Occurence of sclerotinia stem rot of fenugreek caused by <Emphasis Type="Italic">Sclerotinia trifoliorum</Emphasis> and <Emphasis Type="Italic">S. sclerotiorum</Emphasis> in Tunisia

Fenugreek is an annual leguminous crop grown for hay and grains in Tunisia. It is also considered a valuable rotation crop with cereals. Sclerotinia rot was observed in production fields since 2010. The survey conducted in 2013 revealed that the incidence of diseased plants varied between 5 and 20%. The identification of isolates of Sclerotinia obtained from fenugreek plants with symptoms of stem rot was determined using morphological and molecular criteria. The size, shape and abundance of sclerotia in potato dextrose agar (PDA) cultures were used to classify isolates as S. sclerotiorum or S. trifoliorum. A comparison of colony diameter on PDA after 24, 48 and 72 h at 25 °C, showed that one isolate grew faster (36 mm/day) than the other 10 isolates (14.8 mm/day). There was a significant difference in sclerotial size between the fast and the slow growing isolates, but there was no significant difference in the number of sclerotia produced after 3 weeks on PDA. Two of the slow growing isolates exhibited ascospore dimorphism, whereas the fast growing isolate did not. PCR amplification with the primer pair ITS5/ITS4 produced a fragment of 560 base pairs from the fast growing isolate and 1000 base pairs from all of the slow growing isolates. The ITS sequences of the fast growing isolate had 100% homology with S. sclerotiorum, whereas those of the slow growing isolates had 100% homology with S. trifoliorum. Isolates of both species were pathogenic on fenugreek seedlings in the greenhouse assay and there was no significant difference in the percentage of dead plants two weeks after inoculation between the two species.     

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.     

Trypsin from the viscera of Bogue (<Emphasis Type="Italic">Boops boops</Emphasis>): isolation and characterisation

Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS–PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-l-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants K _m and k _cat on BAPNA were 0.13 mM and 1.56 s⁻¹, respectively, while the catalytic efficiency k _cat /K _m was 12 s⁻¹ mM⁻¹. Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.     

Recent sedimentation rates in Garaet El Ichkeul Lake, NW Tunisia, as affected by the construction of dams and a regulatory sluice

Purpose

Ichkeul National Park, NW Tunisia, is a UNESCO Biosphere Reserve. Garaet El Ichkeul Lake is known for its seasonal variability in water level and salinity. In recent decades, the waterbody has been affected by the construction of new hydraulic structures. To reduce the impacts of dams and to maintain the sustainability of the ecosystem, a sluice was built at the outlet of the lake, and it operated for the first time in 1996. This paper describes an investigation of recent sedimentation dynamics in Ichkeul Lake, determined by radiometric dating of sediment cores.

Materials and methods

A sediment core was collected with a UWITEC gravity corer at the deepest, central part of the lake in August 2009. Specific activities of unsupported lead-210 (²¹⁰Pb) and caesium-137 (¹³⁷Cs) were measured in the core, enabling calculation of recent sediment accumulation rates (SAR). Published radiometric data from nearby sediment cores, collected in 1997 and 1982, provide a comparison.

Results and discussion

The measured excess ²¹⁰Pb inventory was 5300?±?500?Bq?m^?2, leading to an estimation of constant flux of 165?±?16?Bq?m^?2?yr^?1, a value higher than the best estimate for local atmospheric fluxes (123?±?12?Bq?m^?2?yr^?1) and the flux estimated from the core collected in 1982 (48?Bq?m^?2?yr^?1). The ¹³⁷Cs inventory was 3550?±?120?Bq?m^?2, two times higher than the historical ¹³⁷Cs atmospheric deposition in the area. The ¹³⁷Cs profile displayed a distinct peak, but the ¹³⁷Cs depth-distribution did not follow the pattern expected from atmospheric deposition. Application of the constant rate of supply (CRS) model, with the reference point method, produced a chronology and SAR values comparable to those found in previous work. The whole ¹³⁷Cs profile was quantitatively reconstructed from the historical records of atmospheric deposition, using the system-time-averaged (STA) model.

Conclusions

The CRS and STA models provide consistent sediment accumulation results for the whole data set, considering the time resolution of the chronology (~6?years) and analytical uncertainties. Results from cores sampled in 1982, 1997 and 2009 reveal an increasing SAR trend, from ~0.25?g?cm^?2?yr^?1 in the early 1940s to ~0.67?g?cm^?2?yr^?1 at present. In the 13?years since installation of sluice gates at Tinja, SAR in the central Ichkeul Lake has not declined. Thus, if siltation continues at the present rate, shallowing of the lake will seriously affect the hydromorphology and ecology of this important lake.     

Ultrasonographic screening for cystic echinococcosis in sheep in Tunisia

Sheep from the areas of Fondouk-Jeddid, Bir Mchergua and El Fahs, located in the Northeast of Tunisia, were examined by ultrasonography between 2001 and 2004 in order to assess their infection with Echinococcus granulosus, the agent of hydatid disease, and to evaluate this method as an efficient aire for hydatid cysts. A total of 1039 sheep, aged between 1 and 14 years was examined. The highest prevalence was found in sheep aged more than 8 years. The least infected animals were aged between 1 and 2 years. All hydatid cysts detected by ultrasound were located in the liver. In all age-groups, the dead cysts were more numerous than viable cysts. Eighteen positive sheep were autopsied and a comparison between ultrasound and autopsy results was performed. The results showed a prevalence of about 40% for the three areas. Ultrasonography allowed the cysts, deep or superficial to localize in the central or left part in relation to the caudal vena cava of the animals. Consequently, all the cysts were not detected with this technique. This work shows that ultrasonography confirms the importance of ovine hydatid cyst in Tunisia and that its use as a mass screening approach for cystic echinococcosis in sheep could be helpful for the monitoring of this disease in a hydatid control program without great stress for the animals.     

Cystic Hydatic Disease in Sheep: Treatment with Percutaneous Aspiration and Injection with Dipeptide Methyl Ester

An in vitro and in vivo study was conducted to show the effect of dipeptide methyl ester on the protoscolices of Echinococcus granulosus and in naturally infected sheep. Easily punctured cysts were located by ultrasonography. A PAIR and PAI method were performed bythe injection of dipeptide methyl ester into these cysts at a final concentration of 110 mmol/L. Follow-up was conducted monthly by ultrasonography. After injection of the compound, the sheep were sacrificed at different times from 6 to 17 weeks. The size and the morphological aspect of treated cysts were noted. Samples were collected for histologyand electron microscopy. In conclusion, these studies revealed significant and rapid detachment of the membrane of the treated cyst and alteration of the inner membrane in less than 5 min after injection of the drug, confirming the effect of the compound on the laminated layer of the parasite.     

Vaccination of calves with an attenuated cell line of Theileria annulata and the sporozoite antigen SPAG-1 produces a synergistic effect

The sporozoite surface antigen, SPAG-1 and the attenuated schizont infected Tunisian line CL1 of Theileria annulata have been shown, in previous studies, to induce variable levels of protection against homologous and heterologous sporozoite challenge, respectively. We report here the result of a vaccination trial comparing the protection level induced by the SPAG-1 antigen (as a recombinant full length His tagged protein) and the attenuated cell line, used singly or in combination. The results, after challenge of immunised calves with a lethal dose of sporozoites, show that SPAG-1 provides limited protection (one out of seven calves surviving), while the attenuated cell line provides moderate protection (three out of seven calves recovered). The combination of SPAG-1 and the attenuated cell line induced the best protection as indicated by the survival of all the vaccinated calves. These results, together with a range of parasitological and clinical parameters, demonstrate the enhanced protection provided by combining sporozoite and schizont antigens in vaccination against tropical theileriosis.     

First serological investigation of peste-des-petits-ruminants and Rift Valley fever in Tunisia

This study, carried out between September 2006 and January 2007, is the first cross-sectional serological investigation of peste-des-petits-ruminants (PPR) and Rift Valley fever (RVF) in Tunisia. The objective was to assess the potential need to develop a dual, recombinant PPR–RVF vaccine and how such a vaccine might be utilised in Tunisia. An overall PPR seroprevalence of 7.45% was determined, a finding supported by the high specificity (99.4%) and sensitivity (94.5%) of the ELISA used. On assessment of the diversity and density of mosquitoes in the sampling area, four species of RVF-vectors of the genus Aedes and Culex were identified. However, no serological evidence of RVF was found despite the use of a highly sensitive ELISA (99–100%). Larger scale investigations are underway to confirm these findings and the continuation of the emergency vaccination program against these two diseases remains valid.     

Genetic structure of Verticillium dahliae isolates infecting olive trees in Tunisia using AFLP,pathogenicity and PCR markers

Since 2006, verticillium wilt of olive induced by Verticillium dahliae has caused considerable economic losses in olive orchards in Tunisia. The genetic structure of V. dahliae isolates collected from different olive growing regions was investigated using virulence tests, vegetative compatibility grouping (VCG) and amplified fragment length polymorphism (AFLP) analyses. In total, 42 isolates of V. dahliae from diseased olive trees were tested. Cluster analysis and principal coordinate analysis revealed that geographic origin was the main factor determining the genetic structure of V. dahliae populations and both methods indicated a genetic separation between the central and coastal isolates. Isolates were divided into two major groups: the AFLP‐I group included all isolates from Sidi Bouzid, Kairouan, Kasserine and Sfax (centre of the country) and the AFLP‐II group included isolates from Monastir, Zaghouane, Sousse, Mahdia (coastal region), and two isolates from Sfax. Analysis of the molecular variance (amova ) indicated a significant level of genetic differentiation among (76%) and within (23%) the two populations. Analyses of both the defoliating (D) and non‐defoliating (ND) pathotypes and VCG markers indicated that most of the isolates belong to VCG 2A and 4B/ND pathotype. The disease severity was highly variable among the isolates tested (P < 0·05) with no evidence of association between aggressiveness and geographical origin of the isolates. Overall, results of this study revealed a clear association between the genetic diversity of the isolates and their geographic origin, but not between genetic diversity and virulence patterns.     

SOIL LIMING AND MINERAL FERTILIZATION FOR ROOT NODULATION AND GROWTH OF FABA BEANS IN AN ACID SOIL IN TUNISIA

Faba beans (Vicia faba L) are grown in northern Tunisia where annual rainfall approaches 1200 mm and where the soil acidity is the most limiting factor for plant growth. Aluminum (Al) and manganese (Mn) toxicities provide a hostile environment to root growth. To alleviate such a problem, farmers use selected acid-tolerant species. However, crop yields remain far below their optimal levels. Liming, a practice to eliminate acidity, has never been tried in this area before. This research aimed to evaluate the impact of lime and mineral fertilizers on root nodulation, plant uptake and yield of faba beans using a pot experiment. Treatments were combinations of two rates of calcium carbonate with three rates of superphosphate and three rates of potassium sulfate. Liming produced significantly higher number and weight of nodules on roots as well as higher nitrogen, phosphorus, and potassium contents in plant tissue. Liming also increased shoot growth and bean yield.