First Study Case of Microbial Biocontrol Agents Isolated from Aquaponics Through the Mining of High-Throughput Sequencing Data to Control Pythium aphanidermatum on Lettuce.

Aquaponics is defined as a sustainable and integrated system that combines fish aquaculture and hydroponic plant production in the same recirculated water loop. A recent study using high-throughput sequencing (HTS) technologies highlighted that microbial communities from an aquaponic system could control one of the most problematic pathogens in soilless lettuce culture, namely, Pythium aphanidermatum. Therefore, this study aims at isolating the microorganisms responsible for this biocontrol action. Based on the most promising genera identified by HTS, an innovative strategy for isolating and testing original biocontrol agents from aquaponic water was designed to control P. aphanidermatum. Eighty-two bacterial strains and 18 fungal strains were isolated, identified by Sanger sequencing, and screened in vivo to control damping-off of lettuce seeds caused by P. aphanidermatum. Out of these 100 isolates, the eight most efficacious ones were selected and further tested individually to control root rot disease caused by the same pathogen at a later stage of lettuce growth. Strains SHb30 (Sphingobium xenophagum), G2 (Aspergillus flavus), and Chito13 (Mycolicibacterium fortuitum) decreased seed damping-off at a better rate than a propamocarb fungicide and a Pseudomonas chlororaphis registered biocontrol agent did. In root rot bioassays, lettuce mortality was prevented by applying strains G2 and Chito13, which were at least as efficacious as the fungicide or biopesticide controls. Lettuce disease symptoms and mortality were eradicated by strain SHb30 in the first bioassay, but not in the second one. These results show that aquaponic systems are promising sources of original biocontrol agents, and that HTS-guided strategies could represent interesting approaches to identify new biocontrol agents.

Mode of action of lactoperoxidase as related to its antimicrobial activity: a review.

Lactoperoxidase is a member of the family of the mammalian heme peroxidases which have a broad spectrum of activity. Their best known effect is their antimicrobial activity that arouses much interest in in vivo and in vitro applications. In this context, the proper use of lactoperoxidase needs a good understanding of its mode of action, of the factors that favor or limit its activity, and of the features and properties of the active molecules. The first part of this review describes briefly the classification of mammalian peroxidases and their role in the human immune system and in host cell damage. The second part summarizes present knowledge on the mode of action of lactoperoxidase, with special focus on the characteristics to be taken into account for in vitro or in vivo antimicrobial use. The last part looks upon the characteristics of the active molecule produced by lactoperoxidase in the presence of thiocyanate and/or iodide with implication(s) on its antimicrobial activity.

Combination of genomic and proteomic approaches to characterize the symbiotic population of the banana aphid (Hemiptera: Aphididae).

Aphids are known to live in symbiosis with specific bacteria called endosymbionts that have positive or negative impacts on their hosts. In this study, six banana aphid (Pentalonia nigronervosa Coquerel) strains from various geographical origins (Gabon, Madagascar, and Burundi) were screened to determine their symbiotic content, using complementary genomic (16S rDNA sequencing and specific polymerase chain reaction) and proteomic (two-dimensional difference gel electrophoresis coupled with protein identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) approaches. Despite the geographical heterogeneity, the combined methods allowed us to identify the same two symbionts in the six aphids strains tested: Buchnera aphidicola and Wolbachia. Although B. aphidicola is found in almost all aphid species, the systematic presence of Wolbachia in banana aphids is particularly interesting, as this bacterium usually has a low prevalence in aphid species. Phylogenetic analyses showed that the Wolbachia sp. strain found in P. nigronervosa was very similar to the strain present in aphids of the genus Cinara, known to have developed a strong and long-term symbiotic association with Wolbachia. The high level of asexual reproduction in P. nigronervosa could be linked to the presence of Wolbachia, but its prevalence also suggests that this symbiotic bacterium could play a more essential role in its aphid host.

First Report of Banana mild mosaic virus Infecting Plantain in Ivory Coast.

Plantain (Musa sp., genomic group AAB) is an important crop for millions of the world's poorest people. In Ivory Coast, it is the second most consumed food and an important source of income for farmers. Between 2010 and 2011, a survey for viruses infecting plantain (AAB) was conducted in 10 major plantain-growing regions located in eastern (Abengourou), middle-western (Bouaflé, Daloa, Issia, Oumé, Sinfra, Zuenoula), central (Yamoussoukro), and southern (Aboisso, Gagnoa) Ivory Coast. Leaf samples showing yellow streaks or mild chlorotic streaks were collected and dried on CaCl2 for storage. A representative sample from each location was selected and tested for the presence of Cucumber mosaic virus (CMV, genus Cucumovirus), Banana streak virus (BSV, genus Badnavirus), Banana mild mosaic virus (BanMMV, family Flexiviridae), and Banana bract mosaic virus (BBrMV, genus Potyvirus). Immunocapture (IC)-PCR was used for the detection of BSV while reverse transcription (RT)-PCR was used for the detection of CMV, BanMMV, and BBrMV. The following primers sets were used: BSV cl1 and BSV cl2 (1), CMV 3' and CMV 5' (3), BanMMV BanCP1 and BanCP2 (4), BBrMV Bract N2 and Bract NR (2). BanMMV was detected as mixed infections with BSV in the 10 tested samples, one of which also contained CMV. To confirm the identity of the amplification products from the BanMMV primers, one cDNA fragment was directly sequenced in the forward direction (Macrogen Inc., Seoul, South Korea). BLAST search in GenBank revealed that the partial coat protein (CP) sequence of the Ivorian isolate shared 80 to 88% nucleotides and 81 to 92% deduced amino acid similarities with BanMMV isolates. In contrast, partial CP sequence of the Ivorian isolate had less than 40% deduced amino acid sequence identity with other Flexiviridae CP sequence. The partial CP sequence of the Ivorian BanMMV isolate was deposited in GenBank under Accession No. JX014304. To further confirm the identification, all the samples were tested by plate trapped antigen (PTA)-ELISA with rabbit polyclonal antiserum specific to BanMMV (obtained from B. E. Lockhart, University of Minnesota, U.S.A.) and anti-rabbit IgG (Sigma-Aldrich, Belgium/A3687). The 10 samples reacted positive for BanMMV by ELISA. CMV and BSV have been reported in Ivory Coast, but to our knowledge, this is the first report of BanMMV in the country. The detection of BanMMV in association with BSV or CMV in mixed infection in 10 locations which are important plantain growing areas is a first step in the evaluation of the impact of virus diseases on plantain production in this country. References: (1) S. Dallot et al. Arch. Virol. 146:2182, 2001. (2) M.-L. Iskra-Caruana et al. J. Virol. Methods 153:224, 2008. (3) M. Sharman et al. J. Virol. Methods 89:77, 2000. (4) P.-Y. Teycheney et al. J. Gen. Virol. 86:3181, 2005.

Effect of a fungal infection on the profile of volatile organic compounds emitted by plant roots.

It is known since few years that the aerial and underground parts of the plants emit volatile organic compounds (VOCs) that can interact with other organisms of the environment. They are involved in the attraction of seed dispersers and pollinators, the repellence of enemies via direct or indirect mechanisms and the induction of defence systems in other parts of the same plant or in other plants in the vicinity (Dudareva et al., 2006). It has been shown previously that the VOCs spectrum emitted by plants hardly depends on their physiological state (Kant et al., 2009). However those phenomenons were poorly studied at the edaphic level. Thus, the Rhizovol project, a multidisciplinary project in Gembloux Agro-Bio Tech was set up to study the emissions of VOCs by plant roots and their interactions with other organisms of the rhizosphere. As a partner of this project, the Plant Pathology Unit of Gembloux Agro-Bio Tech chose to study the effect of a fungal infection on the profile of VOCs emitted by plant roots, based on three model organisms, barley (Hordeum vulgare L.), since it is a major crop in Belgium that can suffer a large range of aggressions, and two pathogenic fungi, Cochliobolus sativus and Fusarium culmorum, responsible for root and foot rots and seedling blight on cereals (Wiese, 1977). Later in the development, C. sativus produces elongate brown-black lesions (spot blotch) and F. culmorum induces head blight and produces mycotoxins that make the grain unsuitable for consumption (Nielsen et al., 2011). The objective of this work was to identify the VOCs emitted during the dual interactions between barley roots and a pathogenic fungus. The study was performed in two steps; first, the independent analyses of the VOCs emitted by each of the partners (C. sativus, F. culmorum and healthy barley roots), then the analyses of the VOCs spectrum emitted during dual interactions.

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes).

AIMS: To determine the effect of water activity (a(w) =0·880-0·960) and temperature (15-35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. METHODS AND RESULTS: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a(w) values (glycerol being added to adjust the a(w)). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a(w), temperature and the a(w) × temperature interaction on mycelial growth (P<0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a(w) and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a(w) and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. CONCLUSIONS: Our results confirm the previous finding that a(w) has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.

Evaluation of three essential oils as potential sources of botanical fungicides.

In previous study, thirty essential oils were evaluated in vitro against two citrus pathogens namely Penicillium italicum Wehmer and Penicillium digitatum Sacc. Essential oils of Cinnamomum zeylanicum, Cinnamomum verum and Eugenia caryophyllus were selected because of their high inhibitory activities against both pathogens. The present study was undertaken to evaluate the in vivo activity of these essential oils. Fresh orange fruits were wounded and treated with different concentrations of essential oil (0.5, 1, and 5%) before being infected at the wound site with conidia suspensions of the tested pathogens. When applied at 5%, essential oils tested controlled totally the infections. Among the three essential oils tested, C. zeylanicum seems particularly interesting because of its high protection activity at 1% compare to the others. It reduced the disease incidence from 40 to 70% and the disease severity from 65 to 82%. Moreover no visible damage burn induced on the orange cuticle or skin was observed up to 5% of essential oil. These results strengthen the potential use of essential oils in postharvest disease management of citrus fruit as alternative to chemical fungicides.

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of 'Candidatus Phytoplasma mali'.

Different PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter- and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB- or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10(5)). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.