Germ Tube Formation Research Articles

Nutrient transport into germinating Trichoderma atroviride conidia and development of its driving force.

The exit from dormancy and the start of growth should be preceded or at least accompanied by the uptake of nutrients. In this work we studied changes in the transport of several nutrients into Trichoderma atroviride conidia. Germination started with a short period of isodiametric growth (conidial swelling), followed by polarized growth (germ tube formation) after about 8 h at 26 °C. The onset of isodiametric growth required the presence of external both phosphate and nitrate. At the same time, an increased uptake of precursors of macromolecules and phospholipids ((14)C- or (3)H-labelled valine, uracil, N-acetylglucosamine and choline) occurred. A low uptake of these precursors was observed also in non-germinating conidia. Concomitantly, this uptake developed an increased sensitivity to the uncoupler 3,3',4',5-tetrachlorosalicylanilide. Expression and activity of H(+)-ATPase started after completing isodiametric growth, suggesting that the proton-motive force (PMF) generated by H(+)-ATPase may be an accelerator of nutrient uptake and metabolism. (14)C-valine uptake was also measured into a mutant with disrupted pma1 gene. This mutant did not form conidia. The mutant also exhibited uncoupler sensitivity of (14)C-valine uptake. These observations showed that a PMF must have been generated by a mechanism(s) other than the H(+)-ATPase activity in the WT before H(+)-ATPase expression and in mycelia with disrupted H(+)-ATPase.

Antifungal activity of the essential oil of Angelica major against Candida, Cryptococcus, Aspergillus and dermatophyte species.

The composition and antifungal activity of the essential oil (EO) of Angelica major and its main components α-pinene and cis-β-ocimene against clinically relevant yeasts and moulds were evaluated. EO from the plant's aerial parts was obtained by hydrodistillation and analysed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The oil showed high contents of α-pinene (21.8%) and cis-β-ocimene (30.4%). Minimum inhibitory concentrations (MICs) were measured according to the broth macrodilution protocols by the Clinical and Laboratory Standards Institute (CLSI). The EO, α-pinene and cis-β-ocimene displayed low MICs and minimum fungicidal concentrations (MFCs) against dermatophytes and Cryptococcus neoformans, with α-pinene being the most active. Regarding Candida species, the EO susceptibility profiles seem to be diverse and not correlated with fluconazole susceptibility patterns. Moreover, an inhibition of yeast-mycelium transition was demonstrated at sub-inhibitory concentrations of the EO, α-pinene and cis-β-ocimene in C. albicans. In addition, their haemolytic activity was low. The activity displayed by A. major EO and its main components associated with low cytotoxic activity confirms their potential as an antifungal agent against fungal species frequently implicated in human mycoses, particularly cryptococcosis and dermatophytosis. The association with commercial antifungal compounds could bring benefits, by the effect on germ tube formation, and be used in mucocutaneous candidiasis treatment.

Effect of Some Non steroidal Anti-Inflammatory Drugs on Growth, Adherence and Mature Biofilms of <i>Candida spp</i><i>.</i>

<i>Candida spp</i>. are the most common cause of fungal diseases and the fourth commonest cause of nosocomial invasive infections which are considered in many cases as life threatening. Among <i>Candida </i>spp., <i>C. albicans </i>is the most common cause of many fungal diseases, but non-albicans spp. infections are in increase. Non-steroidal anti-inflammatory drugs have previously been shown to have antifungal activity. In this study we determine the antifungal activity of the tested NSAIDs using agar well diffusion method, their effect on the dimorphic transition of <i>C. albicans</i> by testing their ability to form germ tube in the presence of human serum. Determining the effect of NSAIDs on the adherence to plastic surfaces and on the mature biofilms formed by the tested <i>Candida</i> spp.. The results indicated that <i>Sodium Diclofenac</i><i> </i>showed lower MIC against <i>C. albicans</i> and <i>C. glabrata</i> while Ibuprofen had lower MIC against <i>C. krusei</i>. upon examining the effect of Diclofenac sodium, Ibuprofen and ketoprofen on biofilm formed on polyurethane segments by Scanning electron microscope (SEM), a damage to membranes of the tested species was observed. <i>Sodium Diclofenac</i><i> </i>showed the highest inhibitory effect on the adherence (51.1-76.9% at MIC and 56.6-83.3% at 2XMIC) of <i>C. albicans</i> and <i>C. glabrata</i> but Ibuprofen showed a higher inhibitory effect against the adherence of <i>C. krusei</i>. For mature biofilms, the highest disruptive effect on mature biofilms formed by all tested <i>Candida Spp</i>. (37.72-59.29% at MIC and 42.68-63.06% at 2XMIC) was observed by Diclofenac sodium. <i>Sodium Diclofenac</i><i> </i>inhibited dimorphic transition of <i>C. albicans</i> but a decrease in germ tube formation was shown by others. In conclusion, the tested drugs showed antifungal, anti-adherent and anti-biofilm activity that make them useful in the treatment of fungal infection and the prevention of biofilm formation on the surface of medical devices.

Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials.

Background:In perspective of the worldwide increase in a number of immunocompromised patients, the need for identification of Candida species has become a major concern. The development of chromogenic differential media, introduced recently, facilitate rapid speciation. However, it can be employed for routine mycology workup only after an exhaustive evaluation of its benefit and cost effectiveness. This study was undertaken to evaluate the benefit and cost effectiveness of chromogenic media for speciation of Candida clinical isolates.Materials and Methods:Sputum samples of 382 patients were screened for the presence of Candida spp. by Gram stain and culture on sabouraud dextrose agar. Candida species were identified using Gram stain morphology, germ tube formation, cornmeal agar with Tween-80, sugar fermentation tests and morphology on HiCrome Candida differential agar. All the Candida isolates were inoculated on HiCrome Candida agar (HiMedia, Mumbai, India).Results:The sensitivity and specificity of HiCrome agar for identification of Candida albicans were 90% and 96.42%, respectively whereas sensitivity and specificity of carbohydrate fermentation test were 86.67% and 74.07%, respectively. Sensitivity and specificity values of HiCrome agar for detection of C. albicans, Candida parapsilosis and Candida glabrata were above 90%.Conclusions:We found HiCrome agar has high sensitivity and specificity comparable to that of the conventional method. In addition, use of this differential media could significantly cut down the turnaround time as well as cost of sample processing.

Candida tropicalis as a Predominant Isolate from Clinical Specimens and its Antifungal Susceptibility Pattern in a Tertiary Care Hospital in Southern India.

The incidence of infections caused by Candida species has increased considerably over the past three decades mainly due to the rise of the AIDS epidemic, an increasingly aged population, higher numbers of immunocompromised patients and the more widespread use of indwelling medical devices. Candida tropicalis is emerging pathogenic yeast among non-albicans species. Recently drug-resistant C.tropicalis was also reported in hospitals. The present study aimed to isolate and speciate C. tropicalis from various clinical samples and to determine its antifungal susceptibility profile. Clinical samples such as urine, blood, exudates and vaginal swab which were submitted to the Microbiology laboratory during the year 2013 were screened for the growth of Candidia species, which then identified as C.tropicalis by the routine microbiological procedures such as germ tube formation, assimilation and fermentation of sugars and colony color on HICHROM Candida agar. Antifungal susceptibility was performed by disc diffusion method with the drugs Amphotericin-B, Itraconazole, Ketaconazole and Fluconazole on C. tropicalis isolates. A total number of 112 Candida isolates were isolated during the year 2012 from various clinical specimens. Among them 61 (54.3%) were identified as C.tropicalis. All the C. tropicalis isolates were sensitive to Amphotericin-B (100%) but 23 isolates (37.7%) were resistant to Fluconazole. We conclude that identification of Candida species is important to know the prevalent species in the clinical setup and routine antifungal susceptibility should be performed to avoid inappropriate treatment.

Fungal Infection in Egyptian Patients with Diabetic Foot Ulcer ( Prevalence , Spectrum , and Antifungal Susceptibility )

Background: Foot ulcers are a very common complication of Type 1 and Type 2 diabetes. Patients having diabetic foot ulcer infection face serious problems including gangrene and amputations. Objective: The aim of this study was to determine the prevalence, spectrum of fungal infections in diabetic foot ulcer and to assess in vitro the antifungal susceptibility. Methodology: This study was conducted on 108 diabetic patients with foot ulcer attending the out-patient clinic of the National Institute of Diabetes and Endocrinology, from May to July 2014. Wound swabs were inoculated to slants of Sabouraud dextrose agar (SDA). Yeast like growth on SDA was identified by Gram stain, germ tube formation, culture on Chrom agar Candida. Results: The susceptibility of the isolated fungi was evaluated by disk diffusion method on Mueller- Hinton agar, using Amphotericin B, ketoconazole, fluconazole and itraconazole, posaconazole and caspofungin disks. Among the 108 patients 51% were females. Fungal foot infection were highest among the age group 51-60 years. 3'.5% of patients were having fungal infection, Candida spp. was the most predominant isolated fungus (94.2%); C. albicans (6'.8%), C. glabrata (''.8%), other Candia spp. (8.8%), C. tropicalis and C. krusei each (5.9%). In vitro susceptibility was highest to ketoconazole (78.'%), the highest resistance was for amphotericin B (87.5%). Candida spp. is a predominant pathogen causing fungal infection in diabetic foot ulcer. Conclusion: High resistance to antifungal drugs is an emerging major problem

Treatment with some anti-inflammatory drugs reduces germ tube formation in Candida albicans strains.

Candida albicans is an opportunistic dimorphic fungus that inhabits various host mucosal sites. It can cause both superficial and serious systemic disease. Conversion from the yeast to the hyphal form has been associated with increased virulence and mucosal invasiveness. The aim of this study was to investigate the effect of sodium diclofenac and aspirin on germs tube formation of different Candida albicans strains. Prostaglandins may play an important role in fungal colonization. Nonsteroidal anti-inflammatory drugs are inhibitors of the cyclooxygenase isoenzymes. These drugs specifically block the biosynthesis of mammalian prostaglandins by inhibiting one or both of cyclooxygenase isoenzymes. In tests for germ tube formation sodium diclofenac reduced the filamentation to the 12.5%- 5.1%. In the presence of aspirin the filamentation was reduced up to 85-45% depending on the tested strain. Our results suggest that cyclooxygenase-depending synthesis of fungal prostaglandins is important for morphogenesis and fungal virulence. Inhibitors of cyclooxygenase isoensymes (aspirin and diclofenac) are effective in decreasing germ tube formation of Candida albicans.

Biofilm formation by Candida albicans is inhibited by 4,4-dichloro diphenyl diselenide (pCl-PhSe)2.

Candida species are the fourth most common cause of nosocomial bloodstream infections. An increase in the frequency of infections which have become refractory to standard antifungal therapyhave been observed. Recently, the effect of different organochalcogenide compounds reducing both growth and germ tube formation by Candida albicans was demonstrated. This work studied the effect of the organochalcogenide compound (pCl-PhSe)2 on both growth and biofilm formation by Candida albicans. A decrease in C. albicans growth in the presence of crescent concentrations of (pCl- PhSe)2 was observed, in a cell density dependent manner. The inhibition of Candida growth by 10µM (pCl-PhSe)2 was ~60, 57, 47 and 24%, in cell densities of 10(3), 10(4), 10(5) and 10(6) cells/ml, respectively. The compound (pCl-PhSe)2 was able to inhibit biofilm formation by Candida albicans, when biofilm was performed using a cell density of 10(6) cells/ml. In addition, an increase in both ROS production (96%) and cell membrane permeability (1.107-fold) by 10 µM (pCl-PhSe)2 was observed in C. albicans.These results demonstrate that the organochalcogenide compound (pCl-PhSe)2 presents a great potential to inhibit both growth and biofilm formation by C. albicans.

Possible mechanism of antifungal phenazine-1-carboxamide from Pseudomonas sp. against dimorphic fungi Benjaminiella poitrasii and human pathogen Candida albicans.

Investigation of antifungal mechanism of phenazine 1-carboxamide (PC) produced by a Pseudomonas strain MCC2142. An antifungal metabolite produced by a Pseudomonas was purified and identified as PC. Human pathogenic fungi such as Candida albicans, Candida glabrata, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus fumigatus and Aspergillus niger were found to be inhibited by PC (MIC90 32-64 μg ml(-1)). Addition of PC (20 μg ml(-1)) during yeast (Y)-hypha (H) transitions inhibited germ tube formation by >90% and >99% in C. albicans National Collection of Industrial Microorganisms (NCIM) 3471 and nonpathogenic model Benjaminiella poitrasii, respectively. After exposure to PC (20 μg ml(-1)), 75-80% yeast cells of B. poitrasii and C. albicans NCIM 3471 showed rhodamine 123 fluorescence indicating high intracellular reactive oxygen species (ROS) production. ROS further led to hyperpolarization of mitochondrial membrane, subsequently induction of apoptosis as evident by externalization of phosphatidylserine, DNA fragmentation, chromatin condensation and finally death in B. poitrasii. In C. albicans NCIM 3471, PC (20 μg ml(-1)) induced apoptosis. The antifungal effect of PC in B. poitrasii and C. albicans may be due to ROS-mediated apoptotic death. Inhibition of Y-H transition of B. poitrasii and C. albicans by PC indicates that it may prove useful in the control of dimorphic human pathogens.

Chemical composition and biological activity of Tanacetum audibertii (Req.) DC. (Asteraceae), an endemic species of Sardinia Island, Italy

The chemical composition and antifungal activity of volatile extracts of Tanaceutum audibertii (Req.) DC., an endemic species of Sardinia and Corsica islands, were investigated.The extract and the essential oil, obtained respectively by supercritical fluid extraction with carbon dioxide (SFE) at pressures of 90bar and temperature of 40°C and by hydrodistillation (HD), were analyzed by GC/FID and GC/MS. Artemisia ketone, trans-linalyl oxide acetate and 1,8-cineole were the main components.The HD oil was evaluated against yeasts and dermatophyte strains, being more active against Cryptococcus neoformans, with MIC and MLC values of 0.64μL/mL. Moreover, the oil revealed an important inhibitory effect on germ tube formation in C. albicans at sub-inhibitory concentrations. At the concentration of 1/16 MIC the inhibition of filamentation was more than 70% in comparison to untreated control cells.

Impact of brief and sequential exposure to nystatin, amphotericin B, ketoconazole, and fluconazole in modulating adhesion traits of oral Candida dubliniensis isolates.

Candida adherence is implicated in the pathogenesis of oral candidosis. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation, and relative cell surface hydrophobicity (CSH) are colonization attributes of candidal pathogenicity. Candida dubliniensis (C.dubliniensis) is allied with recurrent oral candidosis, which can be treated with nystatin, amphotericin B, ketoconazole, and fluconazole. Due to the diluent effect of saliva and the cleansing effect of the oral musculature in the oral cavity C.dubliniensis isolates undergo brief and sequential exposure to antifungal agents during therapy. Thus, in the present study, we evaluated the adhesion to BEC, GT formation, and the CSH of oral isolates of C.dubliniensis following brief and sequential exposure to nystatin, amphotericin B, ketoconazole, and fluconazole. After determining the minimum inhibitory concentration (MIC) of the aforementioned drugs, 20 oral isolates of C.dubliniensis were briefly (1h), and sequentially (10days) exposed to subcidal concentrations of these drugs. Following drug removal, adhesion to BEC, GT formation, and CSH of these isolates were determined. The percentage reduction of adhesion to BEC, GT formation, and CSH of the isolates following exposure to antifungal agents were as follows: nystatin: 53.55%, 33.98%, and 29.83% (P<0.001); amphotericin B: 53.84%, 36.23%, and 28.97% (P<0.001); ketoconazole: 37.43%, 20.51%, and 16.49% (P<0.001); and fluconazole: 8.93% (P<0.001), 1.6%, and 0.63% (P>0.05). Brief and sequential exposure of C.dubliniensis to antifungal agents would continue to wield an antifungal effect by altering its adhesion attributes, and elucidate possible pharmacodynamics by which antifungal agents might operate in modulating candidal adherence.

Antimicrobial Activity and Mechanism of Action of New N-heteroaryl-1H- (benz)Imidazoles

The antimicrobial activity of sixteen new N-heteroarylated 1H-(benz)imidazoles was evaluated against clinically relevant bacteria (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) and fungi (Candida, Aspergillus and dermatophyte) species according to the Clinical and Laboratory Standards Institute guidelines. None of the tested compounds were active against Gram negative bacteria, but only against S. aureus, that was particularly susceptible to N-thianthrenyl- and N-dibenzothienyl imidazole derivatives. Most of the imidazole derivatives showed a broad spectrum of antifungal activity in all tested fungal strains, including fluconazole-resistant species, with a particularly low minimum inhibitory concentration (MIC) for dermatophytes. N-(dibenzofuran-4-yl)-1H-imidazole (1) and N-(dibenzothien-4-yl)-1H-imidazole (3) showed the highest antifungal potential, being most active against C. albicans. Some N-heteroarylated benzimidazoles showed low activity for fungi with the exception of 3-(1H-benzo[d]imidazol-1-yl)quinoline (14) which was selective against dermatophytes (MIC=4-16 µg/mL). The effect of the active compounds in the inhibition of the dimorphic transition, ergosterol biosynthesis and mitochondrial activity was evaluated in Candida albicans. Compounds 1 and 3 showed the capacity to inhibit the germ tube formation in C. albicans, reduced the ergosterol production and impaired the mitochondrial function. Compounds 1 and 3 showed antimicrobial activity and low cytotoxicity, being of interest for further investigation concerning specially the development of new antifungal agents.

New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans

The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa (aerial parts and roots) and Saponaria officinalis (used as a well-known source of plant saponins) against Candida albicans reference and clinical strains, their yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated. Direct fungicidal/fungistatic properties of the tested phytochemicals used alone, as well as their synergy with azoles (probably resulting from yeast cell wall instability) were demonstrated. Here, to the best of our knowledge, we report for the first time the ability of saponin-rich extracts of M. sativa and S. officinalis to inhibit C. albicans germ tube formation, limit hyphal growth, reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the range of concentrations which were active modulators of Candida virulence factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These properties seem to be very promising in the context of using plant-derived SFs as potential novel antifungal therapeutics supporting classic drugs or as ingredients of disinfectants.

Chemical composition and antifungal activity of supercritical extract and essential oil of Tanacetum vulgare growing wild in Lithuania

This article reports the results concerning the qualitative and quantitative composition of the volatile oil obtained from the aerial parts of Tanacetum vulgare, collected in Lithuania, by supercritical fluid extraction (SFE) with CO2 at 90 bar and 40 °C and by hydrodistillation (HD). GC-MS and GC-FID analysis revealed that the oils mainly consisted of 1,8-cineole, trans-thujone, cis-chrysanthenol, borneol and myrtenol. Oils obtained by HD displayed, with respect to the SFE volatile oil, a higher amount of hydrocarbon monoterpenes. The hydrodistilled oil was particularly active against dermatophyte strains and Cryptococcus neoformans, with MIC values in the range of 0.16–0.64 μL/mL. The oil revealed a significant inhibitory effect on germ tube formation in Candida albicans at sub-inhibitory concentrations. At the concentration of 1/16 MIC, the inhibition of filamentation was more than 80% in comparison to untreated control cells.

Saponins of Trifolium spp. aerial parts as modulators of Candida albicans virulence attributes.

The aim was to provide the insight into the biology of C. albicans influenced by undescribed yet properties of saponin-rich (80%–98%) fractions (SAPFs), isolated from extracts of Trifolium alexandrinum, T. incarnatum, T. resupinatum var. resupinatum aerial parts. Their concentrations below 0.5 mg/mL were arbitrarily considered as subMICs for C. albicans ATCC 10231 and were further used. SAPFs affected yeast enzymatic activity, lowered tolerance to the oxidative stress, to the osmotic stress and to the action of the cell wall disrupting agent. In their presence, germ tubes formation was significantly and irreversibly inhibited, as well as Candida invasive capacity. The evaluation of SAPFs interactions with anti-mycotics showed synergistic activity, mainly with azoles. Fluconazole MIC was lowered—susceptible C. albicans ATCC 10231 was more susceptible, and resistant C. glabrata (clinical strain) become more susceptible (eightfold). Moreover, the tested samples showed no hemolytic activity and at the concentrations up to 0.5 mg/mL did not reduce viability of fibroblasts L929. This study provided the original evidence that SAPFs of Trifolium spp. aerial part exhibit significant antimicrobial activity, by reduce the expression/quantity of important Candida virulence factors and have good potential for the development of novel antifungal products supporting classic drugs.

The Effect of Zuccagnia punctata, an Argentine Medicinal Plant, on Virulence Factors from Candida Species

Zuccagnia punctata Cav. has been used as a traditional medicine in Argentina for the treatment of bacterial and fungal infections. In this study, we evaluated the ability of Z. punctata extract (ZpE) and compounds isolated from it to inhibit the growth and virulence factors of Candida species. ZpE showed inhibitory activity against planktonic cells of all assayed Candida species with MIC values of 400 microg/mL and with MFC values between 400 and 1,200 microg/mL. The principal identified compounds by HPLC-MS/MS and UV-VIS were chalcones (2',4'-dihydroxy-3'-methoxychalcone, 2',4'- dihydroxychalcone), flavones (galangin, 3,7-dihydroxyflavone and chrysin) and flavanones (naringenin, 7-hydroxyflavanone and pinocembrine). These compounds were more effective as inhibitors than the extracts upon biofilm formation as well as on preformed Candida biofilm and yeast germ tube formation. Furthermore, ZpE and chalcones are able to inhibit exoenzymes, which are responsible for the invasion mechanisms of the pathogens. All these effects could moderate colonization, thereby suppressing the pathogen invasive potential. Our results indicate that ZpE and chalcones could be used in antifungal therapy.

Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, a marine algae derived bromophenol, inhibits the growth of Botrytis cinerea and interacts with DNA molecules.

Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE) is a bromophenol isolated from marine algae. Previous reports have shown that BDDE possesses cytotoxic and antibacterial activity. In the present study, we demonstrate that BDDE displays broad-spectrum antifungal activities, especially on Botrytis cinerea. BDDE inhibits the growth of B. cinerea cultured on a solid medium of potato dextrose agar (PDA) as well as on the potato dextrose broth (PDB) medium. Moreover, BDDE decreases the incidence of fruit decay and severity of strawberries infected with B. cinerea. Further studies have revealed that BDDE decreases the germination rate and inhibits the mycelial growth of B. cinerea. The inhibition mechanisms are related to the disruption of the cell membrane integrity in B. cinerea spores and newly formed germ tubes. This study also suggests that BDDE possibly interacts with DNA via intercalation and minor groove binding. The studies provide evidence that BDDE has potential application in the control of gray mold after fruit harvest and the compound could serve as a candidate or lead template for rational drug design and for the development of antifungal agents.

A novel partitivirus that confers hypovirulence on plant pathogenic fungi.

Members of the family Partitiviridae have bisegmented double-stranded RNA (dsRNA) genomes and are not generally known to cause obvious symptoms in their natural hosts. An unusual partitivirus, Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), conferred hypovirulence on its natural plant-pathogenic fungal host, Sclerotinia sclerotiorum strain WF-1. Cellular organelles, including mitochondria, were severely damaged. Hypovirulence and associated traits of strain WF-1 and SsPV1/WF-1 were readily cotransmitted horizontally via hyphal contact to different vegetative compatibility groups of S. sclerotiorum and interspecifically to Sclerotinia nivalis and Sclerotinia minor. S. sclerotiorum strain 1980 transfected with purified SsPV1/WF-1 virions also exhibited hypovirulence and associated traits similar to those of strain WF-1. Moreover, introduction of purified SsPV1/WF-1 virions into strain KY-1 of Botrytis cinerea also resulted in reductions in virulence and mycelial growth and, unexpectedly, enhanced conidial production. However, virus infection suppressed hyphal growth of most germinating conidia of B. cinerea and was eventually lethal to infected hyphae, since very few new colonies could develop following germ tube formation. Taken together, our results support the conclusion that SsPV1/WF-1 causes hypovirulence in Sclerotinia spp. and B. cinerea. Cryo-EM (cryo-electron microscopy) reconstruction of the SsPV1 particle shows that it has a distinct structure with similarity to the closely related partitiviruses Fusarium poae virus 1 and Penicillium stoloniferum virus F. These findings provide new insights into partitivirus biological activities and clues about molecular interactions between partitiviruses and their hosts. Members of the Partitiviridae are believed to occur commonly in their phytopathogenic fungal and plant hosts. However, most partitiviruses examined so far appear to be associated with latent infections. Here we report a partitivirus, SsPV1/WF-1, that was isolated from a hypovirulent strain of Sclerotinia sclerotiorum and describe its biological and molecular features. We have demonstrated that SsPV1 confers hypovirulence. Furthermore, SsPV1 can infect and cause hypovirulence in Botrytis cinerea. Our study also suggests that SsPV1 has a vigorous ability to proliferate and spread via hyphal contact. SsPV1 can overcome vegetative incompatibility barriers and can be transmitted horizontally among different vegetative compatibility groups of S. sclerotiorum, even interspecifically. Cryo-EM reconstruction of SsPV1 shows that it has a distinct structure with similarity to closely related partitiviruses. Our studies exploit a novel system, SsPV1 and its hosts, which can provide the means to explore the mechanisms by which partitiviruses interact with their hosts.

Photodynamic inactivation of oropharyngeal Candida strains

Oropharyngeal candidiasis (OPC) is an infection frequent in immunocompromised patients. Photodynamic therapy is an alternative to conventional treatments, based on the utilization of compounds that inhibit or kill microorganisms only under the effect of light, process known as Photodynamic Inactivation (PDI). In the present study, PDI of Candida spp. by the natural product α-terthienyl (α-T) was investigated following the guidelines of CLSI M27-A3, under UV-A light irradiation.The optimal values of two variables, exposure irradiation time (ET) and distance to the irradiation source (DIS) were established by employing Design Expert Software (DES). For this purpose, a panel of Candida strains isolated from OPC (C. albicans, C. tropicalis, C. parapsilosis and C. krusei) was employed and optimal values were 5min (ET) and between 6.06 and 6.43cm (DIS) with a desirability factor of 0.989. α-T plus UV-A light in the optimal conditions caused a complete reduction in viable cells in 5min which was demonstrated by viable cells reduction assays and confocal microscopy after vital staining (propidium iodide/fluorescein diacetate). The germ tube formation of C. albicans was inhibited by α-T at sub-inhibitory concentrations. Results showed that α-T plus UV-A light could constitute an alternative for OPC treatments at the optimal conditions determined here.

Effect of low shear modeled microgravity on phenotypic and central chitin metabolism in the filamentous fungi Aspergillus niger and Penicillium chrysogenum.

Phenotypic and genotypic changes in Aspergillus niger and Penicillium chrysogenum, spore forming filamentous fungi, with respect to central chitin metabolism were studied under low shear modeled microgravity, normal gravity and static conditions. Low shear modeled microgravity (LSMMG) response showed a similar spore germination rate with normal gravity and static conditions. Interestingly, high ratio of multiple germ tube formation of A. niger in LSMMG condition was observed. Confocal laser scanning microscopy images of calcofluor flurophore stained A. niger and P. chrysogenum showed no significant variations between different conditions tested. Transmission electron microscopy images revealed number of mitochondria increased in P. chrysogenum in low shear modeled microgravity condition but no stress related-woronin bodies in fungal hyphae were observed. To gain additional insight into the cell wall integrity under different conditions, transcription level of a key gene involved in cell wall integrity gfaA, encoding the glutamine: fructose-6-phosphate amidotransferase enzyme, was evaluated using qRT-PCR. The transcription level showed no variation among different conditions. Overall, the results collectively indicate that the LSMMG has shown no significant stress on spore germination, mycelial growth, cell wall integrity of potentially pathogenic fungi, A. niger and P. chrysogenum.