Genomic DNA Extraction Kit Research Articles

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

First Report of Leaf Spot Disease caused by Nigrospora oryzae on Nicotiana tabacum in China.

Tobacco (Nicotiana tabacum) is an important economic crop and widely cultivated in rural areas in south of China. A previously uncharacterized disease was observed on field-grown tobacco during 2020 and 2021 around Tongren city, Guizhou province of China (27°59'25.73" N, 108°7'2.43" E). The disease mainly occurred from fast growing period (about 13-16 leaves) to leaf maturity stage. In severely diseased areas, the incidence rate was between 20%-100%. Symptoms first began as yellow-brown necrotic spots on leaves, then merged into larger irregular necrotic spots surrounded by chlorotic halos. Similar lesions were also found on the stems. Ten symptomatic leaf and stem samples were collected from the different infected plants for pathogen isolation. The small pieces of discolored tissues were surface-disinfected with 2% sodium hypochlorite for 3 min and 75% ethanol for 30 s, rinsed three times with sterile water, and blotted on sterile filter paper, placed on potato dextrose agar thenincubated at 28°C in the dark for 3-4 days. The obtained isolates were purified through single-spore culture. Colonies were initially white and fluffy in appearance, later turning gray. Hyphae were smooth, branched, septa, transparent or light brown. Spores were solitary, oblate or nearly spherical, dark brown to black, smooth, 14.3 to 16.1μm × 11.8 to 15.2 μm in diameter. DNA of fungal isolates were extracted using Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China), the internal transcribed spacer (ITS) of the ribosomal DNA, β-tubulin (TUB2) gene and translation elongation factor 1-alpha (TEF1-α) were amplified with primers ITS1/ITS4, βt2a/βt2b and EF1-1728F/EF1-986R, respectively. The resulting ITS, TUB2 and TEF1-α sequences were deposited at GenBank, NCBI under accessions MZ882151, MZ927749, MZ927747, respectively. The sequence identity of ITS, TUB2 and TEF1-α with those of Nigrospora oryzae strains HBN (KU254608), HGUP191068 (MZ724102) and LC7307 (KY019409) were 99.64%, 99.29% and 99.65%, respectively. Based on morphological features and phylogenetic analysis, the pathogen was identified as N. oryzae (Wang et al. 2017). Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of tobacco plants grown at 28°C with 60% humidity in greenhouse. Symptoms appeared on the pathogen inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as N. oryzae based on morphological, molecular and phylogenetic analysis, which were fulfilling Koch's postulates. This pathogen was recently identified from watermelon and kiwifruit in the Guizhou (Far and Rossman, 2021). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Nicotiana tabacum in China.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Molecular Detection of Virulence Genes in Escherichia coli and Salmonella enterica Isolated from Minimally Processed Foods Sold within Kaduna Metropolis

Minimally processed foods are widely consumed in Kaduna, Nigeria and the world at large. However, poor quality control during their processing make them prone to contamination by enteropathogens known to possess multiple virulence genes, possibly causing morbidities and mortalities. Investigating the prevalence and virulence genes of food-borne bacteria is therefore paramount. This study was aimed at detecting virulence genes in enteropathogenic bacteria associated with minimally processed foods sold within Kaduna metropolis. Samples of sliced watermelons, peeled sugarcanes, peeled and sliced pineapples and unshelled coconuts (n=140) were collected from Kaduna North, Kaduna South and Igabi LGAs. Upon enrichment, samples were inoculated onto Eosin-Methylene Blue (EMB) and Cefixime-Tellurite-Sorbitol MacConkey (CT-SMAC) agar and incubated for 18 hours at 35oC. Isolates were identified using MicrobactTM 24E Gram Negative Bacteria Identification System for Enterobacteriaceae. Thereafter, E. coli and Salmonella enterica isolates’ DNA was extracted and purified using AccuPrep Genomic DNA Extraction Kit; used as a template for the PCR amplification of the bundle forming pilus (bfpA) and invasion (invA) genes; and then the PCR products were visualised using agarose gel electrophoresis documentation system. The results of the MicrobactTM analyses showed widespread contamination of the samples with E. coli (22 isolates) and Salmonella enterica (3 isolates). There was no statistically significant difference in the bacterial contaminants isolated from the various sampling areas (one-way ANOVA: p = 0.577); similarly, the type of the minimally processed food samples had no influence on the prevalence of E. coli and Salmonella enterica isolates (p = 0.345). PCR result revealed that the invA virulence gene (284bp) was present in one Salmonella enterica isolate. The presence of invA gene in the Salmonella enterica isolate indicated that the isolate is a virulent strain, which can cause food-borne infections. These results indicated the contamination of the minimally processed fruits with enteropathogens, hence, standards of quality control should be enshrined, towards safer foods and enhanced health of the consuming populace.

PB2254: ASSOCIATION BETWEEN TNF-ALPHA LEVELS AND TNF-ALPHA 238 ALLELES POLYMORPHISMS AND SEVERITY OF ANEMIA AMONG SUDANESE CHILDREN WITH FALCIPARUM MALARIA

Background: Falciparum malaria is the most leading cause of mortality and morbidity in the world especially in Africa among infants and young children due to severe malaria and malaria anemia. TNF-α is plays a central role in malaria pathogenicity either in the cure or complication of malaria. TNF-α levels overproduction and promoter polymorphisms at TNF-α 238 alleles may play a central role in reduced red cell production and malaria-related anemia through suppression of bone marrow erythropoiesis and dyserythropoiesis. Aims: The purpose of this study was to evaluate the TNF-α 238 alleles polymorphism and its association with TNF-α levels in the severe malaria anemia among Sudanese children in Gezira State. Methods: A cross-sectional hospital-based study was included 200 Sudanese children (mean age 8.43 ± 3.80 years; 53% boys; 47% girls). 100 children with severe falciparum malaria (mean age 8.63 ± 3.40 years; 61% boys; 39% girls) and 100 children with uncomplicated falciparum malaria (mean age 8.83 ± 4.20 years; 45% boys; 55% girls). RBCs parameters were determined using the Sysmex XP 300 N automated hematology analyzer. ELISA was further processed for TNF-α level using Human TNF-α ELISA MAX™ Deluxe Sets. DNA extraction was done using G-DEX™IIb Genomic DNA Extraction Kit. PCR and gel running system were used for detecting TNF-α 238 alleles polymorphism (A allele/ G allele). Obtained data were analyzed by SPSS (V 20.0) and Stat disk (V 13.0). Results: TNF-α 238A allele was a common allele (66.8%) among Sudanese children with falciparum malaria, and the G allele was rare (33.2%). While TNF-α 238 GA, AA, and GG account for (54.5, 39.5, and 6% respectively). Falciparum malaria-related anemia accounted for 32%, commonly in SM (55%) compared to UM (9%) giving highly statistically significant differences in TNF-α levels between falciparum malaria-related anemia and falciparum malaria without anemia (P value 0.000). Otherwise, The average of TNF-α levels in mild, moderate, and severe anemia were (190.75 ± 102.55, 189.70 ± 80.35, and 299.75 ± 82.27 pg/ml respectively) giving highly significant differences and strongly significant positive correlated with the severity of anemia (r + 0.309; P value 0.000). TNF-α 238 GA, AA, and GG account for (58, 36, and 6% respectively) in UM; while (51, 43, and 6% respectively) in SM. The TNF-α 238 A allele accounts for 83.6% of malaria anemia (P value 0.000) and 100% severe anemia (P value 0.000); children who have TNF-α 238 A allele, they have ability to acquire falciparum malaria-related anemia with susceptibility to 3.13 folds risk. Summary/Conclusion: Overproduction of TNF-α is essential for the elimination and clearance of falciparum parasite but may be associated with severity of malaria and malaria anemia. Overproduction of TNF-α in children with TNF-α 238 A allele may result in falciparum malaria-related anemia among children (mainly severe anemia). The findings of this study will assist clinicians in diagnosing and better managing severe malaria cases.

Root Rot of Codonopsis tangshen Caused by Ilyonectria robusta in Chongqing, China.

Codonopsis tangshen Oliv. belongs to the Campanulaceae, it is one of the most important economically medicinal materials in China.Which is used in medical and agricultural applications (Wu Q N, et al. 2020). In August 2019, root rot of C. tangshen was firstly observed in Fengjie, Chongqing city, southwest China (30°45' 59″ N; 109°36'36″ E; ), causing approximately 20% yield loss. At the initial stage of the disease, the above-ground stems and leaves turn yellow, and brown to black spots of different sizes appear at the base or root of the stem. With the further development of the disease, the above-ground leaves gradually turn yellow as the diseased spots rot from bottom to top, so that they die, and the diseased spots on the roots expand and begin to rot. Generally, they gradually rot from the bottom up, but the vascular bundles are occasionally normal. If the symptoms of C.tangshen started too late, and the root has not completely rotted by late autumn (late October to early November), the rest part of C.tangshen root will not continue to rot, and it is called half C.tangshen. In the next spring, the halfC. tangshen can continue to sprout, but it will continue to rot, which will seriously affect the yield and quality. In order to identify the pathogen, 25 samples of diseased plants were collected and symptomatic rhizome tissues were surface disinfected with 0.1% HgCl2 solution for 30s, rinsed in sterilized water 3 times, placed on potato dextrose agar (PDA), and incubated at 25℃±1°C in the dark. On the PDA, after seven days of culture, the center appeared light yellow, the edges were white, and the aerial hyphae were felt-like. The surface of the colony was reddish-brown and the margins were white and regular. The conidiophores were simple, usually born on the lateral or apical sides of aerial mycelium, unbranched, or minimally branched. Conidia were abundant, cylindrical, or rod-shaped, straight or slightly curved, usually with 1-3 septa. Macroconidia varied in size depending on the number of cells as follows: one-septate 15.3-26.3×4.2-7.3 μm（n=50）μm, two-septate 20.5-30.5×4.9-7.8μm (n=50), and three-septate 29.3-38.5×5.5-7.4 μm (n=50), round at both ends. For molecular identification, DNA was extracted from a representative isolate using a fungus genomic DNA extraction kit (Solarbio, Beijing, China). The internal transcribed spacer (ITS)(ITS1/ITS4, White, et al. 1990), beta-tubulin (TUB2)(BT2A/BT2B, O'Donnell and Cigelnik 1997), translation elongation factor 1-a (TEF) （ EF446F/EF1035R, Inderbitzin et al. 2005), DNA-dependent RNA polymerase subunit II gene(RPB2, O'Donnell K., et al. 2010 ) and histone H3(HIS3) (CYLH3F/CYLH3R, Crous, et al. 2004b) were amplified. BLAST results indicated that the ITS, TUB2, TEF, HIS3, and RPB2 sequences (GenBank MW392103, MW386994, MW386995 MW392103, and MW915473) showed 96% to 100% identity with Ilyonectria robusta sequences at NCBI (GenBank KU350726, JF335378, MN833103, MN833113, KM232336). The phylogenetic tree was inferred from the combined datasets (ITS, TEF1, TUB, and HIS3) from members of the I. robusta species complex analyzed in this study (Cabral et al. 2012 ). To complete Koch's postulates, a conidial suspension (106 spores/ml) collected from isolate CQ13 was irrigated onto fifteen annual C.tangshen potted plants. Sterile water was used as a negative control, and the pathogenicity assay was repeated three times. Following inoculation, the plants were cultured for 9 days at 75% relative humidity and 25 ℃. The inoculated plants showed symptoms similar to those observed in the field. In contrast, the negative control plants were healthy and unaffected. I. robusta was re-isolated from the infected tissues and identified by morphological characteristics and DNA sequence analysis. To our knowledge, this is the first report of I. robusta causing root rot disease of C.tangshen in China. Our results may help to take appropriate steps to control the disease in the commercial area ofC.tangshen. The authors declare no conflict of interest.

First Report of Cinnamomum japonicum Anthracnose Caused by Colletotrichum fioriniae in Sichuan, China.

The Cinnamomum japonicum Sieb is widely cultivated in urban in China. It's used to make essential, lubricant, soap, and waterproof timber. In September 2019, This new leaf spot was discovered in Chengdu city (30°05'to 31°26'N, 102°54'to 104°53'E), with approximately 61.20% disease incidence. The symptoms started to occur from May to June, the worst from August to September. Firstly, the typical symptom showed round or oval, brown, and slightly sunken necrotic lesions. Gradually, the necrotic lesions increased in number, and expanded; under humid conditions the central part of the spots became black and ruptured, with orange conidial masses emerged at the margin of lesions. Finally, the leaves turn yellow and fall off. Infected tissues from ten samples were cut into small pieces 2 × 2 mm, surface sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL), and incubated at 25°C in a dark. Finally, 8 typical isolates exhibited the morphology described as C. fioriniae (Amelie Grammen et al. 2019). After 5 days, the colony diameter reached 28.6 to 41.2 mm and had white to light grey aerial mycelium, but was pink at the base. Orange conidia masses formed after 6-7 days, conidia were oval, slender and fusiform with acute ends (Figure 1e, f), measuring 8.3 to 19.6 × 2.9 to 7.1 μm (average: 13.9 × 4.8 μm) (Tashiro et al. 2018; Chechi et al. 2019). For molecular identification, DNA was extracted from 8 fungal colonies using a plant genomic DNA extraction kit (Solarbio, Beijing). The 5.8S nuclear ribosomal genes with the two flanking internal transcribed spacer (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial sequences of the actin (ACT), chitin synthase 1 (CHS-1), beta-tubulin (TUB2), histone3 (HIS3), calmodulin (CAL) and glutamine synthetase (GS) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT-512F/ACT-783R, CAL 228F/CAL 737R (Carbone et al. 1999), CHS-354R/CHS-79F (Weir et al. 2012), TUB1F/Bt2bR, CYLH3F/CYLH3R (Crous et al. 2004), and GSF1/GSR1 (Liu et al. 2015), respectively. Sequences were deposited in GenBank (ITS: MT466533, GAPDH: MT460415, ACT: MT460414, CAL: MT954332, CHS-1: MT954330, TUB2: MT460416, HIS3: MT954331, and GS: MT460417). BLAST analysis showed >99.4% identity with several reference sequences of Colletotrichum fioriniae strain CBS 128517 and strain EHS58 (teleomorph of Glomerella fioriniae) previously deposited in GenBank. The conidial suspension (1 × 107 conidia/mL) collected from PDA cultures with 0.05% Tween 80 buffer was used for inoculation by spraying leaves of 5-year-old C. japonicum plants for pathogenicity test. Ten leaves of each plant (10 pots in total) were inoculated with spore suspensions (Approximately 500 μL per leaf). An equal number of control leaves were sprayed with 0.05% Tween 80 buffer to serve as a control. Twenty days later, the inoculated plants showed the similar symptoms as the original diseased plants but the controls remained asymptomatic. The C. fioriniae was re-isolated from the infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of C. japonicum anthracnose caused by C. fioriniae in China.

Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom.

To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method. The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed. When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05). The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples

BackgroundHaemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR.ResultsDNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €).ConclusionsThe Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.

Single Nucleotide Polymorphism of Genes Associated with Metabolic Fatty Liver Disease.

Aims The present study aimed to reveal the relationship between single nucleotide polymorphism (SNP) of PNPLA3, TM6SF2, MBOAT7, GATAD2A, and STAT3 genes and metabolism-related fatty liver disease (MAFLD), so as to provide a research basis for further exploring the diagnosis and treatment of diseases at the molecular level. Methods A total of 564 patients were included in the physical examination center of Xinjiang Karamay People's Hospital. They were divided into an MAFLD case group and a healthy control group. The whole blood DNA of each sample was extracted by a whole blood genomic DNA extraction kit, and the genotypes of PNPLA3 rs738409, MBOAT7 rs64173, STAT3 rs744166, TM6SF2 rs58542926, and GATAD2A rs4808199 were performed; after adjusting for confounding factors, the additive model, dominant model, and recessive model of each gene were analyzed by multivariate logistic regression. Results The CC genotype of the PNPLA3 gene rs738409 and the TT genotype of the MBOAT7 gene rs64173 are risk factors in the occurrence of MAFLD. The AA genotype of the STAT3 gene rs744166 is a protective factor of MAFLD, while TM6SF2 rs58542926 and GATAD2A rs4808199 show no significant correlation with MAFLD.

Bacterial Community and Diversity from the Watermelon Cultivated Soils through Next Generation Sequencing Approach.

Knowledge and better understanding of functions of the microbial community are pivotal for crop management. This study was conducted to study bacterial structures including Acidovorax species community structures and diversity from the watermelon cultivated soils in different regions of South Korea. In this study, soil samples were collected from watermelon cultivation areas from various places of South Korea and microbiome analysis was performed to analyze bacterial communities including Acidovorax species community. Next generation sequencing (NGS) was performed by extracting genomic DNA from 92 soil samples from 8 different provinces using a fast genomic DNA extraction kit. NGS data analysis results revealed that, total, 39,367 operational taxonomic unit (OTU), were obtained. NGS data results revealed that, most dominant phylum in all the soil samples was Proteobacteria (37.3%). In addition, most abundant genus was Acidobacterium (1.8%) in all the samples. In order to analyze species diversity among the collected soil samples, OTUs, community diversity, and Shannon index were measured. Shannon (9.297) and inverse Simpson (0.996) were found to have the highest diversity scores in the greenhouse soil sample of Gyeonggi-do province (GG4). Results from NGS sequencing suggest that, most of the soil samples consists of similar trend of bacterial community and diversity. Environmental factors play a key role in shaping the bacterial community and diversity. In order to address this statement, further correlation analysis between soil physical and chemical parameters with dominant bacterial community will be carried out to observe their interactions.

Brown Culm Rot of Dendrocalamus latiflorus Munro Caused by Diaporthe guangxiensis in Sichuan Province, China.

Dendrocalamus latiflorus Munro, the most widely cultivated bamboo species in southern China, has high ornamental value used in gardens, while culms are also used for buildings and as fibers and edibles (Gao et al. 2011). In June 2020, brown culm rot of bamboo was observed in Yibin city, Sichuan Province, in an area of approximately 1000 hectares. Disease incidence was approximately 60%, of which 30% of the plants had died. At the end of June, the lesions expanded but did not surround the base of the culm. From the end of June to the beginning of September, the lesions expanded upward and formed a streak, of which the color gradually deepened to purple-brown and black-brown. At the same time, the disease spots at the base of the culm also expanded horizontally. After the spots surrounded the base of the culm, the diseased bamboo died. Ten culms showing typical symptoms were collected and cut into 5×5 mm pieces at the junction of infected and healthy tissues. The tissues were sterilized for 1 to 2 min in 3% sodium hypochlorite, decontaminated in 75% alcohol for 3 to 5 min, placed on modified potato glucose agar (PDA) with streptomycin sulfate (50 μg/ml), and incubated at 26°C. Two isolates were obtained by the single-spore method (Sivan et al. 1992). The isolates both produced white round colonies similar to Diaporthe guangxiensis and two types of conidia: one was α type (5.5 to 8.2×1.0 to 2.8 µm, n=30), colourless, single-celled, undivided, and oval, containing two oil droplets; and β type (21.1 to 30.2×0.8 to 1.4 µm, n=30), colourless, single celled and hook shaped. Genomic DNA was extracted from the two isolates by using a fungal genomic DNA extraction kit (Solarbio, Beijing). The products were amplified by polymerase chain reaction (PCR) with primers for the internal transcribed spacer 1 (ITS) region (White et al. 1990), calmodulin (CAL) gene (Carbone and Kohn 1999), translation elongation factor 1-alpha (TEF) gene (Glass and Donaldson 1995) and beta-tubulin (TUB) gene (Soares et al. 2018). The amplified products were sequenced and blasted in GenBank (accession numbers MW380383, MW431318, MW431317 and MW431316 for ITS, CAL, TEF, and TUB, respectively). The ITS, CAL, TEF, and TUB sequences showed 100%, 99.33%, 100%, and 99.80% identity to D. guangxiensis JZB320094 (accession numbers MK335772.1, MK736727.1, MK523566.1, MK500168.1 in GenBank), respectively. To evaluate the pathogenicity of the isolates, five plants were each inoculated with two isolates. The cortex of potted bamboo were injured locally with sterilized needle, and the bamboo culms were inoculated with 100 μl of conidial suspension (105 cfu/ml). The surface of the inoculation wound was covered with gauze soaked with sterilized water. Five plants inoculated with sterile water were used as controls. The treated plants were maintained in a greenhouse at a temperature of 22 to 29°C and relative humidity of 70 to 80%. One month later, of all inoculated plants showed similar symptoms as those observed in the field. D. guangxiensis was re-isolated from all inoculated plants. The pathogenicity test was repeated three times with similar results. This is the first report of D. guangxiensis causing brown culm rot of D. latiflorus in China. These results will facilitate an enhanced understanding of factors affecting bamboo and the design of effective management strategies of the pathogenic species on bamboo and thus to develop corresponding control measures.

Neofusicoccum parvum Causing Stem and Branch Blight Disease of Zanthoxylum bungeanum in Sichuan, China.

The Chinese pepper Zanthoxylum bungeanum Maxim is a special economically important species and a traditional spice in China. It is widely used in medicine, food, timber, tourism, soil and water conservation. In April 2019, A stem and branch blight disease of Z. bungeanum was discovered in Muli, Puge and Yanyuan counties, in Liangshan Prefecture (27°15'20″-27°19'38″N, 101°44'58″-102°04'10″E), causing approximately 15% yield loss in the three counties. Among all fields in Muli County, approximately 41.38%, 10.79% and 2% of Chinese peppers exhibited mild, moderate and severe branch blight, respectively. The symptoms started to occur from March to April. First, red-brown spots on the base of the stem, branches or main trunks of young trees observed but were not obvious. In May, the spots became gray-brown to dark brown ovals and gradually expanded into long strips (Figure 1a, b). When the spots surrounded the branches, the branches above them withered and died, and the spots gradually expanded downward. Around June or July, scattered black dot-shaped fruiting bodies were observed on the lesion. The branches of infected trees were sampled systematically by cutting the branch at the junction of infected and healthy areas in 5×5 mm sections. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before being rinsed three times with sterilized distilled water. The sterile filter paper was used to dry the tissue, and the samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml). Plates were incubated at 25°C in the dark. From the five isolates obtained, four exhibited the morphology described by Yu et al. (2015) for Neofusicoccum parvum. The colonies were white fluffy at first and grew fast (Figure 1c). After five days, the colony diameter reached 75.2-84.8 mm, produced yellow pigment and the mycelium in the middle of the colony began to turn gray (Figure 1d). and the entire colony turned dark gray 7-8 days post culturing as observed previously (Javier-Alva et al. 2009) and formed a black fruiting body at 20 days (Figure 1e). The width of the mycelium measured 2.3-4.8 µm, and with the diaphragm (Figure 1f). The spores were round or fusiform, colorless, transparent, smooth, thin-walled, and measured 6.3-10.6×3.1-5.2 µm (Figure 1g, h), similar to N. parvum (Yu et al. 2013). For molecular identification, DNA was extracted from the mycelia of four fungal isolates using a plant genomic DNA extraction kit (Solarbio, Beijing). Polymerase Chain Reaction (PCR) was performed with the primers ITS1/ITS4 (White et al. 1990), EF446F/EF1035R (Inderbitzin et al. 2005), BTF/BTR2 and HspF3/HspR (Inderbitzin et al. 2010) for the ribosomal internal transcribed spacer region (ITS), elongation factor-1alpha (EF1-alpha), beta-tubulin (TUB) and heat shock protein (HSP) genes, respectively. BLAST searches in the GenBank database indicated that the ITS, TUB, HSP and EF-1α sequences had 100%, 99.0%, 99.7% and 99.7% identity to N. parvum, respectively. Representative sequences were deposited in GenBank (ITS: MT355871; TUB: MT409397; EF-1α: MT409399; HSP: MT460413). A pathogenicity test was performed using N. parvum on ten 2-year-old potted Z. bungeanum plants at 22-28°C and 60% humidity indoors. The conidial suspension (1×107 conidia/ml) collected 25 days old PDA cultures with 0.05% tween buffer was used for inoculation by brushing the wounded area of branch scratched by epidermis with a piece of sandpaper. Ten plants in pots were inoculated with sterile water and served as controls. Thirty days post-inoculation, the plants showed the same symptoms as the original diseased plants, and the controls remained asymptomatic. N. parvum was re-isolated from the infected tissues and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. This fungus is an important pathogen on a variety of woody hosts, and represents a serious problem in the vineyards worldwide (Mélanie, et al. 2017). To our knowledge, this is the first report of N. parvum causing stem and branch blight of Z. bungeanum trees in China.

First report of Podosphaera macularis causing hop (Humulus lupulus) powdery mildew in Brazil.

The hop (Humulus lupulus L.) is a dioecious perennial climbing plant grown commercially worldwide. Wild hops are widely distributed throughout the Northern Hemisphere, Europe, Asia, and North America (Neve, 1991). In the Southern Hemisphere, some of the leading hop-producing countries include South Africa, Australia, and New Zealand. Brazil began hop production less than 5 years ago. In January 2019, amphigenous white powdery circular fungal colonies were observed on the leaves and stems of hop plants (cultivar Chinook) within a 900m2 hop garden in Lages municipality, Santa Catarina State, southern Brazil. The incidence of the disease was present on almost 100 per cent of "Chinook" cultivar plants and diseased foliage was collected to identify the pathogen and used to inoculate healthy plants. Hop powdery mildew lesions with hyaline and septate mycelium with chains of unicellular conidia (n =100) hyaline, barrel-shaped, mean of length/width ± standard deviation 25-27 × 13-18 µm ± 0.980, with fibrosin bodies, and conidiophores erect with cylindrical foot cells, were visible within 10 days. The causal agent was identified as Podosphaera macularis (Wallr.:Fr.) Lind (synonym S. humuli (DC.) Burrill) on the basis of conidial shape, size and host range (Royle 1978; Braun 1987; Mahaffee et al., 2009), complemented with the present molecular analysis. Chasmothecia have not been observed in the field to date. A conidial suspension of 200 ml at concentration of 1.4 x 105 was mixed with 5ul of Tween® 20 for the pathogenicity assay. Ten plants of 9-month-old of hop "Chinook" cultivar, were inoculated with 5 ml of the conidial suspension using a manual spray. The control plot was only sprayed with water. The inoculated plants were maintained at 22ºC ± 1ºC with a 12-hour photoperiod and 65% relative humidity. White mycelia were visible first on the adaxial leaf surfaces of the inoculated younger leaves after 10 days and the disease severity reached between 2 to 5%. No symptoms were observed at the control plot. P. macularis infected most aerial plant tissues of the inoculated plants and caused approximately 50% of cones losses. P. macularis conidia were collected from the infected leaf tissue with a sterile soft camel-hair brush and DNA was extracted using a Wizard Genomic DNA extraction kit. The primers ITS1/ITS4 (White et al., 1990) were used to amplified and sequenced a fragment of the ITS region. PCR products were subjected to Sanger Sequencing to confirm sample species. The resulting 522-bp sequence was deposited into GenBank (accession n°. MN630490). BLASTn showed a 99.81% sequence identity with the CT1 isolate of P. macularis from H. lupulus (MH687414). The presence and identification of P. macularis in hop production regions is a new challenge to growers in Brazil. Research related to the knowledge of the disease cycle, epidemiology, and control strategies for the integrated management should be conducted, as there are no registered fungicides for powdery mildew on hop in Brazil. To our knowledge, this is the first report of P. macularis in Brazil, as well as in South America. References Braun, U. (1987) A Monograph of the Erysiphales (Powdery Mildews). J. Cramer, Berlin, German Democratic Republic. p 113. Mahaffee, W. F., Pethybridge, S.J., Gent, D.H (2009) Compendium of hop diseases and pests. The American Phytopathological Society Press, Saint Paul, Minnesota. Neve R. A (1991). Hops. Chapman and Hall: London. Royle, D. J (1978). Powdery mildew of the hop. Pages 381-409 in: The Powdery Mildews. D. M. Spencer, ed. Academic Press, New York. White, T. J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. pp. 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. Innis, D. Gelfand, J. Sninsky, and T. White, eds. Academic Press, San Diego.

Molecular Detection of Toxoplasma gondii in Helix pomatia in Wasit Province Iraq

DNA of a total of 96 samples of (32) enteric, (32) gastric and (32) pod of Helix pomatia has been obtainedthrough the use of the Genomic DNA Extraction kit and tested by the use of the Real time PCR and specificset of the primers and probe for the detection of the Toxoplasma gondii B1 gene. Six enteric samples and only2 gastric samples have been positive while the others have been negative, that result indicates the presenceof the T. gondii in that snail species, and based upon those results infection can either be acquired from thecontaminated environment or as a result of consuming the grass that has been contaminated with parasiteoocysts. The negative result of pod samples may be due to the failure of filtration the infectious stages ofparasites into the organs that close contacts with environment or inability of the parasites in surviving inthose organs.

First Report of Alternaria spp. Causing Leaf Spot on Sweet Viburnum in China.

Sweet viburnum [Viburnum odoratissimum (L.) Ker Gawl] is an evergreen shrub mainly cultivated along roadsides in urban landscapes and also in parks and residential areas. A foliar disease occurred on about 40% of sweet viburnum plants near Anhui Grand Theatre, Anhui Province of China in June 2019. In early stages of sweet viburnum infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. The spots developed on the upper, middle, and lower leaves of the plant, however, the upper leaves got more severely affected. As the disease develops, the spots enlarged and became rectangular or oval, brown to dark-brown, and their centers became ashen gray. In later stages of infection, the diseased leaves became wilting. Diseased leaves were surface disinfested and three small sections (2-3 mm2) were cut from the margin of the lesions. Sections were placed in 1.5% NaClO for 2 min, submerged in three changes of sterilized distilled water for 1 min each, placed onto potato dextrose agar (PDA) medium amended with 50 μg/ml of ampicillin and kanamycin, and incubated at 25℃ for 3 days. The mycelium from the leading edge of colonies growing from the tissue was sub-cultured onto a PDA plate for 3 days, followed by spore induction (Simmons 2007) and single spore isolation to obtain a pure culture of the putative pathogen. Colonies of one single spore isolate HF0719 were rounded, grayish white with dense aerial mycelium viewed from above and dark brown viewed from below. On potato carrot agar (PCA) medium, conidiophores were branched or occasionally unbranched. On branched conidiophores, conidia were in dwarf tree-like branched chains of 2-5 conidia. On unbranched conidiophores, conidia were simple or in chains of 2-8 conidia. Conidia were light brown or dark brown, ovoid, ellipsoidal to fusiform, and ranged in size from 7 to 26.5 × 4.5 to 11 μm with an average size of 16 × 7 µm based on 500 spore observations, with one beak and 1-7 transverse, 0-3 longitudinal, and 0-3 oblique septa. Beaks were ranged in (1.5-)2-10(-16) μm long. Based on cultural and morphological characteristics, isolate HF0719 was identified as Alternaria spp. (Simmons 2007). For molecular identification, total genomic DNA was isolated from mycelia collected from 7 day-old colonies of isolate HF0719 using the fungal genomic DNA extraction kit (Solarbio, Beijing, China). Fragments of five genes, including those encoding glyceraldehyde-3-phosphate dehydrogenase (gpd), plasma membrane ATPase, actin, calmodulin, and the Alternaria major allergen (Alt a1) regions of isolate HF0719 were amplified and sequenced using primer pairs gpd1/gpd2 (Berbee et al. 1999), ATPDF1/ATPDR1, ACTDF1/ACTDR1, CALDF1/CALDR1 (Lawrence et al. 2013), and Alt-for/Alt-rev (Hong et al. 2005), respectively. The obtained nucleotide sequences were deposited into GenBank as accession numbers: gpd, MT614365; ATPase, MT614364; actin, MT614363; calmodulin, MN706159; and Alt a1, MN304720. Phylogenetic tree using a maximum likelihood bootstrapping method based on the five-gene combined dataset in the following order: gpd, ATPase, actin, calmodulin, Alt a1 of HF0719 and standard strains representing 120 Alternaria species (Lawrence et al. 2013) was constructed. Isolate HF0719 formed a separate branch. On the basis of morphological characteristics and phylogenetic pattern, isolate HF0719 was identified as Alternaria spp.. A pathogenicity test was performed by rubbing 32 healthy leaves of six 5-year-old sweet viburnum plants with a cotton swab dipped in spore suspension containing 2.6 × 106 spores/ml, following leaf surface disinfection with 70% ethanol in the open field. Sterilized distilled water was used as control. The average air temperature was about 28℃ during the period of pathogenicity test. Eleven days after inoculation, 100% of inoculated leaves showed the leaf spot symptom identical to symptoms observed in the field. Control leaves were symptomless. The experiment was done three times. The re-isolated pathogen from the leaf lesion had the same morphological and molecular characteristics as isolate HF0719, thus satisfying Koch's postulates. The genus Alternaria has been reported to cause leaf spot on sweet viburnum in Florida, USA (Alfieri et al. 1984). To our knowledge, this is the first report of Alternaria spp. causing leaf spot on sweet viburnum in China, a highly valued ornamental plant. Our findings will contribute to monitoring and adopting strategies for manage leaf spot disease on sweet viburnum.

Evaluation of the relationship between VRK2, rs4380187 polymorphisms, and genetic susceptibility to schizophrenia in the Chinese Han population.

Schizophrenia (SZ) is a serious hereditary mental disease with a low recovery rate, especially due to the lack of understanding about the cause of the disease. VRK2 is considered to be related to the pathogenesis of schizophrenia. In this study, we analyzed the correlation between VRK2, rs4380187 single-nucleotide polymorphism (SNP), and schizophrenia. Peripheral blood DNA was extracted using a genomic DNA extraction kit. The DNA samples were genotyped using the Agena MassARRAY platform, and four genetic models were applied to compute the odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. The p value was obtained by the chi-square and t test for independent samples. The C allele of rs4380187 SNP was significantly (p = 0.008) associated with decreased risk of SZ. The AA genotype of rs4380187 showed significantly (p = 0.009) lower frequency in cases with SZ than in controls and was associated with decreased risk of the disease. The frequency of the CA genotype of rs4380187 correlated with a 0.73-fold decreased risk of SZ (p = 0.033). In the co-dominant genetic model, the genotype of rs4380187 was associated with a decreased risk of SZ (p = 0.010). We also found that the log-additive model of rs4380187 significantly reduced the risk of SZ disease (p = 0.007). This study provides further evidence that rs4380187 SNP is associated with SZ. This genotype variation could be associated with the psychopathology and cognitive function in SZ.

Comparison of DNA Extraction Methods for Metagenomic Sequencing in Anaerobic Digestion

Objectives:This study was performed to compare three commercial kits for the extraction of genomic DNA from anaerobic digestate for subsequent iSeq 100 sequencing and microbial community analysis.Methods:A full-scale mesophilic biogas plant was sampled, and made into aliquots of identical volumes to extract DNA using three commercial kits: FastDNA spin kit for soil (MP Biomedicals, USA), Exgene stool DNA mini (GeneAll, South Korea) and AccuPrep genomic DNA extraction kit (Bioneer, South Korea). To analyze the microbial communities in the purified DNA, 16S rDNA amplicon sequencing (V3-4 region for bacteria and V4-5 region for archaea) was performed on the Illumina iSeq 100 platform. Quality filtered sequence reads were OTU-clustered for taxonomic assignment conducted using the RDP classifier on-line.Results and Discussion:The microbial community structure visualized on the NMDS plot using the weighted UniFrac distance revealed that both bacteria and archaeal communities have phylogenetic differences depending on the DNA extraction kit used. In addition, the abundance of certain microbial populations was significantly different among the DNA extraction methods. For example, Proteobacteria was the least abundant using the soil kit, while this phylum was the most abundant when the stool kit was used. However, in the case of Thermotogae, this tendency was vice versa. The abundance of archaeal genera Methanomethylovorans and Methanosarcina was also affected by DNA extraction methods.Conclusions:The microbial populations analyzed by 16S based sequencing were affected by DNA extraction methods. To compare microbial community changes in the identical set of research, one DNA extraction method should be chosen and used consistently for the whole experiment.

First Report of Fruit Blotch on Plum caused by Fusarium fujikuroi in China.

Plum is commercially cultivated worldwide for the rich nutrient in its fruit. In May 2019, plum with symptoms of fruit rot were collected from fields located in Liuma town, Guizhou Province, China. The incidence of the disease varied from 10 to 20%, which was observed in 15 plum orchards (18 hectares) surveyed. Estimated yield loss was~5 to 10% for each field. Diseased fruits showed deformity, wilting and sunken lesions, and subsequenly became melanized and rotted. Diseased tissues were surface disinfected with 70% ethanol for 45 s and rinsed with sterile distilled water three times. Four morphologically similar colonies with white fluffy aerial mycelium and a reddish pigment were obtained after 3 days incubation on potato dextrose agar (PDA) at 25°C. Four single-spore isolates produced conidia with 1 to 2 septa that were sickle-shaped, thin-walled with a tapering and curved apical cell, measuring 15.6 to 29.6 × 4.8 to 8.7 μm (average 19.5×5.9 μm, n=50). Based on the cultural and conidial morphology, the isolates were identified as Fusarium (Mun et al. 2012; Leslie and Summerell 2006). DNA of two isolates was extracted using the Ezup Column Fungal Genomic DNA Extraction Kit (Sangon Bioengineering Shanghai, LTD.). To confirm the morphological diagnosis, DNA sequence data from three loci were obtained. PCR amplification was carried out with universal primers ITS1/ITS4 (White et al. 1990), translation elongation factor (EF-1α), EF1-H (5'-ATGGGTAAGGAAGACAAGAC-3') and EF2-T (5'-GGAAGTACCAGTGATCATGTT-3') (O'Donnell et al. 1998) and the second largest subunit of RNA polymerase II (RPB2), 5F2(5'-GGGGWGAYCAGAAGAAGGC-3') and 7cR (5'-CCCATRGCTTGYTTRCCCAT-3') (O'Donnell et al. 2007). Primers ITS1 and ITS4 produced a 559-bp amplicon (GenBank accession. MW085028). BLAST analysis showed 100% sequence identity to sequences of several species, deposited in GenBank, including F. fujikuroi. The EF-1α sequence (MW086868) was 100% identical to that of Fusarium fujikuroi (MN193860.1). The RPB2 primers amplified a fragment (MW086869) that was 99.9% identical to that of F. fujikuroi (MN193888.1). The BLASTn results based on the partial EF-1α and RPB2 sequences suggest isolate HJGF1 is F. fujikuroi. A pathogenicity assay was conducted using an agar disk inoculation method on plum. Fruits were stab inoculated with HJGF1 by piercing 1-mm at 3 points using a sterile needle, and fruits were mock inoculated with sterile PDA, each fruit was inoculated with three disks. (Fig. 1). The treated fruit were maintained in a growth chamber with 90% relative humidity at 25°C, and a daily 12-h photoperiod. After 5 days, the artificially inoculated fruit showed blotches with sunken lesions similar to those observed in the orchards, whereas no symptoms were observed on the control fruit. The experiment was repeated twice with similar results. F. fujikuroi was reisolated from infected tissues and confirmed by sequence analysis. To our knowledge, this is the first report of F. fujikuroi causing fruit blotch of plum in China. Considering the economic importance of plum in China and throughout the world, F. fujikuroi may be an emerging problem for plum cultivation. Thus, further study of fruit blotch of plum is warranted.

A low-cost pipeline for soil microbiome profiling.

Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low‐cost genomic DNA extraction method for soil samples. We also present an Illumina‐compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non‐commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad‐leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column‐based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low‐cost genomic DNA extraction method for soil samples that can be coupled to an Illumina‐compatible simple two‐step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high‐quality genomic DNA at a fraction of the cost of commercial kits and enables cost‐effective, large‐scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics.