Fungal DNA Extraction Research Articles

First Report of Chestnut Blight Caused by Cryphonectria parasitica on Chestnut (Castanea seguinii) in Gansu Province, China.

In China, chestnut blight usually causes insignificant damage to fruit production of Chinese chestnut (Castanea mollissima Blume) and no serious disease epidemics occur, due to the high resistance to Cryphonectria parasitica (Huang et al. 1998). According to recent surveys, chestnut blight was mainly found in sixteen provinces including Shandong, Hebei, Anhui, Hunan, Jiangxi, Beijing, and Fujian, with severe cases occurring occasionally (Guo et al. 2005). The disease incidence has been aggravated with increasing monoculture of newly improved chestnut cultivars in chestnut-producing areas (Yan et al. 2007), though it was not detected in Gansu Province. In September 2021, some chestnut trees (Castanea seguinii) showing symptoms of crown dieback and diffuse sunken cankers on the trunk with swelled margins and subsequent cracking of the outer bark, were collected in mountains of Hui County in Longnan City, Gansu Province (E 104° 15' 5.76″ ，N 35° 11' 30.84″). Symptomatic branches were washed using tap water and dried on sterilized tissue paper. The Junction between diseased and healthy tissue was cut from the bark and sterilized with NaClO (2.5 %) for 2 minutes, then plated on potato dextrose agar (PDA) and incubated at 25 ℃ for 3 to 4 days. After fungal colonies formed, mycelia were transferred and subcultured onto new PDA media and then purified using single spore culture. After 7 days, colonies turned yellow white. Uninucleate conidia were formed in orange pycnidia and the orange pigments could turn purple if in 2% KOH. Conidia were straight or slightly curved, hyaline, with 2.5-3.5 × 1.2-1.5 μm in size. The characteristics of the culture and morphology were similar with those of C. parasitica (Tziros et al. 2016). Perithecia were not found on culture medium. In accordance with previous findings, the sexual stage of C. parasitica appears on diseased trees in late October. For molecular identification, genomic DNA was extracted from mycelium using a Fungal Genomic DNA Extraction Kit (Tsingke Biotech Co. Ltd, Xi'an, China), the ITS region was amplified with primers ITS1/ITS4 (Sorrentino et al. 2019), and the TEF1-α region was amplified with primers TEF-1H/TEF-2T (O'Donnel et al. 1998). Cloning and sequencing of PCR products were carried out by Tsingke Biotech Co. Ltd, Xi'an, China. The resulting sequences were deposited in GenBank (ITS sequence accession number: OM033734, TEF sequence accession number: OM12254). BLAST results revealed that the sequences of ITS and TEF shared identity over 99% with those of C. parasitica strains (GenBank accession number: AY308953, KP524763, KP824756 and KF220299). Based on morphological and molecular characteristic, the fungal isolates were identified as C. parasitica. To verify pathogenicity, thirty 3-year-old chestnut seeding (70 cm high, 1 cm diameter) of Castanea seguinii were used for inoculation. Chestnut branches were wounded (five wounds per sapling) using a hole punch and inoculated with a mycelial plug (5 mm in diameter) from the edge of 7-day-old, actively growing colonies. Pathogen-free PDA plugs were used as controls. To prevent desiccation, inoculated wounds were sealed with parafilm, and saplings were incubated in a greenhouse at 25℃. Each treatment consisted of 5 seedling and the pathogenicity tests were repeated three times. After inoculation for 5 weeks, symptoms of bark cankers were observed on branches similar to those of diseased chestnut trees in the field. Control saplings with sterile PDA discs did not display symptoms. C. parasitica was reisolated from inoculated branches. To our knowledge, this is the first report of C. parasitica causing chestnut blight in Gansu Province, one of the few areas in the China thought to be free of the disease. The specimens were found in the westernmost part of the natural distribution of chestnuts in China. There are more than 2.6 million chestnut trees, which constitute one of the most important economic forests in Hui County Gansu Province (Yang et al. 2005). The occurrence of chestnut blight could be a restricting factor for chestnut forests.

Development of a real-time PCR protocol for the specific detection and quantification of Penicillium digitatum in lemons

Penicillium digitatum is the main species responsible for postharvest losses in citrus fruit and, therefore, rapid and accurate methods are needed for its early detection. For this purpose, a new procedure based on real-time quantitative PCR (qPCR) was developed to detect and quantify P. digitatum in lemons. The procedure included a rapid extraction of fungal DNA using the commercial Quick-DNA Fungal/Bacterial Miniprep Kit; the design of a specific primers pair by selecting the conserved region of the calmodulin A (cmdA) gene; and the design and evaluation of a qPCR system based on SYBR-Green dye detection chemistry. The functionality of the developed method was demonstrated by the high linear correlation of the standard curve constructed over the entire range of fungal DNA concentrations used (R2 > 0.99), indicating precision and accuracy of the qPCR. The protocol was developed aiming to evaluate the biotechnological potential of the biocontrol yeast Clavispora lusitaniae AgL21, by quantifying the fungus P. digitatum in lemon wounds. The yeast was shown to restrict the pathogen growth, reaching fungal DNA concentrations of up to 0.038 ng/wound after 4 days of incubation, compared to 173.06 ng/wound when the fungus was alone. No inhibition of qPCR due to food matrices was observed. The developed qPCR protocol could be considered as a suitable tool to detect P. digitatum in food products and can be used to specifically test the ability of new biological control microorganisms against green mold in lemons.

Gene Replacement by a Selectable Marker in the Filamentous FungusMagnaporthe oryzae.

Magnaporthe oryzaeis a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain. Key features Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

Dimorphism and microcycle conidiation of Pseudocercospora pseudostigmina‐platani conidia from American sycamore

AbstractIn this study, the micromorphology and surface details of Pseudocercospora pseudostigmina‐platani conidia were investigated on American sycamore (Platanus occidentalis) leaves. Sooty films were collected from diseased leaves for fungal DNA extraction and observed using light and field‐emission scanning electron microscopy. Two types of conidia were present on the leaves: (i) hyaline and curved Cercostigmina‐like conidia and (ii) brown and straight Stigmina‐like conidia. The fungal pathogen was identified as P. pseudostigmina‐platani based on morphological characteristics and internal transcribed spacer region sequences. Some non‐glandular trichomes were found near the centre of the conidial masses on the abaxial surface of the naturally infected leaves. Leaf ultrasonication to dislodge conidia revealed sporodochia erupting through the stomata. Hyphae from sporodochia grew on the leaf surface and entered host tissues through the stomata. Conidiogenous cells had terminal rings and tapered towards the flat region where a conidium was attached. The conidia produced secondary conidia directly without hyphae. After dislodging the conidia by ultrasonication, scars were observed. These results indicate that the fungus has dimorphic conidia and microcycle conidiation for simple and rapid asexual reproduction.

P407 Standardization of PCR for the diagnosis of invasive aspergillosis and invasive mucormycosis from blood samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives(1) Standardization of PCR-based diagnosis of invasive aspergillosis and invasive mucormycosis in spiked blood samples. (2) Standardization of PCR-based diagnosis of invasive aspergillosis and invasive mucormycosis in proven cases. (3) Validation of the above-standardized protocol in patients with proven/probable invasive aspergillosis or mucormycosis.Materials and Methods: Blood samples were collected from healthy volunteers and patients with proven mucormycosis, proven invasive aspergillosis, and suspected invasive fungal infection. Standard strains of Mucorales and Aspergillus spp. were collected from the National Culture Collection of Pathogenic Fungi, PGIMER and DNA was extracted using a modified protocol of the fungal DNA extraction method by Lee and Taylor (1990). Mucor-specific primer (ZM13), described by Bialek R et al. (2005) was used for mucormycosis samples. Aspergillus specific primer pair was designed from mitochondrial DNA of A. fumigatus using Primer3 software. Genomic DNA was used to determine the optimum annealing temperature and concentration for ZM13 assay. DNA extracted from whole blood samples of healthy volunteers after spiking with a known quantity of A. fumigatus DNA and only genomic DNA was used separately to optimize annealing temperature and concentration of Aspergillus-specific primers. Similarly, whole blood, plasma, and serum samples from healthy volunteers were spiked with various concentrations of Rhizopus arrhizus and A. fumigatus DNA. DNA extracted from spiked samples was used to determine the analytical sensitivity of PCR assays. DNA extracted from blood samples of ten proven mucormycosis and five invasive aspergillosis patients were used to determine the optimum sample dilution factor and template volume for the assay. The final standardized protocols were validated using 28 proven/probable cases of invasive aspergillosis and invasive mucormycosis and thirty cases of suspected invasive fungal infection.ResultsThe optimum annealing temperature and primer concentrations for the assay were 54.3°C and 0.4μm respectively for ZM13 assay and 53°C and 0.45μm respectively for Aspergillus assay. The limit of detection was 0.06fg/μl for ZM13 assay and 0.08fg/μL for Aspergillus specific assay. For mucormycosis assay, the slope of the standard curve was −3.2687 and the percentage efficiency of the assay was 104% with 0.9936 as the coefficient of determination (R2). Likewise, the slope of the standard curve was −3.2695, the percentage efficiency of the assay 102%, and the coefficient of determination (R2) 0.9858 for aspergillosis assay. The best combination of template volume and sample dilution factor was 2.5 μl and 1000x in whole blood samples and, 2.5 μl and 100x in serum samples for mucormycosis assay. It was 1.0 μl and 10x in serum samples for Aspergillus specific assay. The sensitivity, specificity, positive predictive value, and negative predictive value of mucormycosis qPCR using serum samples were 79.3%, 86.4%, 65.7%, and 92.7% respectively. The assay had a positive likelihood ratio of 5.92 and a negative likelihood ratio of 0.24.ConclusionThe real-time PCR protocols standardized for the diagnosis of mucormycosis and invasive aspergillosis from blood samples showed promising results. However, the assay needs further validation in a larger study population.

Brown leaf spot of Jacaranda mimosifolia Caused by Colletotrichum karstii in Sichuan, China.

Jacaranda mimosifolia D. Don is widely cultivated in southwest China (Yunnan, Sichuan, and other regions). It is widely applied in papermaking, medicine, environmental monitoring, timber, urban and rural afforestation, and soil and water conservation. In October 2020, a new brown leaf spot disease of J. mimosifolia was discovered in Xichang City (27°49' to 27°56'N, 102°16' to 102°11'E), with approximately 66.23% disease incidence. Firstly, the typical symptoms showed deep yellow necrotic lesions in the center or on the margin of the leaves. Gradually, the necrotic lesions expanded and developed into brown spots. Under humid conditions, the edges of necrotic lesions turned dark brown progressively. Finally, the leaves withered, died, and fell off. Infected tissues from ten samples were cut into small pieces of 2.5 × 2.5 mm. The surfaces of infected tissues were sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, and rinsed three times in sterile water. They were then blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) at 25℃ for 3 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 78.2 to 82.7 mm. The colonies were light orange, turned pale pink with light orange beneath. The conidia were single-celled, aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, measuring 12.3 to 16.8 × 4.3 to 5.6 μm (n = 100; average=14.5 × 5.1μm). These morphological characteristics were consistent with the description of C. karstii (Zhao et al. 2021). For molecular identification, the genomic DNA of the representative isolate JM202010 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], calmodulin (CAL) [CL1C/CL2C (Weir et al., 2012)], actin (ACT) [ACT512F/ACT-783R (Carbone & Kohn, 1999)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], β-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: OL454787, CAL: OL518966, ACT: OL518967, CHS-1: OL518968, TUB2: OL518969, and GAPDH: OL518970). BLAST results indicated that the ITS, CAL, ACT, CHS-1, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum karstii sequences at NCBI (GenBank MW494453.1, MW495036.1, MG387951.1, MW495038.1, MW495042.1, and MG602034.1). The conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old J. mimosifolia plants (10 plants) and inoculated for pathogenicity test. Fifteen leaves of each plant (10 pots in total) were inoculated with spore suspensions on both sides of the leaves. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 60 to 68% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum karstii was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on J. mimosifolia caused by C. karstii in China. C. karstii was previously reported as the causal agent of anthracnose on Fatsia japonica (Xu et al. 2020) and Nandina domestica (Li et al. 2017) in China. This finding provides an important basis for further research on the control of this disease.

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.

Optimisation of a bead-beating procedure for simultaneous extraction of bacterial and fungal DNA from pig faeces and liquid feed for 16S and ITS2 rDNA amplicon sequencing

Efficient cell lysis is critical for the extraction of DNA from difficult-to-lyse microorganisms such as Gram-positive bacteria and filamentous fungi. A bead-beating (BB) step is usually included in DNA extraction protocols to improve cell lysis. However, there is no consensus on the duration of BB that is necessary for complete lysis of the microbial communities present in complex microbial ecosystems, but which will still maintain the integrity of DNA released from easy-to-lyse microbes. Another consideration is that most protocols are tailored to one particular target group of microbes, typically either bacteria or fungi, in a given sample matrix. In this study, we investigated the impact of five BB durations (0, 3, 10, 15 and 20 min) during DNA extraction with the QIAamp® Fast DNA Stool Mini Kit, on the bacterial and fungal communities of single pig faecal and liquid feed samples, extracted in triplicate, with the objective of determining a suitable ‘catch-all’ method. Both sample types were subjected to the BB durations in triplicate, followed by 16S (bacterial) and ITS2 (fungal) rDNA amplicon sequencing. The performance of the different BB durations was assessed based on the quantity of total DNA extracted, alpha- and beta-diversity analyses of the resultant microbial communities and differential abundance of bacterial and fungal taxa. Our results suggest that 20 min of BB is most appropriate for maximising the lysis of difficult-to-lyse bacteria and fungi in both pig faeces and liquid feed, while minimising the negative impact on easier-to-lyse microbes. Total DNA yield increased with BB duration for both sample types; however, the yield from faeces decreased after 20 min of BB. Despite this, DESeq2 analysis indicated that changes in the differential abundances of the dominant taxa at this point were limited, which was supported by the Shannon diversity results. Maximising the BB duration appeared to be necessary in order to obtain a representative profile of the Gram-positive bacteria, particularly in liquid feed, and of the filamentous fungi present in both sample types. However, considering the small sample size, along with the reliance on differential as opposed to absolute abundances to validate increases or decreases in taxa, a larger-scale study is necessary to verify the findings of the present study.

Improvement of soil testing techniques for detecting spores of potato wart disease Synchytrium endobioticum using molecular methods

Synchytrium endobioticum (Schilb.) Percival. is a pathogen of potato wart disease and has a restricted distribution on the territory of the Russian Federation. Its main pathways are infected potato tubers and different planting material containing soil particles infected with spores of the fungus. One of the main problems is the use of toxic chemicals during detecting the disease in laboratory methods of direct soil testing to identify resting spores. This paper presents the assessment of molecular methods of soil diagnosis for detection of S. endobioticum by direct extraction of fungal DNA from soil samples using the MetaGen reagent kit. Identification was performed using the Fitoskrin. Synchytrium endobioticum-RT kit. The kit was pre-tested using DNA isolated from potato warts by various commercial kits. It was found that the optimal method of DNA isolation from the warts was using the FitoSorb-Avtomat 48 kit at the Tecan robotic station. Studies have shown that the sensitivity of the direct DNA extraction method from soil samples with various infection levels is the same as that of flotation method using carbon tetrachloride. Moreover, this method makes it possible to work with soil samples of different types, including peaty soils.

A rapid fungal DNA extraction method suitable for PCR screening fungal mutants, infected plant tissue and spore trap samples

Current DNA extraction methods from filamentous fungi take from an hour to a full day. These methods require buffers and tools for grinding fungal tissues. Although commercial extraction kits can reduce the amount of time spent preparing and extracting genomic DNA from fungal samples, these can be expensive.Here we describe a quick and inexpensive sonication technique to extract genomic DNA from filamentous fungi without buffer which can be used to perform PCR in under an hour. DNA was extracted from Zymoseptoria tritici, Fusarium graminearum and Botrytis cinerea in vitro cultures, Z. tritici asexual fruiting bodies and directly from spore trap tapes.

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.

Routine laboratory test enabling the detection of dermatophytes and the identification of Trichophyton rubrum by means of in-house duplex real-time PCR

Dermatophytes are responsible, in majority, for fungal infections of skin, hair and nails, and Trichophyton rubrum is the most frequently isolated dermatophyte in humans. The time for dermatophyte growth in culture requires a total of two to four weeks. Molecular methods were developed to improve time to diagnosis and initiation of treatment. We present here an in-house duplex real-time PCR enabling detection of dermatophytes and simultaneous identification of T. rubrum from mycological samples and cultures. The objective of this work was to optimize the fungal DNA extraction method, the detection of dermatophytes and the identification of T. rubrum on a CFX96® (Real-Time PCR Detection System). In addition, the method comparison showed that this new method is more sensitive than the culture and microscopic observations. To conclude, this routinely used method has been accredited ISO 15189 since January 2020 in our laboratory.

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.

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.

Molecular-based assessment of diversity and population structure of Sporothrix spp. clinical isolates from Espírito Santo-Brazil.

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix species that affects humans and animals. Little information on the genetic diversity and population structure of the pathogen is available for Brazil, which is needed to design effective strategies to tackle the advance of sporotrichosis in endemic areas. We assessed the genetic diversity and mating-type distribution of Sporothrix isolates recovered from human and feline cases of sporotrichosis in Espírito Santo-Brazil to better understand the population structure, epidemiology and diversification of this pathogen, as well as to explore the possible routes of transmission involved in the ongoing outbreaks. In all, 75 Sporothrix isolates were identified with phenotypic characteristics. Then, fungal DNA extraction was performed, and the species-specific PCR technique was applied, using markers directed to the calmodulin gene. The mating-type idiomorph of species was identified by PCR using primers targeting the MAT1-1 and MAT1-2 loci. Among the 75 Sporothrix isolates, 76% were confirmed as S brasiliensis and 24% as S schenckii sensu stricto. S brasiliensis was more prevalent in the metropolitan area and S schenckii s. str. in the mountainous region of the state. In both species, the presence of the two sexual idiomorphs was detected, suggesting that they are heterothallic species. Our data suggest that sporotrichosis takes on an epidemic-urban character involving Sbrasiliensis. This species in Espírito Santo is likely to originate from Rio de Janeiro, as most isolates harbour the same MAT 1-2 locus. We confirm that Sbrasiliensis has significantly broadened its area of occurrence, an essential feature of emerging pathogens.

Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues.

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species levelGap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation.Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues.Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae.Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues.Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

First report of Rhizoctonia solani AG-4 HGI Causing Stem Canker on Fagopyrum tataricum (Tartary buckwheat) in China.

Tartary buckwheat (Fagopyrum tataricum) is an ideal functional food source, which is well known to be gluten-free and rich in proteins, fats, vitamins, minerals, and flavonoids of various pharmaceutical uses, such as rutin, quercitin and epicatechin (Zhou, M et al. 2018). The Rhizoctonia solani AG-4 HGIII causing severe canker disease was first isolated from common buckwheat (F. esculentum) in Inner Mongolia of China (Zhou, H et al. 2015). In 2018, sunken lesion and dark brown symptoms were observed on the root and stem of ten days old Tartary buckwheat in Liangshan (28°21'N, 103°19''E), Sichuan Province and Fenghuang (28°19' N, 109°48' E), Hunan Province in China. In the beginning, water soaked spots appeared on the stem base, where gradually became rotten and necrotic, finally resulting in the damping-off and death of buckwheat seedlings. This disease had over 40% incidence and lead to serious losses to the buckwheat production in 2018. To isolate the pathogens on Tartary buckwheat, ten plants with typical symptoms were collected from each location. The infected tissue was taken and cut into 3mm pieces from the margin between healthy and diseased tissue, surface sterilized with 1% sodium hypochlorite solution for 4 min, washed three times with sterile distilled water, dried on sterilized filter paper and then placed on potato dextrose agar (PDA) with 100 mg/ml streptomycin sulfate. After incubation at 28℃ in the dark for 2 days, mycelial tips of four fungal cultures were transferred to PDA plates for purification. Initially, colonies were pale white, and then turned brown after 2 days incubation. The mycelium was hairy and concentrically whorled in the culture medium. Microscopic observation showed that the hyphae characteristically branched at right angles and had constriction at the base of hyphal branches. Nuclear staining showed that the hyphae cells were multinuclear. These morphological features revealed that the isolates belonged to R. solani (Sneh et al. 1991). Subsequently, the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of one isolate was amplified by PCR (White et al. 1990) and sequenced (GeneBank accession no. MT078642) by Shanghai Majorbio Bio-pharm Technology Co.,Ltd. DNA was extracted by Fungal genomic DNA Extraction Kit, D3390-02, OMEGA. The BLAST similarity analysis showed a 99.96% match with R. solani AG-4 HGI (GenBank accession no. JQ343830) and 99.85% identity to R. solani AG-4 HGI isolate SX-8 (GenBank accession no. KJ170346) (Suli, Sun et al. 2015). Furthermore, the phylogenetic analysis performed by the neighbour-joining method (MEGA 7 software) showed that the isolate was clearly clustered with the group of R. solani AG-4 HGI (Ireland et al. 2015). Pathogenicity was tested in the greenhouse condition to satisfy Koch's postulates. Tartary buckwheat plants of seven days old and fifty days old were respectively inoculated near the base of the stem neck with one mycelial plug contacted directly. Ten plants in a pot were inoculated as one treatment, four pots were used for replicates. Control plants were inoculated with PDA medium plugs without fungi. All the plants were kept at 26℃ with 14 h light, 10 h dark and 96% humidity. After five days (Suli, Sun et al. 2015), over 90% of the inoculated plants exhibited necrotic brown lesions on stems that was similar to those symptoms observed in the field, whereas control plants remained asymptomatic. The visible characteristics and ITS sequence of the pathogen re-isolated from symptomatic plants were in accordance with the original isolate (R. solani AG-4 HGI). Based on disease symptoms in the fields, morphological characteristics, ITS sequence analysis, and pathogenicity assay, we concluded that R. solani AG-4 HGI was the principle cause of Tartary buckwheat blight in Liangshan, Sichuan Province and Fenghuang, Hunan Province in China. Previously, R. solani AG-4 HGI has been identified as a Chinese chive pathogen (Shi, Y et al. 2017). To the best of our knowledge, this is the first report of the natural occurrence of Rhizoctonia solani AG-4 HGI affecting Tartary Buckwheat in China. This finding is helpful for the early diagnosis and identification of the disease, which will be the guiding of effective control methods to the devastating disease at the early stage.

Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens

In immunocompromised patients a colonisation with fungi carries the risk to develop serious invasive fungal infection. An early detection is therefore important, but not optimal hitherto. Fortunately, molecular genetic methods have increased the sensitivity of fungal detection and limited the time, until results are available. However, their success depends on an efficient extraction of genomic DNA from the fungal cell in the given diagnostic specimen.To improve the routine DNA preparation method for yeasts and moulds, the impact of bead beating on fungal DNA release was evaluated. PBS, blood and respiratory rinse were spiked with Candida glabrata or Aspergillus fumigatus. DNA was extracted by mechanical bead beating in addition to the different steps of the DNA preparation protocol, which comprised liquid nitrogen treatment, proteinase K digestion and DNA isolation using the EZ1 DNA Tissue Kit and Workstation. In every method variant tested, treatment with liquid nitrogen did not improve the DNA release. Bead beating once followed by proteinase K digestion and EZ1-work-up led to the highest DNA release from fungus, spiked in PBS, and increased the extracted DNA amount of C. glabrata about 100-fold and of A. fumigatus about 10-fold in relation to sole EZ1-work-up. In fungus-spiked respiratory rinse and blood, highest increase in DNA release was measured after triple bead beating with simultaneous proteinase K digestion. Fungal DNA release of C. glabrata increased for >100-fold in respiratory rinse and for >1000-fold in blood and of A. fumigatus for >10-fold in respiratory rinse and about 5- to 10-fold in blood.The data of this study clearly demonstrate that preparation of fungal DNA from human specimens is optimized by introduction of a bead beating step to the conventional DNA-preparation method without the necessity of a liquid nitrogen step.

Assessment of fecal DNA extraction protocols for metagenomic studies.

BackgroundShotgun metagenomic sequencing has improved our understanding of the human gut microbiota. Various DNA extraction methods have been compared to find protocols that robustly and most accurately reflect the original microbial community structures. However, these recommendations can be further refined by considering the time and cost demands in dealing with samples from very large human cohorts. Additionally, fungal DNA extraction performance has so far been little investigated.ResultsWe compared 6 DNA extraction protocols, MagPure Fast Stool DNA KF Kit B, Macherey Nagel™ NucleoSpin™®Soil kit, Zymo Research Quick-DNA™ Fecal/Soil Microbe kit, MOBIO DNeasy PowerSoil kit, the manual non-commercial protocol MetaHIT, and the recently published protocol Q using 1 microbial mock community (MMC) (containing 8 bacterial and 2 fungal strains) and fecal samples. All samples were manually extracted and subjected to shotgun metagenomics sequencing. Extracting DNA revealed high reproducibility within all 6 protocols, but microbial extraction efficiencies varied. The MMC results demonstrated that bead size was a determining factor for fungal and bacterial DNA yields. In human fecal samples, the MagPure bacterial extraction performed as well as the standardized protocol Q but was faster and more cost-effective. Extraction using the PowerSoil protocol resulted in a significantly higher ratio of gram-negative to gram-positive bacteria than other protocols, which might contribute to reported gut microbial differences between healthy adults.ConclusionsWe emphasize the importance of bead size selection for bacterial and fungal DNA extraction. More importantly, the performance of the novel protocol MP matched that of the recommended standardized protocol Q but consumed less time, was more cost-effective, and is recommended for further large-scale human gut metagenomic studies.

First Report of Leaf Spot on Chenopodium album Caused by Nigrospora pyriformis in China

Chenopodium album Linn. (Chenopodiaceae) is an herbaceous vegetable plant that originates from Western Asia. It is usually grown in gardens and considered as nutritious in the human diet (Poonia and Upadhayay 2015). Moreover, the leaves of C. album harbor several bioactive compounds, which are used in ethnomedical practices (Sikarwar et al. 2017). In July 2018, leaf spots were observed on leaves of C. album cultivated at a production base located in the Shuitang National Forest Park, Hezhang, Guizhou, China (27°04′56″ N, 104°38′36″ E). The emerging occurrence of the disease significantly reduced the yield of C. album at different small-scale farms in the region. At the selected sampling site, the disease incidence was 50 to 60% on a 0.067-ha field. A total of 12 leaf samples with symptomatic spots were collected. They were surface sterilized with 10% sodium hypochlorite solution for 30 s followed by 75% ethanol for 30 s, rinsed three times with sterilized distilled water, air dried, placed on potato dextrose agar, and incubated at 25°C in the dark for 10 days. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after 10 days. Conidia were produced from clusters of conidiophores. The single-cell conidia were initially pale brown with black pigments that accumulated with time, dimorphic, globose to subglobose, 8.2 to 13.3 μm in diameter or pyriform, 14.4 to 21.5 × 8.3 to 14.5 μm. The surface of the conidium was shiny, smooth, and aseptate. Morphological characteristics of the isolates matched the description of the Nigrospora genus (Barnett and Hunter 1998). Genomic DNA of four representative isolates was extracted according to the manufacturer’s instructions (Biomiga Fungal DNA Extraction Kit, U.S.A.). Subsequently, PCR amplifications were performed with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) for the internal transcribed spacer (ITS) region, which also includes flanking rRNA regions and the complete 5.8S rRNA gene (White et al. 1990). The obtained sequences were compared with other ITS sequences following alignment with the GenBank database. BLAST analysis of the ITS region showed more than 97% identity (alignment length: 524 bp) to Nigrospora pyriformis type specimen isolate CGMCC 3.18122 (accession no. NR_153469.1). A representative sequence (isolate CXYL) was deposited in GenBank (accession no. MN044951). In addition, homology was confirmed with a phylogenetic tree constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch’s postulates, conidial suspensions of the representative strain were prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 10⁶ conidia/ml. Leaves of five 4-month-old healthy C. album plants were spray inoculated with the suspension (200 μl per leaf), and an additional five plants were sprayed with equal amount of 0.05% Tween 20 buffer as a control. After 15 days, symptomatic spots appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control. Pure cultures of N. pyriformis were isolated from infected leaves and identified with the methods mentioned above. Recently, Nigrospora leaf spot was reported on watermelon and Photinia serrulata in Guizhou Province, China (Chen et al. 2019). To our knowledge, this study is the first report of N. pyriformis as a pathogen on C. album causing leaf spots. The identification of the pathogen could provide relevant background for its future management.