Identification of Colletotrichum spaethianum causing sugar beet leaf spot in North Dakota, USA

In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 × 18 μm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (O'Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and β-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (β-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (β-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. S. vesicarium spore suspensions (1 × 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in MN. Stemphylium sp. is a major problem of sugar beet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.

In southern Xinjiang, walnut (Juglans regia L.) is the primary source of income for farmers and a key crop in the region's specialty fruit and forestry industries. In September 2024, leaf spotting was observed in several walnut orchards in Aral, Xinjiang. In the early stages of the disease, small brown spots appeared on the leaf tips and margins, which later expanded irregularly across the entire leaf, eventually causing the leaves to wither and necrotize completely. Diseased leaves were collected from four walnut (Xinwen185) orchards in Aral (81.2997°E, 40.5583°N) for pathogen isolation. Leaf sections (4×4 mm) were excised from lesion margins and subjected to surface sterilization through sequential treatment with 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min. They were then rinsed eight times by immersion in 50 mL of sterile distilled water with gentle agitation for 30 s each time. After drying with sterile filter paper, the segments were transferred onto potato dextrose agar (PDA) plates and incubated at 28 ℃ for 5-7 days. Twelve single-spore strains were obtained through single-spore isolation technique. After 6 days of incubation, the isolates produced abundant aerial white mycelia and acquired a purple pigmentation. The hyphae were hyaline with septation. The isolates produced numerous oval unicellular microconidia without septa, 4.8 to 18.6 × 1.5 to 4.2 µm (n = 50) and very few macroconidia with three to four septa (21.6 to 46 × 3.1 to 4.3 µm [n = 30]), narrowed at both ends (Fig.S1). These morphological characteristics are consistent with those described for F. proliferatum (Leslie and Summerell, 2006). Genomic DNA was extracted from a representative isolate KY7 using the cetyltrimethylammonium bromide (CTAB) protocol. Four genetic loci were amplified and sequenced: the elongation factor 1-α (TEF1) gene, internal transcribed spacer (ITS) region, 28S large ribosomal subunit (LSU) rRNA gene, and calmodulin (CAL) gene, along with the second largest subunit of RNA polymerase II (RPB2) gene. Amplification was performed using the following primer pairs: TEF1: EF1-728F/EF1-986R, ITS: ITS1/ITS4, LSU: LR0R/LR5, CAL: CL1/CL2A, RPB2: fRPB2-5F/fRPB2-7Cr. The sequences of the isolate KY7, were deposited in GenBank under the following accession numbers: ITS (PV441515), TEF1 (PV524931), LSU (PV441477), CAL (PV524935), RPB2(PV524933). BLASTn analysis revealed that isolate KY7 showed 99.62% (ITS: EF453150.1), 98.84% (TEF1:KX656210.1), 99.89% (LSU: PP336543.1),99.73% (CAL: AF291057.1) and 99.66% (RPB2: MN193893.1) with F. proliferatum. A maximum likelihood phylogenetic tree was constructed based on concatenated sequences of EF-1α, CAL, and RPB2 using Fast tree 2.2 software. The isolate KY7 clustered within the same evolutionary clade as F. proliferatum. Based on morphological and molecular analyses, isolate KY7 was identified as F. proliferatum (Fig.S2) (Leslie and Summerell, 2006). Three-month-old walnut seedlings were selected for pathogenicity testing of isolate KY7. The leaves were surface-sterilized with 75% ethanol, rinsed three times with sterile water, and then wounded with a sterile needle. For the inoculation group, each plant was sprayed with 5 mL of KY7 spore suspension (1×106 spores/mL), while the control group was sprayed with an equal volume of sterile water. All plants were maintained at 28℃ with 80% relative humidity under a 12 h photoperiod. The experiment consisted of two independent trials, with each trial including five inoculated seedlings and five control seedlings (resulting in a total of ten inoculated and ten control plants). Ten days after inoculation, irregular brown spots consistent with field observations appeared on the inoculated leaves, whereas the control plants remained asymptomatic (Fig.S1). The same pathogen was re-isolated from the diseased leaves, fulfilling Koch's postulates. F. proliferatum has been reported to cause leaf blight in Phoenix dactylifera in Tunisia (Namsi et al., 2021) and leaf spot in Juglans regia in Hebei Province, China (Wang et al., 2022). In Xinjiang, characterized by arid conditions, low rainfall, and abundant sunlight (Chang et al., 2022), the pathogen's ability to infect walnuts has raised concerns among local farmers. In contrast, disease management practices in Hebei Province are likely more targeted. The identification of F. proliferatum in this study enables the development of tailored management strategies, providing a critical foundation for precise disease control. This will assist growers in selecting effective fungicides and formulating optimized application protocols. To our knowledge, this is the first report of F. proliferatum causing walnut leaf spot in Xinjiang, China.

In this study, foliar sprays of Fenton solutions (Fenton reaction, Fenton-like reaction and Fenton complex), titanium dioxide (TiO2) and the recommended fungicide (chlorothalonil) were estimated in the control of sugar beet leaf spot caused byCercospora beticolaunder field conditions in two growing seasons. In addition, the impacts of these treatments on some crop characters (leaf dry weight, root fresh weight, soluble solid content, sucrose content and purity of sugar) were examined. Biochemical and histological changes in the livers and kidneys of treated rats compared to an untreated control were utilized to assess the toxicity of the examined curative agents. Overall, chlorothalonil and Fenton complex were the most effective treatments for disease suppression in both tested seasons followed by Fenton-like reagent, Fenton’s reagent and TiO2, respectively. Growth and yield characters of treated sugar beet significantly increased in comparison to an untreated control. There were mild or no (biochemical and histological) changes in the livers and kidneys of treated rats compared to the control. Fenton solutions and TiO2may offer a new alternative for leaf spot control in sugar beet.

Requirement of the carboxy-terminal domain of RNA polymerase II for the transcriptional activation of chromosomal c-fos and hsp70A genes

As the first step in the analysis of the transcription process in the African trypanosome, Trypanosoma brucei, we have started to characterise the trypanosomal RNA polymerases. We have previously described the gene encoding the largest subunit of RNA polymerase II and found that two almost identical RNA polymerase II genes are encoded within the genome of T. brucei. Here we present the identification, cloning and sequence analysis of the gene encoding the largest subunit of RNA polymerase III. This gene contains a single open reading frame encoding a polypeptide with a Mr of 170 kD. In total, eight encoding a polypeptide with a Mr of 170 kD. In total, eight highly conserved regions with significant homology to those previously reported in other eukaryotic RNA polymerase largest subunits were identified. Some of these domains contain functional sites, which are conserved among all eukaryotic largest subunit genes analysed thus far. Since these domains make up a large part of each polypeptide, independent of the RNA polymerase class, these data strongly support the hypothesis that these domains provide a major part of the transcription machinery of the RNA polymerase complex. The additional domains which are uniquely present in the largest subunit of RNA polymerase I and II, respectively, two large hydrophylic insertions and a C-terminal extension, might be a determining factor in specific transcription of the gene classes.

Baseline Sensitivity of Fusarium Species Associated with Fusarium Diseases to Metconazole, Triticonazole, and Thiabendazole Fungicides

Agricultural industries in small geographical areas with limited acreage tend to be overlooked by those not associated with the growing region or industry. Sugarbeets continue to be produced in a relatively small geographic area and on relatively limited acreage in Minnesota and North Dakota. These factors, along with continued debate over policies affecting domestic sugar industries and recent industry expansions, help justify a continued assessment of the economic importance of the sugarbeet industry to the regional economy. Revenues from sugarbeet production and expenditures by processors to Minnesota and North Dakota entities in fiscal 2011 represented the direct economic impacts from the industry. Expenditure information was provided by sugarbeet processing and marketing cooperatives. Secondary economic impacts were estimated using input-output analysis. The sugarbeet industry, which included the growing regions and processing plants located in the Red River Valley of Minnesota and North Dakota and west central Minnesota planted 652,741 acres and processed 15.5 million tons of sugarbeets in fiscal 2011. Production and processing activities generated $1.7 billion in direct economic impacts. Gross business volume (direct and secondary effects) from the sugarbeet industry was estimated at $4.9 billion. Direct and secondary employment in the industry was 2,473 and 18,830 full-time equivalent jobs, respectively. The industry paid $15.4 million in property taxes and was estimated to generate another $105 million in sales and use, personal income, and corporate income taxes in Minnesota and North Dakota. In real terms, gross business volume of the sugarbeet industry in Minnesota and eastern North Dakota has increased 185 percent since 1987. Increases in business activity from the industry have resulted from increased production, processing, and marketing activities, as well as relatively high sugar prices during fiscal 2011.

Sugar beet is an economically important crop which is contributing 55% of the total sugar in the USA. In June 2018, irregular dark brown somewhat circular spots were observed on sugar beet leaves in Hickson, North Dakota. The symptoms covered approximately 5% on the lower leaves. Symptomatic leaf tissue were excised from the junction of diseased and healthy tissue. Small pieces (5 mm²) were surface sterilized with 10% sodium hypochlorite for 1 min, rinsed thrice with sterile distilled water, air dried and transferred to Potato Dextrose Agar (PDA), and incubated at 24°C with a 12-h photoperiod for 5 days. Dark-green velvety colony appeared in two weeks. Three isolates were developed by the single spore isolation technique. Conidia were club-shaped, two to four transverse septa, and pale brown, without any beak, often in chains (4 to 8 conidia) and or solitary. The dimension of conidia varied from 25-40x7-14 μm [1,2]. Based on the morphological characters, the fungus was tentatively identified as Alternaria species. Genomic Deoxyribonucleic Acids (gDNAs) were extracted from the culture generated from a single spore using Qiagen kit. ITS4/ITS5 were used to amplify the fragments of the Internal Transcribed Spacer (ITS) region. The amplified PCR products were cleaned and sent for Sanger sequencing by GenScript (GenScript, Piscataway, NJ). The sequences from GenScript were congruence to the reference sequence ID MT126620.1. The entire sequences were deposited at NCBI (GenBank accession nos. MK441717). Koch postulates were followed by spraying conidia suspension (5×105 conidia/ml) to 8-week age of 20 plants of and kept in humidity chamber at 28-30 °C, 80-85 % RH. Mock-inoculated seeds were also sown as a control. Three weeks of post inoculation, the similar irregular dark brown symptoms observed in twelve plants. No symptoms were found in the mock. The experiment was conducted twice. The fungus was reisolated from the diseased leaf tissue, as described above. Macroscopic and microscopic analysis indicated the similar dark-green colony and morphology, respectively. Molecular detection performed using the same ITS primers and sent for Sanger sequencing by GenScript, this study further confirmed that the isolate was similar to A. alternata [3]. Another close species of Alternaria was recently reported in sugar beet to cause leaf spot in Minnesota [4,5]. To our best knowledge, this is the first report of A. alternata causing leaf spot on sugar beet in North Dakota.

Triphenyltin hydroxide (TP'I H) has been used exten­ sively for control of Cercospora (Cercospora betiola) leaf spot of sugarbeet (Beta vulgaris) in Minnesota and North Dakota foll owing the development of benz­ imidazole resistant strains in t he early 19808. The discovery oftolerance to TPTH in 1994 prompted ex­ tensive sampling throughout the region in 1995 and 1996. In 1995, 60% of the leaf spots in the southern most district were tolerant to 0.2ppm TPTH and 42% tolerant to Ippm. By 1996 these frequencies had increased to 83 and 60%, respectively. More alarm­ ing than this increase in the southern district was the rapid increase in the occurrence of tolerance fu rther north where the disease is generally less severe and fungicide use is less. In four of the seven factory dis­ tricts the frequency of leaf spots tolerant to O.2ppm exceeded 35% and the frequency tolerant to 1 ppm was greater than 15%, in 1996. Resistance to thio­ phanate-methyl, a benzimidazole-type fungicide, per­ sisted in local populations even though TPTB has been the predominant fungicide for control of Cer­ cospora leaf spot for about 15 years.

Sugar beet leaf spot, caused by Cercospora beticola, is the most important and destructive foliar disease in North Nile Delta of Egypt. However, control of the disease in Egypt is mainly achieved by fungicide treatments. So, the control efficacy of two Sterol demethylation inhibitors (DMI) fungicides, tetraconazol (Eminent) and difenoconazole + propiconazole (Montoro), and one Multi-site activity (MSA) fungicide, benalaxyl + copper oxichloride (Galben), against C. beticola and their impact on sugar beet yield components were tested in this study. Fungicides were tested under natural field infection in four seasons (2008/2009, 2009/2010, 2011/2012 and 2012/2013) in commercial field at Sakha in Kafr El-Sheikh governorate. All the three fungicides suppressed Cercospora leaf spot significantly compared with untreated plots. However, there were significant differences in efficacy among them. The most effective fungicide was Eminint, which provided high levels of efficacy (from 95 to 96.5%) followed by Montoro which showed efficiency from 83 to 86%. Galben provided moderate control efficacy from 53 to 63%. Sprays with Eminint increased root yield, sucrose percentage and gross sucrose more than 90, 56 and 214 % respectively compared with the untreated plots. However, Montoro caused more than 70, 35 and 136% increases in yield respectively. Sprays with Galben provided less increases in yield components (up to 37, 30 and 80% respectively). Since fungicidal application considered as the main tool employed in sugar beet Cercospora leaf spot disease management in Egypt, the obtained results concluded that both DMI fungicides, Eminint and Montoro, were effective in controlling the disease. However, further studies are needed to determine the best application program to avoid appearance of DMI resistance strains of C. beticola.

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.

Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected sugar beet residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed a polymerase chain reaction (PCR) technique for rapid detection of C. beticola in field soils. Total DNA was first isolated from soil amended with C. beticola culture using the PowerSoil DNA Kit. The purified DNA was subjected to PCR in Extract-N-Amp PCR mix with CBACTIN primers over 35 cycles. The amplified products were resolved and compared by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from sugar beet fields near Sidney, MT and Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR and resolved as previously described. The amplicons were purified from the gels and subjected to BigDye Terminator Cycle sequencing. All sequences from field soils samples, C. beticola-amended field soil, and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. Rapid screening for the presence of C. beticola in the soil by PCR will improve research capabilities in biological control, disease forecasting, and management of this very important sugar beet pathogen.

The largest subunit of the RNA polymerase II of Trypanosomacruzi lacks the repeats in the carboxy-terminal domain and is encoded by several genes

Genomic and cDNA clones homologous to the RpII215 gene of Drosophila were isolated from Arabidopsis thaliana and assigned to a single copy gene encoding a transcript of 6.8 kb. Nucleotide sequence analysis of Arabidopsis genomic and cDNAs revealed a striking homology to yeast, Caenorhabditis, Drosophila and mouse genes encoding the largest subunit of RNA polymerase II. The Arabidopsis gene rpII215 contains 13 introns, 12 of which interrupt the coding sequence of a protein of 205 kDa. The position of the first intron is conserved between plant and animal genes, while an intron located in the 3' untranslated region of the rpII215 gene is unique to Arabidopsis. Common domains present in all known largest subunits of eucaryotic RNA polymerase II were identified in the predicted sequence of the Arabidopsis RpII215 protein. Both the order and the position of N-terminal Zn2+ finger and of DNA and alpha-amanitin binding motifs are conserved in Arabidopsis. The C-terminal region of the Arabidopsis protein contains 15 consensus and 26 variant YSPTSPS repeats (CTDs). Highly conserved structure among the various C-terminal domains suggests that the largest subunit of RNA polymerase II in plants may also interact with transcription factors and with protein kinases that control the cell cycle as in other organisms.