the patients with COPD was shown (13.7 ± 13.7%, p = 0.03). The decrease in the PD-L1+ cDC1 ratio was associated with a rapid decline in COPD (p = 0.02) and correlated with the CD4+ T cells (r = −0.33, p = 0.02). This is supported by the NCBI GEO database accession number GSE56766, the researchers of which found that the gene expressions of PD-L1 and CD4, but not CD8 were negatively correlated from PBMC in COPD patients (r = −0.43, p = 0.002). Functionally, the PD-L1 blockade enhanced CD4+ T cell proliferation stimulated by CD3/elastin (31.2 ± 22.3%, p = 0.04) and interleukin (IL)-17A production stimulated by both CD3 (156.3 ± 54.7, p = 0.03) and CD3/elastin (148 ± 64.9, p = 0.03) from the normal PBMCs. The PD-L1 blockade failed to increase IL-17A production in the cDC1-depleted PBMCs. By contrast, there was no significant change in interferon (IFN)-γ, IL-4, or IL-10 after the PD-L1 blockade. Again, these findings were supported by the NCBI GEO database accession number GSE56766, the researchers of which found that only the expression of RORC, a master transcription factor driving the Th17 cells, was significantly negatively correlated to PD-L1 (r = −0.33, p = 0.02). Conclusions: Circulating PD-L1+ cDC1 was reduced in the patients with COPD, and the tolerogenic role was suppressed with susceptibility to self-antigens and linked to rapid decline caused by Th17-skewed chronic inflammation.

First Report of Anthracnose on Wurfbainia villosa var. villosa Caused by Colletotrichum gloeosporioides in China.

Wurfbainia villosa var. villosa is a traditional Chinese herbal medicine under the family Zingiberaceae, and its ripe fruits (called Fructus Amomi) are widely used clinically for the treatment of gastrointestinal disorders (Yang et al. 2023; Chen et al. 2023). In September 2023, plants of W. villosa var. villosa exhibited anthracnose-like symptoms on leaf with a disease incidence of 35% (n = 100 investigated plants) in an approximately 90 m2 field in Guangning, China (N23°42'51.70″, E112°26'35.75″). Light yellowish-green spots (~2 mm diameter) initially appeared on the infected leaves, gradually formed sub-circular or irregular spots, then fused and expanded, resulting in wilting of the leaves. To identify the causal agent, 10 symptomatic leaves were collected and transferred to the laboratory. The symptomatic leaf samples were surface sterilized in 0.5% NaClO for 2 min, and in 70% ethanol for 30 s, then washed three times with sterile water and air-dried on sterile filter paper. The leaf tissues were placed on potato dextrose agar (PDA) medium containing 100 μg mL-1 of ampicillin (Sigma-Aldrich, St. Louis, MO) and incubated for 7 days at 28°C in darkness. Nine isolates with similar colony morphology were isolated from the 10 plated leaves. Three representative isolates (GNAF03, GNAF06, GNAF09 with approximately 3.5 cm in diameter after 3 days of incubation) appeared gray to dark brown with dense aerial hyphae at the front and gray to black colonies on the reverse of the plates. Conidia were cylindrical and measured 21.2 to 29.3 μm long × 7.1 to 9.6 μm wide (n = 50). Appressoria were formed by the tips of germ tubes or hyphae and were brown, ellipsoid, thick-walled, and smooth-margined, measuring 10.2 to 12.3 μm long × 6.4 to 8.2 μm wide (n = 50). Morphologically, the fungal isolates resembled Colletotrichum sp. (Weir et al. 2012). For molecular analysis, genomic DNA was extracted from fresh mycelia of the three isolates, and the primers ACT-512F/ACT-783R, CL1/CL2A, GDF/GDR, and ITS1/ITS4 were used to amplify partial regions of rDNA-ITS, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions, respectively (Weir et al. 2012). The resulting sequences with more than 99% nucleotide identity to C. gloeosporioides were submitted to GenBank (accession numbers PP552725, PP552726, and OR827444 for ACT; PP552727, PP552728, and OR827443 for CAL; PP552729, PP552730, and OR827445 for GAPDH; PP549996, PP549999, and OR841394 for ITS). A phylogenetic tree was generated by the maximum likelihood method using the concatenated sequences of ACT, CAL, GADPH, and ITS by Polysuite software (Damm et al. 2020). Based on morphological and molecular analysis, the three isolates were characterized as C. gloeosporioides. The pathogenicity of the GNAF09 isolate was assessed on W. villosa var. villosa seedling leaves inoculated by spraying with 40 μL of conidial suspension at 106 conidia mL-1 or wounded with a sterile toothpick then inoculated with mycelial agar plugs (5 mm diameter). Control leaves were inoculated with 40 μL of sterile distilled water or agar plugs without mycelia. The inoculated plants were placed in a humid chamber at 28°C with 80% humidity and a 12 h light-dark photoperiod. Symptoms similar to those seen on naturally infected leaves were observed on all inoculated leaves after 7 days inoculation. Re-isolation was performed from 80% of the inoculated leaves and isolates were confirmed as C. gloeosporioides morphologically, confirming Koch's postulates, and by sequencing the ACT, CAL, GADPH, and ITS regions. The control groups remained asymptomatic. In previous studies, C. gloeosporioides has also caused anthracnose on Chinese medicinal plants, including Baishao (Radix paeoniae alba) (Zhang et al. 2017) and Rubia cordifolia L. (Tang et al. 2020). To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. villosa var. villosa in China. The results of our report serve as valuable references for further research on this disease.

Antimicrobial Resistance and ARGs Detection in Treated Final Effluent from STPs: An Upcoming Challenge to the Environment

Aim: The present study analysed the presence of antimicrobial resistance organisms and genes in the final effluent from STPs of hospitals. Study Area and Sampling: Samples for microbiological analysis were collected from two different hospitals in Trivandrum City and carried out further microbiological analysis. Methodology: In this study, MDRB (Multi Drug Resiatant Bacteria) were detected from the hospital effluent samples collected from two hospitals. Antibiotic susceptibility analysis showed that the 90% screened organisms was resistant to different antibiotics -Tetracycline (30µg), Amikacin (30µg), Gentamycin (10µg), Ciprofloxacin (5µg), Colistin (10µg) and Amoxicillin (30µg)). Metagenomic surveillance of effluent helped to assess the efficacy of STPs, at the same time assessing the local clinical antibiotic resistance condition by detection of the presence of antimicrobial resistance towards antibiotics and their genes (ARGs) in the hospital effluent. Results: In this study, a total of 3 antibiotic resistant bacterial strains obtained from hospital STP effluent were identified by16S rRNA sequence analysis. The sequences of E.coli, Klebsiella and Enterobacter were submitted in Genbank with accession number MT784125, OM978270 andMN437586 respectively.The final effluent from Hospital 1 showed100% resistance to Tetracycline and 86% resistance to Amoxicillin followed by sensitivity 28%, 22%, 18% and 10% respectively to Ciprofloxacin, Gentamycin, Amikacin and Colistin. The bacterial strains isolated from final effluent of Hospital 2 showed highest resistance to Amikacin and Colistin which is 100% and 86% and 82% resistance to Gentamycin and Ciprofloxacin. The gene primers used for the respective genes above have been amplified in the sample with a higher efficiency of 16 SrRNA, ermB and ampC primers showing a lower Cq value. Thus, these three genes were detected in the samples at high amount which showed the prominent use of the consecutive antibiotics in the clinical field

A unique Ca2+-inhibited C-type lectin in shrimp Fenneropenaeuschinensis

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight β-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates.

MaGENEgerZ: An Efficient Artificial Intelligence-Based Framework Can Extract More Expressed Genes and Biological Insights Underlying Breast Cancer Drug Response Mechanism

Understanding breast cancer drug response mechanisms can play a crucial role in improving treatment outcomes and survival rates. Existing bioinformatics-based approaches are far from perfect and do not adopt computational methods based on advanced artificial intelligence concepts. Therefore, we introduce a novel computational framework based on an efficient support vector machine (esvm) working as follows: First, we downloaded and processed three gene expression datasets related to breast cancer responding and non-responding to treatments from the gene expression omnibus (GEO) according to the following GEO accession numbers: GSE130787, GSE140494, and GSE196093. Our method esvm is formulated as a constrained optimization problem in its dual form as a function of λ. We recover the importance of each gene as a function of λ, y, and x. Then, we select p genes out of n, which are provided as input to enrichment analysis tools, Enrichr and Metascape. Compared to existing baseline methods, including deep learning, results demonstrate the superiority and efficiency of esvm, achieving high-performance results and having more expressed genes in well-established breast cancer cell lines, including MD-MB231, MCF7, and HS578T. Moreover, esvm is able to identify (1) various drugs, including clinically approved ones (e.g., tamoxifen and erlotinib); (2) seventy-four unique genes (including tumor suppression genes such as TP53 and BRCA1); and (3) thirty-six unique TFs (including SP1 and RELA). These results have been reported to be linked to breast cancer drug response mechanisms, progression, and metastasizing. Our method is available publicly on the maGENEgerZ web server.

Seven draft genome sequences of Streptococcus canis strains, revealing reduced penicillin-G susceptibility.

We report seven draft genome sequences of Streptococcus canis strains revealing reduced penicillin-G susceptibility. The genomes measured 2.054-2.385 Mbp, with G+C contents of 38.8%-39.6%. Amino acid substitutions in penicillin-binding proteins were characterized as compared with those of NCTC 12191(T) genome sequence (GenBank accession number NZ_LR134293.1).

First Report of Anthracnose Caused by Colletotrichum spaethianum on Zephyranthes candida in China.

Zephyranthes candida, an bulbous perennial plant, are planted in almost every park. In October 2023, anthracnose symptoms were observed on Z. candida leaves in Jiangxi Agricultural University (28.75° N, 115.83°E), Nanchang, Jiangxi Province, China, and the incidence of disease were up to 35% (140 of 400 plants). The lesions extended fromtheleafapextothebase, appearing as a dark brown color, and later changed to yellow and became dry. To isolate the pathogen, 20 symptomatic leaves were collected and cut into small pieces (4×4 mm, one pieces per leave), surface-sterilized with 70% ethanol for 10 s and 1% NaClO for 30 s, rinsed thrice with sterile water, placed onto potato dextrose agar (PDA) plates and incubated at 25℃ for 5 days. Fifteen isolates (15 out of 20) with similar morphological characteristics were obtained. The colonies on PDA presented effuse mycelium, initially white and later pale gray. Conidia were hyaline, curved or slightly curved, aseptate, with a truncate base and acute apex, measuring 17 to 23 × 3 to 6 μm (n = 50), and were matched to Colletotrichum species (Damm et al. 2009). To further confirm species, two representative isolates (JFRL 03-2873 and JFRL 03-2874) were selected for molecular identification. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS), histone 3 (HIS3), actin (ACT) and β-tubulin 2 (TUB2) regions were amplified and sequenced by using primers sets ITS5/ITS4, Gpd1/Gpd2, CHS-79F/CHS354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R and T1/Bt2b (Tan et al. 2022), respectively. These sequences were deposited into GenBank with accession number PP425890-PP425891 (ITS), PP437551-PP437552 (GAPDH), PP437549-PP437550 (CHS), PP480643-PP480644 (HIS3), PP437547-PP437548 (ACT) and PP437553-PP437554 (TUB2). A BLASTN search revealed high similarity of 99%-100% to ITS (GU227807, 518 nt/519 nt), GAPDH (GU228199, 525 nt/526 nt), CHS (GU228297, 251 nt/251 nt), HIS3 (GU228003, 372 nt/373 nt), ACT (GU227905, 236 nt/237 nt) and TUB2 (GU228101, 490 nt/490 nt) sequences of Colletotrichum spaethianum CBS 167.49. A maximum likelihood phylogenetic tree was constructed by combining ITS, GAPDH, CHS , HIS3, ACT and TUB2 sequences in IQtree web server (Ngugen et al. 2015). The result indicated that the two representative isolates were clustered together with Colletotrichum spaethianum in a clade with 100% bootstrap support. Based on morphologicalobservationandsequenceanalysis, the isolates were identified as C. spaethianum. To confirm pathogenicity, six surface-sterilized leaves of Z. candida were wounded and inoculated with 1 × 106 conidia/ml conidial suspension of JFRL 03-2873, and control leaves were inoculated with sterile water. They were incubated at 25 ℃ with 12 h photoperiod and 80% humidity, theexperimentwasrepeatedtwice. After five days, all leaves inoculated with JFRL 03-2873 displayed anthracnose symptom, whereas the control leaves remained unaffected. We re-isolated C. spaethianum from the symptomatic leaves and identified it based on morphological and molecular characteristics. Previous studies reported that C. spaethianum caused anthracnose on various common herbaceous plants in China (Vieira et al. 2014, Guo et al. 2013), but to our knowledge, this is the first report of C. spaethianum causing anthracnose on Z. candida in China. Anthracnose disease caused great economic loss to the cultivation of landscape plant Z. candida. Therefore, it is necessary to pay more attention to the anthracnose disease of herbaceous plants caused by C. spaethianum and develop appropriate control strategies.

Molecular Detection of Anaplasma phagocytophilum in Small Mammals and Infesting Ticks in Laikipia County, Kenya.

Anaplasmosis is a set of disease conditions of various mammals caused by bacteria species of the genus Anaplasma. These are sub-microscopic, Gram-negative, obligate intracellular pathogens that infect both vertebrate and invertebrate hosts. Significant species that infect domestic and wildlife animals include Anaplasma marginale, Anaplasma ovis, Anaplasma mesaeterum, Anaplasma platys, and Anaplasma phagocytophilum. Although A. phagocytophilum has a widespread distribution, there are only a few epidemiological reports from sub-Saharan Africa. This study focused on molecular detection and characterization of A. phagocytophilum in small mammals and their infesting ticks in Laikipia County, Kenya. A total of 385 blood and 84 tick archival samples from small mammals (155 females and 230 males) were analyzed. The blood samples were subjected to a nested PCR-HRM melt analysis using species-specific primers to amplify the 16S ribosomal RNA genes. The ticks were also subjected to nested PCR-HRM involving 16S rRNA gene primers. Anaplasma phagocytophilum DNA was detected in 19 out of 385 samples using species-specific 16S rRNA gene primers giving a prevalence of 4.9% for A. phagocytophilum. Analysis of the tick's samples using 16S rRNA gene species-specific primers also detected A. phagocytophilum in 3 samples from Haemaphysalis leachi ticks (3/84) equivalent to prevalence of 3.6%. Sequencing of 16S rRNA PCR products confirmed A. phagocytophilum in small mammals and ticks' samples. Phylogenetic analysis of the haplotype from this study demonstrated a close ancestral link with strains from Canis lupus familiaris, Alces alces, Apodemus agrarius, and ticks (Haemaphysalis longicornis) reported in Europe, China, and Africa. Comparison was also made with a known pathogenic A. phagocytophilum variant HA and a nonpathogenic variant 1 that were clustered into a distinctive clade different form haplotypes detected in this study. All the haplotype sequences for A. phagocytophilum from this study were submitted and registered in GenBank under the accession numbers OQ308965-OQ308976. Our study shows that small mammals and their associated ticks harbor A. phagocytophilum. The vector competence for H. leachi in A. phagocytophilum transmission should further be investigated.