Mustapha Barakate

Actinomycetes of Moroccan habitats: Isolation and screening for antifungal activities

During a search for non-polyenic antifungal antibiotics, 320 actinomycete strains were isolated from several Moroccan habitats. Antibiotic productions of the isolates have been tested at various temperatures and production media. Thirty-two isolates showed strong activity against yeast, moulds and bacteria. The production of non-polyenic antifungal metabolites by active isolates was investigated using some of their biological activities: antibacterial activity, spheroplast regeneration and ergosterol inhibition. Ten active selected isolates were identified as belonging to the genus Streptomyces.

An early Ca2+ influx is a prerequisite to thaxtomin A-induced cell death in Arabidopsis thaliana cells

The pathogenicity of various Streptomyces scabies isolates involved in potato scab disease was correlated with the production of thaxtomin A. Since calcium is known as an essential second messenger associated with pathogen-induced plant responses and cell death, it was investigated whether thaxtomin A could induce a Ca2+ influx related to cell death and to other putative plant responses using Arabidopsis thaliana suspension cells, which is a convenient model to study plant-microbe interactions. A. thaliana cells were treated with micromolar concentrations of thaxtomin A. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements with the apoaequorin Ca2+ reporter protein and by external pH measurement. Involvement of anion and calcium channels in signal transduction leading to programmed cell death was determined by using specific inhibitors. These data suggest that this toxin induces a rapid Ca2+ influx and cell death in A. thaliana cell suspensions. Moreover, these data provide strong evidence that the Ca2+ influx induced by thaxtomin A is necessary to achieve this cell death and is a prerequisite to early thaxtomin A-induced responses: anion current increase, alkalization of the external medium, and the expression of PAL1 coding for a key enzyme of the phenylpropanoid pathway.

In vitro and in vivo antagonism of actinomycetes isolated from Moroccan rhizospherical soils against Sclerotium rolfsii: a causal agent of root rot on sugar beet (Beta vulgaris L.)

Aims:  To evaluate the ability of the isolated actinomycetes to inhibit in vitro plant pathogenic fungi and the efficacy of promising antagonistic isolates to reduce in vivo the incidence of root rot induced by Sclerotium rolfsii on sugar beet.

Control of potato soft rot caused by Pectobacterium carotovorum and Pectobacterium atrosepticum by Moroccan actinobacteria isolates

Pectobacteriumcarotovorum and Pectobacterium atrosepticum are dreadful causal agents of potato soft rot. Actually, there are no efficient bactericides used to protect potato against Pectobacterium spp. Biological control using actinobacteria could be an interesting approach to manage this disease. Thus, two hundred actinobacteria isolated from Moroccan habitats were tested for their ability to inhibit in vitro 4 environmental Pectobacterium strains and the two reference strains (P. carotovorum CFBP 5890 and P. atrosepticum CFBP 5889). Eight percent of these isolates were active against at least one of the tested pathogens and only 2% exhibited an antimicrobial activity against all tested Pectobacterium strains. Four bioactive isolates having the greatest pathogen inhibitory capabilities and classified as belonging to the genus Streptomyces species through 16S rDNA analysis were subsequently tested for their ability to reduce in vivo soft rot symptoms on potato slices of Bintje, Yukon Gold, Russet and Norland cultivars caused by the two pathogens P. carotovorum and P. atrosepticum. This test was carried out by using biomass inoculums and culture filtrate of the isolates as treatment. Among these, strain Streptomyces sp. OE7, reduced by 65–94% symptom severity caused by the two pathogens on potato slices. Streptomyces OE7 showed a potential for controlling soft rot on potato slices and could be useful in an integrated control program against potato soft rot pathogens in the objective to reduce treatments with chemical compounds.

Assessment of summer drought tolerance variability in Mediterranean alfalfa (Medicago sativa L.) cultivars under Moroccan fields conditions

This study was undertaken to assess levels of tolerance to summer drought in Medicago sativa genotypes and to comment on their abilities to adapt to Moroccan conditions. Sixteen alfalfa cultivars originating from the Mediterranean basin were tested at an experimental station in Morocco, located in the semi-arid bioclimatic area. Trials were conducted over three years and consisted of one continuously irrigated treatment and another irrigated treatment in which summer watering was withheld for 9 weeks. Results showed that summer water stress significantly reduced aboveground biomass in all of the cultivars tested. This reduction ranged between 23.9 and 42.6% compared with fully irrigated treatment. At the end of stress period, mean summer leaf senescence rate was 83.3%, with a significant difference between cultivars. Ground cover, estimated at the end of summer in the third year, decreased dramatically and ranged between 12 and 30.7%, depending on the cultivar. High intrinsic water-use efficiency was associated with a decrease in stomatal conductance rather than an increase in photosynthesis.

Resistance to and Accumulation of Heavy Metals by Actinobacteria Isolated from Abandoned Mining Areas

Accumulation of high concentrations of heavy metals in environments can cause many human health risks and serious ecological problems. Nowadays, bioremediation using microorganisms is receiving much attention due to their good performance. The aim of this work is to investigate heavy metals resistance and bioaccumulation potential of actinobacteria strains isolated from some abandoned mining areas. Analysis of mining residues revealed that high concentration of zinc “Zn” was recorded in Sidi Bouatman, Arbar, and Bir Nhass mining residues. The highest concentration of lead “Pb” was found in Sidi Bouatman. Copper “Cu,” cadmium “Cd,” and chromium “Cr” were found with moderate and low concentrations. The resistance of 59 isolated actinobacteria to the five heavy metals was also determined. Using molecular identification 16S rRNA, these 27 isolates were found to belong to Streptomyces and Amycolatopsis genera. The results showed different levels of heavy metal resistance; the minimum inhibitory concentration (MIC) recorded was 0.55 for Pb, 0.15 for Cr, and 0.10 mg·mL−1 for both Zn and Cu. Chemical precipitation assay of heavy metals using hydrogen sulfide technic (H2S) revealed that only 27 isolates have a strong ability to accumulate Pb (up to 600 mg of Pb per g of biomass for Streptomyces sp. BN3).

Pril‐ampicillin‐dextrin‐ethanol agar for the isolation and quantification of Aeromonas spp. from polluted environmental waters

Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples. Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low. When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria. A new medium was developed on the basis of the combination of some positive aspects of several published media, pril‐ampicillin‐dextrin‐ethanol (PADE) agar. The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent. The confirmation rate of typical colonies designated Aeromonas spp. isolated from polluted samples exceeded 90%. Recoveries of stressed aeromonad strains on both PADE agar and a non‐selective medium (TSA) did not show any significant difference (P 0.05). PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus, (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.

Toxic and signalling effects of oxalic acid Oxalic acid—Natural born killer or natural born protector?

Oxalic acid is thought to be a key factor of the early pathogenic stage in a wide range of necrotrophic fungi. We have recently published that oxalic acid induces Programmed Cell Death (PCD) in Arabidopsis thaliana cells. This cell death results from an early anionic efflux which is a prerequisite for the synthesis of ethylene and the PCD. Complementary experiments have been carried out by using seedlings of A. thaliana. The effects of millimolar concentrations of oxalic acid were analysed on A. thaliana seedlings. A treatment with a 3 mM oxalic acid solution does not alter the development of the plants but induces the transcription of defence related genes which are anion channel dependant. Moreover, our results suggest that a pre-treatment of the seedlings with oxalic acid is able to confer the resistance of A. thaliana against Sclerotium rolfsii. Regarding our results, we suggest that oxalic acid plays two distinct roles, depending on the concentration: a high concentration of oxalic acid induces a large PCD and then contribute to the progression of the fungi. However, at low concentration it is able to induce the establishment of a resistance of the plant against the fungi. Addendum to: Errakhi R, Meimoun P, Lehner A, Vidal G, Briand J, Corbineau F, Rona JP, Bouteau F. Anion channel activity is necessary to induce ethylene synthesis and programmed cell death in response to oxalic acid. J Exp Bot 2008; doi:10.1093/jxb/ern166.

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.

Biocontrol Capacities and Plant Growth-Promoting Traits of Endophytic Actinobacteria Isolated from Native Plants of Algerian Sahara

Fusarium root rot is one of the most important plant diseases. Chemical fungicides are efficient but they have been much criticized. The present study investigates endophytic actino-bacteria isolated from native plants of the Algerian Sahara in the aim of assessing their ability to exercise biocontrol over this soil-borne fungus, and to highlight their plant growth-promoting traits on seedlings of tomato cv. Mar-mande. A total of 21 endophytic actinobacteria were isolated from plant roots. Six isolates were first selected for the biocontrol trials on the basis of their antifungal properties. In vivo biocontrol activity was measured by the disease incidence on plants grown in infested soils. Four isolates decreased the disease incidence in both sterilized and non-sterilized infested soils, whereas no significant differences were obtained with the control chemical agent. Indole-3-acetic acid and siderophore production, and phosphate solubilization were investigated as plant growth-promoting traits. The isolate ZL2 showed positive results for these mechanisms. Statistical treatment of the data brought out the potential of this isolate as a promising candidate for the biocontrol of root rot in tomato seedlings and in regard to its plant growth-promoting characteristics. Molecular identification was performed by 16S rRNA gene sequence analysis and indicated that Streptomyces sp. ZL2 was related to Streptomyces caeruleatus (99.6% of similarity). Isolate Streptomyces sp. ZL2 enhanced the plant resistance to F. oxysporum f. sp. radicis lycopersici root rot and promoted the growth of tomato seedlings. These properties open up promising perspectives for the possible application of isolate ZL2 in crop protection.