Développement d’une méthode de traçage mixte pour Aureobasidium pullulans souche Ach 1-1 et souche 1113-5,deux agents antagonistes des maladies fongiques des pommes en conservation

Aureobasidium pullulans strains Ach 1-1 and 1113-5 are two efficient biocontrol agents against Botrytiscinerea and Penicillium expansum on stored apples. In the present work, a monitoring system allowing their identification and quantification has been developed. The used methodology consisted in the development of specific molecular markers for both strains and a semi-selective medium which facilitate the isolation and counting colonies of strains Ach 1-1 and 1113-5. The random amplified polymorphic DNA (RAPD) technique has been applied to a collection of 15 strains of A. pullulans, including the Ach 1-1 and 1113-5 strains. Five specific RAPD fragments have been amplified for strain Ach 1-1 and three others for strain 1113-5. Among them, a fragment of 528 bp specific to strain Ach 1-1 (generated with the OPR-13 RAPD primer) and another one of 431 bp specific to strain 1113-5 (amplified with the OPQ-03 RAPD primer) have been selected, cloned, sequenced, and used to design sequence-characterized amplified region (SCAR) primers. Four different SCAR markers have been amplified: three specific to strain Ach 1-1 (157 bp, 190 bp and 388 bp) and one specific to strain 1113-5 (431 bp). These SCAR primers can clearly identify strains Ach 1-1 and 1113-5 among 14 strains of A. pullulans and among eight yeast strains commonly present on the surface of apples. Their selectivity has been also tested using DNA extracted from epiphytic microflora of the apple surface. As a semi-selective medium, the PDA medium supplemented with 0.5 mg/L Euparen, 1mg/L Sumico, 2.5mg/L Hygromycin B, 30 mg/L Streptomycin sulphate, and 1 mg/L Cycloheximide has been selected. This medium inhibited the development of the air microflora and appeared highly toxic for the epiphytic microflora of apple surface without altering the growth of the targeted strains Ach 1-1 and 1113-5. The combination of the plating technique on semi-selective medium and the identification of the colonies by PCR amplification of SCAR markers of 190 bp and 431 bp, respectively specific to A. pullulans strain Ach 1-1 and strain 1113-5, provides a valuable monitoring tool to specifically identify and quantify our targets strains. This monitoring tool has been used to evaluate their population dynamics on apple surface under cold storage conditions. Using this mixed monitoring method, we have highlighted that the population density of strain Ach 1-1 and strain 1113-5 on apple surface has been respectively 1.43x104 ufc/cm2 and 1.47x104 ufc/cm², two hours after their application on apples at the concentration of 107 ufc/mL. After two months storage at 4°C, these two populations densities were reduced by 58% and 66% respectively for the strain Ach 1-1 and strain 1113-5. This technique allowed also to demonstrate that the natural population of A. pullulans on apples surface represented 1% of the number of A. pullulans recovered on apples treated by strain Ach 1-1 and 8% in the case of strain 1113-5.Aureobasidium pullulans souche Ach 1-1 et souche 1113-5 sont deux agents de biocontrôle efficaces contre Botrytis cinerea et Penicillium expansum, respectivement responsables de la pourriture grise et bleue des pommes en conservation. Dans le présent travail, un système de traçage permettant d’identifier spécifiquement nos deux souches antagonistes et de les quantifier parmi la microflore naturellement présente à la surface des fruits a été développé. La méthodologie utilisée consistait au développement de marqueurs moléculaires spécifiques pour nos deux souches étudiées et d’un milieu de culture semisélectif permettant de faciliter l’isolement et le comptage des colonies des souches Ach 1-1 et 1113-5.La technique de marquage moléculaire RAPD a été appliquée sur une collection de 15 souches d’A. pullulans, y compris les souches Ach 1-1 et 1113-5. Cette technique nous a permis d’identifier cinq fragments RAPD spécifiques chez la souche Ach 1-1 et trois autres chez la souche 1113-5. Parmi eux, un fragment de 528 pb spécifique à la souche Ach 1-1 (généré avec l’amorce RAPD OPR-13) et un autre de 431 pb spécifique à la souche 1113-5 (amplifié avec l’amorce RAPD OPQ-03) ont été sélectionnés, clonés et séquencés. Leurs séquences nous ont servit de base pour concevoir des amorces SCAR. Quatrecouples d’amorces SCAR ont étés sélectionnés pour leur capacité à générer des marqueurs SCAR : trois spécifiques à la souche Ach 1-1 (157 pb, 190 pb et 388 pb) et un spécifique à la souche 1113-5 (431 pb). Ces amorces SCAR peuvent identifier de façon spécifique les souches Ach 1-1 et 1113-5 parmi une collection de 14 souches d’A. pullulans et huit autres souches de levures communément présentes à la surface de pommes. La sélectivité de nos amorces SCAR a été aussi testée sur de l’ADN extrait à partir de la microflore épiphytique des pommes.Comme milieu de culture semi-sélectif, le milieu PDA amendé avec 0,5 mg/L d’Euparen , 1 mg/L de Sumico, 2,5 mg/L d’Hygromycine B, 30 mg/L de Streptomycine sulfate et 1 mg/L de Cycloheximide a été sélectionné. Ce milieu a permis l’inhibition totale du développement de la microflore de l’air et parait très toxique vis-à-vis de la microflore épiphytique de la surface des pommes.La combinaison entre la technique d’étalement sur un milieu de culture semi-sélectif et l’identification des colonies par amplification PCR des marqueurs SCAR de 190 pb et de 431 pb, respectivementspécifiques aux souches Ach 1-1 et 1113-5, nous a fourni un outil de traçage plus précis pour étudier la dynamique des populations de ces deux agents de biocontrôle à la surface de pommes en condition de conservation en chambre froide.L’utilisation de cette procédure de traçage mixte nous a permis de mettre en évidence une densité de population de 1,43x104 ufc/cm2 et de 1,47x104 ufc/cm² respectivement pour la souche Ach 1-1 et 1113-5,deux heures après leur application sur pommes avec une concentration de 107ufc/mL. Après deux mois de stockage à 4°C, ces deux populations ont chuté de 58% et 66 % respectivement pour la souche Ach 1-1 et la souche 1113-5. Cette technique nous a également permis de montrer que la population naturelle en A. pullulans à la surface des pommes représentait 1% du nombre d’A. pullulans récupérées à la surface des pommes traitées avec la souche Ach 1-1 et 8% dans le cas de la souche 1113-5

Development of SCAR markers and a semi-selective medium for the quantification of strains Ach 1-1 and 1113-5, two Aureobasidium pullulans potential biocontrol agents.

peer reviewe

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Genetic polymorphism in Leishmania infantum isolates from human and animals determined by nagt PCR-RFLP

Abstract Background Leishmania infantum is the causative agent of human visceral leishmaniasis (VL) and sporadic human cutaneous leishmaniasis (CL) in the Mediterranean region. The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations. The main objective of this study was to explore the genetic polymorphism in L. infantum isolates from human and animal hosts in different regions of Morocco. Methods The intraspecific genetic variability of 40 Moroccan L. infantum MON-1 strains isolated from patients with VL (n = 31) and CL (n = 2) and from dogs (n = 7) was evaluated by PCR-RFLP of nagt, a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase. For a more complete analysis of L. infantum polymorphism, we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database. Results Moroccan L. infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified. Three of the six genotypes were exclusively identified in the Moroccan population of L. infantum: variant M1 (15%), variant M2 (7.5%), and variant M3 (2.5%). The most common genotype (65%), variant 2 (2.5%), and variant 4 (7.5%), were previously described in several countries with endemic leishmaniasis. Phylogenetic analysis segregated our L. infantum population into two distinct clusters, whereas variant M2 was clearly distinguished from both cluster I and cluster II. This distribution highlights the degree of genetic variability among the Moroccan L. infantum population. Conclusion The nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L. infantum strains isolated from human and canine reservoirs with 6 genotypes identified. Three of the six Moroccan nagt genotypes, have not been previously described and support the particular genetic diversity of the Moroccan L. infantum population reported in other studies

Genetic diversity of Leishmania tropica in Morocco: does the dominance of one haplotype signify its fitness in both predominantly anthropophilic Phlebotomus sergenti and human beings?

International audienc

Additional files 2: of Genetic polymorphism in Leishmania infantum isolates from human and animals determined by nagt PCR-RFLP

Table S1. List of strains of L. infantum and L. donovani previously genotyped as nagt variant by Waki et al. [34] and used in this work. (DOCX 13 kb

Additional files 1: of Genetic polymorphism in Leishmania infantum isolates from human and animals determined by nagt PCR-RFLP

Multilingual abstracts in the five official working languages of the United Nations. (PDF 1009 kb