Phylogenetic,genetic, and phenotypic diversity of Pseudomonas syringae pv. syringae strains isolated from citrus blast and black pit in Tunisia

Citrus blast and black pit caused by Pseudomonas syringae pv. syringae (Pss) is the only bacterial disease reported in Tunisian Citrus orchards. The phylogenetic relationship between Pss strains was studied based on multilocus sequence analysis (MLSA), using partial sequences of housekeeping genes rpoD, rpoB, gyrB, cts, and pfk for 14 representative Pss Citrus strains, including the reference strain LMG5496. The MLSA revealed that the studied Tunisian Citrus strains are closely related to LMG5496 and cluster in phylogroup 02. Based on the cts gene, the majority of Citrus strains clustered in clades “a” and “b”. However, five strains were placed in a newly defined clade “g”. We describe the presence of six different type III secreted effectors (T3SEs). These were found with frequencies of 100% for the effector hopAN1 and the helper hrpK1, 65% for hopT1-2, and 14% for hopN1, hopR1, and hopQ1-2. Investigation of copper resistance showed that 67% of our Pss Citrus strains from Tunisia are resistant to copper sulphate in vitro, and the copper resistance genes copABCDR were detected in 23% of the strains. Our results present new data concerning the genetic diversity and phylogeny, presence of T3SEs, and copper resistance within the Pss populations that affect Citrus in Tunisia.     

Isozymic polymorphism and phylogeny of 10 Lathyrus species

Isozyme variation and phylogenetic relationships between ten annual andperennial Lathyrus species: L.aphaca, L.articulatus, L.cicera, L.hirsutus, L.latifolius, L.nissolia, L.odoratus, L.ochrus, L.sativus and L.sylvestris were studied. Four enzyme systems,leucine-amino-peptidase (LAP), 6-phosphogluconatedeshydrogenase (6-PGD), glutamateoxalo-acetate transaminase (GOT) and phosphoglucoisomerase(PGI) were analyzed, using gel electrophoresis. Fivepolymorphic loci were detected and eleven alleles were identified at these loci.Zymogram data revealed that almost all-present species studied exhibited isozymepolymorphism. L. latifolius andL. sylvestris maintain high levels ofisozyme diversity, which is probably associated with the perenniality of thesetwo species and their predominantly outcrossing reproduction system. Incontrast, the low level of genetic diversity observed in other species isattributed to their breeding systems. These species are annuals and have higherproportions of selfing. The distribution of genetic variation within and amongspecies showed large genetic differences between the analysed species.PGD-1 andPGD-2 loci contributed the most to thedistinction between species. GOT-2,LAP-1 and PGI-1loci contributed to the distinction within species. The low level of gene flowrevealed could be partly related to the high level of autogamy in the majorityof species. The regroupement of species revealed by Nei's genetic similarityagrees only in parte with Kupicha's classification based on morphologicalcharacters. Thus, these isozymic markers are important in germplasm collectionand conservation.     

An integrated multivariate approach to net blotch of barley: Virulence quantification,pathotyping and a breeding strategy for disease resistance

Knowledge of pathotype diversity and virulence in local populations of Pyrenophora teres is a prerequisite to screening for durable resistance to net blotch. The current study aimed to quantify the virulence level of Moroccan isolates, identify and designate existing pathotypes, and select resistant genotypes. We developed a method for virulence quantification of P. teres isolates based on a conversion of infection responses into frequencies for use in correspondence analysis. Coordinates of the first axis of this analysis had a virulence spectrum and ranked isolates from virulent to avirulent. Mixed model analysis was also devised for virulence quantification. Coordinates of the first dimension of correspondence analysis were linearly correlated to BLUPs (Best Linear Unbiased Predictors) of the mixed model. A genotype by genotype by environment model (GGE) coupled with cluster analysis differentiated P. teres isolates into ten and nine pathotypes for net- and spot-forms respectively. Populations of these two forms were dissimilar in terms of classes of virulence. For P. teres f. maculata, avirulent, moderately virulent and highly virulent isolates represented one-third of the population, whereas 90% of P. teres f. teres population was composed of avirulent to moderately avirulent isolates. Barley differential sets were subsequently reduced to two new sets that simplified pathotyping through a key code based on resistant or susceptible reactions. Dendrograms of cluster analysis based on GGE analysis depicted the stability of a genotype’s reactions across all isolates, and using only resistant cultivars as sources of resistance to control net blotch disease would, based on this analysis, fail to control all pathotypes. Therefore, we propose an alternative breeding strategy to control net blotch effectively.     

Efficacy of olive mill waste water and its derivatives in the suppression of crown gall disease of bitter almond

Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in planta for their efficacy against crown gall disease caused by Agrobacterium tumefaciens. OMW and polyphenols displayed a high level of antibacterial activity, however the volatile fraction was less efficient as only a bacteriostatic effect was observed. In pot experiments, the percentage of bitter almond rootstock showing symptoms of crown gall was significantly reduced with the dosage rate of OMW 1% as compared to the control (highly natural infected soil treated with water). Five indigenous bacterial strains isolated from OMW exhibited an antagonistic effect against the bacterium. Based on the gene 16S rRNA sequence analysis, one isolate showed 99.2% similarity to known sequences of Bacillus subtilis, one isolate demonstrated high percentage similarities (99.3%) to the genera Bacillus pumilis, and two isolates were associated with Stenotrophomonas maltophilia and Pseudomonas putida 100% and 99.6% similarities respectively. Among these bacteria, the strain B1 proved efficient against the soil borne pathogen in vitro and pot experiments. Our study in controlled conditions suggested that the addition of OMW to soil exerts significant disease suppressiveness against A. tumefaciens. Thabet Yangui and Ali Rhouma contributed equally to this work and are regarded as joint first authors.     

Does olive mill wastewater stimulate olive plants growth?

Olive mill wastewater (OMW) is annually produced in enormous quantities and it constitutes a serious environmental problem. For several years OMW was used as an amendment in olive orchards which given beneficial effects on growth and soil properties. The present work aimed to study the effect of OMW used after one year from the oil extraction, where phenol content decreased. One-year-old plants of “Chemlali” olive cultivar were subjected to three treatments. C0, C1, and C2 were respectively 0%, 30%, and 45% of OMW used for amendment. Each rate was adjusted with water supplied every 15?days (1?L/plantlet). Results showed significant differences between treatments especially for growth, chlorophyll content of leaves (SPAD), and leaf nutrient content (P, K, Ca, and Na). Always C2 was better than C1. Substrate properties were clearly ameliorated particularly an increase of organic matter but the pH and the electric conductivity were unaffected.     

Chemical Composition and In Vitro Antioxidant and Antimicrobial Activities of the Marine Cyanolichen Lichina pygmaea Volatile Compounds

Volatile compounds from the marine cyanolichen Lichina pygmaea, collected from the Moroccan Atlantic coast, were extracted by hydrodistillation and their putative chemical composition was investigated by gas chromatography coupled to mass spectrometry (GC/MS). Based on the obtained results, Lichina pygmaea volatile compounds (LPVCs) were mainly dominated by sesquiterpenes compounds, where γ-himachalene, β-himachalene, (2E,4E)-2,4 decadienal and α-himachalene were assumed to be the most abundant constituents, with percentage of 37.51%, 11.71%, 8.59% and 7.62%, respectively. LPVCs depicted significant antimicrobial activity against all tested strains (Staphylococcus aureus CCMM B3, Pseudomonas aeruginosa DSM 50090, Escherichia coli ATCC 8739 and Candida albicans CCMM-L4) with minimum inhibitory concentration (MIC) values within the range of 1.69–13.5 mg/mL. Moreover, this LPVC showed interesting scavenging effects on the 2,2-diphenyl-1-picrylhydrazyl radical with an IC₅₀ of 0.21 mg/mL. LPVCs could be an approving resource with moderate antimicrobial potential and interesting antioxidant activity for cosmetics and pharmaceutical applications.     

Water deficit effects on caraway (Carum carvi L.) growth, essential oil and fatty acid composition

The Mediterranean region suffers of drought which affects plant behaviour regarding biochemical responses. Accordingly, the effects of water deficit on growth, essential oil and fatty acid composition of caraway (Carum carvi L.) seeds were investigated. Plants were treated with different levels of water deficit: control, moderate water deficit and severe water deficit. Plant growth (height, fresh and dry matter weight) was significantly reduced by severe water deficit. This last caused also important reductions of the seed yield and yield components. Drought decreased significantly seed total fatty acid contents and particularly the petroselinic ones whose proportions decreased significantly by 12.17 and 18.47%, in comparison with the control, under moderate water deficit and severe water deficit, respectively. Besides, moderate water deficit increased the essential oil yield (expressed as g/100 g on the basis of dry matter weight). The main essential oil constituents were carvone and limonene which showed an increasing of their contents under water deficit levels. Thus, water deficit induced a significant reduction in growth parameters and fatty acid content, and an increase in the essential oil compounds. These bioactive compounds have been required in many industrial products.     

Salt effect on yield and composition of shoot essential oil and trichome morphology and density on leaves of Mentha pulegium

The aim of the present work was to study salt effect on the yield and composition of shoot essential oil (EO) and the structures responsible for its biosynthesis in Mentha pulegium L. Shoot EO was extracted by hydrodistillation and composition was determined by GC–MS method. Apical and basal leaves were taken for microscopy analyses; small fresh samples were observed directly without fixation or metallisation with environmental scanning electron microscope (ESEM) and stereomicroscope (SM). Fresh separate epidermis was used for light microscopy (LM). Salt stress enhanced EO yield by about 2.75 times and affected the percentage of menthone, which is the major compound (51%), increasing that of pulegone. Menthone, pulegone, and neomenthol constituting the monoterpene class were found to be the principal components. The anatomical study showed three types of trichomes: (i) non-glandular, multicellular, simple hairs; (ii) small, capitate glandular trichomes; (iii) and peltate glandular trichomes. In control plants, the density and size of trichomes varied with leaf side (abaxial or adaxial) and developmental stage. Salt stress results in significant modifications affecting trichome distribution and size on both sides.