Corn Meal Agar Research Articles

Endophytic Colletotrichum species from Dendrobium spp. in China and Northern Thailand.

Species of Colletotrichum are commonly found in many plant hosts as pathogens, endophytes and occasionally saprobes. Twenty-two Colletotrichum strains were isolated from three Dendrobium species – D.cariniferum, D.catenatum and D.harveyanum, as well as three unidentified species. The taxa were identified using morphological characterisation and phylogenetic analyses of ITS, GAPDH, ACT and ß–tubulin sequence data. This is the first time to identify endophytic fungi from Dendrobium orchids using the above method. The known species, Colletotrichumboninense, C.camelliae-japonicae, C.fructicola, C.jiangxiense and C.orchidophilum were identified as fungal endophytes of Dendrobium spp., along with the new species, C.cariniferi, C.chiangraiense, C.doitungense, C.parallelophorum and C.watphraense, which are introduced in this paper. One strain is recorded as an unidentified species. Corn meal agar is recommended as a good sporulation medium for Colletotrichum species. This is the first report of fungal endophytes associated with Dendrobiumcariniferum and D.harveyanum. Colletotrichumcamelliae-japonicae, C.jiangxiense, and C.orchidophilum are new host records for Thailand.

Investigation of polyphenol resistance of Aspergillus flavus on Cornmeal media

Cornmeal agar (CMA) is a good to model natural conditions (low C and N, high antioxidants, crude fat) for phytopathogenic fungi. Different CMA media was prepared to model the maize kernel as growth environment for Aspergillus flavus, where stress resistance and aflatoxin B1 (AFB1) production were tested. The CMA medium with high polyphenol and low fatty acid content did not support the mycelial growth and high AFB1 production but the sclerotia development of the cultures. High fatty acid content in the CMA exceeded the inhibitory effect of antioxidant polyphenols of corn and low concentration of AFB1 was detected. Glucose supplement of CMA induced AFB1 production proving the need for free carbon source for the secondary metabolite pathway. The tolerance of the fungus against salt and cell membrane stress was lowered on CMA. At higher fatty acid concentration, the aflatoxin B1 production cannot be hindered by the natural antioxidants and that is important in selection of resistant corn hybrids.

Identification and in Vitro Susceptibility Pattern of Fungal Pathogens in Immunocomprimised Patients with Pulmonary Fungal Infections

AbstractBackground: The frequency of fungal infections of the lung has increased particularly in immunocompromised pa-tients. Early diagnosis and treatment is important to start antifungal therapy and to avoid unnecessary use of toxic antifungal agents. This study aimed to identify the common fungal species causing pulmonary infection in immunocom-promised patients and their in vitro antifungal sensitivity pattern in Assiut University Hospitals (AUH).Subjects and Methods: This was a hospital based descrip-tive study conducted on 135 patients admitted at different Intensive Care Units (ICUs) and Oncology Department at Assiut University Hospitals (AUH). Collected respiratory specimens were subjected to direct microscopic examination and inoculation on Sabouraud Dextrose Agar (SDA). Identi-fication of isolated yeasts was done using phenotypic methods including chromogenic media (Brilliance Candida agar and CHROMagar Candida differential media), germ tube test, cornmeal agar and API candida while mould isolates identifi-cation was mainly dependent on macroscopic and microscopic features. Some isolates had confirmed using rRNA gene sequencing. In vitro antifungal susceptibility testing was done using disc diffusion method.Results: In this study 80/135 (59.3%) of collected samples were positive for fungal infection. The most common fungal pathogens isolated were Candida and Aspergillus species. In vitro sensitivity test showed that the yeast isolates had the highest sensitivity to Nystatin (90.9%) followed by Ampho-tericin B (80.3%) while for mould isolates, the highest sensi-tivity was to Voriconazole (71.4%) followed by Amphotericin B (57.1%).Conclusion: Pulmonary fungal infection appears to be an important problem in immunocomprimised patients with Candida albicans was the most commonly isolated yeast from various clinical specimens; also the increase in the resistance especially to azoles is a major concern.

First Report of Pythium myriotylum Causing Root Rot of Yam in China

Yams (Dioscorea spp.) are widely grown in China as vegetables and for herbal medicine. From 2016 to 2018, water-soaked lesions appeared on the fibrous roots of yam seedlings, and some plants were wilted, up to 15% in Heze city, Shandong Province. Fragments of 1 mm in size were excised from water-soaked roots of diseased plants, dipped in a 0.2% calcium hypochlorite solution for 10 min, placed on a V8 medium, and incubated in dark at 28°C for 5 days. A Pythium sp. was consistently isolated from water-soaked roots of 10 yam plants. The Pythium sp. produced sporangia, oogonia, antheridia, and oospores. Sporangia were terminal or intercalary, filamentous, inflated lobulate. Oogonia were globose, terminal or intercalary, ranging from 20 to 40 µm (average 29.3 µm) in diameter mating with one to five antheridia. Most oospores were globose, smooth, and aplerotic, ranging from 17.8 to 33.5 µm (average 25.3 µm). The antheridia were diclinous and shaped like clavates and crooknecks. Five of the isolates were molecularly identified further. Genomic DNA was extracted from the five isolates of the Pythium sp. using a DNA extraction kit (Omega, U.S.A.). The cytochrome c oxidase subunit I (COI) gene was amplified and sequenced using primers FM55/FM52R (Long et al. 2012). The five COI sequences were determined and identical. BLASTn analysis of COI with a 539-bp sequence (accession no. MH177892) resulted in 100% homology with that of Pythium myriotylum isolate AB292 (accession no. MF374747). Isolates were identified as P. myriotylum based on morphological characteristics (van der Plaats-Niterink 1981) and COI sequences. Koch’s postulates were conducted by first producing inoculum for one of the isolates (SDHZSY-1). Inoculum was produced by first soaking corn kernels for 24 h in water, followed by autoclaving for 2 h at 121°C. Isolate SDHZSY-1 was grown on corn meal agar medium for 10 days, after which agar plugs were transferred to the sterilized corn kernels, which were incubated at 28°C for approximately 15 days, until the corn kernels were covered in white hypha. Ten healthy yam plants were transplanted into a sterilized sandy loam potting soil that was artificially infested with the corn inoculum (3 g of inoculum per 100 g of sandy loam mixture). Inoculated and noninoculated control plants were maintained in a greenhouse at 28°C and 80% relative humidity with a 12-h photoperiod, and the experiment was repeated. All 20 inoculated plants appeared water-soaked within 4 weeks, and P. myriotylum was recovered only from the water-soaked roots of inoculated plants, whereas all 20 control plants remained symptomless in both experiments. To our knowledge, this is the first report of root rot on yams caused by P. myriotylum in China and worldwide. Identification of the pathogen will assist in devising strategies to protect this important plant from the pathogen and to prevent yield losses.

Effect of different nutrient media on radial growth of Alternaria brassicae (Berk.) Sacc. infecting different Brassica host crops

Alternaria brassicae (Berk.) Sacc. is the most common and destructive pathogen of a number of Brassica crops belong to family Brassicaceae. It has been reported from all the continent of the world and causes considerable losses in terms of quality and quantity of crop produce. The present investigation was undertaken in vitro, to know the effect of different nutrient media viz., Potato Dextrose Agar (PDA), V-8 Juice Agar (V8JA), Richard’s Agar (RA), Czapeck’s Dox Agar (CDA) and Corn Meal Agar (CMA) on the growth of A. brassicae isolates collected from the different Brassica host crops i.e. Mustard, Cauliflower, Cabbage and Radish. These isolates were designated on the basis of their hosts as Acae M, Acae Cf, Acae Ca and Acae R, respectively. Radial growth of all isolates was observed after seven days of incubation. The result showed a marked variation in radial growth of A. brassicae isolates. However, maximum growths of all isolates were recorded in V-8 Juice Agar (V8JA) followed by Potato Dextrose Agar (PDA) while the minimum growth of pathogen was observed in Corn Meal Agar (CMA).

2045. Pitfalls in the Use of MALDI TOF Mass Spectrometry for the Identification of Problematic Yeast Isolates from a Historical Collection

BackgroundThe identification of yeast traditionally entails macroscopic/microscopic findings and biochemical testing. Recently, MALDI TOF MS has replaced traditional methods for identification as a proposed new standard. We performed identification of previously unidentifiable yeasts from a collection in the United States.MethodsThe Mycoses Study Group (MSG) collected 2,947 Candida isolates from 1,911 patients as part of two U.S. studies between 1995 and 1999. The identification of the isolates was done in 2002 using API 20 c aux with supplemented standard mycological and biochemical test (corn meal agar, germ tube and the Murex ID system). Ninety-four isolates could not be identified at that time. For this study, the isolates were defrosted, plated on Sabureau Dextrose agar, and incubated at 30°C for 48 hours. We then sub-cultured again in Blood Agar. Isolates when then tested by MALDI TOF MS following the methodology for the Bruker MALDI biotyper.ResultsIn the first attempt, 65/94 (69%) isolates were identified. The remaining 29 samples were re-tested with a yield of 21/29 (72.4%) identified isolates. The remaining isolates had to be identified with another round of MALDI TOF and further biochemical testing. The table below shows the final identification of the isolates.ConclusionMALDI TOF MS is rapidly becoming a reference method for yeast identification. However, in a historical collection of yeast that could not be identified by conventional biochemical methods, it took up to three rounds of MALDI TOF MS with a yield of ~70% per round, and additional biochemical testing, for identification of all isolates. Continuing validation of MALDI TOF MS for identification of difficult yeast isolates is warranted.SpeciesNumberPercentage C. albicans 2021 C. glabrata 2223.40 C. parapsilosis 3436.10 C. tropicalis 88.50 C. krusei 22.10 C. orthopsilosis 33.10 C. nivariensis 11.06 C. Kefyr 11.06 Pichia norvegensis 11.06 C. lusitane 11.06 Trichosporum spp.11.06Total94100Disclosures All authors: No reported disclosures.

Hormetic Effects of Thiophanate-Methyl in Multiple Isolates of Sclerotinia homoeocarpa.

Twenty-eight isolates of Sclerotinia homoeocarpa, causal agent of dollar spot disease in turf, were assessed for fungicide hormesis at sublethal concentrations of thiophanate-methyl (T-methyl). Each isolate was grown in corn meal agar amended with 11 concentrations of T-methyl (30,500 to 0.047 µg/liter), and the area of mycelial growth was determined relative to the control. Three replicates were used per concentration, and the experiment was repeated three to five times for each isolate. Reference isolates (EC50 > 20 µg/liter), with no prior history of T-methyl exposure, were highly sensitive and not stimulated by low doses. Likewise, no stimulation was observed in two highly sensitive isolates (EC50 > 30 µg/liter) that had been preconditioned by exposure to T-methyl, or in four T-methyl-tolerant isolates. Seventeen (81%) preconditioned T-methyl-tolerant isolates (EC50 = 294 to1,550 µg/liter) had statistically significant growth stimulation, in the range of 2.8 to 19.7% relative to the control. These results support that hormesis (low-dose stimulation, high-dose inhibition) is a common dose response in preconditioned S. homoeocarpa, particularly in response to subtoxic doses of T-methyl.

Trichoderma cyanodichotomus sp. nov., a new soil-inhabiting species with a potential for biological control.

During a biodiversity survey of Trichoderma (Ascomycota, Hypocreales, Hypocreaceae) in coastal and lake wetlands of China, a new species, Trichoderma cyanodichotomus, was isolated from Dongting Lake wetland of Hunan province. The strain TW21990-1 was characterized as having two types of conidia and producing a distinct blue-green pigment on potato dextrose agar and cornmeal dextrose agar. The taxonomic position was analyzed using three molecular markers, internal transcribed spacer rDNA, translation elongation factor 1-alpha, and RNA polymerase II subunit B, revealing less than 95.0% homology with all known Trichoderma species. The combined phylogenetic tree further identified T. cyanodichotomus as an independent subgroup belonging to Section Pachybasium, with no close relatives. In vitro antagonistic activity by dual-culture assay exhibited broad inhibition against various plant pathogens, including Botryosphaeria dothidea, Pythium aphanidermatum, Rhizoctonia solani, and Verticillium dahliae. In addition, TW21990-1 demonstrated moderate hydrolase activity of cellulase, chitinase, β-1,3-glucanase, and protease, which might be involved in mycoparasitism. Greenhouse experiments showed strong biocontrol effects against tomato damping-off incited by P. aphanidermatum, together with increased seedling height and weight gain. The identification of T. cyanodichotomus will provide useful information for sufficient utilization of fungal resources.

Distribution and antifungal susceptibility profile of Candida species from candiduria cases at a tertiary care hospital

The present study was conducted to assess the distribution of Candida species in candiduria and to evaluate their antifungal susceptibility pattern. Urine samples were subjected to microscopy and screened for the growth. Candida isolates were identified and speciated by colour on HiCHROM Candida agar, germ tube test, growth in cornmeal agar to study micromorphology, carbohydrate assimilation and fermentation tests according to standard protocols. All isolates were investigated for antifungal susceptibility testing. During the study period, a total number of 2560 urine samples were reported. Out of them 94/2560 (3.7%) urine samples showed significant candiduria. 60 from catheterised patients admitted to the ICU and 34 from patients admitted in different wards. The species-wise distribution among Candida isolates are C.tropicalis53 (56.4%), C.albicans21(22.4%), C.glabrata8 (8.5%), C. krusei 7 (7.4%) and C.parapsilosis 5 (5.3%). Candida species exhibited resistance to Amphotericin B 6 (6.4%), Fluconazole 23 (24.5%) Voriconazole 8 (8.5%), Flucytosine 5 (5.3%) and 4 (4.3%) to Caspofungin.Candidapreviously presumed as non-pathogenic has attained an important clinical role in nowadays circumstances of growing risk factors and developing antifungal resistance. The frequency of isolation of non-albicans Candida was more when compared to Candida albicans and they showed more resistance to antifungal drugs when compared to C.albicans.Caution must be taken in reporting Candida from urine, but it is recommended not to mistreat candiduria. The Candida species identification along with their antifungal susceptibility profile can assist clinicians in choosing better antifungal drugs for the treatment of patients with candiduria.

INFLUENCE OF CULTURE MEDIUM ON THE SPORULATION AND VIABILITY OF ASPERGILLUS SPP. AND TALAROMYCES SPP. ENTOMOPATHOGENIC FUNGI

Influence of Culture Medium on the Sporulation and Viability of Aspergillus spp. and Talaromyces spp. Entomopathogenic Fungi. The purpose of this study was to determine the effect of three kinds of cultures media on the spore production and viability of Aspergillus spp. (AS1, 6, 7, 9) and Talaromyces spp. (AS2–5, 8, 10) entomopathogenic fungi. This study was arranged using Factorial-Completely Randomized Design (CRD) with 2 factors and 3 replications. The first factor was three kinds of cultures media (potato dextrose agar (PDA), corn meal agar (CMA), and sabouraud dextrose agar (SDA)) and the second one was isolates of Aspergillus spp. Or Talaromyces spp.. Data of spore production and spore viability were tested using ANOVA and if there was significantly difference, the data then further analyzed using Tukey‘s Honestly Significant Difference (HSD) test at 5% of significant level. The spore production of Aspergillus spp. were in the range of 0.58 - 14.27 x 108 spores mL-1 (PDA); 0.28 – 2.68 x 108 spores mL-1 (SDA) and 1.85 - 5.33 x 108 spores mL-1 (CMA). The highest spore production was achieved by AS1 isolate that was grown on PDA media. The spore produced by Talaromyces spp. were in the range of 2.15 – 28.62 x108 spores mL-1 (PDA); 0.28 – 29.43 x108 spores mL-1 (SDA); and 1.88 – 16.63 x108 spores mL-1 (CMA). The highest spore production was produced by AS8 isolate which were cultured on PDA. The spore viability among isolates of the two entomopathogenic fungi were not significantly different. The spore viability of Aspergillus spp. was in the range of 95.10 – 97.66% (PDA), 94.02 – 98.45% (SDA) and 92.86 – 98.20% (CMA). The spore viability of Talaromyces spp. was in the range of 95.83 – 100% (PDA), 85.83 – 100% (SDA), and 90.75 – 100% (CMA). Culture medium influenced spore production but not the spore viability. The best culture media used for spore production of both of the entomopathogenic fungi was PDA media.

First Report of Pythium Root Rot Caused by Pythium mastophorum on Parsley in the United States

In California, parsley (Petroselinum crispum [Mill.] Fuss) is a versatile leafy green commodity that is grown for fresh, frozen, dried, and processed products. In May 2017, parsley grown in a commercial field in Santa Barbara County, CA, showed symptoms of an unknown disease. Plants were stunted, wilted, and had chlorotic foliage. The large taproots were discolored, and small feeder roots were sparse and necrotic. Affected plants appeared in clusters, and disease incidence was approximately 5%. To recover possible pathogens, roots were surface sterilized in 0.01% bleach for 30 s and plated on corn meal agar amended with pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP) (Kannwischer et al. 1978). After 3 days, rapidly growing, coenocytic mycelia typical of a Pythium species grew out from all roots. Older cultures showed globose sporangia and ornamented oogonia. Oogonia had an average diameter of 23 µm, and projections were conical, measuring an average length of 3 µm. Five isolates were tested using a recombinase polymerase amplification genus-specific Phytophthora assay (targeting the trnM-trnP-trnM region in the mitochondria) and were found to be negative (Miles et al. 2015). To determine the species, DNA was extracted from mycelial plugs using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and the internal transcribed spacer (ITS) regions 1 and 2 were amplified and sequenced using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 primers (5′-TCCTCCGCTTATTGATATGC-3′) (GenBank accession no. MG593942). The sequences were 99% homologous to Pythium mastophorum strain CBS 375.72 (GenBank accession no. AY598661). To test for pathogenicity, 4-week-old plants of flat leaf (cv. Italian Dark Green Flat) and curly leaf (cv. Moss Curled) parsley were inoculated using a root-dip method for five isolates. Inoculum was made by blending five fully colonized V8 plates with 250 ml of sterile water. Parsley root balls were soaked for 10 to 15 min in the inoculum and then repotted in a 1:2 sand/soil mixture. In 2 to 3 weeks, inoculated plants became stunted and chlorotic and had symptoms that matched those seen in the field. Roots were severely decayed. Roots plated onto PARP resulted in colonies that morphologically and molecularly were identical to the original isolates. Control plants of both cultivars were inoculated with a mixture of sterile V8 media and water using the above protocol. These plants developed no foliar or root symptoms. This test was completed three times with consistent results. We believe this is the first report of P. mastophorum infecting parsley in California. P. mastophorum has been reported on parsley only in southeastern Australia (Petkowski et al. 2013). In California, this species has been reported on celery (Vazquez et al. 1996). Because affected parsley plants were completely unharvestable, this disease could be a significant concern for growers if inoculum spreads throughout this and other parsley growing regions in the state.

Phenotypic and genotypic characterization of Candida species isolated from candideamia in Iran.

Background and Purpose:Candidemia is one of the most important fungal infections caused by Candida species. Infections and mortality caused by Candida species have been on a growing trend during the past two decades. The resistance of yeasts to antifungal drugs and their epidemiological issues have highlighted the importance of accurately distinguishing the yeasts at the species level. The technique applied for yeast identification should be fast enough to facilitate the imminent initiation of the appropriate therapy. Candidemia has not been studied comprehensively in Iran yet. Regarding this, the current study aimed to assess the epidemiology of candidemia at Tehran hospitals and compare the results with the previous findings.Materials and Methods:This study was conducted on 204 positive blood cultures obtained from 125 patients hospitalized in several hospitals located in Tehran, Iran, within a period of 13 months. The yeast isolation and species identification were accomplished using several phenotypic methods (i.e., production of germ tube in human serum, culture on CHROMagar Candida, and Corn meal agar containing Tween 80) and molecular methods, such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In addition, unknown cases were subjected to PCR sequencing. These methods were then compared in terms of accuracy, sensitivity, and speed of identification.Results:According to the results, C. albicans (62.4%) was the most common isolate, followed by C. parapsilosis (n=36, 17.5%), C. glabrata (n=18, 8.8%), C. tropicalis (n=13, 6.3%), Trichosporon asahii (n=3, 1.5%), C. kefyr (n=2, 1.0%), C. lusitaniae (n=2, 1.0%), C. intermedia (n=1, 0.5%), C. guilliermondii (n=1, 0.5%), and C. krusei (n=1, 0.5%), respectively. Conclusion:As the findings indicated, the most common species causing candidemia were C. albicans, C. parapsilosis, and C. glabrata, respectively. Children less than one year old and people with cancer were at higher risk for candidemia, compared to other groups. Moreover, phenotypic and molecular methods resulted in the identification of 65.2% and 96.6% of the isolates, respectively. Consequently, PCR-RFLP could be concluded as a more favorable technique for species identification.

Morphological Identification of Phytophthora A Causal Organism of Pineapple Heart Rot Disease in Uganda

Pineapple (Ananas comosus L. Merr) is an economic horticultural crop in Uganda. However, pineapple production is currently being threatened by the latest outbreak of pineapple heart rot disease in Uganda. Yet, information on pineapple heart rot disease causal pathogen is unknown. Therefore, the objective of this study was to identify the pathogen causing pineapple heart rot disease (PHRD) in Uganda. Samples of pineapple leaves with symptoms of PHRD were collected from four districts of Masaka, Luwero, Mukono and Kayunga. Pathogen isolation was done using corn meal agar (CMA) amended with Pimaricin, Ampicillin, Rifampicin Pentachloronitrobenzene, Hymexazol and Benomyl (PARPHB). Macro- and micro- morphological characteristics of the isolates were assessed using Potato dextrose agar (PDA) and V8 media, respectively. Pathogenicity of the isolates was tested using healthy pineapple plants based on Koch’s postulate. The results showed a significant difference (P0.05) in the growth rates, hyphae diameter and asexual structure dimensions of the isolates. Isolates were characterized by a dense rosette and stoloniferous mycelial growth pattern in PDA media. Although, sporangia were non-caduceus, terminal, papillate and mostly ovoid, obpyriform and limoniform sporangia (32-56μm) with a mean length/breadth ratio of 1.32:1μm were observed. Chlamydospores were spherical and thick-walled (25-42µm diameter) while Sporangiophores branching was sympodial. Based on the morphological characteristics of isolates, it was concluded that Phytophthora nicotianae is the species causing pineapple heart rot disease in Uganda. This study therefore represents the first comprehensive information in Uganda. However, molecular identification of the pathogen is recommended to confirm the genetic identity of the species.

Integrated Metabolomics and Morphogenesis Reveal Volatile Signaling of the Nematode-Trapping Fungus Arthrobotrys oligospora.

ABSTRACTThe adjustment of metabolic patterns is fundamental to fungal biology and plays vital roles in adaptation to diverse ecological challenges. Nematode-trapping fungi can switch their lifestyle from saprophytic to pathogenic by developing specific trapping devices induced by nematodes to infect their prey as a response to nutrient depletion in nature. However, the chemical identity of the specific fungal metabolites used during the switch remains poorly understood. We hypothesized that these important signal molecules might be volatile in nature. Gas chromatography-mass spectrometry was used to carry out comparative analysis of fungal metabolomics during the saprophytic and pathogenic lifestyles of the model species Arthrobotrys oligospora. Two media commonly used in research on this species, cornmeal agar (CMA) and potato dextrose agar (PDA), were chosen for use in this study. The fungus produced a small group of volatile furanone and pyrone metabolites that were associated with the switch from the saprophytic to the pathogenic stage. A. oligospora fungi grown on CMA tended to produce more traps and employ attractive furanones to improve the utilization of traps, while fungi grown on PDA developed fewer traps and used nematode-toxic furanone metabolites to compensate for insufficient traps. Another volatile pyrone metabolite, maltol, was identified as a morphological regulator for enhancing trap formation. Deletion of the gene AOL_s00079g496 in A. oligospora led to increased amounts of the furanone attractant (2-fold) in mutants and enhanced the attractive activity (1.5-fold) of the fungus, while it resulted in decreased trap formation. This investigation provides new insights regarding the comprehensive tactics of fungal adaptation to environmental stress, integrating both morphological and metabolomic mechanisms.IMPORTANCE Nematode-trapping fungi are a unique group of soil-living fungi that can switch from the saprophytic to the pathogenic lifestyle once they come into contact with nematodes as a response to nutrient depletion. In this study, we investigated the metabolic response during the switch and the key types of metabolites involved in the interaction between fungi and nematodes. Our findings indicate that A. oligospora develops multiple and flexible metabolic tactics corresponding to different morphological responses to nematodes. A. oligospora can use similar volatile furanone and pyrone metabolites with different ecological functions to help capture nematodes in the fungal switch from the saprophytic to the pathogenic lifestyle. Furthermore, studies with A. oligospora mutants with increased furanone and pyrone metabolites confirmed the results. This investigation reveals the importance of volatile signaling in the comprehensive tactics used by nematode-trapping fungi, integrating both morphological and metabolomic mechanisms.

Biofilm production by clinically isolated Candida : Comparative analysis based on specimen, methodology, and various Candida species

Abstract Aim and objectives The importance of Candida as a human pathogen has increased, particularly in the hospitalized and immune compromised patients. Candida albicans, the predominant species worldwide, is now being replaced by the more resistant non-albicans Candida. This study compares various biofilm producing clinical isolates of Candida species by three commonly available methods for biofilm detection. Material and methods Clinical samples from patients with suspected fungal/Candida infections were cultured. Candida isolates were speciated on the basis of colony morphology, germ tube formation, chlamydospore formation on corn meal agar and color production on HiCrome Candida agar and confirmed using Vitek-2 Yeast-ID cards. Biofilm production was performed visually by modification of the tube method, spectrophotometrically by Percent transmittance method and Crystal violet assay. Results 20.6% of Candida isolates were obtained. Candida albicans (33.3%) was outnumbered by the non-albicans species (66.7%). Crystal violet micotitre plate assay demonstrated highest positivity (54.8%), followed by percent transmittance microtitre plate method. C. glabrata was the highest biofilm producer, followed by C. tropicalis. Conclusion Biofilm production is very common among Candida isolates, more so for the non-albicans Candida species. Routine testing for biofilm production should be contemplated particularly in serious patients with Candida infection, not responding to typical anti-fungal therapy.

First Report of Pythium myriotylum D., Causing Damping Off and Root Rot in Chili Pepper (Capsicum annum L.) from Punjab, Pakistan

Chilli pepper (Capsicum annuum L.) has significant importance as a cash crop, like other vegetables, and occupies 20% of the total vegetable production area in Pakistan. Pythium spp. are notorious fungal-like agents that may result in 90% plant death either as pre and/or post-emergence damping off. During surveys of nine major chilli growing areas of Punjab, Pakistan in September-November 2015-16, disease incidences ranging from 13.8 to 45.4% was recorded. The symptoms consisted of damping off, and on older seedlings (15 to 30 days post-transplanting) reduced growth, wilting, water-soaking, brown discoloration and root rot. Infected roots were cut into small pieces, surface sterilized using 1% sodium hypochlorite for 3 to 5 min, followed by a rinse with sterile distilled water. Root saps were serially diluted to appropriate dilutions, streaked onto corn meal agar (CMA) medium supplemented with ampicillin (250 mg/l), rifampicin (10 mg/l) and pimaricin (10 mg/l) (Jeffers and Martin 1986) and incubated at 28...

First Report of Pythium cylindrosporum Causing Black Leg on Geraniums in China

Geranium has been widely used in garden landscaping for its rich colors, long florescence, and easy reproduction. During the fall season of 2014 and 2015, the geranium seedlings in a nursery garden in the northern suburbs of Kunming City, Yunnan Province, showed symptoms of black leg. Five cultivars of geranium（Pelargonium×hortorum）, including ‘Horizon’ and ‘Divas’ suffered, showing a maximum morbidity rate of 50%. The basal stems of the plant turned black and were water-soaked, and its leaves turned yellow from the bottom up. As the disease progressed, the affected plants wilted and collapsed. The diseased basal stem tissue was rinsed under running water, blotted dry, and cut into 0.5-cm pieces. The pieces were plated on the semiselective medium for oomycetes (17 g/liter corn meal agar amended with nystatin 15 mg/liter, ampicillin 250 mg/liter, rifampicin 10 mg/liter, and miconazole 1 mg/liter) (Morita and Tojo, 2007) and incubated at 25 °C for 3 days. Colonies resembling Pythium sp. were transferred to ...

Clinico-mycological analysis and antifungal resistance pattern in human immunodeficiency virus-associated candidiasis: An Indian perspective.

Objectives:Candidiasis is a common human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome-associated opportunistic mycoses. The present study ascertained the species spectrum of Candida strains recovered from different clinical samples from symptomatic HIV-positive individuals and determined the antifungal susceptibility profile of the isolates.Materials and Methods:A variety of specimens were collected from 234 symptomatic HIV seropositive individuals depending on their clinical manifestations and subjected to direct microscopic examination. Blood samples were inoculated in biphasic blood culture medium and all other specimens on Sabouraud dextrose agar with chloramphenicol and incubated at 35°C–37°C. Species identification of the recovered Candida isolates was attempted on the basis of germ tube production, micromorphology on corn meal agar, color and morphology on HiCrome Candida Differential agar, and carbohydrate fermentation and assimilation tests. Susceptibility testing of the isolates was performed employing the VITEK 2 system.Results:A total of 167 Candida isolates were obtained; Candida albicans (136), Candida tropicalis (13), Candida krusei (8), Candida parapsilosis (5), Candida glabrata (4), and Candida kefyr (1). Fluconazole resistance was more frequent among nonalbicans species, and significantly higher 5-fluorocytosine resistance compared to C. albicans was also observed. Eight Candida strains (six C. krusei, one C. kefyr, and one C. albicans) were multidrug resistant.Conclusion:Although C. albicans continues to be the leading etiological agent of candidiasis, the incidence of nonalbicans species among HIV-positive Indian individuals is rising. Antifungal resistance was higher among nonalbicans Candida species. Another issue of therapeutic concern is the possible emergence of multidrug-resistant Candida strains among these patients.

Assessment of in vitro growth and predation of Arthrobotrys oligospora to Haemonchus contortus under the influence of media type and age of mycelium

Corn meal agar followed by potato dextrose agar was found the best medium for supporting in vitro growth of Chhattisgarh isolate of Arthrobotrys oligospora. Media type clearly affected the ring structure and trophic hyphal development, though attachment of Haemonchus contortus larvae to adhesive hyphae of A. oligospora was not affected. Initially a part of active thirds stage larvae of H. contortus was attached to hyphae within a period of 4 days in all media types except in nutrient agar at 26°C. However, the capture and immobility of the infective larvae were very sluggish. Growth, capture and development of trophic hyphae inside the H. contortus larval body in water agar indicated that development of trapping devices increased when few energy sources are available. Age of mycelium of A. oligospora isolate had significant influence on deployment of all the trapping structures, where maximum predatory effect was found in one month-old fungal mycelium. In colonies in which nematodes mainly encountered aged hyphae, significant reduction in attachment, capture and development of trophic hyphae was found.

Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods.

Background:Candida dubliniensis is a newly diagnosed species very similar to Candida albicans phenotypically and first discovered in the mouth of people with AIDS in 1995. Among the different phenotypic and genotypic methods, a cost-effective method should be selected which makes it possible to differentiate these similar species.Materials and Methods:Polymerase chain reaction (PCR)-restriction fragment length polymorphism with MspI enzyme and the Duplex-PCR method were done by DNA extraction using boiling. The sequencing of the amplified ribosomal region was used to confirm the C. dubliniensis species. Direct examination and colony count of the yeasts were applied for bronchoalveolar lavage (BAL) samples and the growth rate of the yeasts were studied at 45°C. To understand the ability formation of chlamydoconidia in yeast isolates, they were separately cultured on the sunflower seed agar, wheat flour agar, and corn meal agar media.Results:Fifty-nine (49.2%) yeast colonies were identified from the total of 120 BAL specimens. Twenty-nine isolated yeasts; including 17 (58.6%) of C. albicans/dubliniensis complex and 12 (41.4%) of nonalbicans isolates produced pseudohypha or blastoconidia in direct smear with a mean colony count of 42000 CFU/mL. C. albicans with the frequency of 15 (42.9%) were the most common isolated yeasts, whereas C. dubliniensis was identified in two nonHIV patients.Conclusion:Sequencing of the replicated gene fragment is the best method for identifying the yeasts, but the determination of the species by phenotypic methods such as the creation of chlamydoconidia in sunflower seeds agar and wheat flour agar media can be cost-effective, have sensitivity and acceptable quality.