Dehydrogenase Gene Research Articles

Ocurrence of Colletotrichum siamense Causing Leaf Spots on Soursop (Annona muricata) in Mexico.

Soursop (Annona muricata L: Annonaceae) is a small tropical fruit tree native to South America (Pinto, 2005). The flesh of its fruits is widely used as a main ingredient of pastries, even young fruits are used as a vegetable. In June 2022, leaf spots symptoms were observed on fifty soursop plants in a commercial nursery located in Juan José Ríos (25°45'20"N 108°50'21"W), Ahome, Sinaloa State. The incidence of the disease was 75%, while the severity was 12%. Symptoms were round, small black necrotic spots, that grew up to 6 mm in diameter with brown or gray color at the center. Fungal isolation was done on potato dextrose agar (PDA) and Colletotrichum-like colonies were obtained. Five isolates were recovered and purified by single spore culture and only a single morphotype was observed. One random isolate was selected for pathogenicity tests, morphological and molecular characterization. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Biotic Products Development Center at the National Polytechnic Institute under accession no. IPN 13.0102. Colonies in PDA at 25°C grow at a rate of 9.0-14.0 mm/d. After 14 days, the colony was olive to gray with orange conidial masses, and conidia (n =100) were hyaline, aseptate, cylindrical, and straight with rounded ends, measuring 11.5 to 18.5 and 3.5 to 5.5 μm. Appressoria were melanized and circular or oval in shape, measuring 6.0 to 4.0 μm (n=20). According to the morphological characteristics observed, the isolate was placed tentatively within the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular confirmation, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), partial sequences of actin (ACT) (Weir et al. 2012) and span style="font-family:'Times New Roman'">glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced. Sequences were deposited in GenBank under the accession numbers: ITS, OQ606966; ACT, OQ617292 and GAPDH, OQ617293. A phylogenetic tree including published sequences of the C. gloeosporiodes species complex was constructed according to Talhinhas and Baroncelli (2021) and the isolate IPN 13.0102 was grouped in a clade with the ex-type culture of C. siamense (ICMP18578) and C. pandanicola. However, C. pandanicola was recorded only as an epiphytic fungus occurring on leaves of Pandanus sp. (Pandanaceae) (Tibpromma et al. 2018) and there are no additional reports of this fungus as a plant pathogen on Pandanus or any other plant. Therefore, the isolate IPN 13.0102 corresponds to C. siamense. Pathogenicity was demonstrated by spraying a conidial suspension (1 × 105 conidia/ml) onto four healthy soursop plants, while two control plants were sprayed using sterile distilled water. All plants were kept in a wet chamber for 48 h at 28  2°C and 85% RH. The characteristic symptoms of the disease were observed 14 days after inoculation, while control plants remained healthy. The pathogenicity test was repeated twice obtaining the same results. The morphology of the recovered fungus was consistently identical to that originally isolated from diseased leaves, fulfilling Koch's postulates. Colletotrichum siamense has been previously reported on Anona spp. in Brazil (Costa et al. 2019). To our knowledge, this is the first report of Colletotrichum siamense causing leaf spots on Annona muricata in Mexico. Further studies for monitoring and control strategies of leaf spots on soursop are required.

Absence of Giardia spp. in fecal samples from capybaras (Hydrochoerus hydrochaeris) inhabiting urban areas in the state of Mato Grosso do Sul, Brazil.

Giardiasis is a major cause of diarrhea in humans and animals worldwide. Currently, there are nine species of Giardia, including Giardia duodenalis, which infects most vertebrates. The capybara (Hydrochoerus hydrochaeris) is the largest herbivorous rodent in the world. Although capybaras are hosts of several parasites of public health importance, including helminths and protozoa, there is a paucity of research on their zoonotic potential. We investigated the prevalence of Giardia spp. in populations of capybaras living in urban areas. Fecal samples from 247 capybaras were collected in Lagoa Maior, located in the municipality of Três Lagoas, and in Lago do Amor and Parque das Nações Indígenas, both located in the municipality of Campo Grande, state of Mato Grosso do Sul, Brazil. Fecal samples from capybaras originated from 133 adults (54%), 61 cubs (25%), and 53 juveniles (21%); 183 samples were collected in the rainy season and 64 in the dry season. Giardia spp. DNA was screened by the small-subunit ribosomal RNA (SSU rRNA) targeted PCR. Samples with DNA band sizes suggestive of Giardia spp. amplicons were examined by PCR targeting the glutamate dehydrogenase (GDH) and triosephosphate isomerase (TPI) genes. PCR amplicons were subjected to genetic sequencing. Nested PCR screening of the SSU rRNA gene revealed 16 samples showing faint DNA bands in gel electrophoresis with sizes similar to Giardia spp. amplicons. PCR amplicons of the SSU rRNA gene were analyzed by Sanger sequencing. Most of the sequencing reactions failed, and the chromatogram reads of some samples were ambiguous, suggesting nonspecific amplification. Therefore, all the capybara fecal samples were considered negative for Giardia spp. Two published studies on Giardia spp. in capybaras reported findings similar to ours, i.e., the absence or a low positivity rate for Giardia spp. However, further studies are needed to determine the possible role of capybaras in the epidemiology of giardiasis.

First Report of Colletotrichum siamense Causing Anthracnose on Dioscorea alata in China.

Dioscorea alata is an annual or perennial dicotyledonous plant which is a vegetatively propagated tuberous food crop (Mondo et al. 2021). In 2021, symptoms of leaf anthracnose occurred on D. alata plants at a plantation in Changsha, the Hunan Province of China (28°18' N; 113°08'E). Symptoms initially showed as small, brown water-soaked spots on the leaf surface or margins, and enlarged to irregular, dark brown or black, necrotic lesions, with a lighter center and darker edge. At latter, lesions extended to most of the leaf surface causing leaf scorch or wilting. Almost 40% of the plants surveyed were infected. Symptomatic leaf samples were collected, and small pieces were taken at their disease-healthy junction, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed three times with sterile distilled water, and then placed on potato dextrose agar (PDA) for incubation at 26 °C for 5 days in the dark. Fungal colonies with similar morphology were observed and, in total, 10 isolates were obtained from 10 plants. On PDA, colonies were initially white with fluffy hyphae, and later became light to dark gray, showing faint concentric rings. Conidia were hyaline, aseptate, cylindrical, and rounded at both ends, measuring 11.36 to 17.67 × 3.45 to 5.9 μm (n = 50). Appressoria were dark brown, ovate, globose, measuring 6.37 to 7.55 × 10.11 to 12.3 µm. These morphological characteristics were typical of Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of a representative isolate Cs-8-5-1 were amplified and sequenced using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF/GDR as described previously (Weir et al. 2012). These sequences were deposited in GenBank (accession nos. OM439575 for ITS, OM459820 for ACT, OM459821 for CHS-1, and OM459822 for GAPDH). BLASTn analysis showed 99.59 to 100 % identity to the corresponding sequences of C. siamense strains. A Maximum likelihood phylogenetic tree based on the concatenated ITS, ACT, CHS-1 and GAPDH sequences was generated by MEGA 6. It revealed that the Cs-8-5-1 was clustered with the C. siamense strain CBS 132456 with 98% bootstrap support. For pathogenicity test, conidia suspension (105 spores/ml) was prepared by harvesting conidia from 7-day-old cultures growing on PDA, and 10 uL was dropped onto leaves of potted D. alata plants (8 droplets per leaf). Leaves treated with sterile water were served as controls. All the inoculated plants were placed in humid chambers (with 90% humidity) at 26°C with a photoperiod of 12 h. The pathogenicity tests were performed twice, with each had three replicated plants. Seven days after inoculation, the inoculated leaves showed symptoms of brown necrosis resembling that observed in fields, however, the control leaves remained symptomless. The fungus was specifically re-isolated and identified by morphological and molecular methods, thus fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing anthracnose on D. alata in China. Since this disease might seriously affect the photosynthesis of the plants, which can influence the yield, prevention and management strategies should be adopted to control this new disease. Identification of this pathogen will provide a foundation for the diagnosis and control of this disease.

Hepatocyte-secreted FAM3D ameliorates hepatic steatosis by activating FPR1-hnRNP U-GR-SCAD pathway to enhance lipid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide; however, the underlying mechanisms remain poorly understood. FAM3D is a member of the FAM3 family; however, its role in hepatic glycolipid metabolism remains unknown. Serum FAM3D levels are positively correlated with fasting blood glucose levels in patients with diabetes. Hepatocytes express and secrete FAM3D, and its expression is increased in steatotic human and mouse livers. Hepatic FAM3D overexpression ameliorated hyperglycemia and steatosis in obese mice, whereas FAM3D-deficient mice exhibited exaggerated hyperglycemia and steatosis after high-fat diet (HFD)-feeding. In cultured hepatocytes, FAM3D overexpression or recombinant FAM3D protein (rFAM3D) treatment reduced gluconeogenesis and lipid deposition, which were blocked by anti-FAM3D antibodies or inhibition of its receptor, formyl peptide receptor 1 (FPR1). FPR1 overexpression suppressed gluconeogenesis and reduced lipid deposition in wild hepatocytes but not in FAM3D-deficient hepatocytes. The addition of rFAM3D restored FPR1's inhibitory effects on gluconeogenesis and lipid deposition in FAM3D-deficient hepatocytes. Hepatic FPR1 overexpression ameliorated hyperglycemia and steatosis in obese mice. RNA sequencing and DNA pull-down revealed that the FAM3D-FPR1 axis upregulated the expression of heterogeneous nuclear ribonucleoprotein U (hnRNP U), which recruits the glucocorticoid receptor (GR) to the promoter region of the short-chain acyl-CoA dehydrogenase (SCAD) gene, promoting its transcription to enhance lipid oxidation. Moreover, FAM3D-FPR1 axis also activates calmodulin-Akt pathway to suppress gluconeogenesis in hepatocytes. In conclusion, hepatocyte-secreted FAM3D activated the FPR1-hnRNP U-GR-SCAD pathway to enhance lipid oxidation in hepatocytes. Under obesity conditions, increased hepatic FAM3D expression is a compensatory mechanism against dysregulated glucose and lipid metabolism.

Genetic Polymorphism Study of IDH 1/2 and TET2 Genes in Acute Myeloid leukemia Patients.

Isocitrate dehydrogenase genes (IDH1 and IDH2) encode important enzymes that play pivotal role in cellular metabolism. Mutations in TET2 have been demonstrated to contribute to DNA hypermethylation, either expression of mutant IDH1/2 or TET2 resulted in poor cell differentiation and epigenetic alterations in hematopoietic cells, suggesting a sharing of the oncogenetic impact. In this study, we investigated the frequency of genetic alterations in IDH1/2 and TET2 genes in Egyptian cohort of adult patients with de novo AML, and the association of IDH1/2 and TET2 genetic Polymorphism with AML prognostic criteria and explore prognostic molecular markers with clinical outcome. The SNP assay for IDH1, IDH2 and TET2 genes polymorphism tested with RT-PCR included three polymorphisms that are rs121913500, rs121913503, and rs2454206 respectively, were tested on 141 adult Egyptian patients fulfilling the AML diagnostic criteria. The incidence of IDH mutations is 11/141 (7.8%); 5/141 (3.5%) IDH1 mutant and 6/141 (4.3%) IDH2 mutant. And the incidence of TET2 mutations is 72/141 (51.1%); 15/141 (10.7%) homozygous mutation and 57/141 (40.4%) heterozygous mutations. IDH1, IDH2 and TET2 genes mutations with DFS and OS in AML patients were not significantly correlated. TET2 SNP is common in Egyptian AML patients. Further research on IDH, TET2 and their relationships to other hematological malignancies and leukemogenesis transformation is advised and a study of a larger number of cases is needed for potential statistical significance.

Research of descendants of transgenic plants Triticum aestivum L. with partial suppression of the prolinde hydrogenase gene

Aim. To investigate the level of tolerance to water deficit of seed generations (T1 and T4) of genetically modified winter wheat with partially suppressed expression of the proline dehydrogenase gene (ProDH) based on the analysis of physiological and biochemical indicators and economic characteristics of plants. Methods. Determination of indicators of crop structure and content of free L-proline (Pro). Results. The level of Pro was studied and the main elements of productivity in the offspring of transgenic plants and their original forms under normal and insufficient water supply were analyzed. Conclusions. T1 and T4 biotechnological plants under normal/stress conditions accumulated more Pro than the original genotypes. The level of this amino acid in genetically modified seedlings under optimal cultivation conditions exceeded the initial forms by an average of 1.8 times. Under the influence of water deficit, its content increased by 2.2 and 2.3 times, in relation to normal water supply. In non-transgenic variants, the level of Pro, under similar growing conditions, was lower by 1.9 and 2.0 times, compared to T1 and T4 plants. Under the optimal water regime, the analyzed wheat variants did not differ significantly in terms of productivity. Drought led to their decrease, but the offspring of biotechnological plants were characterized by a less pronounced difference, as well as a higher yield.

Obtaining genetically modified winter wheat plants with partial suppression of the prolin dehydrogenase gene

Aim. Production of genetically modified plants of new promising winter wheat genotypes with partial suppression of the proline dehydrogenase gene in in vitro culture and determination of proline content in transgenic and control plants. Methods. Agrobacterium-mediated transformation in culture in vitro, molecular genetic analysis; biochemical determination of proline content; of mathematical statistics. Results. Using the method of Agrobacterium-mediated transformation of callus cultures of new promising genotypes of winter wheat, transgenic plants carrying a double-stranded RNA suppressor of the proline dehydrogenase gene were obtained. The frequency of transformation with the use of the LBA 4404 strain in the studied genotypes was 1.7-2.0 %, and with the use of the AGL0 strain, it was 2.0-2.3 %. It was established that plants with reduced activity of proline dehydrogenase are characterized by a significantly higher content of free L-proline compared to the control. Conclusions. The relatively greater efficiency of using the AGL0 strain for obtaining transgenic plants of winter wheat genotypes with partial suppression of the proline dehydrogenase gene in in vitro culture is shown. The presence of a double-stranded RNA suppressor of the ProDH gene in transgenic plants leads to an increase in the level of free L-proline accumulation.

The effect of free proline accumulation on the content of photosynthetic pigments in transgenic wheat plants

Aim. To analyze the effect of L-proline accumulation on chlorophyll content in transgenic wheat plants with a double-stranded RNA suppressor of the proline dehydrogenase gene under physiological and stress conditions. Methods. Agrobacterium-mediated transformation in planta; biochemical determination of free proline content; spectrophotometric determination of photosynthetic pigments content; of mathematical statistics. Results. It was shown that the content of free proline in the leaves of transgenic plants under physiological conditions was 1.7-1.9 times higher compared to the original genotype. Under conditions of drought, the content of this aminoacid increased in non-transformed plants of the original genotypes by 2.9-3.1 times, and in transgenic plants – by 4.5-4.9 times. The amount of chlorophyll in the flag leaves of plants of the original genotypes and their transgenic lines under physiological conditions did not differ significantly, while under drought conditions in the first of them it was 1.1-1.2 times less than in the second. Drought reduced the total chlorophyll content in plants of the original genotype to 85-90 %, compared to physiological conditions, while no significant changes were found in transgenic plants. Conclusions. It was established that under conditions of soil drought, the increase in proline content in genetically modified wheat plants compared to non-transgenic ones is accompanied by an increase in the amount of total chlorophyll (by 10-15 %), which indicates a better efficiency of their pigment apparatus under stressful conditions.

Physiological and biochemical characteristics of genetically modified winter wheat

Aim. Analysis of physiological and biochemical characteristics of genetically modified wheat with partially suppressed activity of the proline dehydrogenase gene. Methods. Determination of proline dehydrogenase (PDH) activity, free L-proline (Pro); carbohydrate content (sucrose and fructose). Results. It was established that under conditions of water deficit in genetically modified wheat plants with integrated elements forming the double-stranded RNA suppressor of the pdh gene, there is a partial inhibition of the enzyme activity and an increase in the content of free proline. Changes in carbohydrate metabolism under water deficit stress and in the first hours after rehydration were noted, while the sucrose / fructose ratio in control plants significantly decreases during dehydration and normalizes when water supply is restored, in genetically modified plants this indicator almost does not change under short-term water deficit stress. Conclusions. Partial suppression of the proline dehydrogenase gene leads to an increase in the free proline content, the fluctuations of which contribute to the maintenance of carbohydrate balance.

An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Colletotrichum siamense, a Novel Causal Agent of Viburnum odoratissimum Leaf Blotch and Its Sensitivity to Fungicides.

Viburnum odoratissimum Ker-Gawl is native to Asia and is usually used as a garden ornamental. In September 2022, a leaf blotch on V. odoratissimum was observed in Nanjing, Jiangsu, China. The disease causes the leaves of the plants to curl and dry up and defoliate early. It not only seriously affects the growth of the plants but also greatly reduces the ornamental value. The pathogenic fungus was isolated from the diseased leaves, and the fungus was identified to be Colletotrichum siamense based on morphological features and multilocus phylogenetic analyses of the internal transcribed spacer (ITS) region, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1), Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. Pathogenicity tests were performed by inoculating healthy leaves with conidia. C. siamense can grow at 15-35 °C, with an optimal growth temperature at 25-30 °C. The results of sensitivity to nine fungicides showed that C. siamense was the most sensitive to prochloraz in the concentration range of 0.01 μg/mL to 100 μg/mL. Therefore, spraying prochloraz before the optimum growth temperature of pathogenic fungus can achieve effective control. It provided useful information for future studies on the prevention and treatment strategies of C. siamense. This is the first report of leaf blotch caused by C. siamense on V. odoratissimum in China and worldwide.

Neu Laxova Syndrome—A Terrifying Disease in Two Siblings With a Novel Variant in PHGDH Gene

Introduction Neu-Laxova Syndrome (NLS) is a rare, autosomal, and recessively inherited disease characterized by severe congenital malformations, leading to prenatal or early postnatal mortality. The hallmark clinical features of this syndrome are severe intrauterine growth restriction, central nervous system abnormalities, restrictive dermopathy, and characteristic facial dysmorphia. There is no cure or treatment for this syndrome and the termination of the pregnancy is the suggested approach. Case description Here, we report a new case of 2 siblings with NLS, diagnosed in utero by ultrasonography and genetic examination. Two variants in heterozygosity in the phosphoglycerate dehydrogenase gene were discovered, including a previously uncharacterized variant (c.487C>T p. [Arg163Trp]). Comments Despite all the advances in prenatal diagnosis, NLS remains undiagnosed in many pregnancies. Early and sequential US examinations should be performed in high-risk pregnant women, to establish an early diagnosis.

The Effects of Vitamin D3 Supplementation and Simultaneous Exercise on Aldehyde Dehydrogenase Gene Expression in Endometriosis Female Rats

Background: Endometriosis is an injury caused by the proliferation of endometrial tissue outside the uterine cavity. Exercise and supplements may effectively remove waste materials and produce antioxidant enzymes. Objectives: The present research aimed to investigate the effects of simultaneous exercise and vitamin D3 supplement on aldehyde dehydrogenase 2 (ALDH2A1) gene expression in endometriosis female rats. Methods: The experimental method with a post-test design was used to conduct the research. The statistical population of the study consisted of three-month-old female Wistar rats. After the induction of the endometriosis model, the rats were divided into six groups. The exercise groups performed simultaneous exercises for eight weeks. Vitamin D3 supplement at 50 mg was fed to rats daily for eight weeks. The mean and standard deviation of ALDH2A values were calculated, and two-way analysis of variance (ANOVA) and Bonferroni post hoc test were investigated in female rats in different groups (P

Prevalence and molecular characterization of Giardia duodenalis in dairy cattle in Central Inner Mongolia, Northern China

Giardia duodenalis is a gastrointestinal protozoan ubiquitous in nature. It is a confirmed zoonotic pathogen, and cattle are considered a source of giardiasis outbreaks in humans. This study aimed to evaluate the prevalence and multilocus genotype (MLG) of G. duodenalis in dairy cattle in Central Inner Mongolia. This study was based on the small subunit ribosomal RNA (SSU rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis. DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed on 505 dairy cattle fecal samples collected in 2021 from six sampling sites and four age groups in Central Inner Mongolia to determine the prevalence and MLG distribution of G. duodenalis. The PCR results of SSU rRNA revealed that the overall prevalence of G. duodenalis was 29.5% (149/505) and that the overall prevalence of the diarrhea and nondiarrhea samples was 31.5% (46/146) and 28.5% (103/359), respectively; the difference was not significant (p > 0.05). SSU rRNA sequence analysis revealed that G. duodenalis assemblage E (91.1%, 133/146) was primarily detected and that assemblage A (8.9%, 13/146) was detected in 13 samples. The G. duodenalis—positive samples were PCR amplified and sequenced for gdh, tpi, and bg, from which 38, 47, and 70 amplified sequences were obtained, respectively. A combination of G. duodenalis assemblages A and E were detected in seven samples. Multilocus genotyping yielded 25 different assemblage E MLGs, which formed six subgroups. To the best of our knowledge, this is the first report regarding G. duodenalis infection in dairy cattle in Inner Mongolia, China. This study revealed that Inner Mongolian cattle pose a risk of giardiasis transmission to humans and that the distribution of local cattle G. duodenalis assemblage E MLGs is diverse. The findings of this study can bridge the knowledge gap in the molecular epidemiological investigation of giardiasis in Central Inner Mongolia.

First Report of Apple Brown Rot caused by Monilinia polystroma in Jilin Province in China.

Brown rot caused by Monilinia spp. is an important postharvest disease. It affects fruit quality and can cause serious economic losses. In October 2021, typical brown rot symptoms on fruit were observed at an apple orchard in Xiaobaishan Township, Jilin Province, China (E126°39'10″, N43°44'21″). Over 1200 plants were surveyed in the orchard, and nearly 25% of the plants were infected. In this research, samples from ten different trees showing typical symptoms were isolated and identified. Freshly diseased fruits were surface sterilized with 75% ethanol for 15s, then fungal colonies were isolated from 3 mm diameter diseased tissue samples. The purified colonies were placed on potato dextrose agar (PDA), oatmeal agar (OA) and water agar (WA + Sterilized apple pulp) and incubated at 25 ℃ in a 12 h/12 h light-dark photoperiod for 5 days. The colonies became light to dark brown; they grew faster on PDA with a growth rate of 5.53 mm/d, most densely on OA and slowest on SA + sterilized apple pulp with thin mycelia. After 20 days, some transparent, oval, smooth conidia were observed on the SA + sterile apple culture medium. Conidia sizes were 8.27-16.54 (avg. 11.97) x 2.92-7.09 (avg. 4.52) um (n=50) (Hilber-Bodmer et al. 2012). Pure cultures of eight samples were isolated from single spores, and DNA was extracted with a commercial nucleic acid extraction kit (Omega, cat#D3390-01). Then, fragments of the internal transcribed spacer region (ITS), translation elongation factor 1 alpha (EF1-alpha), beta-tubulin (Bt), and glyceraldehyde-3-phosphate dehydrogenase genes(G3pdh) were PCR amplified using primes ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), BT-2a/BT-2b (Glass and Donaldson 1995), and G3pdhF/G3pdhR (Hu et al. 2011) respectively. The amplicons were sequenced by Takara Bio Inc. Sequences of all isolates were identical, thus one set of sequences were run with BLASTn against the NCBI GenBank nr database. The homology analysis showed that ITS (OQ170786), Bt (OQ179019), EF (OQ834046) and G3pdh (OQ834047) gene fragments were 100% (0/540 nt) , 100% (0/462 nt), 99.01% (3/304nt) and 99.61% (3/767 nt) similar to M. polystroma （NR154198.1, MT038414.1, LT632542.1, MT038415.1）respectively. Based on morphological characteristics and sequence analysis, the fungal isolate was identified as M. polystroma. Koch's postulates were conducted using ten healthy apple fruits that were surface disinfected with 0.2% sodium hypochlorite and repeatedly rinsed with sterile water. The test apples were wounded with a sterile needle, inoculated with mycelial agar plugs (3 mm diameter) on the wounds, and incubated at 25℃, 50% room humidity. The equivalent sterile PDA plugs were used as control and the experiment was repeated three times. After 5 days, brown rot symptoms appeared on the inoculated apples. 10 days later, nearly 1/3 of the inoculated apple surface appeared rotted, but the controls were symptomless. Subsequently, the same strain was re-isolated from the inoculated apples by pure culture and molecular identification. Therefore, M. polystroma (named JL-1) was confirmed as the causal agent of brown rot in Jilin Provincen China. M. polystroma is a typical pathogen of brown rot in the north of China, and only reported on apples in Shandong, apricots in Heilongjiang and pears in Hebei in China (Zhu et al. 2016) but never in Jilin. In addition, it was reported that the contribution of M. polystroma species to brown rot disease on apple and pear in China is 20% out of all the Monilinia spp. species that cause the disease, but M. polystroma virulence is not significantly different from other Monilinia species more widely distributed (Zhu et al, 2016). This is the first report of M. polystroma causing apple brown rot in Jilin Province of China. This finding will provide useful information for future diagnosis and management.

Manipulation of isocitrate dehydrogenase genes affects the anti-autolytic ability of lager yeast

Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast autolysis showed that the citric acid cycle-related genes had a great influence on yeast autolysis. To explore the contribution of isocitrate dehydrogenase genes in autolysis, the IDP1 and IDP2 genes were destroyed or overexpressed in typical lager yeast Pilsner. The destruction of IDP1 gene improved the anti-autolytic ability of yeast, and the anti-autolytic index after 96 h autolysis was 8.40, 1.5 times higher than that of the original strain. The destruction of IDP1 gene increased the supply of nicotinamide adenine dinucleotide phosphate (NADPH) and the NADPH/NADP+ ratio was 1.94. After fermentation, intracellular ATP level was 1.8 times higher than that of the original strain, while reactive oxygen species (ROS) was reduced by 10%. The destruction of IDP2 gene resulted in rapid autolysis and a decrease in the supply of NADPH. Anti-autolytic index after 96 h autolysis was 4.03 and the NADPH/NADP+ ratio was 0.89. After fermentation, intracellular ATP level was reduced by 8% compared with original strain, ROS was 1.3 times higher than that of the original strain. The results may help understand the regulation mechanism of citric acid cycle-related genes on yeast autolysis and provide a basis for the selection of excellent yeast with controllable anti-autolytic performance.

Implementation of upfront DPYD genotyping with a low-cost and high-throughput assay to guide fluoropyrimidine treatment in cancer patients.

Genetic variants in the dihydropyrimidine dehydrogenase (DPYD) gene are associated with reduced dihydropyrimidine dehydrogenase enzyme activity and can cause severe fluoropyrimidine-related toxicity. We assessed the frequency of the four most common and well-established DPYD variants associated with fluoropyrimidine toxicity and implemented a relatively low-cost and high-throughput genotyping assay for their detection. This study includes 457 patients that were genotyped for the DPYD c.1129-5923C>G, c.1679T>G, c.1905 + 1G>A and c.2846A>T variants, either by Sanger sequencing or kompetitive allele specific PCR (KASP) technology. Of these, 172 patients presented toxicity during treatment with fluoropyrimidines (post-treatment group), and 285 were tested before treatment (pretreatment group). Heterozygous DPYD variants were identified in 7.4% of the entire series of 457 patients, being the c.2846A>T the most frequent variant. In the post-treatment group, 15.7% of the patients presented DPYD variants, whereas only 2.5% of the patients in the pretreatment group presented a variant. The KASP assays designed in this study presented 100% genotype concordance with the results obtained by Sanger sequencing. The combined assessment of the four DPYD variants in our population increases the identification of patients at high risk for developing fluoropyrimidine toxicity, supporting the upfront routine implementation of DPYD variant genotyping. Furthermore, the KASP genotyping assay described in this study presents a rapid turnaround time and relatively low cost, making upfront DPYD screening feasible in clinical practice.

First report of leaf anthracnose caused by Colletotrichum camelliae on tea plants (Camellia sinensis) in South Korea.

The tea plant (Camellia sinensis (L.) O. Kuntze) is a popular non-alcoholic beverage crop worldwide. The tea market in South Korea is projected to increase annually by 4.59% (Statista, 2022). Boseong, Hadong, and Jeju Island are the main tea-growing regions in South Korea. Anthracnose is one of the major diseases of tea plants and is responsible for substantial yield loss and poor tea quality. In 2021, anthracnose of tea (disease incidence of 30%) was observed in a garden (33°28'45.5"N 126°42'02.2"E) at Jeju Island, where the Yabukita cultivar has been cultivated. The typical symptoms consisted of round or irregularly shaped lesions with gray-white centers and purple-brown borders. Twelve morphologically similar isolates were recovered from 12 infected leaves using the single spore isolation method on solid potato dextrose agar (PDA) (Cai et al. 2009). Four representative isolates (GT6, GT7, GT8, and GT11) were identified based on morphology, molecular analysis, and pathogenicity tests. The upper side of seven-day-old colonies on PDA (incubated at 25 °C in the dark) was off-white with white aerial mycelia and gray-white with black zonation on their reverse side. Conidia were hyaline, aseptate, cylindrical, with both obtuse ends, and measuring 12.3 - 25.8 µm × 4.4 - 9.3 µm (n = 50). Appressoria were dark brown, irregularly shaped with a smooth edge, and measuring 7.3 -18.8 µm × 6.9 - 11.3 µm (n = 50). According to morphological characteristics, the fungal isolates were tentatively identified as the Colletotrichum gloeosporioides complex, including C. caelliae (Wang et al. 2016; Weir et al. 2012). The genomic DNA was extracted, and the ribosomal internal transcribed spacer (ITS), β-tubulin-2 (TUB2) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, actin (ACT), calmodulin (CAL), and the Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat) genes were amplified and subsequently sequenced using primer sets ITS1/ITS4, BT2a/BT2b, GDF1/GDR1, ACT-512F/ACT-783R1, CL1C/CL2C, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting sequences were deposited in GenBank accession numbers (LC738932-LC738959). All the representative isolates were identified as C. camelliae by constructing the 50% majority rule consensus and maximum likelihood phylogenetic treebased on the combined ITS, TUB2, GAPDH, ACT, CAL, and ApMat sequences using MrBayes v. 3.2.2 and Mega X, respectively (Kumar et al., 2018; Ronquist et al. 2012). The pathogenicity of these isolates was tested on healthy leaves of 2- years-old tea seedlings (the Yabukita cultivar). Onside of unwounded or wounded leaves of seedlings were inoculated with 20 µL of conidial suspension (1 × 106 conidia or spores/ml) per spot (3-4 wounded or unwounded spots per side per leaf). Another side of the leaves received sterile distilled water and served as a control. Each treatment was replicated three times (three seedlings/isolate and four leaves per seedling) and this experiment was repeated twice. All plants were covered with plastic bags, placed in a growth chamber, and incubated at 25 °C with a 12-h photoperiod and 90% relative humidity. Typical anthracnose symptoms appeared on wounded leaves after two days of inoculation. Unwounded and controlled leaves remain asymptotic. To confirm Koch's postulates, fungal isolates were re-isolated from inoculated leaf lesions and identified as C. camelliae based on morphology and ITS sequences. Colletotrichum camelliae is a very common pathogen associated with tea anthracnose worldwide, including China (Liu et al. 2015; Wang et al. 2016).To the best of our knowledge, this is the first report of anthracnose in tea trees caused by C. camelliae in South Korea. The results of this study could help come up with better ways to keep an eye on and deal with this devastating on tea plants. Key words: Tea anthracnose, Colletotrichum camelliae, pathogenicity References Cai, L., et al. 2009. Fungal Divers. 39:183. Kumar, S., et al. 2018. Mol. Biol. Evol. 35:1547. Liu, F. et al. 2015. Persoonia. 35: 63-86. Ronquist, F. et al. 2012. Syst. Biol. 61:539-542. Silva, D. N. et al. 2012. Mycologia. 104:396-409. Statista 2022. Statista Digital Market out Look. Available at www.statista.com. Wang, Y.-C. et al. 2016. Sci. Rep. 6: 35287. Weir, B. S., et al. 2012. Stud. Mycol. 73:115.

Molecular Detection and Characterization of Streptococcus suis Isolated from Pneumonic Lungs of Pigs

Background: Among various diseases, respiratory disease is considered a leading cause of mortality in pigs. Streptococcus suis affects respiratory tract and causes pneumonia as an opportunistic pathogen, that impairs animal health and lowers individual and herd performance in swine. Therefore, it becomes important to have an insight of bacterial pneumonia with special reference to Streptococcus suis in pig, so that control measures could be practiced effectively. Methods: Detailed post mortem examination was carried out on 180 pigs. Bacterial isolation was carried out from 127 (70.55%) lungs samples with pneumonic lesions. Further, suspected colonies of S. suis were subjected to Polymerase Chain Reaction for detection of glutamate dehydrogenase gene. Phylogenetic analysis was carried out for the positive S. suis samples. Results: Bacteria isolated were Pasteurella multocida (34.44%), S. suis (28.89%), E. coli (16.67%), Staphylococcus spp. (14.44%), Klebsiella spp. (3.33%) and Salmonella spp. (2.22%). Out of 26 suspected S. suis colonies subjected to Polymerase Chain Reaction, 17 isolates with amplicon size 688 bp were confirmed to be positive for glutamate dehydrogenase gene. The Phylogenetic analysis of glutamate dehydrogenase gene of S. suis from pigs of Assam showed percent identity above 99% with Germany, Canada and China strain of S. suis.

Effects of 1-Methylcyclopropene Treatment on the Quality and Malic Acid Metabolism of ‘Xiangjiao’ Plum under Low-Temperature Storage

‘Xiangjiao’ plum (Prunus salicina Lindl.) is a stone fruit that is vulnerable to the chilling injury (CI) that is caused by low-temperature stress. The effects of 1-methylcyclopropene (1-MCP) and ethylene absorbent (EA) treatments on the fruit quality and malic acid metabolism of ‘Xiangjiao’ plum stored at 4 °C were compared in this study. Compared with the control check (CK) and EA treatment, fumigation with 1.0 mg·L−1 of 1-MCP for 24 h could more significantly maintain the sensory and physiological quality of the fruit, increase the activity of antioxidant enzymes, and prolong the storage time of plums. Furthermore, 1-MCP treatment can regulate the high expression of the tonoplast dicarboxylate transporter (tDT) and phosphoenolpyruvate carboxylase (PEPC) gene, regulate the high expression of the NAD-malate dehydrogenase (NAD-MDH) gene at the end of storage, and inhibit the expression of the NADP-malic enzyme (NADP-ME) gene. These changes resulted in increased NAD-MDH enzyme activity and decreased NADP-ME enzyme activity, which inhibited the degradation of malic acid that is caused by CI. As a result, 1-MCP can effectively maintain the storage quality of ‘Xiangjiao’ plum, reduce the loss of pleasant sour taste, and improve the edible flavor and commercial value of the fruit.