Protective effect of Bacillus amyloliquefaciens against infections of Citrus aurantium seedlings by Phoma tracheiphila.

Isolate TEB1 an antagonistic endophytic bacterium, obtained from citrus leaves and identified as Bacillus amyloliquefaciens by 16S rDNA sequencing, was used for the biological control of mal secco disease of Citrus aurantium seedlings caused by the mitosporic fungus Phoma tracheiphila. The isolate TEB1 exhibited a good in vitro activity against P. tracheiphila in dual cultures as well as with the well diffusion method. C. aurantium seedlings watered with a suspension of TEB1 cells showed a reduction of 53.61 and 48.63% in disease severity and incidence, respectively. A PCR test with specific primers was performed 365 days after inoculation and P. tracheiphila was detected along the whole stem in inoculated control plant while no amplification product was obtained in TEB1 treated seedlings. Molecular analysis of TEB1 revealed a positive amplification of fenD and ituC genes responsible of the biosynthesis of fengycin and iturin lipopeptides, respectively. Moreover, observations by optical microscope showed that TEB1 reduced by 55% the germination of P. tracheiphila conidia and exhibited a marked effect on mycelia structure. Data suggest that lipopeptides produced by the bacterium interact with the cytoplasmic membrane of the fungus causing pore formation. TEB1 appears a potential candidate for the biological control of citrus mal secco disease.

First Report of Fire Blight Caused by Erwinia amylovora on Pear in Tunisia.

In the spring of 2012 and 2013, symptoms similar to those of fire blight were observed on pear trees (Pyrus communis cv. Alexandrine, Williams) in Tunisia at flowering stages. Disease symptoms appeared in 2012 in the region of Mornag and in the following year extended to the regions of Manouba and Tebourba. More recently, the disease was observed in the regions of Bizerte, Zaghouan, and Beja. The percentages of orchard areas that had infected plants varied from 10 to 40%. Some orchards in Mornag region exhibited more than 75% disease incidence. Symptoms were observed on flowers and young shoots. Blighted blossoms appeared wilted, shriveled, and brown, and dead flowers remained on the stems. Infected shoots wilted rapidly and often formed shepherd's crooks at their tips. Samples of diseased young shoots and flowers were subjected to pathogen isolation and identification. Bacteria were isolated from washed tissues on King's B medium (KB) (1). Colonies with morphology similar to that of Erwinia amylovora were purified by sub-culturing on KB. The strains were first characterized based on morphology and biochemical tests (1). Sixteen strains produced white colonies on KB, were gram-negative, did not produce a fluorescent pigment on KB, did not grow at 35°C, and induced a hypersensitive reaction when infiltrated into tobacco leaves (cv. Xanthi). These strains were identified as E. amylovora by double-antibody sandwich indirect-ELISA and immunofluorescene microscopy using a polyclonal antibody (2) and nested PCR targeting the pEA29 plasmid (3). Pathogenicity was tested using a detached-fruit assay (1). Each strain was inoculated onto three pear fruit (cv. Alexandrine) wounded with a scalpel dipped in a 109 CFU/ml bacterial suspension. The inoculated fruit were incubated at 25°C and 80% relative humidity in plates with sterile 1% agar. Negative controls consisted of fruit wounded with a scalpel dipped in sterile distilled water. Seven days after inoculation, symptoms of discoloration, browning, and production of bacterial ooze appeared at the inoculated points. No symptoms developed on negative controls. Reisolation of bacteria yielded colonies with characteristics of E. amylovora. Purified amplicons from nested PCR were sequenced (KF302525, KF302526) and a BLAST search of the GenBank database revealed 98% homology with E. amylovora strain HF560643.1. References: (1) Anonymous. OEPP/EPPO Bull. 34:159, 2004. (2) M. T. Gorris et al. Acta Hortic. 411:41, 1996. (3) P. Llop et al. Appl. Environ. Microbiol. 66:2071, 2000.

Deoxynivalenol contamination in Tunisian barley in the 2009 harvest.

In Tunisia, barley is commonly used in human consumption in a variety of food forms. In this regard, a high quality of this agricultural product is always demanded by consumers. A survey of the natural occurrence of deoxynivalenol (DON), the most common Fusarium mycotoxin in small grain cereals, in barley harvested in the main cropping regions in Northern Tunisia in the 2009 harvest was conducted. A total of 72 samples were analysed for DON using high-performance liquid chromatography (HPLC) with a UV visible detector set at 220 nm. Between 36% and 100% of the samples were positive for DON with averages ranging from 1.2 to 2.4 mg kg(-1). A positive correlation between DON levels and temperature was seen; on the other side no correlation between DON contents and rainfall was observed. In this study we notably showed the effect of regions on DON contamination.

Biodiversity of Trichoderma strains in Tunisia.

Trichoderma strains were sampled in 4 different bioclimatic zones of Tunisia, a Mediterranean North African country with strong climatic and edaphic variability from north to south, to assess the genetic diversity of endemic species of Trichoderma and their relationship to the bioclimatic zones. In all, 53 strains were isolated and identified at the species level by analysis of their internal transcribed spacers regions 1 and 2 (ITS1 and ITS2) of the rDNA cluster and (or) a fragment of the translation elongation factor 1 (tef1) gene, using an online interactive key for species identification in Trichoderma and ex-type strains and taxonomically established isolates of Trichoderma as references. At least 2 different species were observed in each ecosystem. Trichoderma harzianum clade VI and Trichoderma longibrachiatum were present in forest soils in north Tunisia; Trichoderma atroviride and Trichoderma hamatum were found in cultivated fields in northeast Tunisia; T. harzianum clade VI, a Trichoderma sp. close to the T. harzianum complex, and Trichoderma saturnisporum were isolated from forest soils in central Tunisia; and T. harzianum clade II and T. hamatum were present in oasis soils in south Tunisia.

Biological control of grey mould in strawberry fruits by halophilic bacteria.

AIMS: Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti-Botrytis metabolites and volatiles. METHODS AND RESULTS: Grey mould was reduced in strawberry fruits treated with halophilic antagonists and artificially inoculated with B. cinerea. Thirty strains (20.2%) were active against the pathogen and reduced the percentage of fruits infected after 3 days of storage at 20 degrees C, from 50% to 91.66%. The antagonists were characterized by phenotypic tests and 16S rDNA sequencing. They were identified as belonging to one of the species: Virgibacillus marismortui, B. subtilis, B. pumilus, B. licheniformis, Terribacillus halophilus, Halomonas elongata, Planococcus rifietoensis, Staphylococcus equorum and Staphylococcus sp. The effective isolates were tested for antifungal secondary metabolites. CONCLUSIONS: Moderately halophilic bacteria may be useful in biological control against this pathogen during postharvest storage of strawberries. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of such bacteria may constitute an important alternative to synthetic fungicides. These moderate halophiles can be exploited in commercial production and application of the effective strains under storage and greenhouse conditions.