Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Inducing Effect of Corylus avellana on Cytotoxic Activity in Lung and Breast Cancer Cells via Apoptosis

Turkish hazelnut (Corylus avellana L. cv Tombul) is a widely used nut in the chocolate industry and is also rich in polyphenol content, which promises anticancer effects. The anti-cancer and apoptotic effects of hazelnut leaves extracts examined on lung and breast cancer cells. Sulforhodamine B (SRB) and Adenosine 5’- triphosphate (ATP) assays were carried out for cell viability measurement. The mode of cell death was shown morphologically by the double fluorescence staining. Apoptosis was determined by performing caspase-mediated cytokeratin 18 (M30 ELISA) and western blot analysis. PARP, caspase 3, caspase 8, DR4, and GAPHD (Glyceraldehyde-3-phosphate Dehydrogenase) protein bands were visualized as markers of apoptosis. A wound healing test was employed to measure cell migration. Methanol extract of hazelnut leaf exhibited inhibition of cell growth activities in a dose-dependent manner. IC50 values were determined as 32.17 µg/ml in MCF-7, 32.16 µg/ml in MDA-MB-231, 20.40 µg/ml in A549 and 12.04 µg/ml in H1299 cells for ethanol extract while it was determined as 21.08 µg/ml in MCF-7, 40.16 µg/ml in MDA-MB-231, 22.04 µg/ml in A549 and 5.91 µg/ml in H1299 cells in methanol extract. In comparison, methanol leaf extracts were more effective in H1299 cells (IC50 value was 5.91 µg/ml).In comparison, ethanol leaf extracts were more effective in H1299 cells (IC50 value was 9.722 µg/ml). Western blot analysis demonstrated that hazelnut leaf extract treatment of cancer cells led to cell death via apoptosis and inhibited cell migration in lung and breast cancer cell lines. The cytotoxic effects of hazelnut extract on breast and lung cancer cells might be valuable and promising in elucidating cell death mechanisms for the development of new methods in cancer treatment.

Identification and validation of reference genes for gene expression study using Quantitative Real-Time PCR in shallot (Allium cepa L. Aggregatum group) based on bulb development phase

Abstract. Sulistyaningsih E, Sawitri WD, Handayani VDS, Wicaksono AW, Silalahi CM, Murti RH. 2024. Identification and validation of reference genes for gene expression study using Quantitative Real-Time PCR in shallot (Allium cepa L. Aggregatum group) based on bulb development phase. Biodiversitas 25: 2645-2651. Shallot (Allium cepa L. Aggregatum group) is cultivated vegetatively using bulbs (produce many bulbs) and generatively using TSS (True Seed of Shallot) which tends to produce single bulbs. Bulb aggregation is influenced by activity of genes and the gene expression could be analyzed using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The qRT-PCR analysis requires reference gene data (housekeeping gene) as a comparison gene against our target gene. This study aimed to select and validate three housekeeping genes in shallot as an initial study for qRT-PCR analysis. The research flow began with the preparation of shallot 'TukTuk' leaves and bulb samples in bulb formation from bulb initial phase to mature phase. Furthermore, a specific primer design based on actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences were obtained from gene bank data. The results showed that the actin, tubulin, and GAPDH genes were successfully amplified and the products were 142 bp, 116 bp, and 136 bp in size, respectively and the genes were expressed almost constantly in leaves and bulbs in all phases of bulb formation. The tubulin gene had the potential as a reference gene for qRT-PCR analysis in shallot with the most consistently expressed.

Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures (n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n = 133) of the isolates formed a cluster with a known A. brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

Five novel Curvularia species (Pleosporaceae, Pleosporales) isolated from fairy circles in the Namib desert

The Namib Desert (Namibia) is home to fairy circles which are barren, circular to almost-circular patches of land surrounded by grasses. During a survey of the fungi associated with the most common grass species, Stipagrostis ciliata (Poaceae), and its rhizospheric soils associated with these fairy circles, Curvularia was commonly isolated (80 strains). Curvularia is a cosmopolitan fungal genus that occurs in diverse geographical locations and on a wide range of substrates, but particularly on foliar plants. Curvularia strains were identified based on multilocus sequence comparisons of their internal transcribed spacer rDNA region (ITS), and the partial gene regions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1). The strains belonged to 13 species, including the discovery of five novel Curvularia species. The aim of this paper was to report on the identified species and to formally describe and name the new species as C. deserticola, C. gobabebensis, C. maraisii, C. namibensis, and C. stipagrostidicola.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation.

Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0 ), TATA-binding protein (TBP ), GAPDH , actin beta (ACTB ), tubulin (TUB ), aminolevulinic acid synthase 1 (ALAS1 ), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ ), eukaryotic translational elongation factor 1 alpha (EF1a ), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA ), and beta-2-microglobulin (B2M )] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms’ two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

A fungal-binding agglutinin in the skin slime of Japanese flounder (Paralichthys olivaceus) is glyceraldehyde 3-phosphate dehydrogenase.

Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast-binding protein, which consists of an approximately 35-kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time-of-flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An anti-GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well-known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts.

Selection of Stable Reference Genes for QRT-PCR in Tree Peony 'Doulv' and Functional Analysis of PsCUC3.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes-ACT (Actin), TUB (β-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)-were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

Distribution and Pathogenicity Differentiation of Physiological Races of Verticillium dahliae from Cotton Stems in Western China.

Verticillium wilt, caused by the pathogenic fungus Verticillium dahliae, has emerged as a severe threat to cotton globally. However, little is known about the genetic diversity of this pathogen in an infected single cotton plant. In this study, we isolated three new V. dahliae strains from the disease stems of Gossypium hirsutum from the cotton field in Western China and assessed their pathogenicity to the cotton cultivar Xinnongmian-1 and its two transgenic lines, as well as two laboratory strains, VD592 and VD991. These three new V. dahliae strains were identified using DNA barcodes of tryptophan synthase (TS), actin (ACT), elongation factor 1-α (EF), and glyceraldehyde-3-phosphate dehydrogenase (GPD). Moreover, the haplotype analysis revealed that the three new races had distinct haplotypes at the TS locus. Furthermore, the results of culture features and genetic diversity of ISSR (inter-simple sequence repeat) revealed that there were separate V. dahliae strains, which were strong defoliating pathotypes belonging to race 2 type, as determined by particular DNA marker recognition. The identified strains demonstrated varied levels of pathogenicity by leaf disc and entire plant inoculation methods. Conservatively, these strains showed some pathogenicity on cotton lines, but were less pathogenic than the reference strains. The findings revealed that several strong defoliating V. dahliae pathotypes coexist on the same cotton plant. It indicats the importance of regular monitoring as an early warning system, as well as the detection and reporting of virulent pathogen strains and their effects on crop response.

Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing.

Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3'-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.

First Report of Exserohilum rostratum Causing Brown Spot on Spartina alterniflora in China.

Spartina alterniflora Loisel, a perennial grass, has become an invasive species in China's coastal wetlands (Zhang et al. 2018). In July 2021, brown spot symptoms were observed on S. alterniflora in a coastal wetland (21°45'48″N, 108°44'00″E) in Beihai City, Guangxi Province, China. The disease affected approximately 50% of the plants in the surveyed area (0.2 ha) and was also observed in other regions of Beihai. It caused brown lesions with a gray or whitish center on the leaves and stems of S. alterniflora. As the disease developed, it ultimately led to leaf shedding and plant death. To isolate the causal agent, 18 fragments (~ 5 mm) from six symptomatic plants (3 leaf pieces per plant) were surface sterilized with 1% NaOCl solution for 2 min and rinsed three times with sterilized water. Subsequently, the tissues were placed on potato dextrose agar (PDA) medium supplemented with chloramphenicol (0.1 g/liter) and incubated at 28°C for three days. The hyphal tips were transferred onto fresh PDA to obtain pure cultures. A total of 25 isolates were obtained, 20 of which shared similar morphologies, while the remaining five exhibited distinct morphological characteristics and were non-pathogenic to S. alterniflora. Three isolates (MC16.1.3, MC16.6.2, and MC16.8.3) were randomly selected from the 20 for further investigation. The colonies on PDA were flat with dense aerial mycelia. The colony margins were entire, light brown in the centre, white to grey at the margin; reverse dark brown in the centre, gray at the margin. Conidia were straight to slightly curved, light olive-brown to dark olive-brown, septate, measured 33.5 to 79.1 μm × 10.4 to 18.7 μm (average 52.9 × 14.4 μm, n = 100), with a distinctly protruding hilum swelled from the basal cell. For molecular identification, the genomic DNA was extracted from mycelium on PDA using the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1 alpha (TEF1-α) genes were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1999), and EF1-983/EF1-2218 (Rehner et al. 2005), respectively. A BLAST analysis revealed that the ITS (OR516787-9), GAPDH (OR523686-8), and TEF-α (OR523683-5) had 99.1 to 99.7% identity with those of E. rostratum strains BRIP 11417 (LT837836, LT882553, and LT896656) and CBS 128061 (KT265240, LT715900, and LT896658) (Hernández-Restrepo et al. 2018). Based on the concatenated sequences, a phylogenetic tree generated by PhyloSuite software (Zhang et al., 2020) through Bayesian inference (BI) and Maximum Likelihood (ML) methods placed the isolates within E. rostratum. These morphological characteristics and molecular analyses confirmed the pathogen as E. rostratum (Hernández-Restrepo et al. 2018; Kaboré et al. 2022). To confirm pathogenicity, a conidial water suspension (~ 1 × 106 conidia/ml) of each of the three strains was inoculated on nine healthy S. alterniflora plants that had been grown for six months. Control plants were treated with sterile water. All plants were then enclosed in plastic bags and incubated in a greenhouse at 28°C. Six days after inoculation, the plants exhibited symptoms similar to those observed in nature. The control plants developed no symptoms. These experiments were replicated three times with similar results. To fulfill Koch's postulates, E. rostratum was consistently re-isolated from symptomatic tissue and confirmed by morphology and sequencing, whereas no fungus was isolated from the control plants. In recent years, S. alterniflora has posed a serious threat to the indigenous biodiversity of wetland ecosystems (Zhang et al. 2018). To our knowledge, this is the first report of E. rostratum causing brown spot on S. alterniflora worldwide.

First report of large-berry coffee (Coffea liberica) anthracnose caused by Colletotrichum kahawae in China.

Large-berry coffee (Coffea liberica) is one of the three cultivated coffee species and a precious breeding germplasm in China (Yan et al, 2019). Anthracnose is a damaging epidemic disease on coffee worldwide (Mohammed et al. 2015). Between June and September 2022, anthracnose was observed on coffee plants in Puer area, Yunnan, China and disease incidence (% plants diseased) of 8.5%-28.2% was recorded in the field. The disease symptoms were observed at all growth stages. Lesions on leaves were circular or oval, with a white to gray central zone outlined by a brown margin and surrounded by a chlorotic halo, Φ5.1-18.5 mm; some lesions extended and coalesced later to form large, blighted areas, leading to complete leaf senescence, defoliation and bare blighted branches on heavily infected trees. The spots on coffee berries were oval or fusiform, sunken and brown-black; diseased berries became gray-black and dried-out but remained on the tree. Leaves with typical anthracnose lesions were collected from fields in Simao ( 22.07°E,100.98°N) to isolate the pathogen. Leaf pieces (5×5mm) from the lesion margin were cut, surface-sterilized with 75% ethanol and 2% NaClO, and cultured on PDA at 25°C. Three isolates with the same colony morphology were obtained by hyphal tip purification. Detached and intact leaves of 6-month coffee seedlings were inoculated with Φ5mm mycelial discs of the isolates. Anthracnose lesions developed on the inoculated leaves, with all 3 isolates, 7d after incubation in a growth chamber (25°C, > 90% RH and lighting 8 h/d at 11000 lux). Pathogens with the same colony morphology as those of the original isolates were re-isolated from the infected tissues of inoculated leaves, thus fulfilling Koch's Postulates. The ITS sequence (PP550861) for the isolate was PCR-amplified and Blast-n analyses showed 100 % (554/554bp) identity to Colletotrichum kahawae LWTJ01; so they were the same population and coded as KFTJ02. The actin (ACT), calmodulin(CAL), glyceraldehydes-3-phosphate dehydrogenase (GAPHD) and histone 3 (HIS3) genes (Qiu et al. 2020) were amplified from one of KFTJ02 isolates, sequenced and deposited in NCBI GenBank (OR842543, OR842544, OR842545 & OR842546). A phylogenetic tree was generated based on the concatenated sequences of the four genes and those of related Colletotrichum spp. using MEGA 6.0 and KFTJ02 clustered in the same clade with C. kahawae IMI319418 on the tree (Bootstrap sup.=88%). When cultured at 25°C on PDA for 7 days, its colonies were near round or ovoid, gray-white, contoured, Φ73.2-80.1 (76.2±2.3)mm or growth rate 10.2-11.1(8.1) mm/d (n=10). The hyphae were hyaline, septated, branching at near right angles. Conidial masses formed 14 days after incubation. The conidia were elliptical, hyaline, monocellular, 10.2-15.5 (12.7±1.06)×3.8-5.2 (4.3±0.52) µm (n=50). The appressoria were black-brown, oval or irregular, 7.8-9.3 (8.5±0.81)μm (n= 50). These morphological characteristics were consistent with those of C. kahawae (Bridge et al, 2008). Therefore, KFTJ02 was identified as C. kahawae, which has been found to infect Camellia oleifera, Areca catechu and Ficus microcarpa (Wei et al, 2023; Zhang et al, 2020; Lin 2023). The coffee berry disease pathogen (C. kahawae) is a quarantine species which has not been recorded and so it is first reported on coffee crops in China. Results of the present study provide important references for further studies on this disease.

Network pharmacology study on fermented soybeans for the prevention of Alzheimer's disease in older individuals.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the disruption of synaptic communication among millions of neurons. Recent research has highlighted the potential therapeutic effectiveness of natural polyphenolic compounds in addressing AD. Soybeans are abundant in polyphenols, and their polyphenolic composition undergoes significant alteration through fermentation by Eurotium cristatum. Through comprehensive database searches, we identified active components within fermented soybean polyphenols and genes associated with AD. Subsequently, we utilized Venn diagrams to analyze the overlap between AD-related genes and these components. Furthermore, we visualized the network between intersecting targets and proteins using Cytoscape software. The anti-AD effects of soybeans were further explored through comprehensive analysis, including protein-protein interaction analysis, pathway enrichment analysis, and molecular docking studies. Our investigation unveiled 6-hydroxydaidzein as a major component of fermented soybean polyphenols, shedding light on its potential therapeutic significance in combating AD. The intersection between target proteins of fermented soybeans and disease-related targets in AD comprised 34 genes. Protein-protein interaction analysis highlighted key potential targets, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen synthase kinase 3 beta (GSK3B), amyloid precursor protein (APP), cyclin-dependent kinase 5 (CDK5), and beta-site APP cleaving enzyme 1 (BACE1). Molecular docking results demonstrated a robust binding effect between major components from fermented soybeans and the aforesaid key targets implicated in AD treatment. These findings suggest that fermented soybeans demonstrate a degree of efficacy and present promising prospects in the prevention of AD.

First Report of Leaf Spot Disease on Avocado Caused by Bipolaris setariae in China.

Avocado (Persea americana), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo (22.2019°N, 106.4723°E), Guangxi, China. Approximately 90% of avocado seedlings in the nursery were affected. Symptomatic plant fully expanded leaves showed small brown spots that ranged from 1 to 3 mm, with a yellow halo around (Fig.1). Lesions gradually expanded and became nearly round and dark brown. Finally, leaves withered or curled. For pathogen isolation, 15 symptomatic leaves were randomly sampled from different plants of the nursery, five leaves were selected and four samples size 4×4mm were taken from each leaf and were plated on potato glucose agar. Identical fungus colonies were observed in 80% of the samples, and no bacteria were isolated. Single conidial isolation was performed. After 4 days, the colony diameter reached 74.6 mm, colonies appeared gray, and developed aerial hyphae. Conidiophores were mostly solitary with a few clustered erect or slightly curved, knee shaped, and 3.89 to 5.24 µm wide. Conidia were 39.33 -96.88 × 9.96 - 15.59 µm, slightly curved, rarely straight, light brown to yellowish brown, fusoid or navicular, and truncated at the base with 4 to 10 septa. Based on morphological and cultural characteristics, the fungus was identified as Bipolaris sp. (Manamgoda et al. 2014). An isolate named MP211122 was grown on Sachs' ager at 27℃ under 12-h light/dark for 1 week and consistently with Adhikari et al. (2021) no sexual from was observed. To confirm the tentative identification, genomic DNA was extracted, ITS and GAPDH gene were amplified and sequenced using primers ITS1/ITS4 and GPD/GPD2, respectively (Tan et al. 2022). The ITS sequence (GenBank ON248469) shared 100% identity with B. setariae (MN215632.1), and the GAPDH sequence (ON642344) shared 99.82% identity with B. setariae (MF490833.1, MK144540.1) and B. yamadae (MK026428.1). A maximum likelihood phylogenetic analysis based on GAPDH and ITS sequences using MEGA 7.0 revealed that the isolate clustered with B. setariae with 100% bootstrap support(Fig. 2). Healthy 11-month old potted avocado seedlings from disease-free nursery were selected , the conidial suspension (1 × 105 conidia/mL) of MP211122 isolate was prepared by harvesting conidia from a 10-day-old culture on water agar. Conidia were sprayed onto young leaves of six potted plants. Three additional seedlings sprayed with sterile distilled water served as controls. All plants were covered with plastic bags for 3 days to maintain high humidity and then maintained in a greenhouse at 30℃ with a 12-h/12-h light/dark cycle. After 5 days, typical symptoms of small brown spots were observed on all inoculated leaves (Fig.3). All leaves on control plants remained asymptomatic. The reisolated fungus was morphologically identical to the original isolate used for inoculation, fulfilling Koch's postulates. This is the first report of B. setariae as a pathogen causing leaf spot on avocado in China. This information will facilitate further studies, monitoring and control of the disease as accurate identification of the causal agent is a primary requisite for designing management strategies.

Phloroglucinol ameliorated methylglyoxal induced harmful effects in rats.

Glycation is among the underlying mechanisms attributed to ageing and associated morbidities. There is no drug available to combat this deleterious phenomenon. The present study aimed to explore phloroglucinol (PHL) for its anti-glycation potential at preclinical level. The rats were treated with methylglyoxal (MGO, 17.25 mg/kg, i.p. for 14 days) to induce glvcative stress. The treatment groups received additional administration of test drug (PHL; 0.25mg/kg, 0.5mg/kg, and 1mg/kg) or standard aminoguanidine (AG, 50 mg/kg) or saline (control, 5ml/kg). During 14 days, the weight and food intake was noted. Afterwards, the cognitive function was evaluated using Morris Water Maze (MWM) while hepatic and renal functions were assessed through liver function test (bilirubin, alkaline phosphatase, SGPT, and SGOT) and creatinine respectively, using chemical analyzer. The carboxymethyllysine (CML) levels were quantified in the blood using ELISA technique. Histopathological study was performed on the brain, liver, and kidney using H&E staining. Additionally, the qPCR was used to quantify the expression of TNF-α, RAGE and BACE-1 (brain), RAGE, TNF-α, and glyoxalase-I (liver) and RAGE, TNF-α, and VEGF (kidney), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference housekeeping gene. The data regarding weight and food intake did not reveal significant alterations. In MWM, the MGO treatment caused significant increase in the time to reach target quadrant, while decrease in the time spent in target quadrant and number of crossings through platform position. All these effects were inhibited by both AG and PHL. The navigation maps also exhibit that the retention of spatial memory. Additionally, the MGO-induced alteration in hepatic and renal function indicators was ameliorated by both AG and PHL treatments. The plasma CML levels were found to be elevated following MGO treatment, while the concomitant administration of AG and PHL has resisted this raise. Histopathological assessment revealed no specific pathology in liver kidney and brain tissues. The qPCR data revealed enhanced expression of all genes, especially TNF-α and BACE, which were found to be reduced following both AG and PHL treatments. PHL prevented the brain, hepatic, and renal impairments caused by MGO induced glycative stress. Hence, the PHL, a clinically used anti-spasmodic drug, presents itself as a potential candidate to be repurposed as anti-glycation drug.

Stilbenoids from fenugreek seeds alleviate insulin resistance by regulating the PI3K/AKT/mTOR signaling pathway in a type 2 diabetes zebrafish model

Insulin resistance (IR) is the main cause of type 2 diabetes mellitus (T2DM). The specific targets and underlying mechanisms responsible for the ameliorative effects of the stilbenoid compounds found in fenugreek seeds for ameliorating IR require further study. Here, we were predicted by using the network pharmacology prediction, molecular docking and molecular dynamics simulation approach the targets in common and the potential mechanismsof three stilbenoid compounds (rhaponticin, desoxyrhaponticin, and rhapontigenin) in relation to T2DM and IR. The results showed that the compounds may improve IR through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Molecular docking studies revealed that they exhibit high binding affinity with the structural domains of peroxisome proliferator-activated receptor gamma (PPARG), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PI3K, and AKT. These results suggest that PPARG and GAPDH may be the potential targets for these three compounds in the treatment of T2DM.Subsequently, experiments using the zebrafish T2DM model showed that the stilbenoid compounds had varying degrees of efficacy in improving IR through the PI3K/AKT/mTOR signaling pathway, and rhaponticin had the most promising effects. The findings implicate a potential mechanism of action for the three stilbenoid compounds in enhancing insulin resistance (IR) through modulation of the PI3K/AKT/mTOR pathway.

Anthracnose disease of Ficus benjamina caused by Colletotrichum orchidearum in Iran

Colletotrichum species cause the anthracnose disease that affects a wide variety of fruit, vegetable, and ornamental hosts. During the summer of 2018–2019, infected leaves of Ficus benjamina var. variegata with typical anthracnose symptoms were collected from several greenhouses in Miyandoab (West Azarbaijan Province) and Maraghe (East Azarbaijan Province) counties, Iran, with an incidence of 10–21%. The fungus was isolated and identified as Colletotrichum orchidearum, based on morphological features and multi-gene sequence analyses (ITS‒rDNA, GAPDH, CHS-1, HIS3, ACT and TUB2 genes). Pathogenicity assays were conducted on two-year-old potted plants with conidial suspensions under greenhouse conditions. Typical anthracnose symptoms including fungal conidiomata were developed on the inoculated leaves, while control plants remained symptomless. Re-isolation and re-identification of the inoculated fungi confirmed Koch’s postulates. This study provides the first evidence of C. orchidearum causing anthracnose disease on Ficus benjamina var. variegata in Iran and the world.

First Report of Alternaria alternata Causing Leaf Spot on Catalpa bungei in China.

Catalpa bungei originates from China. It is fast-growing and possesses a vertically aligned trunk, rendering it a commendable construction material and a significant economic species. In July 2022, a serious leaf spot occurred in the LanLake farm (surveyed area of about 700 acres) in Nanyang (33°3'23" N, 112°28'50" E), Henan Province, China. The incidence rate of leaf disease reached 54% (n=100). The disease initially manifested as irregular round spots with a yellowish-brown hue, subsequently extending in all directions. Later, the lesion periphery exhibited a darkening effect, leading to yellowing. Twenty diseased leaves were randomly collected and cut into small pieces at the interfaces between infected and healthy tissues. The tissues were sterilized in a solution of 75% ethanol and 1% NaClO for 30 seconds and 1 minute, respectively. After rinsing in sterile water, the pieces were placed on potato dextrose agar (PDA) plates and incubated at 25°C for 5 days. A total of 29 purified fungal strains were acquired, exhibiting comparable phenotypes in terms of morphological characteristics. Three strains (QS1-1, QS1-2, and QS1-3) were isolated for subsequent investigations. The colony exhibited abundant aerial mycelium with shades ranging from dark green to grey-brown on the reverse side. To analyze the morphological characteristics of conidia, potato carrot agar (PCA) was used as the culture medium and incubated at 25°C with a 12-hour light/dark cycle. Conidia were obclavate or spheroidal, dark brown, with 3 to 5 transverse septa, and 1 to 4 longitudinal septa, measuring 12.4 to 36.7 × 4.4 to 9.0 μm (n=100), with conical beak lengths ranging from 0 to 4.3 μm. These morphological traits suggested that the pathogen shares similarities with the Alternaria species. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (tef1), glyceraldehyde 3-phosphate dehydrogenase gene (gapdh), and RNA polymerase II second largest subunit (rpb2) were amplified for further molecular identification. The resultant sequences were submitted to GenBank with the following accession numbers: OR733559, OR742124, OR761873 (ITS), OR939796, OR939797, OR939798 (tef1), OR939801, OR939802, OR939803 (gapdh), and PP054846, PP054847, PP054848 (rpb2). A Phylogenetic tree was constructed of combined genes (ITS, tef1, gapdh, and rpb2) of sequences, alongside the sequences of the type strains by the neighbor-joining method. The three strains formed a clade with the strains CBS 121456 of Alternaria alternata in phylogenetic trees, being separated from other Alternaria spp. The morphological features and molecular analyses supported the strains as members of Alternaria alternata (Woudenberg et al. 2015). To validate pathogenicity, a conidial suspension (106 conidia ml-1) of all three strains was inoculated onto three healthy leaves of five seedlings, with 50 μl of inoculum absorbed with cotton balls. Another group of five plants received sterile water as a control. All plants were incubated in a climate chamber at 28°C and 90% relative humidity. Four days post-inoculation, lesions resembling natural phenomena were observed, whereas control plants showed no symptoms. Subsequent reisolation produced cultures that were morphologically and molecularly identical to the original strains, fulfilling Koch's postulates. Stem canker of C. bungei caused by Phytophthora nicotianae has been reported in China (Chang et al. 2022). This is the first report of A. alternata causing leaf spots on C. bungei in China. Further research is required on management options to control this disease and the host range still needs to be clarified for accurate disease management.

First Report of Anthracnose on Banana Peppers Caused By Colletotrichum scovillei in New York.

In early August 2023, a disease outbreak on hot banana peppers (Capsicum annuum cv. Golden Dagger) was reported in Cattaraugus County, New York (NY). Disease incidence was at least 60%. Affected developing and mature fruit had at least one tan, soft, sunken lesion with salmon-colored spore masses surrounded by brown, necrotic margins. Microscopic observation of the lesions identified acervuli and setae typical of Colletotrichum spp. Isolations were made from these lesions by spreading conidia from the acervuli on 2% water agar (WA) + 0.02% (w/v) ampicillin. Colonies were hyphal tipped and transferred onto clarified V8 juice agar (CV8) and incubated at 20°C. The isolation frequency was 100% and a total of six isolates were obtained: Coll23Pep001, Coll23Pep003, Coll23Pep005, Coll23Pep007, Coll23Pep008, and Coll23Pep010. After 10 days, colonies were subcultured to potato dextrose agar (PDA) and CV8. On PDA, colonies appeared off-white to dark gray with sparse aerial mycelia. On CV8, the colony was pale gray with acervuli and orange-colored spore masses in the center. Conidia were hyaline, smooth and fusiform to round, and tapered at both ends. Mean conidial dimensions (n = 20) were 20.2 (13.75 to 25) µm long × 4.7 (3 to 6.25) µm wide. To confirm the identity of the isolates, DNA was extracted, and PCR performed to amplify the internal transcriber spacer (ITS) region (primers ITS1/ITS4; White et al. 1990), and actin (ACT) (primers ACT-512F/ACT-783R; Carbone and Kohn 1999) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (primers GDF1/GDR1; Guerber et al. 2003). Pairwise alignment of the sequences showed all isolates had 100% similarity to C. scovillei ex. holotype CBS 126529 (Damm et al. 2012). Sequences from all isolates were deposited in GenBank with accessions PP556967 to PP556972 (ITS), PP565766 to PP565771 (ACT), and PP565772 to PP565777 (GAPDH). For pathogenicity testing, all isolates were grown on CV8 at 20°C in the dark for 10 days. Conidia were harvested by flooding the plates of each isolate with sterile distilled water and filtering the suspension through a double layer of cheesecloth. The concentration of the conidial suspension was adjusted to 5 × 105 spores per ml. Pathogenicity of the six isolates was tested on banana pepper fruit by using a sterile toothpick to pierce the skin at the two opposite ends. A droplet (10 µl) of the conidial suspension was placed on each wound. The same number of fruit were inoculated without wounding, and non-inoculated control fruit received a droplet of sterile distilled water (either wounded or unwounded). The experiment was repeated twice. All fruit were placed in a humid box at room temperature for 7 days. All wounded and inoculated fruit developed sunken lesions filled with salmon-colored conidial masses. Disease did not occur on the unwounded, inoculated fruit nor the non-inoculated controls. C. scovillei was recovered from all inoculated fruit by reisolating onto CV8 media and isolates had similar morphology and conidial dimensions to the original isolates. To the best of our knowledge, this is the first report of C. scovillei causing anthracnose on pepper in NY. C. scovillei has been reported in South Carolina (Toporek and Keinath 2021), Brazil (Caires et al. 2014), eastern Asia (de Silva et al. 2019), and Kosovo (Xhemali et al. 2023). The pathogen is particularly aggressive on pepper and poses substantial threats to pepper production around the world.