Pure Fungal Cultures Research Articles

Potential mycotoxin contamination risks of apple products associated with fungal flora of apple core

During development apples may become infected with mycotoxin-producing fungi without showing surface symptoms at fruit maturity. Cores from five apple cultivars: Ambrosia, Honey Crisp, Golden Delicious, Red Delicious and McIntosh, were aseptically removed from surface sterilized fruit and incubated at 25 °C. The cores were screened for the presence of fungal mycelium and associated mycotoxins. The average percentage of cores with fungal presence was 53.3–58.0%. Twenty five fungal species were identified by sequencing internal transcribed spacers (ITS) of 18S rDNA. The fungi mainly belonged to Alternaria, Penicillium, Aspergillus, Fusarium and Trichoderma species. Fungal pure cultures were inoculated into the apple matrix and incubated for 14 days. Liquid chromatography-mass spectroscopy demonstrated that the isolated fungi had the capacity to produce mycotoxins patulin, alternariol, tentoxin, ochratoxin A and T2-toxin. In addition, mycotoxin-specific genes and/or their transcripts were detected in some of the fungal isolates, but their corresponding mycotoxins were not detected in all isolates containing the genes.

Comparative study of the fungicide Benomyl toxicity on some plant growth promoting bacteria and some fungi in pure cultures.

Six laboratory experiments were carried out to investigate the effect of the fungicide Benomyl on pure cultures of some plant growth promoting bacteria (PGPB) and some fungi. The highest LD50 was recorded for Bacillus circulans and proved to be the most resistant to the fungicide, followed by Azospirillum braziliense, while Penicillium sp. was the most affected microorganism. LD50 values for the affected microorganisms were in 21–240 orders of magnitude lower in comparison with the LD50 value for Azospirillum braziliense. The results indicate a strong selectivity for Benomyl against Rhizobium meliloti and Penicillium sp. when compared to other microorganisms tested. The highest safety coefficient was recorded for Bacillus circulans followed by Azospirillum braziliense, while Rhizobium meliloti, showed the lowest safety coefficient value compared to other bacteria. The lowest toxicity index was recorded for Bacillus circulans and Azospirillum braziliense. The slope of the curves for Bacillus sp. and Rhizobium meliloti was steeper than that of the other curves, suggesting that even a slight increase of the dose of the fungicide can cause a very strong negative effect. In conclusion, Benomyl could be applied without restriction when using inocula based on growth promoting bacteria such as symbiotic nitrogen fixers (Rhizobium meliloti), non-symbiotic nitrogen fixers (Azospirillum braziliense) or potassium solibilizers (Bacillus circulans), given that the fungicide is applied within the range of the recommended field dose.

Investigations of Biodeterioration by Fungi in Historic Wooden Churches of Chiloé, Chile

The use of wood in construction has had a long history and Chile has a rich cultural heritage of using native woods for building churches and other important structures. In 2000, UNESCO designated a number of the historic churches of Chiloé, built entirely of native woods, as World Heritage Sites. These unique churches were built in the late 1700 s and throughout the 1800 s, and because of their age and exposure to the environment, they have been found to have serious deterioration problems. Efforts are underway to better understand these decay processes and to carryout conservation efforts for the long-term preservation of these important structures. This study characterized the types of degradation taking place and identified the wood decay fungi obtained from eight historic churches in Chiloé, seven of them designated as UNESCO World Heritage sites. Micromorphological observations identified white, brown and soft rot in the structural woods and isolations provided pure cultures of fungi that were identified by sequencing of the internal transcribed region of rDNA. Twenty-nine Basidiomycota and 18 Ascomycota were found. These diverse groups of fungi represent several genera and species not previously reported from Chile and demonstrates a varied microflora is causing decay in these historic buildings.

Watershed-Scale Fungal Community Characterization along a pH Gradient in a Subsurface Environment Cocontaminated with Uranium and Nitrate

The objective of this study was to characterize fungal communities in a subsurface environment cocontaminated with uranium and nitrate at the watershed scale and to determine the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution, and diversity of fungi in subsurface groundwater samples were determined using quantitative and semiquantitative molecular techniques, including quantitative PCR of eukaryotic small-subunit rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from the subsurface. Our results demonstrate that subsurface fungal communities are dominated by members of the phylum Ascomycota, and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH <4.5) conditions. Fungal isolates recovered from subsurface sediments, including cultures of the genus Coniochaeta, which were detected in abundance in pyrosequence libraries of site groundwater samples, were shown to reduce nitrate to nitrous oxide. Denitrifying fungal isolates recovered from the site were classified and found to be distributed broadly within the phylum Ascomycota and within a single genus of the Basidiomycota. Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions.

Microbial colonization of Provox voice prosthesis in the Indian scenario.

Tracheoesophageal speech using the voice prosthesis is considered to be the "gold standard" with success rates as high as 90%. Despite significant developments, majority eventually develop dysfunction due to microbial deterioration. We did a pilot study of 58 laryngectomy patients who developed prosthesis dysfunction. A total of 58 laryngectomy patients who had their dysfunctional prosthesis removed were included in this study. Dysfunctional prostheses were removed and examined. Esophageal and tracheal flanges were examined separately. After obtaining pure fungal and bacterial cultures, the yeast strains were identified. Bacteria were identified with the light microscope and gram staining. We analyzed prosthesis lifespan and probable factors affecting it. Central leak was found in 43% cases while in 57% peri-prosthetic leakage was the most common reason for prosthesis replacement. Microbial analysis revealed a combination of yeast and bacteria in approximately 55% culture samples. Out of these, almost 90% had the presence of single yeast species with bacteria. Pure fungal culture was identified in rest of the 45% cultures while none detected pure bacterial forms. Candida tropicalis was the solitary yeast in 81% while Candida albicans was seen in 10% as the solitary yeast. Bacterial isolates revealed Klebsiella pneumonia in 19%, Escherichia coli in 8% while Staphylococcus aureus was grown in 1% cultures. The consumption of curd (P = 0.036, 95% confidence intervals [CI]: 2.292-64.285) to have a significant correlation of the mean prosthesis lifespan. Consumption of curd (P = 0.001, 95% CI: 0.564-2.008) and history of prior radiotherapy (P = 0.015, 95% CI: 0.104-0.909) had a significant bearing on the Provox prosthesis lifespan. Candida is the most common organism grown on voice prosthesis in Indian scenario. Consumption of curd and history of prior radiotherapy significantly affect Provox prosthesis lifespan.

PM 7/117 (1) Hymenoscyphus pseudoalbidus

Specific scopeThis Standard describes a diagnostic protocol for Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea).Specific approval and amendmentApproved as an EPPO Standard in 2013‐09.

Identification and symbiotic ability of Psathyrellaceae fungi isolated from a photosynthetic orchid, Cremastra appendiculata (Orchidaceae)

Photosynthetic orchids found in highly shaded forests are often mixotrophic, receiving part of their carbon energy via ectomycorrhizal fungi that had originally received carbohydrate from trees. A photosynthetic orchid, Cremastra appendiculata, is also found under highly shaded forest, but our preliminary data suggested that its associated fungi were not ectomycorrhizal. We tested whether their relation is an unusual example of a mixotrophic orchid associating with saprotrophic fungi by direct detection of fungal DNAs in conjunction with isolation of the fungus in pure culture and experimental inoculation of orchid seeds with the fungus. • For isolated mycobionts of C. appendiculata plants, two regions of nuclear ribosomal DNA, the internal transcribed spacer (ITS) and the large subunit (LSU), were sequenced, and fruiting bodies of the one isolate, SI1-1 were induced. In addition, two fungal isolates, SI1-1 and KI1-1, were grown in symbiotic cultures with C. appendiculata to verify their status as mycobionts. • In phylogenetic analyses, all isolates clustered with fungi belonging to Coprinellus in Psathyrellaceae of Agaricales. Phylogenetic analyses of these DNA sequences showed that five fungal isolates from C. appendiculata, including SI1-1 and two mycobionts isolated from the mycoheterotrophic orchid Epipogium roseum, have very similar ITS sequences. Isolate SI1-1 was identified as Coprinellus domesticus based on the morphological characteristics of the fruiting body. Isolates SI1-1 and KI1-1 induced seed germination of C. appendiculata as mycobionts. • This report is the first of a mycorrhizal symbiosis between a fungus in Psathyrellaceae and a photosynthetic orchid, revealing a new pathway to full mycoheterotrophy and contributing to our understanding of the evolution of mycoheterotrophy.

Growth Inhibition of Cocoa Pod Rot Fungus Phytophthora palmivora byPseudomonas fluorescence and Bacillus subtilis bacteria

Black pod disease caused by Phytophthora palmivorafungus is one of the important diseases on cocoa crop. Pod rot is the most important disease because it may cause loss of cocoa pod. Until now, the fungal pathogen of cocoa black pod disease is still a crucial problem and there is no fungicide that is really effective against the disease. One alternative to control the cocoa black pod disease is by using biological agents as biofungicide, including utilizing Pseudomonas fluorescenceand Bacillus subtilis bacteria. The research was done by isolation of P. palmivora from infected pods of Kaliwining Experimental Station to obtain pure cultures of fungus and by multiplication of P. fluorescence and B. subtilis. Antagonist test was performed by inoculating P. palmivora into a petri dish in a distance of 3 cm from the edge. P. fluorescenceand B. Subtilis were inoculated into petridishes in three days after the fungal treatment. Control was inoculated with isolate of P. palmivora only. Fungal growth was measured everyday by measuring radius of fungal colonies first time 24 hours after inoculation. Growth of Phytophthora palmivora in the two treatmens were used to calculate the percentage of inhibition. The results of this study indicated that P. fluorescence and B. subtiliswere able to inhibit fungal growth of P. palmivora. Both bacterial antagonists had the same effectiveness in inhibiting the growth of P. palmivora fungus based on the percentage of inhibition and effectiveness criteria. Based on the results of translucent zones indicated that B. subtiliswas more powerfull in inhibiting growth of P. Palmivora compared to P. fluorescence. Key words: Black pod disease of cocoa, biological control, Phytophthora palmivora, Pseudomonas fluorescence, Bacillus subtilis

Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza

Mycorrhizal exchange of nutrients between fungi and host plants involves a specialization and polarization of the fungal plasma membrane adapted for the uptake from the soil and for secretion of nutrient ions towards root cells. In addition to the current progress in identification of membrane transport systems of both symbiotic partners, data concerning the transcriptional and translational regulation of these proteins are needed to elucidate their role for symbiotic functions. To answer whether the formerly described Pi-dependent expression of the phosphate transporter HcPT1.1 from Hebeloma cylindrosporum is the result of its promoter activity, we introduced promoter-EGFP fusion constructs in the fungus by Agrotransformation. Indeed, HcPT1.1 expression in pure fungal cultures quantified and visualized by EGFP under control of the HcPT1.1 promoter was dependent on external Pi concentrations, low Pi stimulating the expression. Furthermore, to study expression and localization of the phosphate transporter HcPT1.1 in symbiotic conditions, presence of transcripts and proteins was analyzed by the in situ hybridization technique as well as by immunostaining of proteins. In ectomycorrhiza, expression of the phosphate transporter was clearly enhanced by Pi-shortage indicating its role in Pi nutrition in the symbiotic association. Transcripts were detected in external hyphae and in the hyphal mantle, proteins in addition also within the Hartig net. Exploiting the transformable fungus H. cylindrosporum, Pi-dependent expression of the fungal transporter HcPT1.1 as result from its promoter activity as well as transcript and protein localization in ectomycorrhizal symbiosis are shown.

First Report of Leaf Blight of Cyperus iria Caused by Fusarium equiseti in India.

In India, rice (Oryza sativa L.) plays a major role in national food security, with total production of 102.75 million t, harvested from 44 million ha during 2011 (1). Weeds are one of the major causes of losses in rice. Cyperus iria, locally known as chatriwala dela (rice flat sedge), is an annual weed in the Cyperaceae that can reach 50 to 60 cm tall. A leaf blight of C. iria was observed during August 2010 in a 20-ha rice field (cv. Basmati 370) at the University Research Farm, Chatha, Jammu (32° 43' N, 74° 54' E). Symptomatic plants were scattered randomly in the field and had water-soaked spots on the upper leaf surfaces initially, which turned brown after 4 days and developed a yellow halo, resulting in a blighted appearance. The diseased leaves shriveled and infected plants died. Infected C. iria leaf pieces with adjacent healthy tissue were collected, surface-sterilized in 0.1% mercuric chloride for 20 s, then rinsed three times in sterilized distilled water. The pieces were plated onto potato dextrose agar (PDA) and incubated at 27 ± 1°C for 4 days. A pure fungal culture was obtained by single-spore technique on 2% water agar and maintained on PDA at 10°C. The fungus initially produced white mycelium that became brown with age. Dark brown spots or flecks of pigment formed in the agar. Macroconidia were long and slender, with tapered apical cells that were elongated or even whip-like. Basal cells of macroconidia were prominent, foot shaped, and elongated. Macroconidia were 39.55 to 56.74 × 3.75 to 4.5 μm with 3 to 5 septa. Conidiophores were compact, penicillately branched, and arose from lateral branches which initially were one-celled and bore 2 to 4 phialides at the apex. Chlamydospores were intercalary, solitary, in chains or in knots, globose, and 7 to 9 μm in diameter. On the basis of morphological characteristics (2), the fungus was identified as Fusarium equiseti (Corda) Sacc. and deposited in the Indian Type Culture Collection, New Delhi (8424.11). The ITS (internal transcribed spacer) region of rDNA was amplified by PCR with primers ITS1/ITS2 and sequenced. BLASTn analysis of the sequence showed 100% homology with the ITS sequence of F. equiseti in the NCBI database (JN596252.1), and the sequence was deposited in GenBank (KC434458). To confirm pathogenicity of the F. equiseti isolate, 10 seeds of C. iria were planted in five clay pots (each 38 cm in diameter) filled with sterilized soil. Three seedlings were used for the experiment and the remaining seedlings removed from each pot. A total of 15 seedlings (5 pots × 3 seedlings per pot) at the two-leaf stage were spray-inoculated with a 50-ml conidial suspension of the isolate (105 cfu/ml) using a hand atomizer. The control treatment included three seedlings treated similarly with sterile distilled water. The spore suspension was prepared in potato dextrose broth using a culture of the fungus incubated for 10 days and then homogenized at 140 rpm. Tween 20 (1%) was added to the spore suspension. Small spots developed 4 days after inoculation, and the lesions then coalesced into large necrotic areas, resulting in leaf blight 10 days after inoculation. F. equiseti was reisolated from inoculated leaves using the method described above, whereas no fungus was reisolated from control plants, fulfilling Koch's postulates. The isolated fungus displayed the same morphological and cultural features as the original isolate. F. equiseti has been reported to infect Echinochloa spp. in Iran (3), but to our knowledge, this is the first report of F. equiseti infecting C. iria in India. Thus, F. equiseti represents a potential biocontrol agent for managing C. iria in rice fields.

First Report of a Leaf Spot on Greenhouse Tomato Caused by Cladosporium oxysporum in China

Tomato (Lycopersicon esculentum Mill.) is an important vegetable crop grown in Liaoning Province, China. In May 2012, dark brown, angular lesions were observed on lower, older leaves of 5-month-old tomato plants of cv. Bi Jiao in commercial greenhouses in Dawa County, which is administered by Panjin City in Liaoning Province. The irregularly shaped lesions varied in size from 1 to 7 mm in diameter. The necrotic lesions were usually surrounded by a yellow halo. Sporulation was rarely seen on the leaf surfaces. This contrasts with tomato leaf mold caused by Passalora fulva, in which the conidia develop as a velvety brown patch in lesions. By July 2012, the same disease was found in research greenhouses of Shenyang Agriculture University and Liaoning Academy of Agricultural Sciences. The incidence of symptoms was 30 to 40%. To identify the pathogen, leaf pieces (3 to 5 mm) with both infected and healthy portions were taken at the edge of lesions and surface-disinfected by placing them in 75% ethanol for 5 s, then transferred to a 0.1% aqueous mercuric chloride solution for 30 s and rinsed with sterilized water three times. The sections were placed on potato dextrose agar (PDA) at 25°C in the dark. Ten pure fungal cultures were obtained from single spores. For studies of microscopic morphology, isolates were grown on synthetic nutrient agar (SNA) in slide cultures. Conidia ranged in shape from subglobose or ovoid (2 to 4 × 2 to 3 μm) to subcylindrical (4.5 to 17.8 × 2.4 to 4.5 μm). Conidiophores were straight to slightly flexuous with typical nodes. The internal transcribed spacer (ITS) region from isolate PJ12-36 was amplified using primers ITS1 and ITS4 and sequenced (GenBank Accession No. KC137278). The 560-bp amplicons had 99% identity to C. oxysporum (JQ775499). On the basis of morphological characteristics (1) and nucleotide homology, the isolate was identified as C. oxysporum. Koch's postulates were fulfilled in the laboratory on fully expanded leaves of 5-week-old tomato plants 'Moneymaker' inoculated with C. oxysporum conidial suspensions (107 conidia ml-1). Eight seedlings were incubated in an illuminated incubator at 25°C for 8 to 10 days with 85% relative humidity. Characteristic lesions that developed on inoculated leaves were similar in appearance to those observed on diseased tomato plants in the greenhouse. C. oxysporum was reisolated from lesions and its identity was confirmed by morphological characteristics. C. oxysporum was previously reported as the causal agent of a leaf spot disease of pepper (2) and greenhouse tomato (4). To our knowledge, this is the first report of C. oxysporum causing disease on tomato foliage in China. It is noteworthy that C. oxysporum has led to a decline in pepper yield in northern regions of China (3). In addition, pathogenicity tests showed that the isolate W10-02 from pepper and the isolate PJ12-36 from tomato could each cause damage to the opposite host, producing symptoms similar to those observed on the host of origin. Studies are needed on strategies to manage C. oxysporum in crops, because its prevalence may cause yield loss both on tomato and pepper in northern regions of China.

A Comparative Study of Caffeine Degradation by Four Different Fungi

ABSTRACT The objective of the present study is to investigate the caffeine-degrading abilities of different fungi and to apply this knowledge to environmental remediation and industrial decaffeination process. Chrysosporium keratinophilum, Gliocladium roseum, Fusarium solani, and Aspergillus restrictus were isolated from the coffee pulp obtained from a coffee estate. Pure cultures of fungi were isolated on standard conventional potato dextrose broth (PDB) medium and authenticated. Pure cultures were subjected to a caffeine tolerance study at different concentrations of caffeine (1–8 g/L) in potato dextrose agar (PDA) and minimal media. On PDA, Fusarium solani could tolerate caffeine concentration up to 8 g/L, whereas Chrysosporium keratinophilum, Gliocladium roseum, and Aspergillus restrictus could tolerate up to 6 g/L. On minimal agar medium containing different concentrations of caffeine (1–8 g/L), Fusarium solani tolerated up to 8 g/L and the other fungi up to 2 g/L. A time-bound caffeine degradation study was undertaken at 1 g/L concentration of caffeine and glucose in nitrogen-containing and nitrogen-free liquid minimal media by subjecting the four fungi to shake flask culture at 120 rpm and 30°C. Degradation of caffeine up to 7 days at 24-h intervals was analyzed by high-performance liquid chromatography (HPLC). Gliocladium roseum followed by Aspergillus restrictus showed maximum degradation of caffeine at 0.47 and 0.3 mg/ml, respectively, by 96 h in nitrogen-containing minimal medium, whereas Fusarium solani showed maximum degradation of caffeine by 48 h (0.35 mg/ml) and Chrysosporium keratinophilum by 72 h (0.29 g/ml). In nitrogen-free minimal medium, Chrysosporium keratinophilum showed maximum degradation of caffeine at 72 h (0.45 mg/ml), followed by Gliocladium roseum, Fusarium solani (0.3 mg/ml), and Aspergillus restrictus (0.25 mg/ml) at 96 h. Overall, Chrysosporium keratinophilum showed a comparatively higher rate of caffeine degradation in minimal medium with or without a nitrogen source as compared with the other three fungi, indicating that nitrogen affects caffeine metabolism.

Fungal polymerase chain reaction testing in equine ulcerative keratitis

To assess the diagnostic utility of fungal polymerase chain reaction (PCR) in forty-three horses with naturally acquired corneal ulcers presenting to a private practice. Routine evaluation of cytologic, histologic, and microbiologic samples was performed. Two PCR approaches were compared - generic and specific fungal nested PCR followed by sequencing and quantitative PCR (qPCR). PCRs were applied to pure control fungal cultures, corneal tissue from ulcerated eyes and in a subset of 9 horses, to swabs from contralateral normal eyes. The expected fungus was identified by nested PCR and qPCR in all control fungal cultures. In all fungal culture-positive affected eyes (10/43), one or more fungi were identified by nested PCR and 4/10 were positive by qPCR. In 6/10 animals, the same fungus was identified by nested PCR and culture. Of these 6, only three were positive by qPCR. Fungal agents were identified by morphology in 8/10 horses. Diagnosis of fungal keratitis was reserved for only those cases in which the same fungus could be identified by PCR, culture, and morphology (5 horses). In 33/43 culture-negative affected eyes and in 6/9 unaffected eyes, one or more fungi were identified by nested PCR in 26 samples and by qPCR in 2 samples. Apart from Aspergillus spp, similar fungi were identified in affected and control eyes. Most eyes harbored mixed bacterial and fungal agents. Nested PCR results confirmed all cytologically positive cases of fungal keratitis. Nested PCR identified a greater spectrum of agents than either culture or qPCR.

First Report of Southern Blight of Mexican Petunia (Ruellia brittoniana) Caused by Sclerotium rolfsii in Taiwan.

Mexican petunia (Ruellia brittoniana) is an herbaceous flowering perennial with strikingly colored flowers, widely cultivated commercially as a potted plant and a popular garden plant. In July of 2010, root and stem rot that caused death was observed on Mexican petunia at the flower nursery of the Council of Agriculture & Chiayi County in Taiwan. Plants had rotted and girdled stem bases. Necrotic areas were covered with fans of white mycelium as well as abundant spherical sclerotia. A fungus was isolated from infected tissue and sclerotia, and maintained on potato dextrose agar (PDA) plates incubated at 25°C without light. Colonies were white, cottony, often forming fans; pure cultures were prepared by transferring hyphal tips to PDA. Sclerotia formed after 10 days, initially white becoming dark brown with age, and 0.5 to 0.6 mm in diameter. To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA region of the causal fungus was amplified using the primers ITS4 and ITS5 (2) and sequenced. The resulting sequence of 687 bp was uploaded in NCBI. The sequence was 98% similar to sequences of Athelia rolfsii (Sclerotium rolfsii) in NCBI (Accession No. JN543691.1). Koch's postulates were performed using two inoculation techniques. The soil near the base of healthy Mexican petunia plants (four plants per pot) were exposed to recently matured sclerotia (10 sclerotia per plant) developed from pure fungal cultures or 10-mm-diameter agar plugs of mycelium (one plug per plant). Noninoculated plants, in a separate pot, were used as a control. All plants were incubated in a growth chamber at 28 to 33°C. Disease symptoms occurred on all inoculated plants by 5 to 7 days and included yellowing of leaves, basal stem rot, and wilt. Ten days after inoculation, inoculated plants were dead whereas control plants remained healthy. The pathogenicity test was repeated twice with similar results and S. rolfsii was reisolated from infected plants in each test. The pathogen has been reported to cause substantial loss of Mexican petunia in Louisiana (1). The disease is becoming more common in Taiwan and could cause losses in Mexican petunia production. To our knowledge, this is the first report of disease on Mexican petunia caused by S. rolfsii in Taiwan.

Simultaneous monitoring of two fungal genotypes on plant roots by single nucleotide polymorphism quantification with an innovative KASPar quantitative PCR

An innovative quantitative PCR-based method derived from the Kompetitive Allele Specific PCR Assay Reagent (KASPar) system was developed to quantify the genomic DNA from two coexisting genotypes on the same tissues of a host-plant. For this purpose, the classical end-point KASPar method was evolved to a real-time method thanks to the addition of an adapted measurement step after each PCR cycle. It was applied to the quantification of the two genotypes G1 and G2 of the Gaeumannomyces graminis var. tritici (Ggt) soilborne fungus, pathogenic on wheat roots. Specific primers targeting a single nucleotide polymorphism from the ITS sequence were used allowing simultaneous quantification of both genotypes in the same reaction. The assays were applied to quantify fungal DNA of each genotype, aside or mixed together, after DNA extraction from fungal pure cultures and from single or co-inoculated roots in artificial medium or in soil. The detection and quantification lower limits for the two genotypes were 1.25 pg and 5 pg for DNA from fungal pure cultures, and 1.8 pg and 7 pg for DNA from fungal inoculated roots. The advantages of this cost-effective method are the high levels of specificity, sensitivity and reproducibility. Moreover, the accuracy of the method is independent of the copy numbers of the target sequences. The method is the first one to adapt the non-quantitative genotyping KASPar system to a quantitative application of two known genotypes of a species simultaneously and is suitable for simultaneous genotype-specific quantification of any other organisms (fungi, bacteria, plants).

Development and application of a loop-mediated isothermal amplification assay for rapid identification of aflatoxigenic molds and their detection in food samples

Aflatoxins are the most thoroughly studied mycotoxins. They are produced by several members of the genus Aspergillus in section Flavi with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being frequently isolated from contaminated food sources. In this work, we describe the development and evaluation of loop-mediated isothermal amplification (LAMP) assays for rapid detection of the three species in separate analyses. The acl1-gene of A. flavus and amy1-genes of A. nomius and A. parasiticus were used as target genes. The detection limits were 2.4, 7.6 and 20pg of pure DNA/reaction for A. flavus, A. nomius and A. parasiticus, respectively. For specificity testing, DNA extracted from mycelia of representative strains of 39 Aspergillus species, 23 Penicillium species, 75 Fusarium species and 37 other fungal species was used as a template for the specific LAMP primer sets developed for the three target species. The LAMP assay was combined with a DNA extraction method for the analysis of pure fungal cultures as well as artificially contaminated Brazil nuts, peanuts and green coffee beans. It is suggested that the developed LAMP assay is a promising tool in the prediction of a potential aflatoxin risk in food and food raw materials and may therefore be suitable for high throughput analysis in the food industry.

First Report of a Leaf Spot on Pepper Caused by Cladosporium oxysporum in China

Pepper (Capsicum annuum L.) is an important vegetable crop grown in Liaoning Province of China. In June 2009, leaf spotting was observed on hot pepper cv. 37-74 in Wafangdian County of Liaoning Province. By August 2011, the disease had spread to Pulandian and Donggang Counties. Symptoms initially appeared on both sides of leaves as pinpoint chlorotic spots that enlarged and developed into irregular, brown lesions, 1 to 8 mm in diameter. To identify the pathogen, leaf pieces (3 to 5 mm) taken at the edge of lesions with both infected and healthy portions were surface disinfected by placing them in 75% ethanol for 5 s, then transferred to a 0.1% aqueous mercuric chloride solution for 30 s and rinsed with sterilized water three times. The sections were placed on potato dextrose agar (PDA) at 25°C in the dark. Ten pure fungal cultures were obtained from single spores. For growth rate determination and morphological description of colonies, single conidial isolates were inoculated on PDA, malt extract agar (MEA), and oatmeal agar (OA), and incubated at 25°C for 14 days in darkness. For studies of microscopic morphology, isolates were grown on synthetic nutrient agar (SNA) in slide cultures. Colonies grew up to 77 to 80 mm in diameter on PDA, 62 to 66 mm on MEA, and 58 to 60 mm on OA after 14 days. Conidiophores were straight to slightly flexuous. Conidia ranged in shape from subglobose or ovoid to subcylindrical. Macronematous conidiophores measured 40 to 670 × 3 to 5.5 μm with swellings, micronematous conidiophores 15 to 137 × 1.5 to 3.5 μm, terminal conidia 2 to 4 × 2 to 3 μm, and intercalary conidia 4 to 10 × 2.5 to 4 μm. Ramoconidia were rarely observed. Secondary ramoconidia were zero- to one-septate and measured 5.2 to 14.8 × 2.4 to 4 μm. On the basis of these characteristics, the isolates were identified as Cladosporium oxysporum (1). The internal transcribed spacer (ITS) region from isolate W10-02 was amplified using primers ITS1 and ITS4 and sequenced (GenBank Accession No. JQ775499). The 525-bp amplicons had 99% identity to C. oxysporum (GenBank Accession No. EF029816). On the basis of morphological characteristics and nucleotide homology, the isolate was identified as C. oxysporum. Koch's postulates were fulfilled in the laboratory on pepper leaves inoculated with C. oxysporum conidial suspensions (107 conidia ml-1). Eight inoculated 4-week-old seedlings were incubated under moist conditions for 8 to 10 days at 25°C. All leaf spots that developed on inoculated leaves were similar in appearance to those observed on diseased pepper in the field. C. oxysporum was reisolated from lesions and its identity was confirmed by morphological characteristics. C. oxysporum was first reported as a pathogen of pepper in the Sultanate of Oman (2). To our knowledge, this is the first report of C. oxysporum causing leaf spot on pepper in China. The outbreak and spread of this disease may decrease the yield of pepper in the northern regions of China. More studies are needed on the management strategy including the resistance of pepper cultivars against C. oxysporum.

Clonal Spread ofGeomyces destructansamong Bats, Midwestern and Southern United States

To the Editor: Bat geomycosis (white nose syndrome) is caused by the psychrophilic fungus Geomyces destructans, which has rapidly spread in the United States and Canada since it was first reported from Albany, New York (1,2). In 2011, a single genotype of G. destructans was found in bats with geomycosis in different parts of New York (3). The findings raised the possibility of clonal spread of a new pathogen with serious implications for the survival of the affected bat populations (4). To provide information for devising conservation measures, we explored whether this emerging infectious disease is caused by a novel pathogen (5). To do so, we genotyped G. destructans isolates from the midwestern and southern United States. During 2010 and 2011, a total of 11 cultures of G. destructans were isolated and identified: 1 each from Pennsylvania and Ohio, 3 from North Carolina, and 6 from West Virginia (Figure). The cultures came from 8 little brown bats (Myotis lucifugus) and 3 tri-colored bats (Perimyotis subflavus). Two recent G. destructans isolates from New York and 1 G. pannorum isolate were included as controls. Genomic DNA was prepared from fungal growth by the conventional glass bead treatment, phenol–chloroform extraction, and ethanol precipitation. PCR amplifications of 8 G. destructans gene fragments (ALR, Bpntase, DHC1, GPHN, PCS, POB3, SRP72, and VPS13) were performed as described (3). The amplicons were sequenced and nucleotides were aligned by Sequencher 4.8 (www.genecodes.com); phylogenetic analyses were done using PAUP*4.0 software (www.sinauer.com). Figure A) Consensus maximum-parsimony tree of 8 concatenated gene fragments of Geomyces destructans. Data were derived from 13 G. destructans test isolates. G. destructans M1379 and G. pannorum M1378 were used as controls in this study; they were described in ... A total of 4,722 nt sequences were obtained from 8 gene fragments of 13 G. destructans isolates (GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:JQ029780-JQ029883,start_term:JQ029780,end_term:JQ029883,start_term_id:380040511,end_term_id:380040717}}JQ029780-JQ029883) and 1 G. pannorum isolate (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:HQ834330,term_id:386776025,term_text:HQ834330}}HQ834330, {type:entrez-nucleotide,attrs:{text:HQ834347,term_id:386776059,term_text:HQ834347}}HQ834347, {type:entrez-nucleotide,attrs:{text:HQ834364,term_id:386776093,term_text:HQ834364}}HQ834364, {type:entrez-nucleotide,attrs:{text:HQ834381,term_id:386776127,term_text:HQ834381}}HQ834381, {type:entrez-nucleotide,attrs:{text:HQ834398,term_id:386776161,term_text:HQ834398}}HQ834398, {type:entrez-nucleotide,attrs:{text:HQ834415,term_id:386776195,term_text:HQ834415}}HQ834415, {type:entrez-nucleotide,attrs:{text:HQ834432,term_id:386776229,term_text:HQ834432}}HQ834432, and {type:entrez-nucleotide,attrs:{text:HQ834449,term_id:386776263,term_text:HQ834449}}HQ834449). Multiple alignments of these sequences showed 100% identity, and the aligned nucleotides matched perfectly with those of earlier G. destructans sequences for the same gene fragments analyzed from New York isolates (3). The nucleotide alignments of 8 sequences showed differences from those obtained from the closely related fungus, G. pannorum. Maximum-parsimony trees were generated by using sequences from each gene fragment. These trees showed a single clade of G. destructans strains distinct from G. pannorum; similar topologies were obtained when different phylogenetics methods were used for analysis (details not shown). A consensus maximum-parsimony tree derived from the 8 concatenated gene fragments also showed a single clade of G. destructans isolates from New York and the midwestern and southern United States (Figure). The data obtained in this study strongly indicate further clonal spread of G. destructans from its origin near Albany, New York. The locations in which G. destructans was detected in the current study were spread across 5 states, which were >800 miles from Albany. The test isolates were compared with a New York isolate from 2008, which provided a 4-year temporal variation in our sampling. Bats of 2 species were positive for G. desctructans in the current samples, and they yielded the same G. destructans genotype. Thus, there is evidence for host-independent spread of a single clone of G. destructans. These data would support the novel-pathogen hypothesis for the origin of bat geomycosis (5). However, these conclusions are based on limited sampling because isolations of G. destructans from affected bats are uncommon. The demonstration of pure fungal culture in the affected animals is still not the standard for geomycosis diagnostics, and most geomycosis is confirmed by bat morphologic appearance or histopathologic examination. Additionally, our phylogenetics analyses were limited to ≈5 kbp of fungal genomes, which could lead to sampling bias (3). Ideally, a large number of G. destructans isolates, including isolates from Europe, and additional polymorphic markers would be needed to determine the novel or local origin of this pathogen (6,7). The environmental factors that led to introduction or reemergence of G. destructans in mines and caves remain unknown, and their contribution in the spread of the fungus through air, water, and soil is yet to be determined (8). Although no direct evidence has emerged, a role for anthropomorphic activities (occupational or recreational) in this spread is a distinct possibility (9). We provide genetic evidence for further spread of a single genotype of G. destructans from Albany, New York, to locations in the midwestern and southern United States. Experimental transmission of geomycosis from infected bats to healthy bats by direct contact has recently been confirmed (10). Therefore, G. destructans might be rapidly spreading along summer and winter migration routes of bats, which present ample opportunities for mixing of healthy and diseased animals.

Species richness and nitrogen supply regulate the productivity and respiration of ectomycorrhizal fungi in pure culture

Abstract The effects of biodiversity of aboveground organisms have been widely investigated in a range of ecosystems, yet whether similar responses are also seen in belowground microbial communities, such as ectomycorrhizal (EM) fungi, are little understood. We investigated, in vitro , the effects of a gradient of 1–8 species of EM fungi interacting with substratum carbon:nitrogen (C:N) ratio on biomass production and CO 2 efflux. The model experimental systems enabled us to recover and measure biomass of individuals within communities and calculate net selection and complementarity effects. Both biomass and CO 2 efflux increased with species richness particularly under high N concentrations. Moreover, net biodiversity effects were largely positive, driven by both selection and complementarity effects. Our results reveal, in pure culture, the implications of EM species richness on community productivity and C cycling, particularly under high N conditions, and constitute the basis for future experiments under natural conditions.

Evaluation of electrolyzed oxidizing water for phytotoxic effects and pre-harvest management of gray mold disease on strawberry plants

Near neutral (pH = 6.3–6.5) electrolyzed oxidizing water (EO water) has been demonstrated to inactivate fungi in pure culture and to mitigate infection on fruit surfaces. One possible alternative or supplement to traditional pre-harvest crop management practices that currently rely on the use of large quantities of fungicides is near neutral EO water. In the present work, treatment of Botrytis cinerea or Monilinia fructicola with near neutral EO water (50 or 100 ppm total residual chlorine (TRC)) in pure culture resulted in a 10 6 reduction and 100% inactivation as evidenced by negative broth enrichment. When applied in concert with 50 or 100 ppm EO water, treatments of Captan 50WP (captan), Rovral (iprodione), Iprodione 4LAG (iprodione), or Switch 62.5 WDG (cyprodinil and fludioxonil) effectively inhibited fungal growth of B. cinerea as evidenced by a 10 6 reduction on the direct plate and negative broth enrichment. Treatments of Captan 50WG (captan), Rovral (iprodione), Iprodione 4LAG (iprodione), Switch 62.5 WDG (cyprodinil and fludioxonil), Captan 80 WDG (captan), or Captevate (captan and fenhexamide) when applied in concert with 50 or 100 ppm EO resulted in a 10 6 reduction of M. Fructicola and 100% inactivation as evidenced by negative broth enrichment. Strawberry plants sprayed with EO water (pH = 6.3–6.5) at concentrations of 50 and 100 ppm TRC once per week, did not result in significant ( P > 0.05) phytotoxicity relative to a water (0 ppm TRC) treatment. In this study, the application of 100 ppm EO water (pH = 6.3–6.5) twice per week to strawberry plants infected with B. cinerea was more effective ( P ≤ 0.05) than a once per week Captan application and as effective as a once per week captan/once per week EO treatment. The once per week captan/once per week EO treatment was significantly more effective ( P ≤ 0.05) than the captan once per week treatment. Dip treatments of strawberries in near neutral EO solutions (50 and 100 ppm TRC; pH = 6.3–6.5) did not leave a chlorine residue on the fruit relative to a water dip. The results from this study suggest that near neutral EO solutions could be used to manage infection of B. cinerea on strawberry plants in the field and also as a disinfection solution for harvesting equipment, greenhouses, packing houses and in commercial facilities to prevent or manage infections of B. cinerea and M. fructicola.