Chlamydospore Formation Research Articles

A study on candiduria in neonates and infants from a tertiary care center, North India.

Candida albicans is the major cause of fungal UTI in neonates and infants but nowadays non albicans Candida is also increasing and these are mostly multidrug resistant. So it's important to know the species of candidal UTI for the proper management. This study was undertaken to determine the Candida species distribution in UTI along with their susceptibility pattern and outcome in infants and neonates admitted in different wards and ICU of our hospital. We also assess the incidence rate of candiduria in ICUs. Urine samples were collected from infants and neonates presented in pediatrics and neonatal ICU (intensive care units) and clinical wards with a clinical suspicion of candiduria and infants at risk of invasive candidiasis were also included in the study. Identification of Candida sp. was done by Gram's staining, germ tube test, chlamydospore formation on corn meal agar, color appearance on CHROM agar and also confirmed by MALDI-TOF Assay. Antifungal susceptibility was performed by using broth microdilution method as per the CLSI M27-A3/M27-S4. Urine samples were received from 219 infants, and Candida was isolated from samples from 52 infants (isolation rate 23.75%), of which 30 were admitted in pediatric or neonatal ICU and 22 in the wards. The incidence rate of candiduria in ICU was 3.25%. Candida albicans was the most frequently isolated species from the samples of infants in the wards (13/22 i.e. 59%), while Candida tropicalis was most frequently isolated from samples of infants in the ICUs (13/30 i.e. 43.34%). Candida glabrata was the least commonly isolated species and was only encopuntered in the ICU. There was no discrepancy between the results of conventional methods of identification and MALDI-TOF. Antifungal susceptibility was performed for 18 randomly selected isolates. All were found to be susceptible to caspofungin, micafungin, itraconazole, voriconazole, fluconazole, amphotericin B. High suspicion of candiduria is needed especially in ICU admitted infants and identification of candida at species level along with the susceptibility pattern is important for the better management of patients.

FoRSR1 Is Important for Conidiation, Fusaric Acid Production, and Pathogenicity in Fusarium oxysporum f. sp. ginseng.

The root rot disease caused by Fusarium oxysporum f. sp. ginseng is one of the most destructive diseases of ginseng, an economically important herb. However, little is known about the pathogen's toxin biosynthesis or the molecular mechanisms regulating infection of ginseng. In this study we identified and functionally characterized the FoRSR1 gene that encodes a Ras-related (RSR) small GTPase homologous to yeast Rsr1 in F. oxysporum f. sp. ginseng. Disruption of FoRSR1 resulted in a significant reduction in mycelial dry weight in liquid cultures, although vegetative growth rate was not affected on culture plates. Notably, the Forsr1 mutant exhibited blunted and swollen hyphae with multi-nucleated compartments. It produced fewer and morphologically abnormal conidia and was defective in chlamydospore formation. In infection assays with ginseng roots, the Forsr1 mutant was significantly less virulent and caused only limited necrosis at the wounding sites. Deletion of FoRSR1 also affected pigmentation, autophagy, and production of fusaric acid. Furthermore, the expression of many candidate genes involved in secondary metabolism was significantly downregulated in the mutant, suggesting that FoRSR1 is also important for secondary metabolism. Overall, our results indicated that FoRSR1 plays important roles in conidiation, vacuolar morphology, secondary metabolism, and pathogenesis in F. oxysporum f. sp. ginseng.

Propyl (E)-3-(furan-2-yl) Acrylate: a synthetic antifungal potential with a regulatory effect on the biosynthesis of ergosterol in Candida Albicans

The genus Candida represents the main cause of infections of fungal origin. Some species stand out as disease promoters in humans, such as C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. This study evaluated the antifungal effects of propyl (E)-3-(furan-2-yl) acrylate. The minimum inhibitory concentration of the synthetic compound, amphotericin B and fluconazole alone against four species of Candida ranged from 64 to 512 μg/mL, 1 to 2 μg/mL, and 32 to 256 μg/mL, respectively. The synergistic effect of the test substance was observed when associated with fluconazole against C. glabrata, there was no antagonism between the substances against any of the tested strains. The potential drug promoted morphological changes in C. albicans, decreasing the amount of resistance, virulence, and reproduction structures, such as the formation of pseudohyphae, blastoconidia, and chlamydospores, ensuring the antifungal potential of this substance. It was also possible to identify the fungicidal profile of the test substance through the study of the growth kinetics of C. albicans. Finally, it was observed that the test compound inhibited the ergosterol biosynthesis by yeast.

Anaerobic conditions are a major influence on Candida albicans chlamydospore formation.

Candidiasis now represents the fourth most frequent nosocomial infection both in the USA and worldwide. Candida albicans is an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad-spectrum antibiotics, and other immune defects. Unfortunately, these infections carry unacceptably high morbidity, mortality rates and important economic repercussions (estimated total direct cost of approximately 2 billion dollars in 1998 in US hospitals alone). This pathogen can grow both in yeast and filamentous forms and the pathogenic potential of C. albicans is intimately related to certain key processes including filamentation. Chlamydospores are considered to be a dormant form of C. albicans that remain understudied. Chlamydospores have been widely used as a diagnostic tool to separate C. albicans and C. dubliniensis from other Candida species. More recently, media have been developed that use chlamydopsore formation to separate C. albicans and C. dubliniensis from each other. Chlamydospore formation can be stimulated by hypoxic conditions but only on limited specific media types. Here, we show that anaerobic conditions are enough to drive chlamydospore formation in C. albicans on the surface of nutrient-rich agar.

Cultural, Molecular and Genetic Variability of Ustilaginoidea virens Causing Rice False Smut Disease

Rice false smut disease is caused by Ustilaginoidea virens, an ascomycetous fungi. Thirty isolates of U. virens showed well defined colonies on potato sucrose medium (PSA). The maximum colony diameter was found to be 85.68 mm with a growth rate of 2.85 mm in the UV23 isolate and the minimum colony diameter was found to be 10.14 mm with a growth rate of 0.33 mm, which was observed in the UV15 isolate. However, thirty isolates exhibit various cultural and morphological characteristics like colony color, growth pattern, elevation, and chlamydospore formation. In cluster analysis, two major groups (I) and (II) were formed. The first major group (I) contains UV15 isolates and the second major group (II) contains 29UV isolates. Furthermore, the division of group II includes subgroup IIa, which contains 22 isolates, and subgroup IIb, with 7 isolates. The PCR amplification was done with a species-specific primer which yielded products of 380 bp and 230 bp, respectively. The results of the phylogenetic tree analysis revealed that cluster I contained 5 isolates and cluster II contained 28 isoates from China and Japan. The MAT1-1-1 primer yielded a product of 250 bp that contains 18 isolates. Whereas the MAT1-2-1 primer yielded a product of 220 bp with 12 isolates. The nucleotide divergence analysis results revealed 7 polymorphic sites (s), 10 mutations (Eta), 0.76515 nucleotide diversity (k) and -2.13885 for the tajimas test value. Likewise, haplotype analysis revealed 10 haplotype(h)groups, with a haplotype diversity (hd) of 0.4773. With 24 isolates, hap-1 was the most common. Similarly, clear nucleotide variation was observed between Indian and Chinese isolates as well as Japanese ones. The genetic similarity coefficient matrix revealed that thirty Indian isolates showed maximum similarity (0.9). whereas the UV32 isolate showed 0.6, the UV31 isolate exhibited 0.5, and the UVA33 isolate had 0.4,.These results indicate a genetic variability between the Indian UV isolates and the isolates from China.

First Report of Crown Rot Caused by Fusarium algeriense on Wheat in Kyrgyzstan.

Fusarium crown rot of wheat is an economically important disease that leads to significant yield and quality losses, especially in many arid and semi-arid wheat-growing areas worldwide. In June 2020, winter wheat (Triticum aestivum L.) plants exhibiting crown rot symptoms were identified in a commercial field located in the Tokbay location (43.033719°N, 74.325623°E), Chuy Province, Kyrgyzstan. The diseased plants were stunted and had brown discoloration on internodes of the stem bases and roots. Disease incidence was about 3%. A total of 10 plants were sampled at the ripening stage from the field to identify the causal agent. Symptomatic tissues were excised, surface disinfected with 1% NaOCl, rinsed three times with distilled water, and placed on one-fifth strength potato dextrose agar (PDA) followed by incubation at 23°C in the dark for 5 days. A total of 8 Fusarium isolates were recovered from tissues and purified by the hyphal tips method onto fresh PDA and Spezieller-Nährstoffarmer agar (SNA) plates (Leslie and Summerell 2006). Sequence analysis of the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were performed with primers EF1 and EF2 (O'Donnell et al. 1998), and 5f2 (Reeb et al. 2004) and 7cr (Liu et al. 1999), respectively. The sequences of three isolates showed 100% identities with the corresponding sequences of the strain NRRL 66652 of Fusarium algeriense Laraba & O'Donnell (TEF1: MF120515 and RPB2: MF120504), and the sequences of a representative isolate (KyrFa01) were deposited in GenBank (TEF1: OM135603 and RPB2: OM135604). On PDA, fungal colonies were initially yellowish-white but gradually turned yellowish-brown. Ellipsoidal microconidia produced in false heads on monophialides were usually aseptate (8.30 ± 1.17 μm, n = 50) and occasionally one-septate (21.89 ± 2.01 μm, n = 50). Sporodochial macroconidia were mostly 3-4 septate measuring 43.41 ± 2.83 μm (n = 50), slightly curved and formed generally on monophialides on SNA. No chlamydospores formation was detected after 15 days on SNA or PDA. Morphological characteristics described above were consistent with the morphology of F. algeriense, as reported by Laraba et al. (2017). To confirm pathogenicity, seeds of wheat cultivar Seri 82, Fusarium crown rot susceptible, were treated in 1% NaOCl for 2 min, rinsed twice, and placed in plates containing a piece of sterile filter paper saturated with water to induce germination for 3 days. Five pregerminated seeds were placed on the soil surface for each 9-cm-diameter pot, which was filled with a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v). A 1-cm-diameter mycelial plug taken from the margin of actively growing colonies (PDA) of the representative isolate KyrFa01 was contacted with each seed, and then seeds were covered with the same potting mix. The seeds in control pots were treated with sterile PDA plugs. The experiment was conducted in a growth chamber in a completely randomized design with five replicated pots at 23°C with a 12-h photoperiod. Disease assessment was made after 4 weeks of fungal inoculation. The isolate KyrFa01 induced discoloration on the crown and root tissues of inoculated plants similar to those observed in the field-grown plants, whereas no symptoms were observed on plants grown in the control pots. The pathogen was successfully reisolated from the symptomatic tissues, confirming Koch's postulates. To the best of our knowledge, this is the first report of crown rot caused by F. algeriense on wheat in Kyrgyzstan. Fusarium algeriense was firstly described within the Fusarium burgessii species complex by Laraba et al. (2017) as a crown rot pathogen of wheat in Algeria. The pathogen was secondly reported from wheat-growing areas in Azerbaijan (Özer et al. 2020a) and thirdly from Kyrgyzstan in this report. Özer et al. (2020b) confirmed the coexistence of this pathogen with other Fusarium species. The result warrants the need to further investigate the potential of this species in the Fusarium crown rot complex of wheat.

P425 Approach to identify and isolate C. dubliniensis in laboratory and comparison of various methods for same

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMIntroduction: C. dubliniensis, a newly recognized fungal pathogen, is clinically significant as it develops a stable fluconazole-resistant phenotype on exposure to this antifungal in vitro. C. dubliniensis is overlooked and misidentified as it shares many phenotypic traits with C. albicans. Molecular methods developed to distinguish the two species are not readily applicable. So, an easy-to-perform and an accurate phenotypic test would be a valuable tool.Aim: This study first of its kind from Kashmir Valley (conducted in SKIMS-1200 bedded tertiary care hospital) evaluated seven phenotypic tests with PCR-RFLP as a gold standard. The best methodology for routine identification of this emerging pathogen was developed.Materials: A total of 206 Candida spp. were tested which included 186 in-house C. albicans strains and 20 C. dubliniensis strains. The in-house 186 C. albicans strains were isolated from cancer patients. A total of 500 patients with various malignancies were screened, patients on antifungal drugs and screened once were excluded. All strains were sensitive to both fluconazole and voriconazole. Among 22°C dubliniensis strains, 14 were provided by Dr. Ziauddin Khan (Professor and Chairman, Department of Microbiology, Kuwait University) and 6 by VPCI institute Delhi.Reference strains included in the study: C. albicans 90028; (National culture collection of pathogenic fungi (NCCPF), Department of Medical Microbiology PGIMER Chandigarh) C. dubliniensis (CD36) and (CBS 7987); provided by Dr. Ziauddin KhanMethodsPCR-RFLP using BlnI (AvrII) enzyme was done to confirm the identification of all isolates (Fig. 1). Seven phenotypic tests that were evaluated include Growth on hypertonic Sabouraud Dextrose Agar (SDA), Colony color on Crome Candida Differential Agar, Growth at 45°C, Assimilation of xylose (XYL), Colony color and Chlamydospore formation on Tobacco Agar, Germ tube formation at 39°C and Fluorescence on methyl blue SDA. The variety of tests applied makes our study one of the most detailed studies for the identification of C. dubliniensis till date.ResultsNo C. dubliniensis was found among the isolates recovered from cancer patients by PCR-RFLP.Phenotypic tests that showed the best results wereGrowth on Hypertonic SDAColony color on Crome Candida Differential AgarRelative growth at 45°C.Assimilation of xylose (XAM as well as DISCs)Germ tube formation at 39°C was most rapid. Phenotypic methods proved to be very cost-effective than PCR-RFLP. Based on our results of growth on Hypertonic SDA and XAM and also knowing the fact that 5% of C. dubliniensis assimilate xylose and give false results of xylose assimilation, we developed a novel medium ‘Hypertonic xylose agar medium’ (HXAM). HXAM proved to be 100% accurate, simple, easy to perform, and cost-effective.ConclusionFor routine identification of C. dubliniensis we recommend colony color on Crome Agar and growth on HXAM as they are simple, reliable, inexpensive, reproducible, and readily applicable methods. For retrospective evaluation of stored cultures, we recommend HXAM.Based on our observations, we propose a scheme for the identification of C. dubliniensis (Fig. 2).

P025 A study on candiduria in neonates and infants from a tertiary care center, North India.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjective Candida albicans is the major cause of fungal UTI in neonates and infants but nowadays incidence of other species of Candida is also increasing, and these are mostly multidrug resistant. It is, therefore, important to determine the causative Candida species in fungal UTI for appropriate management. This study was undertaken to determine the Candida species distribution in UTI along with susceptibility patterns and outcomes in infants and neonates admitted to various wards and intensive care units (ICUs) of our hospital. The incidence rate of candiduria in ICUs was also assessed.MethodUrine samples were collected from infants and neonates presented in pediatric and neonatal ICUs and clinical wards with a clinical suspicion of candiduria. Infants at risk of invasive candidiasis were also included in the study. Identification of Candida species was done by Gram's staining, germ tube test, chlamydospore formation on corn meal agar, color of colonies on CHROMagar, and confirmed by Matrix Assisted Laser Desorption-Time of Flight (MALDI-TOF). Antifungal susceptibility was performed by using the Broth microdilution method as per the latest CLSI guidelines (M27-A3/M27-S4).ResultUrine samples were received from 219 infants, and Candida was isolated from samples from 52 infants (isolation rate 23.75%), of which 30 were admitted to pediatric or neonatal ICU and 22 in the wards. The incidence rate of candiduria in ICU was 3.25%. Candida albicans was the most frequently isolated species from the samples of infants in the wards (13/22 ie, 59%), while Candida tropicalis was most frequently isolated from samples of infants in the ICUs (13/30 ie, 43.34%). Candida glabrata was the least commonly isolated species and was only encountered in the ICU. The species distribution of isolates is given in Table 1. There was no discrepancy between the results of conventional methods of identification and MALDI-TOF.Antifungal susceptibility was performed for 18 randomly selected isolates. All were found to be susceptible to caspofungin, micafungin, itraconazole, voriconazole, fluconazole, and amphotericin B. MIC distribution for various isolates is given in Table 2.ConclusionHigh index of suspicion of candiduria is necessary for early diagnosis of fungal UTI and initiation of antifungal treatment, especially in critically ill infants requiring intensive care. Species other than C. albicans are also encountered more frequently nowadays and these species often have higher MICs for commonly used antifungal drugs, which may lead to delayed or failed response to routine antifungal therapy and necessitate prolonged use and or higher doses of antifungals. Identification of Candida isolates at species level along with analysis of the susceptibility patterns is therefore important for successful outcomes in candiduria in neonates and infants.

P367 Candida sepsis in neonates a neglected pathogen: study from neonatal intensive care unit tertiary care hospital

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM IntroductionSignificance of Candida species in neonatal intensive care units (NICU) is increasingly being recognized. It is the third most common cause of late-onset sepsis in NICU patients and accounts for 9 to 13% of bloodstream infections (BSI) in neonates. Although Candida albicans has historically been the most frequently isolated species, recently non-albicans Candida (NAC) have emerged as an important opportunistic pathogen, notably Candida tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C auris. This changing trend and the increasing use of azoles agents is now a matter of concern due to the emerging resistance of the non-albicans species to azoles.AimTo evaluate the Candida sepsis in neonates from neonatal intensive care units from a tertiary care hospital.Material and MethodsA cross-sectional study was conducted in the Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, East Delhi, India, during the period of July 2021 to December 2021. Overall, 30 suspected Candida sepsis patients from neonatal intensive care units were enrolled in the present study. The Candida spp. Isolated was identified as per standard mycological techniques. Preliminary identification was done by colony morphology on SDA, chromogenic media (Hichrome, Himedia Pvt. Ltd.), grown at 45°C, germ tube test, chlamydospore formation, and was confirmed by carbohydrate fermentation and assimilation tests. Antifungal susceptibility testing (AFST) was performed for Fluconazole (25 μg), and amphotericin B (AMB, 100 units) using the disc diffusion method on Muller-Hinton agar supplemented with 2% glucose and methylene blue (5 mg/ml). Zone diameters were interpreted as per the approved Clinical Laboratory Standards (CLSI-M44-A) guidelines. Quality control for AFST was performed using C. albicans-ATCC 90028 and C. parapsilosis-ATCC 22019.Statistical analysisThe statistical analysis was done using Statistical Package for Social Sciences (SPSS) version. The P-value ˂0.05 were considered as statistically significant.ResultOut of 30 Candida species from suspected septicemia neonates, non-albicans Candida (NAC) species accounted for (87%) compared to C. albicans (13.4%). Candida tropicalis was predominant at 9 (30%) followed by C. glabrata 8 (27%), C.parapsilosis 4 (13.3%), and C. krusei 3(10%), Pichia species (4.2%), and C.auris (3.4%). Antifungal sensitivity of the NAC isolates demonstrated >85% resistance to fluconazole amongst C. glabrata, C. parapsilosis, though amphotericin B had 100% sensitivity in all Candida species. The risk factor for candidemia as observed in our study were low birth weight 26(87%), use of broad-spectrum antibiotics 25 (83.4%), presence of an orogastric tube 18 (60%), and total parental nutrition 9 (30%) were significantly associated statistically (P <.05, paired t-test). The overall mortality amongst these neonates was 16.6%.ConclusionFungal BSI has emerged as an important cause of morbidity and mortality in neonates. Reporting of fungal bloodstream infections and the spectrum of species involved are essential measures in neonatal intensive care units in order to implement appropriate preventive and therapeutic strategies.

A six-year hospital-based surveillance study on burden of esophageal candidiasis in Gangtok, Sikkim.

Esophageal candidiasis once thought to be restricted amongst immunocompromised patients is being increasingly reported among non-immunocompromised individuals. It is debilitating and if not treated well may cause chronic long-lasting infections. The objective of this study was to identify the various species of Candida causing esophageal candidiasis and analyse their antifungal susceptibility pattern. This was an observational, prospective study. Total of 108 patients who attended the Gastroenterology Department of Sir Thutob Namgyal Memorial Hospital, Govt of Sikkim, Gangtok, India between July 2012 - May 2018 were included in the study. They had complaints of upper gastrointestinal disturbances and chronic dyspeptic symptoms that required an endoscopy. Esophageal biopsy and brushings were taken and were transported to Microbiology Department. They were subjected to microscopic observation, fungal culture on Sabourauds dextrose agar. Preliminary species identification was done by chlamydospore formation and growth characteristics on CHROMagar Candida. Species confirmation and antifungal susceptibility testing was done on VITEK 2 system at Microbiology Department, Kasturba Medical College and Hospital, MAHE, Manipal, Karnataka, India. A total of 108 patients were screened among which 73 samples were positive for Candida species and species identification and antifungal susceptibility was performed. Forty fiveisolates were found to be C. albicans, 8 were C. glabrata, 4 were C. tropicalis, 3 were C. lusitaniae 2 were C. krusei, 2 were C. lipolyticaand 1 was C. parapsilosis. Eight isolates could not be identified and were recorded as Candida spp. C. albicans isolates were predominantly sensitive strain with susceptibility of 95% for both amphotericin B and fluconazole and 100% for caspofungin. C. glabrata showed high resistance to fluconazole with one isolate showing intermediate resistance to caspofungin. Upper gastrointestinal symptoms even in non-immunocompromised patients need to be screened by endoscopy to rule out esophageal candidiasis. With the emergence of drug resistant non albicans Candida species diagnostic testing laboratories should include Candida species identification and antifungal susceptibility testing facility to provide effective patient care.

Assessing the Candida in Healthy Subjects and Subjects with Oral Submucous Fibrosis: A Comparative Clinical Study

One precancerous condition with a high potential to turn into malignancy and pose a high risk is OSMF (oral submucous fibrosis) which mainly affects the young subjects globally as well as in India. The etiology of OSMF is attributed to the gutkha chewing habit. Another predominant etiologic factor governing the etiopathogenesis of malignant and premalignant lesions is the Candida species. Our aim of the present clinical study was conducted to assess and compare the intensity and incidence of Candida species in healthy subjects and subjects with oral submucous fibrosis. This study included 24 healthy controls and 24 subjects with a confirmed diagnosis of OSMF. A detailed history was recorded and a clinical examination was done for all the study subjects. All the samples were obtained from the oral rinse and were cultured using Sabouraud’s agar medium followed by identification and counting of isolated yeast species on sugar assimilation test, chlamydospore formation, and germ tube test. In the OSMF group, Candida species were isolated from 41.66% (n=10) of subjects. However, in the control group, Candida species were isolated from 16.66% (n=4) of the collected samples. This identification difference showed that higher species were isolated from the OSMF group compared to the control group (p=0.07). In the OSMF group, CFU/ml had a range of 500-970 with mean CFU values of 721.27±143.88. In the control group, the CFU/ml range was 60-320, whereas, the mean CFU/ml value was 186.64±130.10. The mean value was higher in the OSMF group compared to the control group. This difference was statistically significant with a p-value of 0.005. The present study concludes that the intensity and incidence of C. Albicans, candida species was higher in subjects with OSMF compared to healthy controls, however, these species were in the normal range of 3% to 47%. Hence, Candida cannot be considered an etiologic factor for transforming OSMF into a malignant lesion. However, further evaluation is needed to evaluate if betel quid chewing in subjects with OSMF promotes or inhibits the invasion and adherence of Candida species.

PhcA and PhcR Regulate Ralsolamycin Biosynthesis Oppositely in Ralstonia solanacearum.

Ralsolamycin, one of secondary metabolites in Ralstonia solanacearum, is known to be involved in crosstalk between R. solanacearum and fungi. Ralsolamycin formation is catalyzed by two-hybrid synthetases of RmyA (non-ribosomal peptide synthetase) and RmyB (polyketide synthase). A methyltransferase PhcB catalyzes formation of 3-OH MAME or 3-OH PAME, signals for the quorum sensing (QS) in R. solanacearum, while PhcB positively modulates ralsolamycin biosynthesis. A two-component system of PhcS and PhcR can response these QS signals and activate phcA expression. Here, we experimentally demonstrated that deletion of phcA (ΔphcA) substantially impaired the ralsolamycin production and expression of rmyA and rmyB in R. solanacearum strain EP1, and failed to induce chlamydospore formation of plant fungal pathogen Fusarium oxysporum f. cubense (stran FOC4). However, deletion of phcR significantly increased ralsolamycin production and expression of rmyA and rmyB, and phcR mutants exhibited enhanced ability to induce chlamydospore formation of FOC4. Results of the electrophoretic mobility shift assay suggested that both PhcA and PhcR bind to promoter of rmy operon. Taken together, these results demonstrated that both PhcA and PhcR bind to promoter of rmy operon, but regulate ralsolamycin biosynthesis in an opposite way. It could extend our knowledge on the sophisticated regulatory networks of ralsolamycin biosynthesis in R. solanacearum.

The Velvet Protein UvVEA Regulates Conidiation and Chlamydospore Formation in Ustilaginoidea virens.

Rice false smut, caused by Ustilaginoidea virens, is a serious disease of rice worldwide, severely reducing the quantity and quality of rice production. The conserved fungal velvet proteins are global regulators of diverse cellular processes. We identified and functionally characterized two velvet genes, UvVEA and UvVELB, in U. virens. The deletion of these genes affected the conidiation of U. virens but had no effect on the virulence of this pathogen. Interestingly, the ΔUvVEA mutants appeared in the form of smaller false smut balls with a reduced number of chlamydospores compared with the wide-type strains. In addition, the deletion of UvVEA affected the expression of some transmembrane transport genes during chlamydospore formation and rice false smut balls development. Furthermore, the ΔUvVEA mutants were shown to be defective in the utilization of glucose. These findings proved the regulatory mechanism underlying the formation of rice false smut balls and chlamydospores and provided a basis for the further exploration of the mechanism of these processes.

Phytophthora tropicalis: Causal agent of persimmon fruit rot in Brazil

AbstractPersimmon fruit (Diospyros kaki L.) from commercial orchards located in southern Brazil, in Curitiba, Paraná, showed dark firm rot. The incidence in the field was about 2% in an evaluation in the 2016 growing season, and from these infected fruit, three Phytophthora sp. isolates were obtained. Therefore, this study aimed to identify the causal agent of the disease through morphophysiological and molecular analysis of the isolates. Initially, the pathogenicity test was performed on persimmon fruit to fulfil Koch's postulates. Mycelial growth at eight temperatures ranging from 8°C to 35°C was evaluated for taxonomic purposes. Next, the morphological characteristics of sporangia, chlamydospores and oospores were evaluated. For molecular characterization, sequencing of the ITS‐rDNA region and portions of the COXI and TEF1α genes and phylogenetic analysis were performed. All isolates were pathogenic, causing symptoms of firm and dark rot similar to those observed in the orchards. Mycelial growth was not observed at 35°C. There was an abundant production of ellipsoid, papillate, deciduous sporangia with long pedicels and the formation of globose chlamydospores. The isolates were heterothallic, all belonging to group A1, in which the production of plerotic oospores with amphigenous antheridia was verified. The morphophysiological and molecular characterization allowed the identification of the isolates as Phytophthora tropicalis. To our knowledge, this is the first report of persimmon fruit rot caused by P. tropicalis.

Peering Into Candida albicans Pir Protein Function and Comparative Genomics of the Pir Family.

The fungal cell wall, comprised primarily of protein and polymeric carbohydrate, maintains cell structure, provides protection from the environment, and is an important antifungal drug target. Pir proteins (proteins with internal repeats) are linked to cell wall β-1,3-glucan and are best studied in Saccharomyces cerevisiae. Sequential deletion of S. cerevisiae PIR genes produces strains with increasingly notable cell wall damage. However, a true null mutant lacking all five S. cerevisiae PIR genes was never constructed. Because only two PIR genes (PIR1, PIR32) were annotated in the Candida albicans genome, the initial goal of this work was to construct a true Δpir/Δpir null strain in this species. Unexpectedly, the phenotype of the null strain was almost indistinguishable from its parent, leading to the search for other proteins with Pir function. Bioinformatic approaches revealed nine additional C. albicans proteins that share a conserved Pir functional motif (minimally DGQ). Examination of the protein sequences revealed another conserved motif (QFQFD) toward the C-terminal end of each protein. Sequence similarities and presence of the conserved motif(s) were used to identify a set of 75 proteins across 16 fungal species that are proposed here as Pir proteins. The Pir family is greatly expanded in C. albicans and C. dubliniensis compared to other species and the orthologs are known to have specialized function during chlamydospore formation. Predicted Pir structures showed a conserved core of antiparallel beta-sheets and sometimes-extensive loops that contain amino acids with the potential to form linkages to cell wall components. Pir phylogeny demonstrated emergence of specific ortholog groups among the fungal species. Variation in gene expression patterns was noted among the ortholog groups during growth in rich medium. PIR allelic variation was quite limited despite the presence of a repeated sequence in many loci. Results presented here demonstrate that the Pir family is larger than previously recognized and lead to new hypotheses to test to better understand Pir proteins and their role in the fungal cell wall.

Autophagy-related protein UvAtg7 contributes to mycelial growth, virulence, asexual reproduction and cell stress response in rice false smut fungus Ustilaginoidea virens

Autophagy is a conserved mechanism for nutrient and cytoplasmic components recycling in eukaryotic cell, in which E1-like enzyme Atg7 activates ubiquitin-like conjugation in the autophagy pathway. In plant pathogenic fungi Ustilaginoidea virens, UvAtg7, an ortholog of AAtg7 in baker’s yeast was identified and functionally investigated. UvAtg7 was confirmed to be essential for autophagy, because the disruption of UvATG7 gene in U. virens completely blocked the fusion of autophagosome-like into vacuoles and catalytic degradation of GFP-UvAtg8 under N-starving condition. The fluorescent signal indicated UvAtg7 protein was dispersed in cytoplasma, but spatially coordinated with core autophagy protein UvAtg8 on occasion. Interestingly, disruption of UvATG7 in U. virens caused slightly reduction in mycelial growth, but resulted in a considerable decrease in virulence, conidia production in YT broth and chlamydospore formation on rice false smut balls. Moreover, the UvATG7 deletion mutants exhibited increased sensitivity to cell wall integrity stress caused by congo red and calcofluor white, meanwhile the UvATG7 deletion mutants showed decreased sensitivity to osmotic stress, cell membrane stress and reactiveoxygen stress caused by sorbitol, sodium dodecyl sulfate and H2O2, respectively. All of these defects in UvATG7 deletion mutants could be partially or completely restored by gene complementation. In general, our study indicates that UvAtg7 is essential in autophagy pathway and contributes to mycelial growth, virulence, asexual reproduction and cell stress response in U. virens.

Prevalence of Candida Carriage and in Vitro Evaluation of Phospholipase and Haemolysin Activity of Oral Candida albicans among Tobacco Users and Smokers in Dharan, Nepal

Objectives: The main objective of this study was to determine the prevalence of Candida carriage among tobacco users and smokers along with in vitro evaluation of phospholipase and hemolysin activity of Candida albicans. Methods: A laboratory based cross-sectional study was carried out in Dharan Sub-Metropolitan city, Eastern Nepal from June 2018 to November 2018. During the study 150 oral rinse samples were obtained from smokers (50), smokeless tobacco consumers (50) and non-tobacco users (50) as control group. The participants were provided 10 ml of normal saline and were asked to oral rinse for 1 minute. Oral rinse was collected in a sterile screw capped container and was transported to microbiology laboratory by maintaining the cold chain. The oral rinse sample was inoculated onto the Sabouraud dextrose agar with chloramphenicol and was incubated at 37°C for 3-4 days. The number of colonies of Candida was counted and C. albicans were identified by cultural characteristics, staining, germ tube test and chlamydospore formation test. Results: The prevalence of Candida carriage was reported to be 22 (44%) in smoker group, 26 (52%) among smokeless tobacco users and 13 (26%) among control groups. The prevalence of Candida carriage was found to be significantly higher in the study group associated with tobacco chewers (P=0.008). However, the Candida carriage among smoker’s group was not found to be statistically significant (P=0.059). Isolation of Candida albicans was higher among smokeless tobacco users 15 (30%), smokers 5 (10%) and non-users 6 (12%). Conclusion: Colonization and carriage of Candida in the oral cavity of smokers and tobacco chewers were found to be higher than in controls. In addition, individuals with poor oral hygiene increase the risk of Candida colonization and infection under host debilitated condition.

Discovering the chlamydospore regulatory network in Candida albicans

A normal resident of healthy humans and warm-blooded animals, C. albicans is a commensal fungus that is also among the most common opportunistic pathogens of humans. C. albicans forms unique morphological structures called chlamydospores, which are large, spherical, thick-walled structures formed at the ends of hyphae that have unknown biological function. My goal is to discover the regulatory network controlling chlamydospore formation in C. albicans. By determining this network, we can gain insight into the biological roles of chlamydospores in the C. albicans lifestyle, better understand C. albicans morphological transitions, and determine the selective advantage (if any) provided by chlamydospores to this pathogenic fungus. To determine this regulatory network, I have screened a library of 211 C. albicans transcription factor (TF) homozygous deletion mutants to assay for their abilities to form chlamydospores under standard chlamydospore-inducing growth conditions. I have identified seven TF mutants that fail to produce any chlamydospores andthree TF mutants that produce high levels of chlamydospores relative to WT. To characterize the transcriptional changes occurring during chlamydospore formation, I have performed RNA sequencing (RNA-seq) on these identified regulator mutants to uncover the differentially regulated target genes of each chlamydospore regulator. I will use genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) on epitope-tagged versions of these regulators to determine which genes are directly under the control of each TF. RNA-seq coupled with ChIP-seq will allow me to determine the regulatory network controlling chlamydospore formation in C. albicans.

A comparative study for production of germ tube in Candida albicans of various pulmonary samples, by different methods in a tertiary care hospital of south west Rajasthan

Background: Systemic candidiasis is associated with a high crude mortality rate, even with first line antifungal therapy. C. albicans is the predominant cause of invasive fungal diseases which is a serious public health issue. The main objective was to assess the reliability of different media for germ tube production in Candida albicans isolated from various clinically diagnosed pulmonary samples.Methods: All Candida isolates were identified and speciated by conventional methods such as Gram’s staining, germ tube test, chlamydospore formation on corn meal agar, sugar fermentation test, sugar assimilation test, and growth on Hi-chrome candida agar.Results: Out of 108 clinical isolates of Candida albicans, 5 different methods were used for germ tube production. Pooled human sera showed 93/108 (86.1%) was the most sensitive method wherein YEPD (yeast extract peptone dextrose) broth 91/108 (84.7%) was the reliable and easy method for detection of germ tube, followed by trypticase soy broth 81/108 (81.4%); peptone water 80/108 (74.7%) and 2% sucrose 71/108 (65.7%).Conclusions: YPED broth is found to be a better serum free substrate and subsequently for the presumptive differentiation of C. albicans from non-albicans candida (NAC), without the extensive time required for the preparation and testing of pooled human serum. Furthermore, this medium is commercially available, more stable, effective, and is not bio hazardous.

Quantification of Oral Candidal Carriage Rate and Prevalence of Oral Candidal Species in HIV Patients with and Without Highly Active Antiretroviral Therapy.

Background:Most documents review decrease in the prevalence of HIV related oral lesions to the tune of 10-50% following the advent of HAART. However long term use of HAART on oral health status of HIV infected subjects is poorly documented. Also antifungal agents can effectively treat mucosal candidiasis. However their use can lead to colonization with less susceptible strains among normal susceptible strains.Aims and Objectives:To know the candidal carriage rate (i.e. presence/absence of candidal growth), candidal density(CFUs/ml) & species variations (species diversity) in HIV positive individuals with and without highly active anti-retroviral therapy (HAART), attending the regional voluntary Counselling and Confidential Testing Centre (VCCTC).Materials and Methods:The study population were categorized into 3 groups. Method followed were Germ tube test, Chlamydospore formation test, CHROM-Agar test.Results:Quantification comparison study of candidal carriage rate, density with detection of various candidal species in the oral cavity of HIV-positive individuals with and without HAART therapy was conducted.Conclusion:HIV positive individuals with HAART therapy treatment prooved higher candidal carriage rate and lower density than Non-HAART category.