Lassaad Belbahri

Insight into trade???off between wood decay and parasitism from the genome of a fungal forest pathogen

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

Phylogenetic analysis and Real Time PCR detection of a presumbably undescribed Peronospora species on sweet basil and sage

Downy mildew of sweet basil (Ocimum basilicum) has become a serious disease issue for the producers of sweet basil in Switzerland since it was first recorded in 2001. Reported in Africa in Uganda as early as 1933, major outbreaks of this disease in Europe were first noted in Italy in 1999 and in the USA from 1993. Previous reports have named the pathogen as Peronospora lamii. Its preferential hosts belong to the Lamiaceae family including basils (Ocimum spp.), mints (Menta spp.), sages (Salvia spp.) and other aromatics. This study investigated the taxonomic status of the downy mildew pathogen, using both morphological characters and molecular analysis of the ITS region of the rDNA. The inherent variability of conidial dimensions made species differentiation difficult. Sequence homology and phylogenetic analysis of nine collections of the Peronospora on sweet basil showed unique ITS sequences distinct from those of P. lamii and any other sequenced Peronospora species. This paper describes and illustrates the morphology of this presumably undescribed species of Peronospora. Its taxonomic position and relationships with other related species in the same genus are presented and discussed. In addition to this work, PCR primers for real time PCR analysis have been developed for the specific detection of this downy mildew pathogen from infected tissues or seeds. It is shown that these primers can also be used in classic PCR.

SSU rRNA reveals major trends in oomycete evolution

Oomycetes are a group of heterokonts that have a huge impact on the environment as well as on human welfare, due the parasitic nature of many species. However, their evolutionary patterns are still not well understood, due in part to the lack of molecular markers suited to resolve the deep phylogeny of this group. Here, we propose a phylogeny of the whole clade based on the nuclear ribosomal small subunit gene, that comprises both culture and environmental studies derived sequences. Our analysis shows notably that i) plant pathogenesis occurred only rarely in oomycete evolution in comparison to animal parasitism ii) obligate symbiosis happened only in a few derived groups and iii) transitions from soil/freshwater to marine environment (and viceversa) are common unlike for most eukaryotic groups. This study illustrates the complexity of evolutionary patterns and will help to better understand the emergence of pathogenicity in the different oomycete groups.

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.

Application of response surface methodology to optimize decolourization of dyes by the laccase-mediator system.

Response surface methodology (RSM) was applied to optimize the decolourization of 3 dyes belonging to 3 dye families such as reactive black 5 (diazoic), indigo carmine (indigoid) and aniline blue (anthraquinonic). Crude laccase from Trametes trogii and the laccase-mediator 1-hydroxybenzotriazole (HBT) were used in this study. Box-Behnken design using RSM with six variables namely pH, temperature, enzyme concentration, HBT concentration, dye concentration and incubation time was used in this study to optimize significant correlation between the effects of these variables on the decolourization of reactive black 5 (RB5), indigo carmine (IC) and aniline blue (AB). The optimum of pH, temperature, laccase, HBT, RB5 and reaction time were 4.5, 0.5 U ml(-1), 0.5 mM, 100 mg ml(-1) and 150 min respectively, for a maximum decolourization of RB5 (about 92.92% ± 7.21). Whereas, the optimum decolourization conditions of both IC (99.76% ± 7.75) and AB (98.44% ± 10) were: pH 4.5, temperature of 45 °C, enzyme concentration of 0.1 U ml(-1) and 0.5 U ml(-1), HBT concentration of 0.9 mM and 0.5 mM, dye concentration of 60 mg l(-1) and reaction time of 150 and 90 min, respectively. The experimental values were in good agreement with the predicted ones and the models were highly significant, the correlation coefficient (R(2)) being 0.864, 0.663 and 0.776 for RB5, IC and AB, respectively. In addition, when the kinetic parameters for the three dyes decolourization were calculated according to Hannes-Wolf plot, the following values were obtained: Km of 268.4, 47.94 and 44.64 mg l(-1) then V(max) of 35.58, 10.43 and 9.23 mg l(-1) min for the RB5, IC and AB decolourizations by laccase, respectively.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and β-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

Olive oil mill wastewaters: Phenolic content characterization during degradation by Coriolopsis gallica

Olive mill wastewaters (OMW) pose a serious environmental concern owing to high polyphenol content. Decolorization and degradation of phenolic compounds (PC) by Coriolopsis gallica was demonstrated in our laboratory as a potential biotreatment of OMW in solid and liquid media. High performance liquid chromatography coupled to electrospray time-of-flight mass spectrometry was used to analyze the evolution of the main phenolic compounds during the C. gallica biodegradation process. Amongst total the compounds characterized in methanolic extracts of OMW, 12 were unknown, 15 were from different polyphenolic families, and 27 were other non-phenolic compounds. The evolution of PC content during the degradation process indicated that, despite the complexity of the OMW phenolic fraction, C. gallica was able to grow on OMW-based media using PC as sources of carbon and energy, particularly acids, alcohols, lignans and flavones. Complete dephenolization of OMW was obtained.

Green Fluorescent Protein (GFP) as a Reporter Gene for the Plant Pathogenic Oomycete Phytophthora ramorum

ABSTRACT. Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2‐based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron, one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non‐labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum. Limitations of the technology are discussed.

Direct PCR for DNA Barcoding in the Genera Phytophopthora and Pythium

ABSTRACT A protocol for direct polymerase chain reaction (DPCR) amplification of commonly used phylogenetic markers of the genera Pythium and Phytophthora from mycelia was developed. This has proven useful for direct PCR amplification from hypha, zoospores and infected plant material, which is quicker and simpler, than other proposed attempts. Optimal reaction conditions are described allowing amplification of commonly used phylogenetic markers with a size of up to 1 kbp. This approach proved successful for amplification of selected markers from hundreds of Phytophthora, Pythium and fungal isolates and has been so far used in routine genotyping identification of oomycota and fungi in our laboratory.

Multiple barcode assessment within the Saprolegnia-Achlya clade (Saprolegniales, Oomycota, Straminipila) brings order in a neglected group of pathogens.

The Saprolegnia-Achlya clade comprises species of major environmental and economic importance due to their negative impact on aquaculture and aquatic ecosystems by threatening fishes, amphibians, and crustaceans. However, their taxonomy and phylogenetic relationships remain unresolved and suffer from many inconsistencies, which is a major obstacle to the widespread application of molecular barcoding to identify pathogenic strains with quarantine implications. We assessed phylogenetic relationships of major genera using three commonly used markers (ITS, SSU rRNA, and LSU rRNA). A consensus tree of the three genes provided support for nine clades encompassing eleven documented genera and a new clade (SAP1) that has not been described morphologically. In the course of this study, we isolated a new species, Newbya dichotoma sp. nov., which provided the only culture available for this genus. In parallel, we attempted to summarize the evolution of traits in the different genera, but their successive reversals rendered the inference of ancestral states impossible. This highlights even more the importance of a bar-coding strategy for saprolegniacean parasite detection and monitoring.