Gpd Gene Research Articles

First report of leaf spot of maize caused by Curvularia verruculosa in India.

Curvularia leaf spot affects maize plants worldwide and is commonly caused by Curvularia lunata, C. geniculata, and C. pallescens (Manzar et al. 2022; Manzar et al. 2021; Choudhary et al. 2011). In February 2017, leaf spot symptoms were observed in a Deogaon, (25.74 N, 82.99 E) in Uttar Pradesh, India, with disease incidence of less than 10% of the plants in maize fields. On the leaves and sheaths, variously shaped yellow spots were developed. The spots were 2.5 mm in diameter and frequently grew larger, reaching a diameter of 1 cm. They were encircled by a chlorotic halo with dark borders. The symptomatic tissue showing leaf spots of 10 plants was taken and cut into pieces (4 mm2) then surface sterilized with 1% sodium hypochlorite for 1 min, and rinsed three times with distilled water. The cut leaf tissue was placed on the Petri plate containing potato dextrose agar medium amended with streptomycin sulfate (125 ppm). Then incubated at 25±2°C with a 12-h light and dark period, after 5 days of incubation, five pure cultures were obtained using the hyphal tip technique. The pure culture was incubated at 26±2°C for 10 days. The upper surface of the colony was dark grayish black with fluffy mycelia, and the reverse colony was dark brown. The conidia have three septa, are light brown to dark brown in color, straight to curved, ellipsoidal to fusiform, and have two bigger, darker central cells than terminal cells. On average, conidia are between 27.22 to 31.21 mm long and 10.61 to 12.62 mm wide (n=30). The morphological description is similar to the Curvularia verruculosa morphological traits described by Tandon & Bilgrami (Ellis 1966). Molecular identification was done in addition to supporting morphological identification. The nucleopore GDNA Fungus Kit (Genetix Brand, India) was used to extract the genomic DNA of the E40 isolate. The ITS rDNA region (White et al. 1990) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene (Berbee et al. 1999) were amplified through PCR(Manzar et al., 2022).The amplicons were bidirectional sequenced through the Sanger sequencing method. The similarity percentage of E40 isolate matched 100% with MH859788 (CBS444.70 ) of Curvularia verruculosa strain for ITS, and 100% with LT715824 (CBS150.63) of Curvularia verruculosa strain for gpd after Blastn analysis. The gene sequences were deposited to GenBank and accession no. OR262893 for ITS, and LC773704 for gpd were assigned. As a result, C. verruculosa was determined to be the presumed pathogen by both morphology and molecular characteristics. The pathogenicity of E40 isolate was performed twice by spraying (106 conidia/ml in sterile water) onto the leaves of 25 days old maize plant cv. Kanchan (n = 10). Uninoculated healthy maize plants (n=5) were sprayed only with autoclaved water. All pots are kept in a glass house at 25°C±2°C with 90% relative humidity. After 15 days of pathogen inoculation the foliar spots with chlorotic halo, enlarger upto 1cm, and from these spots the identical fungus was reisolated. The reisolated fungus showed similar morphological characteristics to C. verruculosa. Control plants showed no symptoms. C. verruculosa has been previously reported as a causative agent of leaf spot disease in Common beans (Wei et al., 2022), Cotton (Shirsath et al., 2018). To our knowledge, this is the first report of leaf blight caused by C. verruculosa on maize in India.

Endophytic Alternaria and Fusarium species associated to potato plants (Solanum tuberosum L.) in Iran and their capability to produce regulated and emerging mycotoxins

Endophytic fungi live inside virtually every plant species, without causing any apparent disease or damage to the host. Nevertheless, under particular conditions, mutualistic lifestyle of endophytes may change to pathogenic. In this study, the biodiversity of Alternaria and Fusarium species, the two most abundant endophytic fungi isolated from healthy potato plants in two climatically different regions of Iran, Ardebil in the north-west and Kerman in the south-east, was investigated. Seventy-five Fusarium strains and 83 Alternaria strains were molecularly characterized by multi-locus gene sequencing. Alternaria strains were characterized by the sequences of gpd and caM gene fragments and the phylogenetic tree was resolved in 3 well-separated clades. Seventy-three strains were included in the clade A, referred as Alternaria section, 6 strains were included in clade B, referred as Ulocladioides section, and 4 strains were included in clade C, referred as Infectoriae section. Fusarium strains, identified by sequencing the translation elongation factor 1α (tef1), β-tubulin (tub2) and internal transcribed spacer (ITS) genomic regions, were assigned to 13 species, viz. F. brachygibosum, F. clavum, F. equiseti, F. flocciferum, F. incarnatum, F. nirenbergiae, F. nygamai, F. oxysporum, F. proliferatum, F. redolens, F. sambucinum, F. solani and F. thapsinum. Twenty-six selected strains, representative of F. equiseti, F. nirenbergiae, F. oxysporum, F. nygamai, F. proliferatum, and F. sambucinum, were also tested for production of the mycotoxins deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), T-2 toxin (T-2), beauvericin (BEA), enniatins (ENNs), fumonisins (FBs), fusaric acid (FA) and moniliformin (MON). None of the tested strains produced trichothecene toxins (DON, NIV, DAS and T-2). Two out of 2 F. equiseti isolates, 1/6 F. oxysporum, 1/3 F. proliferatum, and 1/9 F. nygamai did not produce any of the tested toxins; the rest of strains produced one or more BEA, ENNs, FBs, FA and MON toxins. The most toxigenic strain, F. nygamai ITEM-19012, produced the highest quantities of FBs (7946, 4693 and 4333 μg/g of B1, B2, and B3 respectively), along with the highest quantities of both BEA (4190 μg/g) and MON (538 μg/g). These findings suggest that contamination of potato tubers with mycotoxins in the field or at post-harvest, due to a change in lifestyle of endophytic microflora, should be carefully considered and furtherly investigated.

First Report of Exserohilum rostratum Causing Leaf Blight on Broccoli (Brassica oleracea var. italica) in China.

Broccoli (Brassica oleracea var. italica) is not only an important crop worldwide with a large amount of production and consumption annually, but also rich in biologically active compounds (Surh et al., 2021). In November 2022, an unknown leaf blight was observed in the Broccoli planting area, Wenzhou City of Zhejiang Province (28.05 °N, 120.31 °E). Symptoms initially occurred at the leaf margin with yellow to gray lesions that were irregular and wilting. Approximately 10% of the surveyed plants were affected. To determine the pathogen, leaves with blight were collected randomly from five B. oleracea plants. Tissue blocks (3×3 mm) from diseased leaf portions were disinfected with 75% ethanol, rinsed three times with sterilized water, placed aseptically onto potato dextrose agar (PDA) medium, and incubated for 5 days at 28℃ in darkness. Seven fungal isolates with the same morphology were obtained using the spore method. The observed colonies were circular, taupe, pewter in color with light gray edging and many cottony aerial mycelia. Conidia were straight, curved or slightly bent, ellipsoidal to fusiform, and septate (typically 4-8 septa per conidium), with the size of 50.0-90.0 μm × 10.0-20.0 μm (n=30). The conidia had a slightly protruding and truncate hilum. These morphological characteristics were consistent with Exserohilum rostratum (Sharma et al., 2014). To further identify the pathogen, isolate WZU-XLH1 was chosen as a representative and the internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase-like (GAPDH) gene were amplified and sequenced using primer pairs ITS1/ITS4 (White et al., 1990) and Gpd1/Gpd2 (Berbee et al., 1999), respectively. The ITS and gpd gene sequences of isolate WZU-XLH1 were deposited in the GenBank database with accession numbers OQ750113 and OQ714500, respectively. BLASTn analysis showed matches of 568/571 (MH859108) and 547/547 (LT882549) with Exserohilum rostratum CBS 188.68. A neighbor-joining phylogenetic tree was constructed by combining the two sequenced loci, this isolate in the E. rostratum species complex clade at 71% bootstrap support.To verify the pathogenicity of the isolate, ten healthy Broccoli (cultivar 'You Xiu') seedlings with at least five leaves were divided into two groups: one group was inoculated with the isolate, while the other group served as a control. After surface disinfection with 75% ethanol and wiping with sterile water, tiny wounds were made on two leaves (two wounds in one leaf) using an inoculation needle. Fungal culture plugs cut from the isolate were placed on the wounds, while sterile PDA plugs served as the control. The leaves were sealed in wet airtight bags to retain moisture at room temperature with natural light (Cao et al., 2022). After five days, all leaves inoculated with isolate WZU-XLH1 showed symptoms identical to those observed in the field, with no symptoms present in the control group. The pathogenicity was confirmed by repeating the test in triplicate, and fungi re-isolated from symptomatic leaves were identified as E. rostratum by the morphological and molecular methods described above. To the best of our knowledge, this is the first report of E. rostratum causing leaf blight on broccoli in China. This study contributes to our understanding of B. oleracea leaf blight and establishes a basis for future studies on E. rostratum to develop management strategies.

First report of leaf blight on Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa caused by Alternaria alternata in China.

Ligusticum jeholense (Nakai et Kitagawa) Nakai et Kitagawa is one of the sources of Chinese herb "Gao-Ben". It is widely distributed in the Northeastern China. L. jeholense has antipyretic, antibacterial and anti-inflammatory effects (Zhang et al. 2021). In September 2021, a serious leaf blight was found in a 1.2 ha plantation of L. jeholense in Harbin, Heilongjiang Province, and the incidence was about 85%. The foliar symptoms were grayish-brown lesions, surrounded by a yellow margin at the edge of the leaf. In serious cases, the lesions extended into the middle of the leaf, and finally the whole leaf withered. A total of 12 samples (5×5mm) from symptomatic and healthy junction of 12 infected leaves from 6 different plants of L. jeholense with typical symptoms were cut and surface disinfected in 75% ethanol, and with 7% NaClO for 1 min, then rinsed three times with sterilized water. These tissues were placed onto Potato dextrose agar (PDA) plates at 28℃ in the dark. The colonies cultured for 7 days were obtained and transferred onto new PDA and potato carrot agar (PCA) plates by single spore method to further purify. After 7 days, the colonies on PDA were 63 to 75 mm in diameter, circular, grayish, with white aerial hyphae on the edge, the back of the colonies were grayish green. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear, 2 to 6 transverse septa, 0 to 3 longitudinal or oblique septa, 16.5 to 67.5 × 8.5 to 20.5 μm. The beaks were conical or cylindrical, 2.5 to 25.3 × 2.0 to 3.0 μm. Conidiophores were grayish brown, erect or bent, separated, 57.0 to 137.0 × 5.1 to 13.7 μm. Morphological characteristic showed the 12 isolates were the same fungus and similar to Alternaria sp. (Simmons 2007). Two typical strains (LGB and LGB2) from twelve isolates were randomly selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates on PDA by modified CTAB method, and internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit (RPB2) and Alternaria major allergen (Alt a 1), translation elongation factor 1-alpha (TEF) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified and sequenced with the primers ITS1/ITS4, RPB2-5F2/RPB2-7CR, Alt-F /Alt-R, TEF-F/TEF-R and gpd-F/gpd-R (Woudenberg et al. 2015). The obtained sequences were deposited in GenBank (ITS: OM319506, OM943431; RPB2: OM393721, OM984854; Alt a 1: OM649816, OM984853; TEF: OM238108, OM984852; gpd: OM296228, OM984851). The phylogenetic analysis of maximum-likelihood tree by MEGA7 showed the LGB and LGB2 had 100% identity with A. alternata CBS 916.96. For pathogenicity test, conidial suspension (1 × 106 spores/mL) of the strain LGB and LGB2 was sprayed on 10 healthy 40-day-old L. jenholense plants and five plants with sterile water as control. The plants were incubated at 25℃. After 28 days, grayish withering appeared on the leaves. The test was repeated three times. The same fungi were re-isolated from the inoculated leaves and with the same morphological and molecular characteristics as A. alternata, fulfill the Koch's postulates. No symptoms and fungi were found in the control group. This is the first report of leaf blight on L. jenholense caused by A. alternata. Leaf blight could reduce the yields of L. jenholense. This study provides a reference for the prevention and treatment to the leaf blight of L. jenholense.

First report of leaf spot disease caused by Alternaria alternata on Lonicera caerulea L. in China.

Relatively few studies investigating plant diseases in blue honeysuckle (Lonicera caerulea L.) have been conducted in China. In September 2021, a leaf spot disease was observed on approximately 10% of blue honeysuckle 'Lanjingling' leaves in a 0.02 ha field plantation in Tiekuang Street (124.36°E, 40.12°N) in Dandong City, Liaoning Province, China. The main symptom consisted of leaf spots with black mildew centers typically surrounded by yellow halos. Small pieces (3-4 mm) of the infected leaves were plated onto potato dextrose agar (PDA) medium as described by Wang et al. (2020) and six purified cultures were obtained through single spore culture on PDA. The observed conidia, consistent with the morphology of Alternaria alternata, were obpyriform and dark brown, measuring 5.8 to 15.3 μm × 7.9 to 42.5 μm, with 1-6 transverse septa and 0-3 longitudinal septa (n = 50) (Simmons 2007). For molecular verification, genomic DNA was extracted from a representative isolate LD-8. The ITS (GenBank OL454815), GPD (GenBank OL601993), TEF (GenBank OL538256), RPB2 (GenBank OL601966), and Alt (GenBank OL538257) genes were partially amplified with the respective primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Woudenberg et al. 2015), EFl-728F/EFI-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-7CR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005). BLAST analysis revealed that these genes shared 99%-100% identity with OK345332, MK451977, MN756011, KU933459, and MN655781, respectively. A greenhouse experiment was conducted using six, healthy two-year-old blue honeysuckle 'Lanjingling' plants to observe disease development (Mirzwa-Mróz et al. 2018). After 10 d, we noted typical leaf spot symptoms on inoculated leaves sprayed with a conidial suspension (106 spores/mL) while no symptoms were detected on uninoculated leaves. The same isolate, reisolated from infected leaves with the same morphological and molecular traits, was also identified as A. alternata, confirming Koch's postulates. The fungus was previously reported in cockscomb plants in Heilongjiang Province, China (Wang et al. 2020). To our knowledge, this is the first report of leaf spot disease caused by A. alternata in blue honeysuckle grown in China. This study will provide a basis for future development of effective protection strategies against blue honeysuckle leaf spot in China.

Phylogenetic Analysis and Toxigenic Profile of Alternaria Species Isolated from Chickpeas (Cicer arietinum) in Argentina

Chickpeas are a very important legume due to their nutritional richness and high protein content and they are used as food for humans and as fodder for livestock. However, they are susceptible to fungal infections and mycotoxin contamination. The Alternaria genus was among the main fungi isolated from chickpea samples in Argentina. The species within this genus are able to produce several mycotoxins such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA). So, the objectives of this study were to identify the Alternaria spp. found in the chickpea samples and to determine their toxigenic potential in vitro. A phylogenetic analysis of 32 Alternaria strains was carried out based on the combined sequences of the tef1, gpd, and Alt a1 genes. All Alternaria strains clustered into the section Alternaria and were identified as A. alternata and A. arborescens. Further, the toxigenic profile of each strain was determined in a ground rice–corn steep liquor medium and analysed by HPLC. Most strains were able to co-produce AOH, AME, and TA. These results indicate a potential risk for human health when consuming chickpeas since this legume could be contaminated with Alternaria and its mycotoxins, which are not yet regulated in food.

S3.4e Unraveling the role of DOG genes in a novel alternative pathway of glycerol biosynthesis in Candida albicans and its influence on virulence

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMDOG genes, encoding for 2-deoxyglucose-6-phosphate phosphatase are low molecular weight phosphatases, with an unknown biological function. In contrast to Saccharomyces cerevisiae which has two DOG homologs, C. albicans only has one DOG gene. We hypothesized that DOG1 plays an important role under osmotic or ionic stress by biosynthesizing glycerol, which is known to be vital for biofilm formation and virulence of this pathogenic yeast, via a novel alternative pathway. The known classical pathway of glycerol production begins when the glycolytic intermediate molecule dihydroxyacetone phosphate (DHAP) is converted into glycerol-3-phosphate (G-3-P) by a pair of glycerol-3-phosphate-dehydrogenases, Gpd1 and Gpd2. G-3-P is further dephosphorylated into glycerol by glycerol-3-phosphate-phosphatases, Gpp1 and Gpp2. However, an alternative pathway, where DHAP is dephosphorylated into DHA, which is subsequently converted into glycerol has been proposed, but the enzymes involved in this process have not yet been described. We recently showed that in Saccharomyces cerevisiae, the DOG enzymes are involved in the production of DHA from DHAP, thereby allowing the synthesis of glycerol in the absence of the classical pathway. Overexpression of the DOG genes restored the osmo-tolerance of the gpp1Δ gpp2Δand gpd1Δ gpd2Δ double deletion strains. Furthermore, purified DOG enzymes could also convert DHAP into DHA (Awasthy et al., submitted). Since DOG1 has a potential role in biosynthesizing glycerol via an unconventional route, we are interested to determine its contribution in influencing virulence and biofilm formation in Candida albicans. This putative pathway has been overlooked for the past two decades, leaving behind an evident knowledge gap. We have now generated multiple deletion strains, using CRISPR Cas9, for the C. albicans counterparts of the GPP, GPD, and DOG genes as well as multiple DOG1 overexpression strains in which we observed the restoration of osmotic stress tolerance phenotypically and via growth curves. We also have NMR data showing the accumulation of various metabolites of central metabolism in these strains. Additionally, we have determined the possible role of DOG1 on virulence factors like morphogenesis, adhesion, and biofilm formation in vitro as well as in vivo, the latter with our catheter-based biofilm subcutaneous mouse model system. We also linked DOG1 and its role in glycerol biosynthesis to the survival of C. albicans inside macrophages. Finally, we would be setting up a high throughput small compound screening for this phosphatase as a potential antifungal drug target.

First Report of Stemphylium lycopersici Causing Leaf Spot on Polygonatum kingianum in China.

Polygonatum kingianum, a member of the Liliaceae, is valued in traditional medicine and as a vegetable food crop. In July 2019, more than 50% of P. kingianum growth was suppressed in several field nurseries in Simao, Mojiang, Jingdong and Lancang County, Puer City, China. At the early stage of infection, symptoms manifested as a small circular brown spot. As the lesion matured, the spot gradually enlarged, forming an oval to irregular lesion with reddish-brown and dark green borders. In serious cases, the leaves were withered, and became brittle with cracks. The infected plants were collected from six major fields. The tissues of diseased leaves were soaked in 75% ethanol for 10 s, 0.1% mercuric chloride for 2 min, rinsed with sterilized water, and placed on potato dextrose agar (PDA) at 25℃ for 7 days. On PDA, four strains were isolated, and the colony was gray to dark yellowish-brown, flocculent, regular with concentric growth rings. Strain PKLS06 produced a dark red to brown pigment in the agar medium. On lesions, the conidiophores were solitary or in fascicles, straight or slightly curved, brown, with a conical apex, with three to five septa. The conidiogenous cells were pale-brown and swollen at the apex. On PDA, spores were solitary, oblong, bluntly rounded or sometimes with a point at the apex, with two to five transverse septa and one to two longitudinal septa with contractions at the main transverse septum. Morphological characteristics were consistent with published descriptions of Stemphylium lycopersici (Kee et al. 2017; Xie et al. 2018). For molecular identification, rDNA internal transcribed spacer (ITS) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified and sequenced (ITS accessions: MW243098, MW243099, MW243100, MW243101; gpd accessions: MW246803, MW246804, MW246805, MW246806) using published primers (White et al. 1990; Câmara et al. 2002). A phylogenetic tree was developed by Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). These four isolates fall into the S. lycopersici clade with strong support and all isolates were distinguished clearly from other species. Pathogenicity tests were performed using these four isolates. Each isolate was cultured on PDA and shake-cultured in V-8 juice broth (Nasehi et al. 2014). Conidia were resuspended in sterilized water (1×106 conidia/mL) and inoculated on intact leaves with injury of 1-year-old P. kingianum. The plants were incubated at 25℃ with a 12 h photoperiod and 90% humidity. A small spot began to appear after 3 days, and symptoms were similar to the those observed in the nursery after 10 days. Interestingly, the pathogenicity of strain PKLS06 was relatively weaker. Control plants treated with sterile water showed no disease symptoms. Re-isolated strains had the same morphological characteristics and the same ITS and gpd sequences as the original isolates, thus fulfilling Koch's postulates. S. lycopersici, an important pathogen, is widely distributed, and can cause a variety of plant diseases. It is noteworthy that the disease was observed on a plant in the Liliaceae, expanding the host range of S. lycopersici, which previously was reported to primarily infect plants in the Solanaceae. Based on the results presented above, P. kingianum is a new host plant of S. lycopersici in China. This disease is a threat for nursery production of P. kingianum, leading to a reduction in yields and economic losses. References Kee, Y. J., et al. 2017. Plant Disease 102 (2): 445-446 Xie, X. W., et al. 2019. Canadian Journal of Plant Pathology-Revue Canadienne De Phytopathologie 41 (1): 124-128 White, T. J., et al. 1990. PCR Protocols: A Guide to Methods and Applications PCR Protocols: A Guide to Methods and Applications 18: 315-322 Câmara M. P. S., et al. 2002. Mycologia 94 (4): 660-672 Nasehi A., et al. 2014. Archives of Phytopathology & Plant Protection, 47 (14): 1658-1665.

First Report of Brown Leaf Spot of Rice (Oryza sativa) Caused by Bipolaris sorokiniana in Pakistan.

Brown leaf spot of rice is one of the major seed-borne diseases and can diminish grain production up to 52% (Barnwal et al. 2013). In 2018, infected leaf samples showing the typical symptoms of brown spots were collected from the vicinity of the University of Agriculture, Faisalabad (31°26'10.3"N 73°03'35.1"E). The symptoms were brown-dark spots, with gray-light gray or brown centers surrounded by dark margins and with chlorotic halos and of oval or cylindrical shapes (5 to 9 mm in diameter). Disease incidence averaged 61% across the seven fields observed. Leaves were collected from the seven infected fields and symptomatic leaf tissues of 5 mm2 were excised from representative necrotic spots in each. These tissues were surface disinfected with 70% ethanol, rinsed with sterile distilled water (SDW), dried by blotting on paper, and placed on potato dextrose agar medium. For pathogen growth, the plates were placed at 25oC (±2oC) with a 12-hour photoperiod for 5 days. Five samples from each of the infected fields were taken for pathogen isolation and among them ten isolates were sub-cultured and purified by using the single spore method. The resulting fungal colonies were fluffy and ranged in color from grayish black/black to light brown. Fifteen conidia were measured that are olivaceous-brown to dark brown in color, elliptical to oblong with narrow (tapered) ends, with 3-10 septa and 35.6-65.4 µm in length x 13.1-25.7 µm in width. Conidiophores were yellowish-brown, geniculate, and solitary (Pratt 2003). For molecular studies, rDNA of the internal transcribed spacer (ITS) region, translation elongation factor (tef), RNA polymerase II second largest subunit (rpb2) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified by using the primers ITS1F/ITS4R (White et al. 1990), EF1-983F/EF1-2218R (Rehner and Buckley 2005), 5F2/7CR (O'Donnell et al. 2007), and GPD1/GPD2 (Berbee et al. 1999) respectively. The sequence of all the amplified gene regions of one SUL-1 isolate was deposited into GenBank with accession numbers MN314844 (ITS), MN326866 (tef), MN990457 (rpb2) and MN990456 (gpd). BLASTn queries of the obtained sequences (ITS, tef, rpb2 and gpd) showed 99-100% homology with the corresponding nucleotide sequences of B. sorokiniana (GenBank accession nos. GU480767, MF490855, LT715652 and MK558818 respectively). To fulfill the Koch's postulates, twenty rice plants (cv. Basmati-385) were sprayed at 2 to 3 leaf stages by using the two representative isolates with a spore suspension of 105 spores/ml. SDW was sprayed on ten control plants. The plants were covered with polyethylene bags to keep the moisture contents and incubated at 25oC (±2oC) for 7 days. After a week, same symptoms as those described above were observed. In the repeated experiment, B. sorokiniana was re-isolated from the infected rice leaves and confirmed morphologically; fulfill the Koch's postulates. With grave worry, the other species of the genus Bipolaris (B. oryzae, and B. victoriae) have also been found to the cause brown leaf spot of rice (Motlagh and Kaviani 2008). To our knowledge, this is the first report of Bipolaris sorokiniana causing brown leaf spot of rice in Pakistan. Because rice is highly consumable grain in Pakistan, so the rapid spread of this disease in the rice farming areas is of a serious concern.

First Report of Exserohilum rostratum Causing Leaf Spot on Maize (Zea mays) in Henan, China.

In August to September of 2019 and 2020, a leaf spot was observed on maize at growth stages R1 through R5 in Henan. Leaf samples (N = 63) were collected from 152 fields from 50 counties. Symptomatic plants had light-yellow to brown elliptic lesions (0.6 to 5.0 cm × 0.2 to 0.8 cm) with gray. Incidence rates were 1 to 15% in individual fields. Symptomatic leaf samples (3 to 5 cm2) were surface sterilized (75% ethanol, 30 s; 3% NaClO, 2 min), rinsed three times in sterile distilled water, and allowed to dry. Small sections from the samples were placed on potato dextrose agar (PDA) medium and incubated at 25°C for 4 days. Observed colonies were circular, taupe, pewter in color with light gray edging and many cottony aerial mycelia. Single spores (N = 45) were inoculated on water agar medium with wheat straw (TWA + W). Conidiophores on wheat straw were single, erect or slightly curved, in clusters, septate, sympodial, geniculate and 5 to 7 μm thick. The basal cell was slightly swollen and was dark brown. The basal septum was darker and thicker than other septa, with a distinctly protruding truncate hilum. The conidia belonged to two types (Lin et al. 2011). Type A were fusiform, cylindrical, pyriform to oval, dark brown, 2 to 7 septate, 16.5 to 70.5 µm × 10.2 to 24.7 µm (N = 50). Type C were straight or slightly bent, ellipsoidal or long ellipsoidal, light brown, 6 to 13 septate, 36.3 to 106.8 µm × 10.5 to 16.0 µm (N = 50). All single spore isolates were identified morphologically as Exserohilum rostratum (Drechs.) Leonard & Suggs (Ahmadpour et al. 2013; Sivanesan et al. 1987). Four isolates (ML154-1, ML300-2, ML598-4 and ML626-1) were arbitrarily selected for molecular identification and inoculation testing. The internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase-like (gpd) gene were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990) and Gpd1/Gpd2 (Berbee et al. 1999), respectively. Sequences MW440488, MW440489, MW440491 and MW440492 of ITS and MW448214, MW448215, MW448217 and MW448218 of gpd were submitted to GenBank. BLASTn analysis revealed a high match (ITS: 598/600 bp; gpd: 527/527 bp) to E. rostratum isolate BRIP11422 (ITS: LT837464; gpd: LT883541). A phylogenetic tree was constructed based on concatenated sequences of ITS and gpd gene in MEGA-X. All four isolates formed a single clade with E. rostratum reference isolates CBS732.96, BRIP11422 and BRIP11416. To test pathogenicity in the greenhouse, a conidial suspension of each of the four strains (1 × 104 conidia/ml) was sprayed on the leaves of the common maize hybrids, Zhengdan 958 and Xianyu 335 (V6- V7). Control plants were sprayed with sterile water. Plants were incubated in plastic bags to retain high relative humidity for 24 h at 25~27°C. Three days after inoculation, the leaves began to show symptoms, and 7 days later, symptoms of the leaves were identical to those observed in the field. Control plants developed no symptoms. The pathogen was re-isolated from the infected leaves and was confirmed to be E. rostratum. The first E. rostratum isolate was isolated from the leaves of Eragrostis in China (Jiang et al. 1959). Thereafter E. rostratum has been reported as the causal agent of leaf spot in Ananas comosus (He et al. 2011), Musa paradisiacal (Luo et al. 2012), Hevea brasiliensis (Zhang et al. 2016) and many gramineous grass weeds (Liu et al. 2016) in China. To our knowledge, this is the first report of E. rostratum causing leaf spot disease on maize in Henan, China. Since this is an economically important pathogen of maize throughout the world, disease management strategies are likely to be needed.

First Report of Maize Ear Rot Caused by Exserohilum rostratum in Hainan Province in Southern China.

Maize (Zea mays L.) is one of three major grain crops in China, with production reaching 261 million tons in 2019(NBS, 2020). Some fungi cause maize ear rot which lead to significant yield and quality losses. In 2016, about 5% of maize ears were dark brown and covered with a white mould in seed production fields in Lingshui, Hainan Province, China. These ears were brought back to the laboratory for analysis. Molded kernels were surface sterilized in 75% ethanol for 3 min and in 10% sodium hypochlorite for 3 min, subsequently rinsed three times in sterile-distilled water, placed onto potato dextrose agar (PDA), and incubated at 28°C in the dark for 3 days. mycelia tips grown from kernels were transferred into fresh PDA plates. Seven fungal isolates with similar morphology characteristics were obtained, and three of them were identified by morphology and molecular identification. The colonies grew rapidly. The aerial mycelia turned white to black with age. Conidia were straight to slightly curved, oval, pyriform or geniculate, brown to dark brown, and had 2 to 7 septa, with both basal and caudal septa thicker and darker than others, 39.47 to 78.66 ×13.96 to 22.78 μm, with a distinctly protruding hilum swelled from the basal cell. Conidiophores were dark brown, with geniculate tip and many septa. For molecular identification, genomic DNA of isolate was extracted from mycelia. The internal transcribed spacer (ITS), 1,3,8-trihydroxynaphthalene reductase (Brn) and glyceraldehyde-3-phosphate dehydrogenase-like (Gpd) genes were amplified with primers ITS1/ITS4 (White et al. 1990), Brn01/Brn02 (Shimizu et al. 1998) and gpd1/gpd 2 (Berbee et al. 1999) , respectively. BLASTn analysis showed that high identities with Exserohilum rostratum (ITS, LT837845.1, 100%; Brn, AY621165.1, 99.87%; Gpd, LT882543.1, 99.75%). Sequences of ITS, Brn and Gpd were deposited in GenBank with accession numbers MW362495, MW363953 and MW363954, respectively. Based on morphological characteristics and molecular analysis, the isolate was identified as E. rostratum (Hernández-Restrepo et al. 2018). Koch's postulates were completed by using ears of maize inbred line Huangzaosi and Chang7-2 growing in the experimental field of Baoding, Hebei Province. Three days post-silk emergence, each of the four maize ears was injected with 2 ml conidial suspension (1×106 conidia/ml) of isolate ZBSF005 through the silk channel. In the control groups, three ears were inoculated with an equal amount of sterile-distilled water. The inoculated ears grew under natural conditions for 30 days, the diseased kernels and ear tips were black brown and the surface covered with white or gray black mildew layer. The kernels with severe infection were wizened. But the bract could not be infected by the pathogen. Meanwhile, the control remained asymptomatic. The same fungus was successfully re-isolated from the inoculated kernels, and its identity was confirmed by morphological and molecular biology approaches, thus fulfilling Koch's postulates. E. rostratum has been reported to cause leaf spots in a wide range of hosts, such as Calathea picturata, Lagenaria siceraria, Saccharum officinarum, Ananas comosus, Hevea brasiliensis, Zea mays and so on (Chern et al. 2011; Ahmadpour et al. 2013; Choudhary et al. 2018), and it was also reported to cause root rot in Lactuca saliva (Saad et al. 2019). To our knowledge, this is the first report of E. rostratum causing maize ear rot in China. The pathogen was simultaneously inoculated to 8 maize inbred lines in Hebei province, but the disease only occurred in some varieties and the incidence area was large. Therefore, attention should be paid to the prevention and treatment of ear rot caused by this pathogen in the breeding process.

Phylogenetic evaluation of Bipolaris and Curvularia species collected from turfgrasses

AbstractThe dothideomycete genera Bipolaris and Curvularia are important pathogens of grasses (Poaceae) and can associate with host tissues without disease symptoms. Species of both genera are responsible for leaf spot epidemics that commonly occur on turfgrass throughout the United States transition zone and southern regions. Recent species typifications with DNA sequences have made a broad examination of Bipolaris and Curvularia species obtained from turfgrass possible. Isolates for analysis were obtained from culture collections or isolated from symptomatic turfgrass samples. Phylogenies based on ITS, GPD, and TEF gene sequences were constructed using maximum likelihood and Bayesian analyses and used to determine intraspecific and intrageneric relationships among Bipolaris and Curvularia species isolated from turfgrasses. Similar to previous studies, Bipolaris and Curvularia species formed separate, well‐supported clades. Morphological identifications of genera and species based on published host‐based keys often disagreed with molecular groupings. For example, C. hawaiiensis (= B. hawaiiensis) could not be differentiated from B. cynodontis while C. australiensis (= B. australiensis) could not be differentiated from C. spicifera (= B. spicifera). Additionally, phylogenies suggest B. coffeana and B. cynodontis are synonymous. However, the number of pseudosepta per conidium was the only consistent morphological character. The delineation of species causing leaf spot diseases of warm‐season turfgrasses should improve disease management decisions and help identify potential pathogens in asymptomatic tissues.

Cloning and functional characterization of gpd and α-tubulin promoters from Annulohypoxylon stygium, a companion fungus of Tremella fuciformis

Abstract Annulohypoxylon stygium is an ascomycete that helps Tremella fuciformis produce the fruiting body in wild state or artificial cultivation. Although the interaction between these two fungi is well known, the underlying molecular mechanism is limited. In this study, the 981 bp and 886 bp promoter sequences of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and α-tubulin gene have been cloned, respectively. Sequence analysis showed that gpd promoter contained nine CAAT boxes, and single TGACG-motif, TATA box, ABRE motif, STRE motif, MYB motif, and W box. The α-tubulin promoter included eight CAAT boxes, three STRE, two TATA boxes and MYB, single Box 4, CAT-box, CCAAT-box, TGA-element, and ABRE. Subsequently, we evaluated the promoters' function by constructing four vectors pGEH, pGRH, pTEH, and pTRH to drive fused enhanced green fluorescent protein and hygromycin B phosphotransferase (egfp-hph) or red fluorescent protein and hygromycin B phosphotransferase (rfp-hph) expression under the control of gpd or α-tubulin promoters in A. stygium. The integration of the transformed DNA into A. stygium genome was verified by PCR, Southern blot, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). All the results indicated that the two promoters could drive egfp-hph and rfp-hph expression. This result could provide help in gene functional studies by using gpd and α-tubulin promoters to direct gene over-expression or build dual promoter silencing systems.

Ripening proteolysis profile of soft cheese enriched with Nigella sativa seed oil

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.

A family with X-linked recessive hypohidrotic ectodermal dysplasia due to EDA c.895G > A mutation

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.

Deletion of glycerol-3-phosphate dehydrogenase genes improved 2,3-butanediol production by reducing glycerol production in pyruvate decarboxylase-deficient Saccharomyces cerevisiae

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.

Differential expression of two gpd genes in the cultivated mushroom Pleurotus eryngii using RNA sequencing analysis

Abstract Transcriptional analysis showed that Pegpd1 was constitutively and highly expressed in various developmental stages of P. eryngii. The expression level of Pegpd2 was much lower than Pegpd1. The promoter of Pegpd1 contained the conserved cis-regulatory elements and could be used to drive heterologous gene expression in genetic transformation of P. eryngii.

Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora

A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.

First report of Stemphylium lycopersici causing leaf spot on hot pepper in China

Hot pepper is an exceedingly popular vegetable crop in China and is cultivated on 737 hectares in Beijing. A severe leaf disease with typical symptoms of spots with grey centres and dark brown borders was observed on hot pepper plants in Beijing in the winter of 2016. More than 50% of all plants in the region were infected. Fungal cultures were isolated from naturally infected leaf tissue, and identified as Stemphylium lycopersici based on morphological features, cultural characteristics and molecular identification by sequencing the ITS, gpd and cmdA genes. Pathogenicity was determined by inoculating healthy pepper plants with hyphal suspensions, and the fungus was re-isolated from developing lesions on the inoculated plants, thus fulfilling Koch’s postulates. This is the first report of natural infection by S. lycopersici causing leaf spot on hot pepper in China.

Cloning and Function Verification of gpd Promoter from Hypoxylon sp.

In order to construct the transformation system and study the gene function of Hypoxylon sp.,a highly active promoter element was necessary for the transformation system. In this experiment,the gpd gene and the sequence of about 1000 bp upstream of the gene were cloned by PCR. The results of Neural Network Promoter Prediction,Signal Scan Search Request and PLANTCAREA database analysis showed that the upstream region of the gpd gene contained not only the characteristic core required by the promoter,such as TATA-box,CAAT-box,G-box,GC-box,CT-rich,etc,but also contained a high score transcription start site. To further verify the promoter function of this sequence,the gpd promoter element was ligated with the enhanced green fluorescent protein gene(egfp)and hygromycin gene(hph)to construct a fusion expression vector,which was transformed by Agrobacterium tumefaciens-mediated transformation. Hygromycin resistance screening,green fluorescent protein detection results showed that the gpd promoter of Hypoxylon sp. successfully drove the expression of hygromycin resistance gene and enhanced green fluorescent protein gene,and Agrobacterium tumefaciens-mediated transformation was successful. It laid a foundation for the study of exogenous gene expression and gene function in Hypoxylon sp.