El Hassan Achbani

Molecular typing of Pectobacterium carotovorum isolated from potato tuber soft rot in Morocco

Twenty-two isolates of Pectobacterium carotovorum were collected from soft-rotted potato tubers originating from different production areas in Morocco, and their phenotypic and genetic characteristics were investigated. All of the isolates were identified as P. carotovorum by API 20E (identification kit) system, and yielded a 434-bp DNA fragment of the pectate-lyase-encoding pel gene in PCR experiments. Collected strains were also evaluated for their susceptibility to 11 different antibiotics: Sulfamethoxazole/trimethoprim (SXT), Ceftazidime (CAZ), Ciprofloxacin (CIP), Cefotaxime sodium (CTX), Imipenem (IPM), Gentamicin (CN), Amikacin (AK), Colistin sulphate (CT), Netilmicin (NET), Ampicillin (AMP) and Cephalothin (KF). Of the total P. carotovorum strains isolated, 5 were resistant to only two antibiotics: Ampicillin and Cephalothin. Evaluation of genetic diversity was performed on all isolates by molecular typing with Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). Results showed the isolates to be distributed on 2 major clusters subdivided into many subclusters. The genetic diversity within the potato strains of P. carotovorum subspecies is shown. No correlation between ERIC-PCR analyses, suscebtibility patterns, geographic areas and year isolated was observed. These results may indicate that the tuber could be the main source of contamination. In conclusion, the ability to distinguish Pectobacterium carotovorum strains from diseased potato with PCR based assay will be useful for strain relatedness studies of this pathogen in Morocco.

Phenotypic and genotypic diversity of Pectobacterium carotovorum subsp carotovorum causing soft rot disease of potatoes in Morocco

The species Pectobacterium carotovorum include a diverse subspecies of bacteria that cause disease on a wide variety of plants. In Morocco, approximately 95xa0% of the P. carotovorum isolates from potato plants with tuber soft rot are P. carotovorum subsp carotovorum. The aim was to monitor this Moroccan population in time and to continue the work carried out to understand the genetic diversity of these subspecies. The diversity of 30 strains of P. carotovorum subsp carotovorum collected between 2012 and 2013 from potato soft rot from different Moroccan regions was evaluated. On the basis of phenotypic characteristics, the isolates were classified into four different biovars and in three groups based on the aggressiveness traits. Moreover, the genetic variability was also investigated by fingerprint analysis. The typing by ERIC and REP sequences of the new collection compared to ERIC-PCR and phylogenetic analysis of pmrA sequences, demonstrated that there is a considerable genetic diversity in P. carotovorum subsp. carotovorum strains, which can be divided into two distinct groups, independently to their source of isolation, by their pathogenicity.

Essential oils of Origanum compactum and Thymus vulgaris exert a protective effect against the phytopathogen Allorhizobium vitis

Allorhizobium (Agrobacterium) vitis is a host-specific pathogenic bacterium that causes grapevine crown gall disease, affecting vine growth and production worldwide. The antibacterial activities of different aromatic plant essential oils were tested in vitro and in planta against A. vitis. Among the essential oils tested, those of Origanum compactum and Thymus vulgaris showed the most significant in vitro antibacterial activities, with a MIC of 0.156 and 0.312xa0mg/mL, respectively. A synergistic effect of these two essential oils (1:1) was observed and confirmed by the checkerboard test. Carvacrol (61.8%) and thymol (47.8%) are, respectively, the major compounds in the essential oils of O. compactum and T. vulgaris and they have been shown to be largely responsible for the antibacterial activities of their corresponding essential oils. Results obtained in vitro were reinforced by an in planta pathogenicity test. A mixture of O. compactum and T. vulgaris essential oils (1:1), inoculated into the injured stem of a tomato plant and a grapevine at 0.312xa0mg/mL as a preventive treatment, reduced both the number of plants developing gall symptoms and the size of the tumors.

Insecticidal effect of essential oil and powder of Mentha pulegium L. leaves against Sitophilus oryzae (Linnaeus, 1763) and Tribolium castaneum (Herbst, 1797) (Coleoptera: Curculionidae, Tenebrionidae), the main pests of stored wheat in Morocco

Abstract Stored grains are threatened by several insects, leading to losses in quality and quantity. Several studies have revealed the risks of using chemicals that can cause serious health problems for humans. It is in this perspective that the objective of our work should be seen: It was to assess the effect of the essential oil and leaf powder of Mentha pulegium against Sitophilus oryzae and Tribolium castaneum adults attacking post-harvest cereals. The insecticidal activity of the essential oil against these two insects was evaluated by three methods: contact, inhalation and ingestion. The ingestion method was used in the case of the leaf powder. The essential oil and leaf powder of Mentha pulegium exhibited insecticidal activity against Sitophilus oryzae and Tribolium castaneum adults (Mentha pulegium essential oil caused up to 100% mortality of both insects). Both insects were influenced affected by the dose, exposure time and the method by which the insecticidal activity of the essential oil was demonstrated. Adults of Sitophilus oryzae were more sensitive to different concentrations of essential oil and leaf powder than those of Tribolium castaneum. The essential oil has no effect on the germination rate of soft wheat grains (Triticum aestivum). On the contrary, it acts positively by reducing the damage caused by these major stock pests. The data from this study could present an alternative solution for replacing synthetic insecticides for the protection of stored commodities.

Rapid and sensitive methods for detection of Allorhizobium vitis, causal agent of grapevine crown gall

A rapid method and sensitive methods for extraction of bacterial DNA from pure culture and directly from plant materiel were compared in polymerase chain reaction with specific primers VCF3/VCR3 to see the reliable method that can used in the detection of tumorigenic strain of Allorhizobium vitis causal agent of grapevine crown gall. From the three tested methods of DNA extraction from pure culture, the alkaline method is the most effective technique for the extraction presenting a high sensitivity with a detection threshold equal to 5.104 CFU/ml. Five different protocols for extracting bacterial DNA from plant tissues of infected tomato, based on the use of an extraction buffer, were tested to see its usefulness in detecting pathogenic strain of A. vitisS4. Two protocols based on the use of Triton X-100 and Tween 20 were efficient for detecting A. vitis S4 directly from tomato tumors with a sensitivity of 103 CFU/ml for the both protocols. Consequently, these protocols were proposed as specific protocols for the detection of tumorigenic strain of A. vitis from symptomatic and asymptomatic plants.