First report of Leaf Wilt caused by Fusarium proliferatum and F. brachygibbosum on Date Palm (Phoenix dactylifera) in Tunisia.

Since 2017, a new leaf wilt syndrome was observed in plantations of date palm in Tunisia. Its incidence increases sharply from year to year, especially in 'Deglet Nour' trees, aged between 5 and 15 years. In severe cases, the large number of dried leaves per tree can lead to complete cessation of date production. Symptoms appear on one or more leaves in the center of the crown. Whitening and drying start at the top of the leaflets and proceed to their base, while the midrib remains green. Then the whole leaf dies. Small white-creamy leaflet fragments and roots were collected from five different regions in the Djerid Oases. They were disinfected with diluted bleach (0,8 % NaOCl) and ethanol (80%) (each 2 min), rinsed with sterile distilled water, dried and finally plated in Petri dishes containing Potato Dextrose Agar (PDA) amended with 50mg/l neomycin. After incubation for 7 days at 25ºC±2, emerging fungal colonies were single-spored by serial dilution. They were transferred to PDA, Carnation Leaf Agar (CLA) and Spezieller Nahrstoffarmer Agar (SNA) for morphological identification. Based on the colony color on PDA, conidial morphology and phialide structures on CLA and/or SNA, of the 85 Fusarium isolates, around 90% were identified as F. proliferatum and around 10% as F. brachygibbosum (Leslie and Summerell, 2006). Fusarium proliferatum colonies rapidly developed white aerial mycelium that became purple in old cultures. Microconidia were abundant in the aerial mycelium and formed chains of variable length, on monophialides and polyphialids, a characteristic that distinguishes F. proliferatum from F. verticilloides. Less often, they were observed in false heads. Chlamydospores were absent. On CLA, microconidia were mostly 2 × 15 µm in size, a large number of sickle shaped macroconidia (2 × 25 µm) had one septum, some were larger (2 × 50 µm) with 3 septa and tips at both ends. Molecular identification was carried out based on elongation factor (EF-1α) gene sequencing. The region between the EF1 and EF2 primers (O'Donnell et al., 1998) was amplified and the sequences were compared to Fusarium reference sequences (GenBank). The sequences of the isolates Fus 1953 (539 bp), Fus 1962 (618 bp), and Fus 1965 (605 bp) shared respectively 100%, 99.51% and 99.51% homology with that of F. proliferatum JF740713.1 and were deposited in GenBank with the following accession numbers: MT630418, MT630419, and MT630420, respectively. The sequences of isolates 7F, 28F, Fus 1955 and Fus 1956 shared 100 % homology with that of F. brachygibbosum (GQ505418.1) while those of Fus 1955 and Fus 1956 showed 99.02 and 98.91 % identity, respectively, with F. brachygibbosum JX118981.1. The sequences of 7F (535 bp), 28F (535 bp), Fus 1955 (608 bp), and Fus 1956 (647 bp) were deposited in GenBank with the following accession numbers: MT630409, MT630410, MT630411, and MT630412, respectively. Two ml suspension of 106 conidia / ml of each isolate was sprayed separately or in combinations on in vitro cloned 'Deglet Nour' plants, placed in a greenhouse at 28°±2 °C and 70% R.H.. Isolates of F. proliferatum led to dryness and wilting leaflets after 3 weeks. Fusarium brachygibbosum only induced mild leaf yellowing, while in combination they were more virulent. Fungal isolates of both species were re-isolated and their identity confirmed to be the same of those isolated from leaflets infected in the open field, confirming Koch's postulates. Control plants lacked symptoms. Fusarium proliferatum is known as date palm pathogen in many countries (Saleh et al. 2017), however, to our knowledge, this is the first report of F. proliferatum and also F. brachygibbosum causing Leaf Wilt symptoms on P. dactylifera in Tunisia.

Chemical composition and biological activities of Tunisian Cupressus arizonica Greene essential oils.

The chemical composition of the essential oils obtained by hydrodistillation of leaves, stems, and female cones of Cupressus arizonica Greene, grown in Tunisia, was studied by GC-FID and GC/MS analyses. Altogether, 62 compounds were identified, 62 in the leaf oil, 19 in the cone oil, and 24 in the stem oil. The cone and stem oils were mainly composed by monoterpene hydrocarbons (96.6 and 85.2%, resp.). In the leaf oil, the total sesquiterpene fraction constituted 36.1% and that of the monoterpene hydrocarbons 33.8% of the total oil composition. The three oils were evaluated for their in vitro herbicidal activity by determining their influence on the germination and the shoot and root growth of the four weed species Sinapis arvensis L., Lolium rigidum Gaudin, Trifolium campestre Schreb., and Phalaris canariensis L. At the highest doses tested (0.8 and 1.0 mg/ml), the leaf essential oil inhibited either totally or almost completely the seed germination and the shoot and root growth of S. arvensis and T. campestre. The oils were also tested for their antifungal activity; however, their effects on the fungal growth were statistically not significant.

Essential Oils and Biological Activities of Eucalyptus falcata, E. sideroxylon and E. citriodora Growing in Tunisia.

Many plants are able to synthesize essential oils (EOs), which play key roles in defense against weeds, fungi and pests. This study aims to analyze the chemical composition and to highlight the antioxidant, antimicrobial and phytotoxic properties of the EOs from Eucalyptus falcata, E. sideroxylon and E. citriodora growing in Tunisia. EOs were analyzed by gas chromatography coupled to mass spectrometry (GC/MS) and their antioxidant properties were determined by total antioxidant capacity (TAC), DPPH and ABTS assays. The phytotoxic potential was assessed against weeds (Sinapis arvensis, Phalaris canariensis) and durum wheat crop (Triticum durum) and compared to chemical herbicide glyphosate. The antifungal activity was investigated in vitro against eight target fungal strains. All EOs displayed a specific richness in oxygenated monoterpenes (51.3-90%) and oxygenated sesquiterpenes (4.8-29.4%), and 1,8-cineole, citronellal, citronellol, trans-pinocarveol, globulol, spathulenol and citronellyl acetate were the main constituents. Eucalyptus EOs exhibited remarkable antioxidant activity and E. citriodora oil exhibited significant activity when compared with E. falcata and E. sideroxylon EOs. The phytotoxic potential of the tested oils had different efficacy on seed germination and the growth of seedlings and varied among tested herbs and their chemical composition variability. Their effectiveness was better than that of glyphosate. At the post-emergence stage, symptoms of chlorosis and necrosis were observed. Furthermore, a decrease in chlorophyll and relative water content, electrolyte leakage and high levels of MDA and proline were indicators of the oxidative effects of EOs and their effectiveness as bioherbicides. Moreover, all the EOs exhibited moderate fungitoxic properties against all the tested fungal strains. Therefore, according to the obtained results, Eucalyptus EOs could have potential application as natural pesticides.

An endophyte of Macrochloa tenacissima (esparto or needle grass) from Tunisia is a novel species in the Fusarium redolens species complex.

Here, we report on the morphological, molecular, and chemical characterization of a novel Fusarium species recovered from the roots and rhizosphere of Macrochloa tenacissima (halfa, esparto, or needle grass) in central Tunisia. Formally described here as F. spartum, this species is a member of the Fusarium redolens species complex but differs from the other two species within the complex, F. redolens and F. hostae, by its endophytic association with M. tenacissima and its genealogical exclusivity based on multilocus phylogenetic analyses. To assess their sexual reproductive mode, a polymerase chain reaction (PCR) assay was designed and used to screen the three strains of F. spartum, 51 of F. redolens, and 14 of F. hostae for mating type (MAT) idiomorph. Genetic architecture of the MAT locus in the former two species suggests that if they reproduce sexually, it is via obligate outcrossing. By comparison, results of the PCR assay indicated that 13/14 of the F. hostae strains possessed MAT1-1 and MAT1-2 idiomorphs and thus might be self-fertile or homothallic. However, when the F. hostae strains were selfed, 11 failed to produce perithecia and one only produced several small abortive perithecia. Cirrhi with ascospores, however, were only produced by 8/28 and 4/84 of the variable size perithecia, respectively, of F. hostae NRRL 29888 and 29890. The potential for the three F. redolens clade species to produce mycotoxins, pigments, and phytohormones was assessed by screening whole genome sequence data and by analyzing extracts on cracked maize kernel cultures via liquid chromatography-mass spectrometry.

Chemical composition, phytotoxic and antifungal properties of Ruta chalepensis L. essential oils.

The chemical composition, and phytotoxic and antifungal activities of the essential oils isolated by using hydrodistillation from the aerial parts of Tunisian rue were evaluated. Significant variations were observed among harvest periods. The analysis of the chemical composition by gas chromatography/mass spectrometry showed that 2-undecanone (33.4-49.8%), 2-heptanol acetate (13.5-15.4%) and α-pinene (9.8-11.9%) were the main components. The antifungal ability of rue essential oils was tested by using disc agar diffusion against ten plant pathogenic fungi. A high antifungal activity was observed for the essential oil isolated at flowering developmental phase. Furthermore, rue essential oils showed high level of herbicidal activity against several weeds.

Chemical composition and biological activities of essential oils of Pinus patula.

Essential oils isolated from needles of Pinus patula by hydrodistillation were analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography mass spectrometry (GC-MS). Thirty-eight compounds were identified, representing 98.3% of the total oil. The oil was rich in monoterpene hydrocarbons (62.4%), particularly alpha-pinene (35.2%) and beta-phellandrene (19.5%). The in vitro antifungal assay showed that P. patula oil significantly inhibited the growth of 9 plant pathogenic fungi. The oil, when tested on Sinapis arvensis, Lolium rigidum, Phalaris canariensis and Trifolium campestre, completely inhibited seed germination and seedling growth of all species. Our preliminary results showed that P. patula essential oil could be valorized for the control of weeds and fungal plant diseases.

Trichothecene chemotypes of Fusarium culmorum infecting wheat in Tunisia.

Fusarium culmorum is a major pathogen associated with Fusarium head blight (FHB) of wheat in Tunisia. It may cause yield loss or produce mycotoxins in the grain. The objectives of the present study were threefold: to evaluate by PCR assays the type of mycotoxins produced by 100 F. culmorum isolates recovered from different regions in Northern Tunisia, to determine the amount of mycotoxin production by HPLC analysis, and to analyse for correlations between the amount of mycotoxin produced and the aggressiveness of isolates. PCR assays of Tri5, Tri7, Tri13, and Tri3 were used to predict whether these isolates could produce nivalenol, 3-acetyl-deoxynivalenol, or 15-acetyl-deoxynivalenol. Two of the isolates were predicted to produce NIV, whereas the others were predicted to produce 3-AcDON. Trichothecene production was confirmed and quantified by high pressure liquid chromatography (HPLC) in 28 isolates, after growth on wheat grains, and in a liquid Mycotoxin Synthetic medium (MS). All strains produced DON/3-AcDON at detectable levels ranging from 21 microg/g to 11.000 microg/g of dry biomass on MS medium and from 10 microg/g to 610 microg/g on wheat grain. The evaluation of the relationship between 3-AcDON production and aggressiveness of 17 strains revealed a significant difference in aggressiveness among the isolates. Moreover, only a significant correlation was revealed between aggressiveness and the amount of 3-AcDON produced on MS medium (r=0.36). Chemotyping of F. culmorum isolates is reported for the first time for isolates from Tunisia, and highlights the important potential of F. culmorum to contaminate wheat with 3-AcDON trichothecenes.