Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa.

BACKGROUND: In Tunisia a first SARS-CoV-2 confirmed case was reported in March 03, 2020. Since then, an increase of cases number was observed from either imported or local cases. The aim of this preliminary study was to better understand the molecular epidemiology and genetic variability of SARS-CoV-2 viruses circulating in Tunisia and worldwide. METHODS: Whole genome sequencing was performed using NGS approach on six SARS. CoV-2 highly positive samples detected during the early phase of the outbreak. RESULTS: Full genomes sequences of six Tunisian SARS-CoV-2 strains were obtained from imported and locally transmission cases during the COVID-19 outbreak. Reported sequences were non-identical with 0.1% nucleotide divergence rate and clustered into 6 different clades with worldwide sequences. SNPs results favor the distribution of the reported Tunisian sequences into 3 major genotypes. These SNP mutations are critical for diagnosis and vaccine development. CONCLUSIONS: These results indicate multiple introductions of the virus in Tunisia and add new genomic data on SARS-CoV-2 at the international level.

Identification of two novel hepatitis C virus subtype 2 from Tunisia (2v and 2w).

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity. Eight genotypes and 90 subtypes are currently described. Genotypes are clinically significant for therapeutic management and their determination is necessary for epidemiological studies. METHODS: Tunisian patients plasma samples (n = 6) with unassigned HCV-2 subtype using partial sequencing in the NS5B and Core/E1 regions were analyzed by realizing whole-genome sequencing analysis. Phylogenetic analyses were performed to assign subtypes. RESULTS: Phylogenetic analysis of the full genome sequences of Tunisian strains shows two subtypes within HCV-2. These later were genetically distinct from all previously established HCV-2 subtypes with nucleotide divergence greater than 15% (20% -31%). These two subtypes are proposed as new subtypes 2v and 2w. CONCLUSIONS: The discovery of two new HCV-2 subtypes circulating in the Tunisian population confirms the great diversity of HCV-2 viruses and increases the total number of HCV-2 subtypes from 21 to 23.

Seroprevalence of hepatitis A virus infection in Central-West of Tunisia.

Hepatitis A infections still represent a major global health concern. During the past years, a transition pattern of the hepatitis A epidemiology was noted in many parts of the world. In Tunisia, there is not a recent survey on age-specific hepatitis A virus seroprevalence. This study aimed to investigate the seroprevalence of hepatitis A virus infection in Central-West Tunisia, representative of regions with lowest socioeconomic level in the country, before vaccine implementation. Sera obtained from the blood samples of subjects were screened for the detection of hepatitis A virus. The seroprevalence was evaluated by detection of total antibodies to hepatitis A virus using commercially available immunoassay kits. A total of 1379 subjects, aged 5-75 years (mean age: 29.0 ± 17.3 years) were studied. The global anti-hepatitis A virus seroplevalence was 84.7% (95% confidence interval: [82.6-86.5]). A higher hepatitis A virus seroprevalence was showed in subjects aged 10-14 years compared to those aged less than 10 years (50.0% vs. 31.0%). In subjects aged 20-29 years, a rapid increase in the hepatitis A virus prevalence was noted; it reached 97.0%. The seroprevalence of anti-hepatitis A virus differed by zone of residence (81.1% in rural area vs. 72.4% in urban area, p = .005) and increased significantly with lower level of education (p = .019). There was no statistical significant seroprevalence difference between male and female: 84.2% versus 85.2%, respectively. Our study confirm the transition pattern of the hepatitis A virus endemicity in Tunisia from high to intermediate and provide an evaluation of the hepatitis A virus epidemiological situation before vaccine implementation.

Hepatitis a virus infection in Central-West Tunisia: an age structured model of transmission and vaccination impact.

BACKGROUND: The epidemiological pattern of hepatitis A infection has shown dynamic changes in many parts of the world due to improved socio-economic conditions and the accumulation of seronegative subjects, which leads to possible outbreaks and increased morbidity rate. In Tunisia, the epidemiological status of hepatits A virus is currently unknown. However, over the past years higher numbers of symptomatic hepatitis A virus infection in school attendants and several outbreaks were reported to the Ministry of Health, especially from regions with the lowest socio-economic levels in the country. The aim of this study was to investigate the current seroprevalence of hepatitis A virus antibodies in central-west Tunisia and assess the impact of hepatitis A virus vaccination on hepatitis A epidemiology. METHODS: Serum samples from 1379 individuals, aged 5-75 years, were screened for hepatitis A virus antibodies. Adjusted seroprevalence, incidence and force of infection parameters were estimated by a linear age structured SEIR (Susceptible-Exposed-Infectious-Recovered) compartmental model. A vaccine model was then constructed to assess the impact on hepatitis A virus epidemiology of 3 scenarios of vaccination strategies: one dose at 12-months of age, one dose at 6-years and one dose at 12-months and another at 6-years of age during 6 years. RESULTS: A rapid increase in anti-hepatitis A virus seroprevalence was noted during infancy and adolescence: 47% of subjects under 10-years-old are infected; the prevalence increases to 77% at 15-years and reaches 97% in subjects aged 30-years. The force of infection is highest between 10 and 30-years of age and the incidence declines with increasing age. The vaccine model showed that the 3-scenarios lead to a significant reduction of the fraction of susceptibles. The two doses scenario gives the best results. Single-dose vaccination at 6-years of age provides more rapid decrease of disease burden in school-aged children, as compared to single-dose vaccination at 12-months, but keeps with a non-negligible fraction of susceptibles among children < 6-years. CONCLUSIONS: Our study confirms the epidemiological switch from high to intermediate endemicity of hepatitis A virus in Tunisia and provides models that may help undertake best decisions in terms of vaccinations strategies.

Interleukin IL-1B gene polymorphism in Tunisian patients with chronic hepatitis B infection: Association with replication levels.

Approaches based on association studies have proven useful in identifying genetic predictors for many diseases, including susceptibility to chronic hepatitis B. In this study we were interested by the IL-1B genetic variants that have been involved in the immune response and we analyzed their role in the susceptibility to develop chronic hepatitis B in the Tunisian population. IL-1B is a potent proinflammatory cytokine that plays an important role in inflammation of the liver. Polymorphic gene IL-1 (-511, +3954) was analyzed in a total of 476 individuals: 236 patients with chronic hepatitis B from different cities of Tunisia recruited in Pasteur Institute between January 2017 and December 2018 and 240 controls. Genomic DNA was obtained using the standard salting-out method and genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For -511C>T polymorphism a significant association was found between patients and controls when comparing the genotypic (P = 0.007; χ2 = 9.74 and odds ratio [OR] = 0.60; confidence interval [CI] = 0.41-0.89) and allelic (P = 0.001; χ2 = 10.60) frequencies. When the viral load was taken into account a highly significant difference was found (P = 9 × 10-4 ; χ2 = 10.89). For +3954C>T polymorphism a significant association was found between patients and controls when comparing genotypic (P = 0.0058; χ2 = 7.60 and OR = 1.67; CI = 1.14-2.46) and allelic (P = 0.0029; χ2 = 8.81) frequencies. T allele can be used as a strong marker for hepatitis B virus disease for both polymorphisms.

Hepatitis viruses take advantage of traditional practices to increase the burden of hepatocellular carcinoma in Tunisia.

Hepatocellular carcinoma (HCC) is a major public health issue in Africa. In Tunisia, hepatitis B virus (HBV) is known to be an important risk factor for HCC in the south of the country, but the role played by hepatitis C virus (HCV) still remains unclear. The aim of the current case-control study was to identify risk factors for HCC development in the northern part of the country. Clinical and biological data including viral hepatitis status (serological and molecular) and non-infectious risk factors from 73 patients with HCC and 70 control subjects without hepatic diseases were collected. The mean age of the patients was 63 ± 10 years, and the ratio of males to females was 1.1. HCC occurred in cirrhotic liver in 72.0% of the cases. HCV infection was the dominant risk factor (64.3% of cases); the presence of HBV was observed in 53.4% of the cases. Occult hepatitis B and C were implicated, respectively, in 30.1% and 9.6% of the cases. HCV genotype 1b was predominant. Patients originating from western Tunisia formed a homogeneous group, characterized by significantly higher rates of tattoos or scarifications (83%) and HCV infection (80%) than those from other parts of the country. Chronic HCV infection is currently the primary risk factor for HCC in Tunisia; HBV infection remains frequent in its overt or occult infection forms. Traditional esthetic practices apparently contribute to increasing the burden of terminal liver diseases in western Tunisia.

Patulin and patulin producing Penicillium spp. occurrence in apples and apple-based products including baby food.

INTRODUCTION: Patulin has raised the international attention because of its health risk. In fact, it has mutagenic, neurotoxic, immunotoxic, genotoxic and gastrointestinal effects in animals. In the present work, patulin and patulin-producing Penicillium spp. in apple and apple-based products marketed in Qatar were analysed. METHODOLOGY: Sampling was carried out using apple fruits and apple-based products. Fungi were isolated from undamaged apples, apple juice and baby apple food. DNA extraction was carried out with DNeasy Plant Mini Kit (QIAGEN, Valencia, USA). The molecular identification of fungal isolates was carried out using ITS1-ITS4 PCR. PCR products were sequenced and blasted. Patulin was extracted and analyzed by LC/MS/MS, then quantified using Agilent 1290UHPLC coupled to 6460 triple quadruple mass spectrometer. RESULTS: Forty-five samples of undamaged fresh apple fruits, apple juice and apple-based baby food products sold in different markets in Qatar were surveyed for both fungal and patulin contamination using Liquid Chromatography Tandem Mass Spectrometery (LC/MS/MS). Twenty-five Penicillium spp. isolates were selected, including 23 P. expansum and one isolate each of P. brevicompactum and P. commune. All the tested Penicillium spp. isolates produced patulin in vitro (from 40 to 100 µg/g on Malt Yeast Extract agar medium). Patulin was detected in 100% of apple juice samples at levels ranging from 5.27 to 82.21 µg/kg. Only 5 samples contained patulin levels higher than European Union recommended limit (50 µg/kg). The average patulin contamination was 30.67 µg/kg and 10.92 µg/kg in baby apple juice and in baby apple compote, respectively.

Molecular detection and characterization through analysis of the hexon and fiber genes of Adenoviruses causing conjunctivitis in Tunisia, North Africa.

Human adenoviruses (HAdVs) are common causes of conjunctivitis. This study describes the epidemiological features and characterizes by phylogenetic analysis HAdVs isolated from patients with conjunctivitis in Tunisia, North Africa. Data on out-patients presenting with conjunctivitis during 2 years (2012-2013) were analyzed. Conjunctival swabs obtained from 240 patients were assessed for the presence of HAdVs by PCR amplification on the fiber and hexon genes. Positive PCR products, together with those of nine viral isolates from previous years, were sequenced and analyzed phylogenetically. Conjunctivitis represented 11.5% of all reasons of consultations with a slight increase between mid-March and mid-June. Sixty-five percent of samples (n = 156) revealed positive by at least one PCR test. PCR amplification in the hexon gene was slightly more sensitive as compared to the fiber gene. Genotyping in the two genomic regions gave concordant results for almost all isolates. HAdV-D8 was the most predominant genotype (87.6%) and was detected continuously from 2000 to 2013. Minor co-circulating genotypes including HAdV-E4, HAdV-B3, HAdV-B55, and HAdV-D37 were identified; most of them were detected by amplification in the hexon gene. In conclusion, this work reports molecular data on adenoviral conjunctivitis from a region where such information is scarce and contributes to a better knowledge of the worldwide distribution of causative genotypes. It revealed a predominance and endemic circulation of HAdV-D8, a genotype that was mainly reported from epidemic keratoconjunctivitis. It shows that PCR amplification in two different genomic regions enhances the sensitivity of HAdV detection in clinical samples and the identification of minor genotypes. J. Med. Virol. 89:304-312, 2017. © 2016 Wiley Periodicals, Inc.

Phylogenetic Analysis and Epidemic History of Hepatitis C Virus Genotype 2 in Tunisia, North Africa.

HCV genotype 2 (HCV-2) has a worldwide distribution with prevalence rates that vary from country to country. High genetic diversity and long-term endemicity were suggested in West African countries. A global dispersal of HCV-2 would have occurred during the 20th century, especially in European countries. In Tunisia, genotype 2 was the second prevalent genotype after genotype 1 and most isolates belong to subtypes 2c and 2k. In this study, phylogenetic analyses based on the NS5B genomic sequences of 113 Tunisian HCV isolates from subtypes 2c and 2k were carried out. A Bayesian coalescent-based framework was used to estimate the origin and the spread of these subtypes circulating in Tunisia. Phylogenetic analyses of HCV-2c sequences suggest the absence of country-specific or time-specific variants. In contrast, the phylogenetic grouping of HCV-2k sequences shows the existence of two major genetic clusters that may represent two distinct circulating variants. Coalescent analysis indicated a most recent common ancestor (tMRCA) of Tunisian HCV-2c around 1886 (1869-1902) before the introduction of HCV-2k in 1901 (1867-1931). Our findings suggest that the introduction of HCV-2c in Tunisia is possibly a result of population movements between Tunisia and European population following the French colonization.

Genetic variability of human adenovirus type 8 causing epidemic and sporadic cases of keratoconjunctivitis.

Human adenovirus type 8 (HAdV-8) is a main aetiological agent of keratoconjunctivitis. It has been reported from both epidemic and sporadic cases. The aim of our study was to investigate the genetic characteristics and chronological pattern of HAdV-8 strains that have been circulating in Tunisia over a 14-year period. Fourteen HAdV-8 isolates from a keratoconjunctivitis outbreak that occurred in 2000 and from sporadic cases between 2001 and 2013 were studied. Nucleotide sequences from the hexon, fiber and penton base genes were determined, including hypervariable regions of the hexon (loops 1 and 2), the fiber (knob) and the penton base (HVR 1 and RGD loops). The sequences were compared to each other and to those of HAdV-8 strains. The Tunisian sequences were unique when compared to the previously published sequences. Also, despite a relatively low degree of genetic variation in the three genomic regions, phylogenetic analysis and alignment of amino acid sequences showed that the sequence from the year 2000 and two other sequences from the year 2013 were similar to each other and differed from the isolates that circulated in the intervening year by two main amino acid changes in the loop 1 hexon gene and the knob-fiber gene. Our results confirm the genetic variability of HAdV-8 and document the chronological changes of circulating genetic variants.

Genotype Distribution and Prevalence of Human Pegivirus among High-Risk Populations in Tunisia.

OBJECTIVE: A recently discovered non-A-E hepatitis virus has been designated as human Pegivirus (HPgV). HPgV is prevalent in high-risk groups such as patients with hepatitis C virus (HCV), and it is of interest for patients who are at risk for transmitted infections. The aim of this study was to evaluate the prevalence of HPgV as well as the genotype distribution among patients in the Tunisian population who are infected with HCV and also in multitransfused patients. METHODS: A total of 144 patients were screened using RTPCR/nested PCR of the 5'-untranslated region (UTR); 14 cases were sequenced and phylogenetically analyzed. RESULTS: Seven (14.9%) subjects from the multitransfused group and 7 (7.2%) patients infected with HCV, respectively, were found positive for HPgV RNA. Sequencing and phylogenetic analysis of the 14 cases revealed that genotype 2a was the main genotype circulating in Tunisian patients. Genotype 2b was found in the amplified samples of 2 HCV-infected patients. CONCLUSION: This study enriches the limited data on HPgV prevalence in Tunisia, and shows, for the first time, the molecular epidemiology of the circulating strains in this country.

Molecular epidemiology of hepatitis B and Delta virus strains that spread in the Mediterranean North East Coast of Tunisia.

BACKGROUND: Tunisia is classified as an area of middle endemic for hepatitis B virus (HBV) infection, however little is known about hepatitis Delta virus (HDV) infection. OBJECTIVES: This study aimed to address the prevalence of HDV infection, to identify possible risks factors, and to analyze the genetic diversity of HDV strains that are spreading in Tunisia. STUDY DESIGN: A retrospective large-scale study including 1615 HBsAg positive patients, native of the North East coast of Tunisia, recruited from Gastroenterology departments, was conducted. Demographic, epidemiological, ethnical, clinical and biological data were recorded. HBV and HDV serological analyses and DNA and RNA viral load quantification were performed. Genotyping of HBV and HDV strains was performed using nucleotide sequencing followed by phylogenetic analyses. RESULTS: The study population included 819 (50.7%) men and 796 (49.3%) women; aged 12-90 years (mean age 41±13 years). A very low prevalence of HDV infection, 2% was observed. No risk factor, except a history of hospitalization for surgery was found. All HDV strains belonged to genotype 1, with a wide distribution within the HDV-1 group. They all share the African amino acid marker, a serine at position 202 of the large Delta protein. HBV genotypes were distributed as follows: HBV/D1 (56.8%), HBV/D7 (40.9%), and HBV/A2 (2.3%). CONCLUSION: Tunisia is a low endemic region for HDV infection, due to an efficient policy of HBV infection control. HDV-1 is the sole genotype found, with a high diversity within this group. Further studies are ongoing in order to better characterize and manage the HBV/HDV-infected patients according to the genetic variability of the viral strains.

Biocontrol activity of four non- and low-fermenting yeast strains against Aspergillus carbonarius and their ability to remove ochratoxin A from grape juice.

Aspergillus spp. infection of grape may lead to ochratoxin A (OTA) contamination in processed beverages such as wine and grape juice. The aim of the current study was to evaluate the biocontrol potential of two non-fermenting (Cyberlindnera jadinii 273 and Candida friedrichii 778) and two low-fermenting (Candida intermedia 235 and Lachancea thermotolerans 751) yeast strains against the pathogenic fungus and OTA-producer Aspergillus carbonarius, and their ability to remove OTA from grape juice. Two strains, 235 and 751, showed a significant ability to inhibit A. carbonarius both on grape berries and in in vitro experiments. Neither their filtrate nor their autoclaved filtrate culture broth was able to prevent consistently pathogen growth. Volatile organic compounds (VOCs) produced by all four selected yeasts were likely able to consistently prevent pathogen sporulation in vitro. VOCs produced by the non-fermenting strain 778 also significantly reduced A. carbonarius vegetative growth. Three yeast strains (235, 751, and 778) efficiently adsorbed artificially spiked OTA from grape juice, while autoclaving treatment improved OTA adsorption capacity by all the four tested strains. Biological control of A. carbonarius and OTA-decontamination using yeast is proposed as an approach to meet the Islamic dietary laws concerning the absence of alcohol in halal beverages.

Subtyping genotype 2 hepatitis C viruses from Tunisia: identification of two putative new subtypes.

HCV variants were classified into six genotypes (1-6) subdivided into several subtypes with different geographic distribution worldwide. Previous studies conducted in Tunisia showed that genotype 1 counts for more than 80 % of circulating HCV genotypes and most of the isolates belong to subtype 1b. Genotype 2 comes in the second position, however, few sequences have been analyzed and published. In the present study, 89 isolates from Tunisian patients, typed as genotype 2 by the InnoLIPA commercial probe hybridization test, were sequenced in the NS5B and Core/E1 regions. All the isolates, clustered with the genotype 2 reference sequences, in the NS5B and in the Core/E1 region and the phylogenetic analyses in the two genomic regions were perfectly concordant: subtype 2c was the most frequent (58 out of 89, 65.1 %) and few isolates belonged to subtypes 2k(n = 10), 2i(n = 5), and 2b(n = 1). Fifteen isolates did not match with any of the reference sequences representing the genotype 2 subtypes, identified up-to-date. They divided into 2 separate clusters with high bootstrap values in both genomic regions. This study shows perfect concordance between the NS5B and the Core/E1 region suggesting that any of the two regions can be used for genotyping and that intergenotypic and intragenotypic recombinants are not very frequent, at least for HCV isolates from genotype 2. The present study also shows a predominance of subtype 2c among genotype 2 HCV isolates circulating in Tunisia, the co-circulation of minor subtypes (2k, 2i, and 2b) and proposes the possible existence of two other new subtypes.

Frequency and clinical significance of core promoter and precore region mutations in Tunisian patients infected chronically with hepatitis B.

Genetic variability of hepatitis B virus (HBV) in the C gene and its association with the different stages of chronic liver disease has been studied inadequately with controversial results. The objectives of the current study were to determine the frequency of core promoter and precore mutations in chronic hepatitis B in Tunisia and to evaluate their impact on viral replication and disease progression. Sequencing was performed in upstream regulatory sequence (URS), pre-core (PreC) and basal core promoter (BCP) regions for 123 chronic infected patients by HBV genotype D at different status of disease. Mutations were detected in 98.4% of cases, affecting URS, BCP and Pre-C in 95.1%, 95.9% and 87.8% respectively. Multi-mutations increased significantly from asymptomatic carrier to advanced liver disease status. G1896A (74.8%), G1764A/T/C (71.5%), G1899A (54.4%) and T1678C (52%) were the most common. Special attention should be paid to A1703T, T1678C/G-A1703T, and A1652G-A1679G mutations probably specific of Tunisians sequences; they were observed in 40.6%, 41.5% and 30.1% respectively. A1679G/C, T1753C/G/A, A1762T/G and A1762T-G1764A were more prevalent in older patients. High DNA levels were associated with G1899A or G1764T/C-C1766G-C1799G and advanced liver disease with mutations at positions 1762, 1764 and/or 1899 alone or in double or triple mutations. It was also shown that substitutions at nucleotides 1762, 1764 and 1899 have an impact on the disease progression. It is the first report for specific mutations in the URS region for genotype D. It should be completed by studying eventual correlation with clinical progression and the response to treatment.

Ecological basis of the interaction between Pseudozyma flocculosa and powdery mildew fungi.

In this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology of Pseudozyma flocculosa and its biocontrol activity. For this purpose, we compared the development of P. flocculosa to that of a close relative, the plant pathogen Ustilago maydis, under different environmental conditions. This approach was further supported by measuring the expression of cyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression of cyp1 remained unchanged over time in P. flocculosa and was undetected in U. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves, P. flocculosa induced a complete collapse of the pathogen colonies, but glycolipid production, as studied by cyp1 expression, was still comparable to that of controls. In complete contrast, cyp1 was upregulated nine times when P. flocculosa was applied to Botrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth of P. flocculosa on powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such as U. maydis do not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity of P. flocculosa toward Erysiphales does appear to be more complex than simple antibiosis.

Proteomic analysis of the metabolic adaptation of the biocontrol agent Pseudozyma flocculosa leading to glycolipid production.

The yeast-like epiphytic fungus Pseudozyma flocculosa is known to antagonize powdery mildew fungi through proliferation on colonies presumably preceded by the release of an antifungal glycolipid (flocculosin). In culture conditions, P. flocculosa can be induced to produce or not flocculosin through manipulation of the culture medium nutrients. In order to characterize and understand the metabolic changes in P. flocculosa linked to glycolipid production, we conducted a 2-DE proteomic analysis and compared the proteomic profile of P. flocculosa growing under conditions favoring the development of the fungus (control) or conducive to flocculosin synthesis (stress). A large number of protein spots (771) were detected in protein extracts of the control treatment compared to only 435 matched protein spots in extracts of the stress cultures, which clearly suggests an important metabolic reorganization in slow-growing cells producing flocculosin. From the latter treatment, we were able to identify 21 protein spots that were either specific to the treatment or up-regulated significantly (2-fold increase). All of them were identified based on similarity between predicted ORF of the newly sequenced genome of P. flocculosa with Ustilago maydis' available annotated sequences. These proteins were associated with the carbon and fatty acid metabolism, and also with the filamentous change of the fungus leading to flocculosin production. This first look into the proteome of P. flocculosa suggests that flocculosin synthesis is elicited in response to specific stress or limiting conditions.

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa.

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.