Glyceraldehyde 3-phosphate Dehydrogenase Gene Research Articles

Selection and validation of optimal reference genes for RT-qPCR analyses in Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae).

Aphidoletes aphidimyza is a predator that is an important biological agent used to control agricultural and forestry aphids. Although many studies have investigated its biological and ecological characteristics, few molecular studies have been reported. The current study was performed to identify suitable reference genes to facilitate future gene expression and function analyses via quantitative reverse transcription PCR. Eight reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, β-actin, RPL32, and elongation factor 1 alpha (EF1-α) were selected. Their expression levels were determined under four different experimental conditions (developmental stages, adult tissues, sugar treatment, and starvation treatment) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that GAPDH, RPL32, and EF1-α were ranked as the best reference gene combinations for measuring gene expression levels among different developing stages and in various starvation treatments. RPL8 and RPS3 were recommended to normalize the gene expression levels among different adult tissues. RPL32, β-actin, and EF1-α were recommended sugar-feeding conditions. To validate the utility of the selected reference pair, RPL8, and RPS3, we estimated the tissue-biased expression level of a chemosensory protein gene (AaphCSP1). As expected, AaphCSP1 is highly expressed in the antennae and lowly expressed in the abdomen. These findings will lay the foundation for future research on the molecular physiology and biochemistry of A. aphidimyza.

First Report of Anthracnose Caused by Colletotrichum spaethianum on Iris lactea in Daqing, China.

Iris lactea Pall. has high ornamental value and strong drought resistance, which can be used as a useful sand-stabilization and ornamental plant. In September 2021, typical anthracnose symptoms on I. lactea were found in the campus of Heilongjiang Bayi Agricultural University (125°10'45.31"E, 46°35'36.88"N), Daqing, China. Disease incidence varied from 37 to 51% at four survey sites (~20 m2 per site) of approximately 1,000 plants. Initially brown lesions with a yellow halo, and gray in the center were observed on the leaves. As disease progressed, the lesions expanded rapidly, resulting in dieback. To identify the pathogen, symptomatic tissues were excised from four infected leaves of four individual plants, surface sterilized for 1 min in 75% ethanol, washed twice with sterile water, plated on potato dextrose agar (PDA) contain 0.5 g/L streptomycin sulfate, and incubated at 25℃ for 3 to 4 days under dark conditions. Four morphologically similar fungal isolates (Irs-1 to -4) were obtained by using a single-spore isolation. After growth on PDA for 17 days at 25℃, a circular like zonation colonies with a color of greyish to gray green were observed. On the reverse side, circular like with zig-zag zonation colonies light brown to light black from the margin to the center. Conidiophores were pale brown, septate. Conidia were hyaline, aseptate, curved or slightly curved, round, or somewhat acute apex, base truncate, unicellular, whose sizes ranged at 18.5 ± 2.3 × 4.6 ± 0.7 μm (n = 170), with length/width ratio 4.04. Appressoria were in oval, star, or irregular shape, with brown color with sizes approximately at 13.3 ± 2.1 × 10.4 ± 2.1 μm (n = 30). These morphological characteristics are consistent with Colletotrichum spaethianum with curved conidia (Damm et al. 2009). For molecular identification, primers ITS1/ITS4 (Schoch et al. 2012), GDF1/GDR1 (Guerber et al. 2003), ACT-512F/ACT-783R and CHS-354R/CHS-79F (Carbone and Kohn 1999) were used to amplify the partial region of rDNA-ITS, a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), and chitin synthase 1 (CHS-1) from fresh mycelia of the four isolates (Irs-1 to -4), respectively. The sequences were submitted to GenBank (ITS: OM004553, OP804624, OR029392, and OR029391; GAPDH: OP883938, OP883939, OR060963, and OR060962; ACT: OP883940, OP883941, OR060965, and OR060964; CHS: OP868844, OP868845, OR046504, and OR046503). Phylogenetic analysis based on the ITS, GAPDH, ACT and CHS gene sequences indicated that isolates obtained in this study were all clustered with C. spaethianum. Pathogenicity test was conducted by inoculating mycelial plugs on the I. lactea seedlings (2 seedlings with 4 to 5 leaves per isolate). Two seedlings inoculated with sterilized PDA plugs were used as control. All inoculated plants were maintained in humid chamber at 25℃ under dark, and typical anthracnose symptoms were observed up to 6 days after inoculation, while the control leaves were asymptomatic. The same pathogen was successfully re-isolated and phenotypically identical to the original isolates to fulfill Koch's postulates. C. spaethianum has been described on Hemerocalis flava (Vieira and Michereff 2014) and Allium fistulosum (Santana et al. 2016) in Brazil, Polygonatum cyrtonema in China (Ma et al. 2020), Iris germanica in Japan (Sato et al. 2012). To our knowledge, this is the first report of C. spaethianum causing anthracnose disease on I. lactea in Daqing, China.

First report of Anthracnose caused by Colletotrichum theobromicola on Cyclamen persicum in South Carolina, USA.

Florist's cyclamen (Cyclamen persicum) is an herbaceous perennial native to the Mediterranean region and has become an increasingly popular plant around the world. Leaves of these plants are cordate-shaped with varying green and silver patterns. Flowers vary in color from white through different shades of pink, lavender, and red. In September 2022, symptoms of anthracnose including leaf spots and chlorosis, wilting, dieback, and crown and bulb rot were observed on 20 to 30% of approximately 1,000 cyclamen plants in an ornamental production nursery in Sumter County, SC. Tissue samples surrounding the necrotic crowns were excised and sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto acidified potato dextrose agar (APDA), and incubated at 25°C with 24-h photoperiod. A total of five Colletotrichum isolates, 22-0729-A, 22-0729-B, 22-0729-C, 22-0729-D, and 22-0729-E were obtained by transferring hyphal tips to new plates. The morphology of these five isolates was identical, observed as gray and black with aerial gray-white mycelia and orange-colored spore masses. Conidia (n=50) measured 19.4 ± 5.1 mm (11.7 to 27.1 mm) in length and 5.1 ± 0.8 mm (3.7 to 7.9 mm) in width. Conidia were tapered with rounded ends. Setae and irregular appressoria were infrequently observed in aged cultures (> 60-day-old). These morphological features resembled those of members of the Colletotrichum gloeosporioides species complex (Rojas et al. 2010; Weir et al. 2012). Sequence of the internal transcript spacer (ITS) region of a representative isolate 22-0729-E (GenBank accession No. OQ413075) is 99.8% (532 / 533 nt) and 100% (533 / 533 nt) identical to those of the ex-neotype of Co. theobromicola CBS124945 (JX010294) and the ex-epitype of Co. fragariae (= Co. theobromicola) CBS 142.31 (JX010286), respectively. Its glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequence is 99.6% (272 / 273 nt) identical to those of CBS124945 (JX010006) and CBS 142.31 (JX010024). Its actin (ACT) gene sequence shares 99.7% (281 / 282 nt) and 100% (282 / 282 nt) identities with those of CBS124945 (JX009444) and CBS 142.31 (JX009516), respectively. Lastly, its beta-tubulin 2 (TUB2) gene sequence is 99.6% (704 / 707 nt) and 100% (707 / 707 nt) identical to those of CBS124945 (JX010447) and CBS 142.31 (JX010373), respectively. The causal agent causing anthracnose on cyclamen in SC was identified as Co. theobromicola. To confirm the pathogenicity, cyclamen 'Verano Red' plants grown in 2.5-inch pots were used in two pathogenicity assays using different inoculation methods. In the first assay, three plants were inoculated by spraying a conidial suspension (1 × 106 conidia per ml; 30 ml per plant) of isolate 22-0729-E onto the foliage. Three non-inoculated control plants were sprayed with distilled water. All six plants were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 7 days to maintain humidity. Early symptoms including small spots, marginal necrosis, and chlorosis were observed on leaves and flowers 8 days after inoculation (DAI) and the entire aboveground tissues of inoculated plants were blighted 13 to 21 DAI. Non-inoculated plants remained asymptomatic. In the second assay, sterile toothpicks were used to slightly wound the crown and bulb surface of three plants and secure a mycelial APDA plug of isolate 22-0729-E (5×5 mm2) onto each wound (three wounds per plant). Three control plants were wounded in the same manner, while sterile APDA plugs were used in place of mycelial plugs. All six plants were maintained in the same manner as in the first assay. Apparent leaf yellowing and wilting symptoms appeared as early as 13 DAI. On 21 to 28 DAI, severe crown rot on inoculated plants caused the entire foliage to collapse. At least one third of the inner crown and bulb tissues of each inoculated plant were rotten, while those of non-inoculated plants appeared healthy. Each assay was repeated once. Colletotrichum isolates resembling morphological characters of 22-0729-E were recovered from leaves and inner crown tissues of all inoculated plants in both assays, respectively, but not from non-inoculated control plants. Anthracnose diseases on Cyclamen persicum caused by Co. theobromicola (syn. Co. fragariae) have been reported in NC, USA (Lui et al. 2011) and Israel (Sharma et al. 2016). This is the first report of anthracnose on cyclamen in SC, USA. Colletotrichum gloeosporioides (teleomorph Glomerella cingulate) species complex on cyclamen has also been reported in Argentina (Wright et al. 2006), South Africa, and several other U.S. states (Farr and Rossman 2022). However, it remains unknown whether these previous reports in fact attributed to Co. theobromicola due to lack of molecular identification (Weir et al. 2012). Colletotrichum theobromicola can cause diseases on at least 30 other agricultural and horticultural crops such as strawberry, cacao, and boxwood (Farr and Rossman 2022). It may pose a threat to cyclamen in greenhouse and nursery productions. Therefore, management strategies are warranted in the future.

Leaf Spot caused by Alternaria alternata on Macleaya cordata in China.

Macleaya cordata is a perennial herb that belongs to the Papaveraceae and is typically prescribed as a traditional antibacterial medicine in China (Kosina et al. 2010). The extract from M. cordata has been widely used in the manufacturing of natural growth promoters as an alternative to antibiotic growth promoters in the livestock industry (Liu et al. 2017), and the products are marketed in 70 countries such as Germany, China, etc (Ikezawa et al. 2009). During the summer of 2019, symptoms of leaf spot were observed on M. cordata (cv. HNXN-001) in two commercial fields (approximately 1, 300 m2 and 2, 100 m2) of Xinning county, Shaoyang City, Hunan Province, China, where approximately 2 to 3% of the plants were affected. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to leaf blight. Six symptomatic basal leaf sections from six plants from two fields were surface disinfested in 0.5% NaClO for 1 min, then 75% ethanol for 20 s, rinsed in sterile water three times, air dried, and placed onto potato dextrose agar (PDA), one dish for samples from a single leaf. Plates were incubated at 26°C in darkness. Nine strains with similar morphological characters were isolated, and one representative isolate ( BLH-YB-08) was used for morphological and molecular characterization. The colonies on PDA were grayish-green with white round margins. Conidia were typically obclavate to obpyriform, brown to dark brown, and 12.0 to 35.0 × 6.0 to 15.0 μm, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa (n=50). Isolates were identified as Alternaria sp. on the basis of mycelial characteristics, color, and conidial morphology. To confirm identity of the pathogen, DNA was extracted from isolate BLH-YB-08 with the DNAsecure Plant Kit (TIANGEN, Biotech, China). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), actin (ACT), 28S nrDNA (LSU), 18S nuclear ribosomal DNA (SSU), histone 3 (HIS3), internal transcribed spacer (ITS) region of ribosomal DNA, and translation elongation factor 1-α (TEF) genes ( Berbee et al. 1999; Carbone and Kohn. 1999; Glass and Donaldson. 1995; White et al. 1990.) were amplified and sequenced. Sequences were deposited into the GenBank database. They were 100% sequence identity of GAPDH (OQ224996) with A. alternata strain AA2-8 (MH65578; 578/578bp), 100% sequence identity of RPB2 (OQ190460) with A. alternata strain SAX-WN-30-2 ( MK605877; 933/933bp), 100% sequence identity of ACT (OQ923292) with A. alternata strain FCBP0352 (OL830257; 939/939 bp), 100% sequence identity of LSU (OQ891167) with A. alternata XL14 (MG839509 ; 908/908 bp), 100% sequence identity of SSU (OQ139544) with A. alternata strain BJ19.4.1(OM736063; 1,067/1,067 bp), 100% sequence identity of HIS3 (MT454856) with A. alternata YJ-CYC-HC2 (OQ116440 ; 442/442 bp), 100% sequence identity of ITS (MT212225) with A. alternata CS-1-3 (OQ947366; 543/543bp), and 100% sequence identity of TEF (OQ190461) with A. alternata strain YZU 221185 (OQ512730; 252/252 bp). To test pathogenicity, the isolate BLH-YB-08 was cultured on PDA for 7 days to prepare conidial suspensions and the spore concentration adjusted to a final concentration of 1×106 spores/ml. The leaves of five potted 45-day-old M. cordata (cv. HNXN-001) plants were sprayed with conidial suspensions, and five control potted plants were wiped with 75% alcohol and washed five times with sterile distilled water. They were then sprayed with sterile distilled water. Plants were placed in a greenhouse at 25 to 30°C with 90% relative humidity. Pathogenicity tests were conducted twice. Fifteen days after inoculation, lesions were found on inoculated leaves, and the symptoms were the same as those in the field, whereas the controls were healthy. A fungus was consistently isolated from the inoculated leaves and identified as A. alternata by DNA sequencing of the GAPDH, ITS, and HIS3 genes, fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on M. cordata caused by A. alternata in China. Understanding its etiology may help to control this fungal pathogen, thus reducing economic losses. Funding: Hunan Provincial Natural Science Foundation General Project (2023JJ30341) Hunan Provincial Natural Science Foundation Youth Fund (2023JJ40367) Seed Industry Innovation Project of Hunan Provincial Science and Technology Department Special project for the construction of Chinese herbal medicine industry technology system in Hunan Province "Xiangjiuwei" Industrial Cluster Project of the Ministry of Agriculture and Rural Affairs.

Analysis of the stability of the reference genes GAPDH, SDHA and RPL-19 in sheep from a semi-arid region infected by gastrointestinal nematodes

Analyzing the stability of reference genes already described as universal is an important methodology to lead gene expression analysis because different studies have shown that the expression of universal reference genes may vary between experimental treatments. In this sense, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Succinate dehydrogenase complex subunit A (SDHA) and Ribosomal Protein L-19 (RPL-19) reference genes (already described in other studies with sheep from different regions, breeds and infectious agents or in organisms evolutionarily close to sheep) were investigated in the abomasum, small and large intestines of resistant and susceptible crossbred sheep groups to gastrointestinal nematode infections in the Semi-arid region in Northeast of Brazil. The animals were naturally infected to determine the resistance or susceptibility status by counting eggs per gram (EPG) of feces from the gastrointestinal tract after 33 weeks of observations of infection evolution. Relative gene expression was performed by RT-qPCR methodology using Sybr green and relative gene expression stability was tested by different software programs such as REST, BestKeeper, geNorm and Normfinder. Our results showed the susceptible animals had increase in egg counts per gram of feces than resistant animals (p < 0.001), and both groups showed a mixed infection by nematodes of the genus Haemonchus, Trichostrongylus, Oesophagostomum and Trichuris. Furthermore, we show the importance of analyzing different genes in different software programs and the importance to choose ideal reference genes. In this sense, GAPDH was the most stable gene in the abomasum, whereas SDHA was the most stable in the small and large intestines. In addition, we discuss about variables which can interfere in relative expression such as breed, species, climate and tissue. However, utilizing other reference genes already described in other studies with the same and different variables should be performed.

The role of sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the development of pathologies-from asthenozoospermia to carcinogenesis.

The review considers various aspects of the influence of the glycolytic enzyme, sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) on the energy metabolism of spermatozoa and on the occurrence of several pathologies both in spermatozoa and in other cells. GAPDS is a unique enzyme normally found only in mammalian spermatozoa. GAPDS provides movement of the sperm flagellum through the ATP formation in glycolytic reactions. Oxidation of cysteine residues in GAPDS results in inactivation of the enzyme and decreases sperm motility. In particular, reduced sperm motility in diabetes can be associated with GAPDS oxidation by superoxide anion produced during glycation reactions. Mutations in GAPDS gene lead in the loss of motility, and in some cases, disrupts the formation of the structural elements of the sperm flagellum, in which the enzyme incorporates during spermiogenesis. GAPDS activation can be used to increase the spermatozoa fertility, and inhibitors of this enzyme are being tried as contraceptives. A truncated GAPDS lacking the N-terminal fragment of 72 amino acids that attaches the enzyme to the sperm flagellum was found in melanoma cell lines and then in specimens of melanoma and other tumors. Simultaneous production of the somatic form of GAPDH and sperm-specific GAPDS in cancer cells leads to a reorganization of their energy metabolism, which is accompanied by a change in the efficiency of metastasis of certain forms of cancer. Issues related to the use of GAPDS for the diagnosis of cancer, as well as the possibility of regulating the activity of this enzyme to prevent metastasis, are discussed.

First report of leaf spot caused by Curvularia muehlenbeckiae on Fraxinus rhynchophylla Hance in China.

Fraxinus rhynchophylla Hance, is a deciduous trees cultivated on a commercial scale focused on medicinal and wood production. In September 2021, leaf spot was observed on F. rhynchophylla in Heilongjiang Province (127.34°E, 45.19°N), China. These symptoms were observed on 100% F. rhynchophylla plants and the incidence of diseased leaves per plant reached 70% in fields measuring 90 ha. Disease symptoms were small yellow flecks initially, and then turned to gray necrotic spot. Ten diseased leaves were collected randomly from 5 plants and surface disinfested. Tissue samples (2 × 2 mm) were cut at the disease-health junction of the leaves, surface sterilized in 75% ethanol for 30 s, submerged in a 7% NaOCl solution for 3 mins, and rinsed three times with sterile water. Leaf segments were placed onto potato dextrose agar (PDA) and incubated at 26℃ for 5 days. After isolation and purification of monospore, the colonies of the all isolates were inky black, with aerial fluffy mycelium, and concentric whorls on PDA. The conidiophore is septate, single-branched, brown, smooth and 35 - 313 × 2 - 5 μm in size (n = 50), while the conidia are brown, bow-shaped, mostly four cells, with three septa and 12 - 385 × 5 - 20 μm in size (n = 150). The morphological characters matched those of Curvularia muehlenbeckiae (Madrid et al. 2014). DNA was extracted from isolates HQLa and HQLb and used for PCR amplification of RNA polymerase II gene (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequences using the primer fRPB2-SF/fRPB2-7Cr (Schoch et al. 2009), gpd1/gpd2 (Berbee et al. 1999), respectively. The RPB2 (OM984674, OM984675) and GAPDH (OM984672, OM984673) were deposited in GenBank. The phylogenetic tree was constructed by combining other published sequences of RPB2 and GAPDH genes using the maximum likelihood method, and the results showed that the obtained isolates clustered into the same clear branch as C. muehlenbeckiae CBS 144.63 (HG779180, HG779108), with 100% bootstrap support. Combining morphological characteristics and phylogenetic analysis of the fungus, the obtained isolates were identified as C. Muehlenbeckiae. To fulfill the Koch's postulates, pathogenicity tests were carried out on newly grown leaves of F. rhynchophylla. Conidia of the selected isolates grown on PDA plates were flooded with sterile distilled water. Spore suspension was adjusted to 105 spores/mL with the hemocytometer. Three leaves of each plant were disinfected with 1% NaOCl for 2 min, washed with sterilized distilled water three times, and dried with sterile paper towels. Three plants were randomly selected for inoculation under field conditions and each leaf was sprayed with 2 mL of the spore suspension for a total of nine leaves, then the plants were bagged and moistened for 48 h. However, control leaves were sprayed with distilled water. Symptoms were observed nine days after inoculation. No symptoms were observed on control leaves. The same fungus was successfully re-isolated from the lesions. The experiment was replicated three times with the same results and C. muehlenbeckiae identification was confirmed by morphological observations and RPB2 and GAPDH sequencing, indicating that the fungus is the causal pathogen of leaf spot disease on F. rhynchophylla. This is the first report of C. muehlenbeckiae determined as fungal pathogens on F. rhynchophylla plant in China. The results of the study laid the foundation for the future occurrence and epidemiological pattern of the disease and scientific control.

First report of Colletotrichum plurivorum causing anthracnose on pecan (Carya illinoinensis) in China.

Pecan (Carya illinoinensis) is an economically important nut crop worldwide (Xiao et al 2021). Anthracnose symptoms were found on pecan fruits and leaves in plantations in Anhui and Jiangsu provinces, China in August 2019. Irregular, dark brown or black spotted lesions firstly appeared on the surface and inside of fruits, and spread to all leaves. The symptoms resulted in 30% to 50% leaf drop and nearly a half of fruit decay in almost all trees of the susceptible cv. Wichita. The causal agent were isolated from fruits with target symptoms following the steps: surface disinfected with 75% ethanol (2×, 30 s), rinsed with sterile deionized water (3×), ~ 0.5 cm small fragments of the fruits excised and plated on potato dextrose agar (PDA) medium and incubated at 28 °C in dark for 3-d. Mycelium of each colony was picked and incubated on fresh PDA at 25 °C with a 12-hour light/dark cycle for 6-d to induce conidia formation. One 5-mm hyphal plug produced from each single spore isolate was transferred onto fresh PDA to obtain the pure cultures. Koch's postulates was employed for pathogenicity determination of the isolates. Non-wounded healthy leaves from seedlings of the disease susceptible cv. Pawnee were disinfected with 1% NaClO and inoculated with 5-mm 5-d hyphal of each isolate at 25 ℃. Tiny lesion spots were visible on the leaves after 2 days post inoculation (DPI) with isolate W-6 (the only pathogenic one among all isolates), and expanded over time until to the leaves withered, while the control leaves and leaves inoculated with other isolates remained asymptomatic. The pathogenicity of W-6 were confirmed using leaves and fruits of living Pawnee trees growing in Linglong Mountain Plantation, Lin'an, Hangzhou, China (119⁰38'51″E, 30⁰12'39″N, elevation: 119m). Three experimental replicates were conducted separately with three bio-replicates for all pathogenetic testing. The same symptoms were observed on both detached leaves and leaves and fruits of living trees.. The colony of W-6 have round cottony mycelium with complete edges and showed the fastest growth rate 3 - 4 DPI. After 7 DPI, white aerial mycelium turned yellowish brown and formed Acervulus in the mycelium. Conidia (n=50) one-celled, 12.0 - 20.0 μm × 3.5 to 6.0 μm width. Hyaline cylindrical with slightly rounded ends and two or three large guttulate at the centre. Most Acervulus dark brown and slightly irregular in shape, 12.70 × 18.79 μm (n=10). Setae were dark brown in color with average length around 34.10 μm (n=10). These characteristics matched previous descriptions of Colletotrichum orchidearum species complex, including C. plurivorum (Damm et al 2019). The identity of W-6 was confirmed by multi-locus phylogenetic analysis using the internal transcribed spacer (ITS) rDNA region and partial sequences of the conserved genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin 2 (TUB2), and chitin synthase (CHS). The sequences of W-6 were used for Basic Local Alignment Search Tool (BLAST) against NCBI GenBank and the sequences with 100% identity to that of W-6 were achieved, respectively. The concatenated sequences of the ACT-CHS-GAPDH-ITS-TUB2 was used for building a phylogenetic tree. The molecular analyses allowed the identification of the pathogen as C. plurivorum. It was known that 9 of the 11 Colletotrichum species causing pecan anthrax worldwide were reported in southern China (Brenneman 1989; Oh et al 2021). This is the first report of C. plurivorum as causal agent of pecan in China.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Wisteria sinensis in China.

Wisteria (Wisteria sinensis) is a well-known ornamental plant for environmental protection in the garden, which also has a high value for medicinal use. In December 2021, leaf spots were observed on W. sinensis plants growing on the campus of Jiangxi Agricultural University in Nanchang, Jiangxi Province (28.45° N, 115.49° E), with a incidence rate of 40% plants were infested (n = 100 investigated plants). Initially leaf spots were small and pale brown (Approx. 2 mm in diameter), which gradually expanded into round or irregular dark brown spots as disease progressed, and lesions developed greyish-white necrotic tissues in the center at later stages, eventually causing the leaves to rot. To isolate the pathogen, tissues (5 × 5 mm) at the margin of lesions were cut from ten symptomatic leaves, surface disinfected with 75% ethanol for 30 s followed by 2% sodium chloride (NaClO) for 1 min, rinsed three times with sterile distilled water, and the dried tissues were cultured on potato dextrose agar (PDA) at 28 ± 1℃ in darkness for 3 days. After culture purification, five isolates were obtained and the representative single spore isolate (ZTTJ1) was used for subsequent identification tests. After 10 days of incubation on PDA medium, colonies had dense aerial mycelium with a gray center and dark gray-green mycelium outward, with orange-red conidial masses distributed in a ring on the surface. The underside of the colonies was light gray to dark gray. Conidia were cylindrical, with ends obtuse-rounded, 11.83 to 20.74 × 3.34 to 5.33 μm (av=16.11 μm × 4.26 μm, n = 50) in size. These morphological characteristics were consistent with Colletotrichum gloeosporioides (Shi et al, 2019). Six conserved regions of isolate (ZTTJ1), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), β-tublin (TUB), actin (ACT), and chitin synthase 1 (CHS1) gene regions were amplified using ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Li et al, 2018), Bt2a/Bt2b (Prihastuti et al, 2009), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999) primers, respectively. Using BLAST, ITS, GAPDH, CAL, TUB, ACT, and CHS1 gene sequences (GenBank Accession No. OP703312, OP713773, OP713775, OP713776, OP713772, OP713774, respectively) were over 99% identical to C. gloeosporioides (GenBank Accession No. MK967281, MH594288, MT449307, MN624110, MN107239 and MN908602, respectively). A maximum likelihood (ML) phylogenetic analysis based on ITS-ACT-GAPDH-CHS1-CAL-TUB2 sequences using MEGA7.0, placed isolate (ZTTJ1) within C. gloeosporioides. To complete Koch's postulates, 10 μL spore suspension (1.0 × 106 conidia/mL) of ZTTJ1 (7-day-old culture on PDA medium) was dropped onto 10 leaves wounded with a sterilized needle and 10 non-wounded leaves, respectively. Ten wounded leaves were inoculated with sterile water as controls. All leaves were incubated at 28 ± 1 °C and 90 % relative humidity (12 h/12 h light/dark). After 7 days, all wounded leaves inoculated with C. gloeosporioides developed symptoms as previously observed, while the control and non-wounded leaves remained healthy. The fungus re-isolated from the inoculated leaves were identified as C. gloeosporioides by morphological and molecular identification; the pathogen causing disease in W. sinensis was determined to be C. gloeosporioides. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. sinensis in China. This work has identified the pathogenic species of the disease, which helps to take targeted measures to control its spread, providing a basis for the prevention and treatment of the disease.

Leaf Spot Disease on Punica granatum Caused by Alternaria alternata in China.

Pomegranate (Punica granatum L.), which is native to central Asia, is considered as one of the most renowned commercial fruit trees in the world. The planting area in China is roughly 120 thousand hectares. In June 2020, symptoms of leaf spot on P. granatum appeared in Nanyang City (32º40´34˝N, 111º44´20˝E), Henan Province, with an incidence rate of 35% in several 3.3-hectare orchards. Initially, the lesions showed as round or subrounded brown spots on affected leaves. The spots then progressively developed into irregular lesions with distinct yellow halos surrounding them. Parts of the lesions were weakly zonate, which finally led to leaf withering and falling off. Diseased tissues were cut into 5×5 mm2 pieces, which were surface sterilized with 75% ethanol solution for 30 s, washed 3 times in sterilized water, and put on potato dextrose agar (PDA) plates supplemented with 50 μg ml-1 streptomycin. A total of 16 purified fungal isolates with similar phenotypic features were obtained. Three randomly chosen isolates SLY11, SLY24, and SLY25 were utilized for the investigation. Fungal colonies on PDA were first white to gray and later mycelium became olive green to blackish brown. To examine the morphological properties of conidia, we utilized potato carrot agar (PCA) culture medium and incubated it at 23°C under a 12-hour light/dark alternation. Conidia were obclavate or spheroidal, dark brown, with 3 to 5 transverse septa and 1 to 4 longitudinal septa. Conidiophores were septate, solitary, and measured 22.7 (±4.64) × 10.6 (±2.15) μm (n=50), with a conical beak length of 0 to 5.5 μm. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (TEF1), β-tubulin gene (TUB), and glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and GDF1/GDF2 from genomic DNA. Sequences were submitted to GenBank with accession numbers OL840230, OL840231, OL840232 for ITS, OL982540, OL982541, OL982542 for TEF1, OL982543, OL982544, OL982545 for TUB, OL862608, OL862609, OL862610 for GAPDH sequences of isolates SLY11, SLY24, and SLY25, respectively. BLASTn analysis of ITS (OL840230), TEF1 (OL982540), TUB (OL982543), GAPDH (OL862608) sequences indicated 100, 99.59, 99.68, and 100% similarity to the sequences of Alternaria alternata strain HC-2 (MT644140), BJFA-1 (MK895958), CS36-5 (KY814630), and ag1 (KP057228) in GenBank. Isolates SLY11, SLY24, and SLY25 formed a clade with the type strains CBS 130265 and CBS 130258 of A. alternata in phylogenetic trees established, clearly seperating from other Alternaria spp. The morphological features and molecular analyses supported the isolates as members of A. alternata. To validate the pathogenicity of the isolates, ten healthy leaves of 3-year-old potted pomegranate trees were utilized for testing and inoculated with conidial suspension (106 conidia ml-1), 20 µl each leaf. Control plants were inoculated with sterilized water. An additional pathogenicity test was repeated on wounded leaves. The inoculated plants were placed at 28°C in a greenhouse (12 h light per day and 90% relative humidity) for 5 days. The pathogenicity testing was conducted three times. Distinct lesions were found on the nonwounded and wounded leaves of inoculated plants after 3 to 5 days. The morphology and ITS sequences of the fungi that were reisolated from each of the inoculated plants were similar to that of the inocula, fulfilling Koch's postulates. Fruit rot of pomegranate induced by A. alternata was not identified in our investigation. A. alternata is reported to induce leaf spot disease on P. granatum in India (Zakir et al. 2009), Israel (Ezra et al. 2010), Spain (Garcia-Jimenez et al. 2014). To our knowledge, this is the first report of A. alternata causing leaf spot disease on P. granatum in China. Severe leaf disease caused by A. alternata can lead to reduced pomegranate yields in the harvest stages. This note will aid in pathogen identification and disease control.

Genome-wide identification and analysis of glyceraldehyde-3-phosphate dehydrogenase family reveals the role of GmGAPDH14 to improve salt tolerance in soybean (Glycine max L.).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential key enzyme in the glycolytic pathway and plays an important role in stress responses. Although GAPDH family genes have been found in different plant species, the determination of their gene family analysis and their functional roles in soybean are still unknown. In this study, gene sequence and expression data were obtained using online tools, and systematic evolution, expression profile analysis, and qRT-PCR analysis were conducted. Here a total of 16 GmGAPDH genes were identified on nine chromosomes, which were classified into three clusters. Additionally, all GmGAPDH genes harbor two highly conserved domains, including Gp_dh_N (PF00044) and Gp_dh_C (PF02800). The qRTPCR analysis also showed that most GmGAPDH genes significantly responded to multiple abiotic stresses, including NaHCO3, polyethylene glycol, cold, and salt. Among them, GmGAPDH14 was extraordinarily induced by salt stress. The GmGAPDH14 gene was cloned and overexpressed through soybean hair roots. The overexpressed transgenic soybean plants of the GmGAPDH14 gene have also shown better growth than that of control plants. Moreover, the overexpressed transgenic plants of GmGAPDH14 gene had higher activities of superoxide dismutase but lower malonaldehyde (MDA) content than those of control plants under salt stress. Meanwhile, a total of four haplotypes were found for the GmGAPDH14 gene, and haplotypes 2, 3, and 4 were beneficial for the tolerance of soybean to salt stress. These results suggest that the GmGAPDH14 gene might be involved in the process of soybean tolerance to salt stress. The results of this study will be valuable in understanding the role of GAPDH genes in the abiotic stress response of soybean.

Research on the oncogenic role of the house-keeping gene GAPDH in human tumors.

As an internal reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis. While increasing evidence suggests that GAPDH plays a crucial role in tumorigenesis of some cancers, no systematic analysis of GAPDH has been conducted. Here, we sought to analyze the expression of GAPDH and its oncogenic processes in pan-cancer. GAPDH was investigated in The Cancer Genome Atlas (TCGA) tumor types using several bioinformatic tools including Tumor IMmune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), University of ALabama at Birmingham CANcer (UALCAN), cBio Cancer Genomics Portal (cBioPortal), and Search Tool for Recurring Instances of Neighbouring Genes (STRING) for the expression and relationships with prognosis and immune infiltration separately. Through our analysis, we measured the higher expression of GAPDH across the majority of TCGA tumors. GAPDH overexpression predicts poor survival in patients with tumors expressing a high level of GAPDH. Moreover, the genetic changes in GAPDH contributed to an increased mRNA expression. Additionally, GAPDH expression was negatively correlated with immune infiltration involving cancer-associated fibroblasts, neutrophil cell and endothelial. The house-keeping gene GAPDH might be a promising biomarker for pan-cancer prognosis. And GAPDH is not suitable as an internal reference gene for most cancer research, whether RNA or protein analyses.

First report of Curvularia penniseti causing leaf blight of Bajra Napier hybrid grass in India.

Bajra Napier hybrid (Pennisetum glaucum x Pennisetum purpureum) is a perennial, high yielding grass and is widely grown for fodder in India. During August-2021, Bajra Napier hybrid germplasm line (NBN 15-2) showed severe leaf blight symptoms at ICAR-Indian Grassland and fodder research institute, Jhansi (25.527890 N, 78.5451400 E). Symptoms were initial irregular yellow spots on the leaf lamina, which later became brownish, coalesced and gave blighted appearance to the leaf surface. Disease severity recorded was 55 to 60 percent. To isolate the pathogen, 10 symptomatic leaf samples were cut into small pieces (~4 mm2), surface-sterilized with 70% ethanol for 30 seconds and rinsed with sterile water. Sterilized leaf pieces were transferred to potato dextrose agar (PDA) and incubated at 28°C for 7 days. Four similar fungal isolates (BNHCP-1 to BNHCP-4) were obtained from the affected portions. The colonies were grayish-brown with dark brown margins. Conidia were mostly clavate, elongated, straight or bent at the terminal cell, with 2-3 septa with dimensions of 17.5 to 30 μm × 10 to 12.5 μm (avg. 24 μm × 12 μm; n=40). The third cell from the base was broader and darker. These morphological characteristics were consistent with previous descriptions of Curvularia penniseti (Mitra) Boedijn (Ellis, 1971). To confirm the species, BNHCP-1 was chosen as representative isolate for further studies. Internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of isolate BNHCP-1 was amplified with primers ITS1F/ITS4R (White et al. 1990) and GDF/GDR (Templeton et al. 1992), and sequenced. The sequences were deposited in GenBank (ITS: OM073980; GAPDH: OM103702.2). BLASTn analysis showed 99.6% and 98% similarity of ITS and GAPDH gene respectively with GenBank accession numbers MH859833.1 (548 bp/550 bp) and MN688838.1 (130 bp/133 bp) of C. penniseti. A maximum-likelihood phylogenetic analysis based on concatenated sequences of ITS and GAPDH gene using MEGA X placed the isolate BNHCP-1 within a clade comprising C. penniseti. Pure culture of BNHCP-1 was deposited in National Agriculturally Important Microbial Culture Collection (NAIMCC), Maunath Bhanjan (Uttar Pradesh) with accession number NAIMCC-F-04251. For pathogenicity, root slips of Bajra Napier hybrid germplasm line NBN 15-2 were transplanted in pots (6 pots; 2 root slips per pot) and kept for fresh growth in a growth chamber at 25 0C for 21 days. Bajra-Napier hybrid plants were sprayed until runoff with conidial suspension (5 × 105 conidia/ml) made from 2-week old fungal colony grown on PDA petri dish. The pots were covered with plastic bag for 48 h to maintain humidity. Inoculated plants displayed small, brown, oval-shaped lesions within seven days on the lamina and edges of the leaf which later enlarged and gave blighted appearance to the leaf. Control plants were asymptomatic. The pathogen was re-isolated from the inoculated leaves and confirmed morphologically, fulfilling Koch's postulates. C. penniseti has been reported earlier from Pennisetum americanum, P. clandestinum, Sorghum and Triticum sp. from different parts of the world (Sivanesan, 1987). However, there is no report of C. penniseti in Bajra Napier hybrid. Thus, to the best of our knowledge, this is the first report of C. penniseti from Bajra-Napier hybrid grass in India. Further studies on economic impact of this disease on Bajra-Napier hybrid production and its presence on commercial cultivars are needed.

Brown leaf spot of Prunus serrulata Caused by Colletotrichum fioriniae in Sichuan, China.

Prunus serrulate Lindl is widely cultivated in urban areas of China. It is mainly used for wood cultivation and urban landscaping. In May 2021, new leaf spot disease was observed in Chengdu city (30°42' to 30°45'N, 103°51' to 104°7'E), with 69.3% disease incidence, which could inhibit leaf growth and reduce their biomass. A planting area of more than 1000 square meters was investigated. The diseased leaves were mostly concentrated in the lower position of plants, where the humidity was higher. The disease infected P. serrulata leaves and occurred in the field from March to October, with the highest incidence in early May. The typical symptoms initially appeared as brown necrotic lesions on the margin of the leaves. The lesions then enlarged gradually and developed into reddish brown spots, eventually coalescing into large irregular, necrotic lesions with dark brown margins. Finally, the diseased leaves withered and died. Conidiomata were not formed on the diseased tissue. Ten symptomatic leaves were collected from 5 different trees in the planting area. Infected tissues from ten samples were cut into small pieces of 3 × 3 mm. The infected tissues were surface-sterilized by 3% sodium hypochlorite and 75% ethanol respectively for 30s and 60s, and rinsed three times in sterile water. Then they were blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL), and incubated at 25°C for 4 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 67.5 to 78.6 mm. The colonies were initially white, cottony, then became light pink to misty rose at the center, and the reverse side of the colony turned dark red to red and had pale yellowish borders. The conidia were straight, smooth-walled, colorless, fusiform with acute ends, measuring 8.2 to 16.7 × 3.1 to 5.9 μm in size (n = 100). For molecular identification, the genomic DNA of the representative isolate RBWY202105 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], histone3 (HIS3) [CYLH3F/CYLH3R (Crous et al. 2004)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], actin (ACT) [ACT512F/ACT (Carbone & Kohn, 1999)], β-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: ON000436, HIS3: ON014581, CHS-1: ON014579, ACT: ON014583, TUB2: ON014582, and GAPDH: ON014580). BLAST results indicated that the ITS, HIS3, CHS-1, ACT, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P sequences at NCBI (GenBank MW497230 (561/582), MT740312 (415/415), KU736865 (258/258), MK680659 (246/246), MK967342 (757/757), and MW656269 (263/263)). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 4-year-old P. serrulata plants for pathogenicity test. Fifteen leaves of each plant were inoculated with spore suspensions on the leaves (600 μl per leaf). The same amount of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under a 16 h/8 h photoperiod and 67 to 78% relative humidity. After ten days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum fioriniae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis (The ITS, HIS3, TUB2, CHS-1, GAPDH and ACT genes). The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on P. serrulata caused by C. fioriniae in China. The identification of C. fioriniae could provide relevant information for taking management strategies and further research on the Prunus serrulata disease.

Re-engineering of bicistronic plasmid pGPD/IFN to construct fusion gene co-expressing Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita (Hamilton) and its in vitro functional analysis

Edwardsiella septicemia disease in the cultured Indian major carps is caused by the fish pathogen Edwardsiella tarda and it is preventable by DNA vaccination. Here, we tried to develop a bicistronic DNA vaccine pGPD/IFN expressing the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita. The vaccine showed high protective efficiency in our previous studies; however as a limitation of bicistronic construct the expression of gene cloned in second frame (B) is poor. To overcome this limitation we re-engineered the construct and designed a fusion gene co-expressing the GAPDH and IFN-γ genes as one frame with an aim to get the optimum expression of both the genes. For this purpose, a fusion insert comprising GAPDH and IFN-γ coding sequences was cloned in to pcDNA3.1(+) plasmid vector. The fusion genes' in vitro expression was confirmed in the striped snakehead fish cell line (SSN-1). Successful expression of the re-engineered fusion gene DNA vaccine in the cell line was achieved at 48h post-transfection, which was confirmed by amplifying the expression transcripts of GAPDH and IFN-γ genes. Thus, the study concludes that the re-engineered fusion vaccine pcGPD/IFN (pcDNA3.1(+) plasmid having fusion GPD/IFN) is functional and can be effectively utilized to vaccinate rohu (Labeo rohita) as it contains the species-specific immune gene (IFN-γ) as an adjuvant.

A comparative assessment of RNF38 and P53 genes expression in the sperm samples obtained from males with normozoospermia and asthenospermia: A case-control study.

Infertility is considered as a common problem appears in about 10-12% of couples in their reproductive ages. Ring finger protein 38 (RNF38) gene is a ubiquitin-protein ligase that can regulate Protein 53 (P53) and affect cellular motility. Considering the role of P53 on cellular motility and RNF38 on the regulation of P53, the present study aimed to assess the difference between RNF38 and P53 genes expression in normozoospermic and asthenospermic samples as a diagnostic biomarker in males. The present study was conducted among 21 asthenospermicsand 63 healthy individuals. First, the real-time polymerase chain reaction technique was applied to measure the expression level of the P53 and RNF38 genes extracted from sperm samples, and the glyceraldehyde-3phosphate dehydrogenase gene was selected as the reference gene. An increase and a decrease occurred in the level of P53 and RNF38 genes expressions in asthenospermic and normozoospermic samples, respectively. In addition, a significant difference was observed between increasing P53 gene expression (p 0.001), reducing RNF38 one, and decreasing sperm motility (p 0.001) in asthenospermic cells compared to that of normozoospermic ones. Based on the results, an increase in the expression of the P53 gene and a decrease in the expression of the RNF38 gene had a significant relationship with asthenospermia in men. Therefore, it is expected that an effective step should be adopted to diagnose the asthenospermia expression pattern by using these results.

Glyceraldehyde-3-phosphate dehydrogenase Gh_GAPDH9 is associated with drought resistance in Gossypium hirsutum.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the central enzyme of glycolysis and plays important regulatory roles in plant growth and development and responses to adverse stress conditions. However, studies on the characteristics and functions of cotton GAPDH family genes are still lacking. In this study, genome-wide identification of the cotton GAPDH gene family was performed, and the phylogeny, gene structures, promoter progenitors and expression profiles of upland cotton GAPDH gene family members were explored by bioinformatics analysis to highlight potential functions. The functions of GhGAPDH9 in response to drought stress were initially validated based on RNA-seq, qRT‒PCR, VIGS techniques and overexpression laying a foundation for further studies on the functions of GAPDH genes. This study is the first systematic analysis of the cotton GAPDH gene family, which contains a total of 84 GAPDH genes, among which upland cotton contains 27 members. Quantitative, phylogenetic and covariance analyses of the genes revealed that the GAPDH gene family has been conserved during the evolution of cotton. Promoter analysis revealed that most cis-acting elements were related to MeJA and ABA. Based on the identified promoter cis-acting elements and RNA-seq data, it was hypothesized that Gh_GAPDH9, Gh_GAPDH11, Gh_GAPDH19 and Gh_GAPDH21 are involved in the response of cotton to abiotic stress. The expression levels of the Gh_GAPDH9 gene in two drought-resistant and two drought-sensitive materials were analyzed by qRT‒PCR and found to be high early in the treatment period in the drought-resistant material. The silencing of Gh_GAPDH9 based on virus-induced gene silencing (VIGS) technology resulted in significant leaf wilting or whole-plant dieback in silenced plants after drought stress compared to the control. The content of-malondialdehyde (MDA) in cotton leaves was significantly increased, and the content of proline (Pro) and chlorophyll (Chl) was reduced. In addition, the leaf wilting and dryness of transgenic lines under drought stress were lower than those of wild-type Arabidopsis, indicating that Gh_GAPDH9 is a positive regulator of drought resistance. In conclusion, our results demonstrate that GAPDH genes play an important role in the response of cotton to abiotic stresses and provide preliminary validation of the function of the Gh_GAPDH9 gene under drought stress. These findings provide an important theoretical basis for further studies on the function of the Gh_GAPDH9 gene and the molecular mechanism of the drought response in cotton.

Detection of Anthracnose in Strawberry and Methods of Etiological Diagnosis

Anthracnose of strawberry caused by Colletotrichum fungi is a dangerous disease associated with serious damage in berry plantations. Colletotrichum nymphaeae, C. lineola, and C. godetiae have been found in Russian and international planting material of strawberry plants. The cultural and morphological characteristics are described for the isolates and nucleotide ITS1-5.8S-ITS2 sequences of fragments received are identified. It is shown that the fragments of glyceraldehyde 3-phosphate dehydrogenase and actin genes can be used to efficiently differentiate the C. lineola species from the closely related С. dematium. Two diagnostic test systems for acutatum complex identification are compared. The studied test systems do not demonstrate any false-positive results; the prepared set of С. acutatum complex-RT (ZAO Sintol) shows specificity only for the C. nymphaeae and C. fioriniae species and turned out to be nonspecific to the C. godetiae species included in the acutatum complex. The test system elaborated by Garrido et al. is found to be highly sensitive and specific to the target species of the acutatum complex.

Study of Pathogenesis Using Fluorescent Strain of Cordyceps farinosa Revealed Infection of Thitarodes armoricanus Larvae via Digestive Tract.

Cordyceps farinosa is often utilized as a biocontrol agent because of its wide host range, strong lethality, and safety for mammals. Artificial rearing of Thitarodes armoricanus larvae is a prerequisite for the artificial cultivation of Chinese cordyceps, and C. farinosa is the most lethal pathogenic fungus during the rearing process. However, the infection process of C. farinosa is still unclear. In this study, we cloned the promoter of the C. farinosa glyceraldehyde 3-phosphate dehydrogenase gene, constructed the EGFP expression cassette, and integrated it into the C. farinosa genome via Agrobacterium transformation. We obtained a fluorescent strain for better observation of the infection process. Using two different inoculation methods of the fluorescent strain, we observed the traditional infection process through the body surface as well as through the digestive tract via feeding. Both infection modes can lead to larval death and mummification. Our findings demonstrated that during the artificial rearing of T.&amp;nbsp;armoricanus, preventing C. farinosa pollution should be an important part of the disinfection of the rearing environment.

Selection and validation of reference genes for qPCR analysis of differentiation and maturation of THP-1 cells into M1 macrophage-like cells.

For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ), after which it differentiates and polarizes into proinflammatory M1-like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription-quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP-1 cells stimulated with LPS and IFN-γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), UBC (ubiquitin C), B2M (0β2-microbulin), ACTB (β-actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP-1 monocyte model system.