Universal Fungal Primers Research Articles

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips.

A simple model system for identifying arbuscular mycorrhizal fungal taxa that actively colonize rice (Oryza sativa L.) roots grown in field soil

ABSTRACTEven a few centimeters of roots in field soils can be colonized by genetically diverse arbuscular mycorrhizal (AM) fungi. The DNA sequences of AM fungi in roots suggest the fungal identities; however, it is difficult to determine which AM fungal taxa are physiologically active. In this study, we took advantage of the characteristics of rice (Oryza sativa L.) mycorrhizal roots, in which active colonization in roots is easily detected via histochemical staining of fungal succinate dehydrogenase activity (vital staining) and individual active colonization regions (infection units) in roots rarely coalesce. Root segments (< 3 mm) containing an active infection unit were dissected and squashed, large subunit (LSU) ribosomal RNA genes (rDNAs) were amplified using fungal universal primers and the sequences were directly determined by Sanger sequencing. All obtained sequences of colonization regions were of glomeromycotan origin. Phylogenetic analysis revealed that the levels of LSU-rDNA heterogeneity within an active colonization site were different among different clades. The methodology presented in this study offers researchers a novel tool for investigating the DNA information of physiologically active AM fungi in roots, whereas the factors that affect genetic diversity among active colonization remain to be clarified.

The phylogenetic relationship and non‐specific symbiotic habit of mycorrhiza fungi from a terrestrial orchid (Cymbidium)

To investigate the specificity of the symbiotic relationship between Cymbidium plants and their mycorrhiza fungi, thirty mycorrhiza fungi were isolated from roots of six terrestrial Cymbidium species. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) with universal fungal primers ITS1/ITS4. All fungal strains isolated from natural roots of orchids were inoculated into the rhizomes of in vitro Cymbidium goeringii. Phylogenetic analysis indicated fungal isolates of different cluster could be obtained from a special terrestrial Cymbidium species. Observation of light microscope and scanning electron microscope showed that fungi entered the cortical tissue by destroying cell wall of epidermal cells, where they formed hyphal knots in the cortical cells and were digested gradually. A large number of small protuberances were visible on cross sections of the rhizome. There was no strict inter‐species specificity between the isolated mycorrhiza fungi and terrestrial Cymbidium.

Direct colony polymerase chain reaction for rapid identification of yeasts isolated from blood specimen

Context: Bloodstream infections (BSIs) caused by yeasts have an increasing frequency due to the growing population of immunosuppressed individuals. Among yeasts, Candida remains the most prevalent species with the increase in the incidence of non-albicans Candida species. Apart from Candida, other yeasts are also involved in causing BSI. High mortality associated with Candida and other yeast infection can be reduced by prompt and appropriate antifungal therapy. Hence, rapid identification and speciation of yeasts isolated from blood play a significant role in the management of the patients. Since conventional methods used for speciation of Candida and other yeasts are laborious, time-consuming and often unclear, rapid and accurate molecular techniques are required. Materials and Methods: Instead of using purified genomic DNA as template for polymerase chain reaction (PCR), we used yeast colony and cell suspensions in water and 0.10M potassium hydroxide as template for PCR. Candida albicans, Trichosporon and Cryptococcus neoformans were used as reference strains. Further, a total of 100 yeast isolates were also tested. All reactions were performed using the universal fungal primers ITS1 and ITS4; the PCR products were then digested with restriction enzyme (Msp1). Results: Direct colony PCR (DCPCR) produced sharp and distinct bands compared to the cell suspensions with the reference strains. All the 100 clinical isolates tested also produced distinct bands. Conclusion: DCPCR approach not only reduces the DNA template preparation time but is also easy, rapid and reduces the cost of PCR.

Laboratory approach for detection of Non-invasive fungal Rhinosinusitis: A case-control study

Noninvasive fungal rhinosinustis is problematic being resistant to traditional medical treatment. Mycology laboratory work helps solving this issue. This case control study was designed to supplement the lacking information about the frequency of noninvasive fungal rhinosinusitis in our locality and identify fungal species responsible for this condition in Zagazig University Hospitals. In addition, to evaluate the role of microscopic examination, antigen detection and PCR in comparison to culture technique in diagnosis. Sinus material was collected from seventy eight cases represented clinically and radiologically with noninvasive fungal rhinosinusitis from June 2013 to September 2015. A control group 78 subjects with healthy sinuses from whom nasal smears were obtained. Samples were processed in Mycology Unit and examined microscopically in 10% KOH preparations. Lactophenol cotton blue slide preparations were examined for microscopic structures as hyphae and conidia. PCR amplification of the extracted DNA was performed using fungal universal primers for amplification of 28 S rDNA genes. Results: Microscopic examination revealed hyphae and fruiting bodies in 37 (47.4% of the cases). Culture diagnosed 36 FRS patients. Aspergillus fumigatus was the most frequently isolated from fungal rhino sinusitis (52.7 %) of cases, followed by Penicillium spp. in 22.2%. PCR amplification exhibits the same sensitivity and specificity as those demonstrated by microscopic examination (100% and 97.3% respectively). ELISA of Aspergillus galactomannan (GM) antigen lacked sensitivity (58.3%), with a higher specificity (100%). Conclusion: It is concluded that an experienced mycological confirmation especially, direct microscopic examination of clinically suspected noninvasive FRS cases is necessary for a final diagnosis. Key words: Rhinosinusitis; fungus; KOH; galactomannan; PCRRunning Title: Fungal rhinosinusitis mycology lab

Etiological Analysis of Fungal Keratitis and Rapid Identification of Predominant Fungal Pathogens.

Fungal keratitis is a worldwide-distributed refractory and potentially blinding ocular infection caused by various fungi. It is necessary to investigate the etiological and epidemiological characteristics of this disease and establish a rapid and specific pathogenic identification method. Here, we isolated and identified fungal pathogens of 275 patients with presumed fungal keratitis from Jilin Province, China, and conducted statistical analyses of epidemiological information. The positive rate of fungal culture was 72.0 %. Fusarium sp. was the most common genus among 210 fungal isolates. The predominant species were Fusarium solani, Aspergillus fumigatus, and Candida glabrata, which accounted for over 50 % of the isolated organisms. Corneal trauma and previous use of drugs were the most important predisposing factors. In addition, a multiplex polymerase chain reaction (PCR) was designed with species-specific primers of the three species that could identify them with amplicons of approximately 330 bp from F. solani, 275 bp from A. fumigatus, and 230 bp from C. glabrata. Additionally, PCR with fungal universal primers and multiplex PCR were performed using DNA prepared by an improved DNA extraction method from corneal scrapings. With this method, fungal pathogens from corneal scrapings could be specifically and rapidly identified within 8 h. The culture-independent rapid identification of corneal scrapings may have great significance for the early diagnosis and treatment of fungal keratitis.

PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus

Aspergillus species are important agents of life-threatening infections in immunosuppressed patients. Proper speciation in the Aspergilli has been justified based on varied fungal virulence, clinical presentations, and antifungal resistance. Accurate identification of Aspergillus species usually relies on fungal DNA sequencing but this requires expensive equipment that is not available in most clinical laboratories. We developed and validated a discriminative low-cost PCR-based test to discriminate Aspergillus isolates at the species level.The Beta tubulin gene of various reference strains of Aspergillus species was amplified using the universal fungal primers Bt2a and Bt2b. The PCR products were subjected to digestion with a single restriction enzyme AlwI. All Aspergillus isolates were subjected to DNA sequencing for final species characterization.The PCR-RFLP test generated unique patterns for six clinically important Aspergillus species, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus and Aspergillus nidulans.The one-enzyme PCR-RFLP on Beta tubulin gene designed in this study is a low-cost tool for the reliable and rapid differentiation of the clinically important Aspergillus species.

Morphological and Molecular Characterization of Ectomycorrhizal Amanita Species Associated with Pinus wallichiana A. B. Jacks.

Worldwide genus Amanita is represented by more than 500 ectomycorrhizal (ECM) species. These species form mutualistic associations with a number of host trees among which the conifers predominate. Set out to explore the ECM associations of Amanita in Kashmir Himalayas, Amanita calyptroderma, Amanita pantherina and Amanita vaginata show symbiotic associations with the roots of Pinus wallichiana. Besides morphological characterization of mycorrhizal roots and sporocarps, molecular characterization of rDNA ITS region was performed for identification of species. ITS-rDNA was used for molecular analysis. The target region of rDNA was amplified using universal fungal primers (ITS1 and ITS4). The sequencing of amplified products and their subsequent blast analysis confirmed the identification of species by comparing the sequences of these species with sequences present in GenBank. Phylogenetic analysis also confirmed the identification of species.

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

BackgroundIntra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate.MethodsWe developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples.ResultsBased on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR.ConclusionsWe developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

Use of Restriction Fragment Length Polymorphism to Rapidly Identify Dermatophyte Species Related to Dermatophytosis

Background:Dermatophytes are a group of keratinophilic fungi worldwide, which can infect the skin, hair and nails of humans and animals. This genus includes several species that present different features of dermatophytosis. Although, laboratory diagnosis of dermatophytes is based on direct microscopy, biochemical tests and culture, these manners are expensive, time consuming and need skilled staff. Therefore, molecular methods like PCR-RFLP are the beneficial tools for identification, which are rapid and sensitive. Thus, dermatophyte species are able to generate characteristic band patterns on agarose gel electrophoresis using PCR-RFLP technique, which leads to successful identification at the species level within a 5-hour period.Objectives:The purpose of this study was to study inter- and intraspecific genomic variations for identification of clinically important dermatophyte species obtained from clinical specimens in Isfahan, Iran using PCR-RFLP.Materials and Methods:From March 2011 to August 2012, 135 clinical isolates were collected from infected patients at Isfahan, Iran. ITS1-5.8S-ITS2 region of rDNA was amplified using universal fungal primers. Subsequently, amplified products were digested by the MvaI restriction enzyme. Using discriminating band profiles on agarose gel, dermatophyte species were identified. However, DNA sequencing was used for unidentifiable strains.Results:The specimens were obtained from skin scrapings (70.3%), nail (24.4%) and hair (5.1%) clippings. Most patients were between 21 - 30 years and the ratio of male to female was 93/42. Trichophyton interdigitale was the commonest isolate (52.5%) in our findings, followed by Epidermophyton floccosum (24.4%), T. rubrum (16.2%), Microsporum canis (2.2%), T. erinacei (1.4%), T. violaceum (1.4%), T. tonsurans (0.7%) and M. gypseum (0.7%) based on PCR-RFLP.Conclusions:Combination of traditional methods and molecular techniques considerably improves identification of dermatophytes in the species level in clinical laboratories, which can lead to properly antifungal therapy and successful management of infections. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.

First Report of Aspergillus niger Causing Root Rot of Peanut in China.

Peanut (Arachis hypogaea) is one of the most important oil crops and food legumes worldwide. China sows approximately 3.5 million hectares each year and produces 40% of the world's peanuts. Fungal diseases are among the main biotic stresses affecting peanut production. Root rot is a serious disease caused by several fungi. Pythium spp., Fusarium spp., and Rhizopus spp. are some of the root rot fungi that have been reported in China. In 2012 and 2013, root rot symptoms were observed in several fields in Laixi District, Qingdao City, Shandong Province, China. The first symptoms appeared in July. Initial symptoms of the disease were brown spots on the stem base and root. Affected plants were stunted, with leaf chlorosis, reduced growth, or sudden wilting. As disease progressed, the infected tissues showed brown discoloration and rot, and abundant dark brown and black powdery spores were visible on the surfaces of affected parts. Eventually, affected plants collapsed and died. To isolate the causal organism, roots and stems were cut into sections, which were surface-disinfected with 70% ethanol solution (v/v) for 20 s, soaked in 0.1% mercuric chloride solution for 50 s, rinsed with sterilized water three times, dried, placed on Czapek's Dox agar supplemented with chloramphenicol (100 μg/ml), and incubated at 28°C for 7 days. Fungal colonies were white initially and then covered with a dense layer of dark brown or black conidial heads. The conidial head was radiate; vesicles were nearly spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose (3.0 to 5.5 μm in diameter), dark brown to black, with rough cell walls. Total genomic DNA was extracted from mycelia using the EasyPure Genomic DNA Kit (TransGEN, Beijing, China). The rDNA-ITS region was amplified using PCR with the universal fungal primers ITS1 and ITS4 (2). The purified products were separately sequenced in both directions using the same primer pair. The sequences (GenBank Accession No. KJ848716) obtained were 99% similar to the ITS sequence of isolates of Aspergillus niger. This, together with the morphological characters (1) described above, suggested that the microorganism we had isolated was A. niger. Koch's postulates were completed in the laboratory by inoculating peanut. Thirty Huayu20 peanut seeds were placed in a 500-ml sterile pot with 300 g of autoclaved soil. Twenty days after seedling emergence, 15 peanut plants were wounded with a needle and inoculated with 5 ml of conidia suspension (106 ml-1). The same number of peanuts were similarly wounded and inoculated with 5 ml of sterile distilled water to serve as controls in the same pot. All peanuts were kept in a randomized complete block design at 30°C under a 12-h photoperiod. After 7 days, disease symptoms similar to those observed in the field appeared in all inoculated but not in non-inoculated peanuts. The tests were repeated three times in the greenhouse. Koch's postulates were satisfied after re-isolating the A. niger from inoculated peanuts using the method described above. To our knowledge, this is the first report of A. niger causing root rot in peanut in China.

Identification and Characterization of Ectomycorrhizal Cortinarius Species (Agaricales, Basidiomycetes) from Temperate Kashmir Himalaya, India, by ITS Barcoding

The coniferous forests of Kashmir Himalayas provide a sustainable habitat for wide varieties of ectomycorrhizal fungi. The identification and characterization of many of these fungi however largely involves morphological descriptions of sporocarps alone, thus sometimes raising questions about the authenticity of these studies. The present study was carried out to identify and characterize ectomycorrhizal fungi from the coniferous forests of Kashmir Himalaya using both morphological and molecular methods. Herein we report on the identification and characterization of three potential ectomycorrhizal Cortinarius fungal species, namely, Cortinarius flexipes (Pers. Ex Fr.), Cortinarius fulvoconicus M. M. Moser, and Cortinarius infractus (Pers.) Fr., from Kashmir Himalaya, India, on the basis of their morphological and molecular characterization. Morphological characteristics of all species were measured and compared with standard taxonomic literature. ITS-rDNA (the fungal molecular marker) was used for molecular analysis. The target region of rDNA (ITS1 5.8s ITS2) of these species was amplified using universal fungal primers (ITS1 and ITS4). The sequencing of amplified products and their subsequent blast analysis confirmed the identification of species by comparing the sequences of these species with respective species sequences present in GenBank. Phylogenetic analysis also confirmed the identification of species.

Could fungi be detected in the fluid of persistent otitis media with effusion?

Abstract Otitis media with effusion (OME) often is considered a direct extension of the inflammatory process that occurs during long-lasting or recurrent episodes of acute otitis media. The observations above suggest that OME has an infectious etiology. Most bacterial and viral cultures of middle ear fluid that had been performed were often negative suggesting that other infectious agents may be involved such as fungi. Thirty patients (group A) suffering from chronic secretory otitis media (OME) were enrolled in this study. Three samples were collected and investigated using PCR assay with universal fungal primers and Sabouraud agar. The first sample was obtained from the fluid of the middle ear before insertion of the ventilation tube; the second sample was obtained from nasal secretions; and the third sample was obtained from the ipsilateral peritubal area of the nasopharynx. Thirty patients (group B) with comparable age group without history of ear diseases scheduled for tonsillectomy or adenotonsillectomy were added as a control group. Samples from peritubal area of the nasopharynx of patients and nasal secretion were tested using PCR assay with universal fungal primers and Sabouraud agar. PCR examination of the middle ear aspirate in group (A) cases was positive in 7 cases (23.3%), in nasal secretions samples 2 cases only (13.3%) were positive and no positive cases were detected in nasopharyngeal swab samples. In group (A), Sabouraud agar culture was positive for fungal culture of middle ear aspirates in 5 cases (16.6%) but in no cases for nasal secretion samples. Group A showed also negative (N0) growth in 30 (100%) patients for nasopharyngeal swab on Sabouraud agar. In group B, the findings of nasopharyngeal swab were negative (N0) growth in all examined samples on Sabouraud agar, and nasal secretions were also negative for fungal DNA detection using PCR assay. In this study, fungal DNA could be detected in the middle ear fluid in seven (23.3%) of 30 patients with persistent OME using PCR assay, and fungi could be detected in five (16.6%) patients on Sabouraud agar. A significant relationship was found between detection of fungi in the middle ear fluid and the duration of the disease, associated adenoid, and history of asthma.

Composition of the sooty blotch and flyspeck complex on apple in Norway is influenced by location and management practices

Sooty blotch and flyspeck (SBFS) is a complex of at least 80 fungal species in 24 genera within the Dothideomycetes order Capnodiales. These epiphytes embed in the waxy epicuticle of apple fruit and colonies become visible in mid- to late summer. The blemished fruit are not marketable as fresh produce. Previous surveys of orchards in central and eastern USA and southern Europe found SBFS assemblages that were unique to geographic region. In the USA, SBFS species diversity was higher in orchards that received no fungicide sprays than those receiving fungicide sprays, suggesting that orchard management was an important determinant of SBFS species distribution and prevalence. To test the hypothesis that the composition of the SBFS complex is affected by geographic region and orchard management practices in Norway, 35 sites with a range of different management practices and cultivars were sampled. Colonies on each apple were counted according to the mycelial type, and subsamples of each mycelial type were amplified and sequenced using either primers specific to the rDNA of Capnodiales or a universal fungal primer pair for the large subunit of rDNA. Results indicated that a single genus was associated with each mycelial type. Seven genera known to cause SBFS were identified: Microcyclospora, Dissoconium, Peltaster, Microcyclosporella, Phaeothecoidiella, Schizothyrium, and Geastrumia. The percent composition of each site’s SBFS assemblage was estimated to genus. Significant differences were found in SBFS assemblages among geographic districts and management practices, and there was a significant interaction of district and management. Organic orchards tended to have the least SBFS severity and genus diversity, whereas higher SBFS diversity tended to occur in abandoned than managed orchards. This is the first survey of SBFS fungi in apple orchards in Scandinavia.

ITS2 REGION-BASED MOLECULAR IDENTIFICATION OF FUNGAL PATHOGENS IN EQUINE CORNEAL ULCERATION

This study aimed to assess the utility of polymerase chain reaction (PCR) and DNA sequencing in diagnosing fungal ulcerative keratitis in horses and compare their sensitivity with conventional microbiologic techniques used in laboratories. Conjunctival swabs from 12 horses with corneal ulcerations admitted to the Veterinary Teaching Hospital, College of Veterinary Medicine, PurdueUniversity were collected for examinations. 14 conjunctival swabs were analyzed by the conventional culture method and by generic PCR using universal fungal primers to amplify the internal transcribed spacer 2 (ITS2) genetic region followed by DNA sequencing. The conventional culture method revealed that two samples exhibited fungal growth that identified as Aspergillus sp. and Penicillium sp. while three conjunctival samples contained bacterial growth which was identified as Staphylococcus intermedius, Staphylococcus epidermidis and Streptococcus zooepidemicus. Interestingly, PCR followed by DNA sequencing of the biological specimens on the swabs identified fungal pathogens in 9/14 samples and Ascomycete species in 3/14 samples. In 2/14 samples, no fungal pathogen was identified using PCR. Fungal pathogens detected included Aspergillus sp. in six eyes, Penicillium sp. in one eye, Fusarium sp. in one eye and Cladosporium sp. in one eye. Our findings demonstrate a limitation of the conventional culture method as a diagnostic tool as a fungal pathogen was detected in only two samples by this method as compared to nine samples using PCR and DNA sequencing. Therefore, PCR followed by DNA sequencing is able to identify a greater spectrum of agents including fastidious organisms or organisms present in lower numbers within mixed infections than can be identified by culture. In conclusion, PCR combined by DNA sequencing not only proved to be an effective and rapid method for the diagnosis of fungal keratitis, but was also a more sensitive diagnostic tool compared to the conventional culture method. Our findings demonstrate that PCR, in particular semi-nested PCR, is a promising tool for faster diagnoses of fungal keratitis in affected horses.

Ectomycorrhizal fungal species of Kashmir Himalaya: identification and characterization by ITS analysis

Most vascular plants in terrestrial ecosystems form mutualistic mycorrhizal associations, among which ectomycorrhizal (ECM) associations predominate. Although coniferous forests of Kashmir Himalaya harbor wide diversity of such ECM fungi, their identification remains largely obscure. The present study was carried out to explore the diversity of ECM fungi from coniferous forests of Kashmir Himalaya. Herein we report four new potential ECM fungal species from Kashmir Himalaya, India on the basis of their morphological and molecular characterization. Morphological characteristics of all species were measured and compared with standard taxonomic literature. Internal transcribed spacer-ribosomal DNA (ITS-rDNA); the fungal molecular marker) was used for molecular analysis. The target region of rDNA (ITS1 5.8S ITS2) of these species was amplified using universal fungal primers (ITS1 and ITS4). The sequencing of amplified products and their subsequent Basic Local Alignment Search Tool analysis confirmed the identification of species by comparing the sequences of these species with respective species sequences present in GenBank. Phylogenetic analysis also confirmed the identification of species.

First Report of Root Rot of Watermelon Caused by Ceratobasidium sp. in Sonora, Mexico.

Watermelon is one of the major crops grown in Mexico and represents 4% of the total cultivated area with fruits in this country. In 2013, Sonora State was ranked second in the production of watermelon at a national level. Fungal and oomycete diseases are among the main biotic factors affecting watermelon production, particularly those caused by species of the genera Fusarium, Phytophthora, Pythium, and Rhizoctonia. During the spring of 2013, wilting or death symptoms were confirmed in approximately 50% of ungrafted watermelon plants grown in four sampled fields along the coast of Hermosillo and Guaymas Valley in Sonora, Mexico. On both roots and stems of infected plants, localized lesions were found that were 0.2 to 2.0 cm long, reddish brown, and slightly sunken on the stem base. In some cases, the discolorations encompassed nearly 90% of the root system. One-centimeter pieces from the edge of lesions on stems and roots were superficially disinfected with 1% sodium hypochlorite, then rinsed with sterile distilled water, placed onto petri dishes containing potato dextrose agar (PDA), and incubated at 25°C for 3 days. Fungal colonies were white initially, then turned brown, and septate hyphae were 3.7 to 4.3 μm in diameter and branched at right angles with a constriction at the origin of the branch point. These characteristics are typical of the genus Rhizoctonia. Binucleate cells from five isolates were observed using a lactophenol aniline blue solution stain, according to Ceratobasidium morphological descriptions. Mycelia from five isolates grown on PDA was used for DNA extraction. The rDNA-ITS region was amplified using PCR with the universal fungal primers ITS1 and ITS4 (3). The purified products were separately sequenced in both directions using the same primer pair. The sequences obtained were 99% similar to those of Ceratobasidium sp. AG-F and AG-Fa isolates (accessions KC193238.1 on Tagetes erecta, HQ168370.1 on Musa spp., and JX913821.1 on soy-rice-weeds, respectively) from GenBank (2,4). The pathogenicity of the fungus was tested under growth chamber conditions. Sets of seven healthy watermelon seedlings of the Sugar red variety were inoculated with five isolates of Ceratobasidium. Three disks (8 mm in diameter) of mycelia grown on PDA were placed around the roots of each plantlet. The pots were maintained at 27 ± 0.1°C for 14 days with a photoperiod of 12 h. Seven uninoculated seedlings were used as a control. Initial symptoms showing water-soaked lesions developed on all inoculated seedlings within 6 to 7 days, while typical disease symptoms appeared after 10 to 14 days after inoculation. Seedlings without inoculum were free from infection. The fungus was re-isolated from the inoculated seedlings on PDA, and identified as Ceratobasidium sp., confirmed using morphological characteristics. A similar disease has been reported recently in Italy and Arizona (1); however, this report is the first description of a Ceratobasidium sp. causing root rot of watermelon in Sonora, Mexico. Agricultural areas where the study was carried represent 90% of the total area cultivated with watermelon in this state, so it is necessary to evaluate the impact of this pathogen in the crop.

CHANGES IN BACTERIAL COMMUNITIES IN DAIRY MANURE DURING NINE MONTHS OF COMPOSTING AS DETERMINED BY DENATURING GRADIENT GEL ELECTROPHORESIS

ISHS International Symposium on Growing Media and Soilless Cultivation CHANGES IN BACTERIAL COMMUNITIES IN DAIRY MANURE DURING NINE MONTHS OF COMPOSTING AS DETERMINED BY DENATURING GRADIENT GEL ELECTROPHORESIS

First Report of Sclerotinia sclerotiorum Causing Sclerotinia Blight on Peanut (Arachis hypogaea) in Northeastern China.

Peanut, Arachis hypogaea L., is an important source of vegetable oil and protein in China with a planting area of 5 million ha and annual production of 16.2 million metric tons. In September of 2012, typical symptoms of Sclerotinia blight were first observed on peanut in Shuangcheng, Heilongjiang Province, China. Approximately 50% of the plants in a field were infected. Symptoms began as a chlorotic wilt on the foliage and developed into necrosis of basal stems and developed very quickly in the field. In advanced stages of the disease, stems and branches became bleached and eventually died. White, fluffy mycelium and black irregular sclerotia (3.5 to 5.4 mm diameter) were observed on the infected stems. Infected branches and pegs were shredded, and most pods dropped on the soil during harvest. To isolate the causal agent of the disease, sclerotia were collected from the field. Twenty sclerotia were surface disinfected in 1% NaOCl for 3 min, rinsed three times with sterile water, placed on potato sucrose agar (PSA) with 100 μg/ml streptomycin, and then incubated at 22°C in the dark for 10 days. Fungal DNA was extracted from mycelia with a TIANGEN DNAsecure Plant Kit (Beijing) and amplified by PCR with the universal fungal primer ITS1 and ITS4. PCR products of five replicates were sequenced and subjected to an NCBI BLAST search. The BLAST search revealed that our sequences (GenBank Accession No. KC935388) had 100% identity with reported sequences of Sclerotinia sclerotiorum. The isolates were identified as S. sclerotiorum (Lib.) de Bary based on mycelia, sclerotia, and rDNA sequence analysis. To conduct pathogenicity tests, nine potted peanut plants (1 month old) were each inoculated with a 5-mm-diameter disk of colonized PSA by placing the inoculum on the base of the stem. Plants were inoculated with a plug of non-colonized PSA as controls. All inoculated plants were covered with a plastic bag for 5 days to maintain high humidity and incubated at 22 to 24°C in the growth chamber with a 12-h photoperiod. After 2 days, the inoculated plants showed water-soaked brown symptoms on the stem base and whole plants wilted after 5 days under high moisture conditions. The symptoms were identical to those observed on peanut plants in Shuangcheng, Heilongjiang Province, whereas the control plants remained symptom-free. Re-isolation of the fungus from the inoculated plants confirmed that the causal agent was S. sclerotiorum. S. sclerotiorum has been reported on peanut in the United States and Argentina (1,2,3). To our knowledge, this is the first report of S. sclerotiorum as a pathogen causing Sclerotinia blight on peanut in northeastern China.

Morphological and molecular analyses of fungal endophytes of achlorophyllous gametophytes ofDiphasiastrum alpinum(Lycopodiaceae)

To understand the early evolution of mycorrhizal symbioses, it is important to know the fungal partners of gametophytes and sporophytes for basal lineages of vascular plants. Subterranean mycotrophic gametophytes of the clubmoss Diphasiastrum alpinum found at three localities gave an opportunity to study their morphology and anatomy and to identify and describe their hitherto unknown fungal endophytes. In addition, sporophytes were screened for fungal partners. Gametophytes with attached young sporophytes were excavated, and their anatomy and their associated fungi were studied by light microscopy. DNA was isolated and amplified with both universal and group-specific fungal primers for the ITS region, the large subunit and small subunit of the nuclear rDNA, respectively, to identify the fungal partner. Gametophytes were uniformly colonized by a fungus with septate hyphae forming coils and vesicles. Its morphology resembles that of the sebacinoid genus Piriformospora. Both ITS and LSU sequences were identified as Sebacinales group B, a basal clade of the Agaricomycetes (Basidiomycota). This fungus was detected in 11 gametophytes from two localities and in rootlets of adjacent Calluna vulgaris (Ericaceae) plants, but was absent in roots of sporophytes. In addition, several ascomycetes and glomeromycetes were found by DNA sequencing. Our study suggests a fungus belonging to Sebacinales group B as the main fungal host of the D. alpinum gametophytes. However, Sebacinales group B fungi occur as well in adjacent Ericaceae plants; therefore, we assume the mycoheterotrophic gametophyte to be epiparasitic on Ericaceae, which would explain the steady association of these plants.