Identification, phylogénie et caractérisation du profil des mycotoxines des espèces pathogènes Fusarium et Alternaria associées aux syndromes de déclin soudain et aux symptômes de flétrissement et de tâches foliaires du palmier dattier en Tunisie

In heel Noord-Afrika wordt op grote schaal dadelpalmen gekweekt. In Tunesië neemt de dadelpalm een strategische plaats in de sociaal-economische stabiliteit van semi-aride en aride gebieden. Ver-scheidene biotische en abiotische factoren kunnen de dadelpalmteelt in gevaar brengen. In 2017 observeerden we uitgebreide symptomen van plotselinge achteruitgang, bladverwelking en bladvlek-ken op dadelpalmen in zuidelijk Tunesië. Fusarium was het meest waargenomen schimmel geslacht. De sequenties van TEF1, Calm1 en RPB2 werden gebruikt om de Fusarium stammen te identifice-ren. De voornaamste gedetecteerde soort was F. proliferatum, en in veel mindere mate F. brachygib-bosum, F. caatingaense, F. clavum, F. incarnatum, en F. solani. Alle Fusarium soorten waren patho-geen en voldeden aan Koch's postulaten. De F. proliferatum stammen produceerden vooral fumo-nisinen (FB's), beauvericine (BEA), en, in mindere mate, enniatinen (ENN's) en moniliformine (MON). Alle F. brachygibbosum stammen produceerden lage gehalten BEA, diacetoxyscirpenol, en neosolaniol; twee stammen produceerden ook T-2 toxine, en een enkele stam HT-2 toxine. Fusarium caatingaense, F. clavum en F. incarnatum produceerden alleen BEA. De F. solani stammen produceerden MON, BEA, en ENNs. Voor het derde syndroom, bladvlekken, worden Alternaria soorten worden echter gerapporteerd als de meest frequente ziekteverwekkers. Vijftig stammen werden geïsoleerd en geïdentificeerd als Alternaria en Curvularia. Door sequentie-bepaling van de allergen-alt-1a, GPD en calm1 genen konden we 35 stammen, geïdentificeerd als A. arborescens, A. tenuissima en A.alternata soorten, en 10 stammen, geïdentificeerd als A. consortialis. Op basis van de analyse van de seqentie van de gecombineerde Internal Transcribed Spacer, GPD en Elongation factor, werden alle 5 Curvularia stammen geïdentificeerd als C. spicifera. Alle Alternaria en Curvularia soorten die op dadelpalm plantjes getest werden, bleken pathogeen te zijn, en voldeden aan Koch's postulaten. Het vermogen om in vitro mycotoxinen te produceren werd geanalyseerd. Geen van de A. consortialis stammen produceerde enig mycotoxine, terwijl meer dan 80% van de stammen uit de Alternaria sectie zeer wisselende hoeveelheden van meerdere mycotoxi-nen produceerde, zoals AOH, AME, TEN en TA. Bovendien produceerde 40% van de stammen van de Alternaria sectie ook ALT. We testten ook de productie van FB's. Enkel de C. spicifera stammen vertoonden het vermogen fumonisinen te produceren. Dit werk rapporteert voor het eerst een uitgebreide multidisciplinaire studie van schimmel pathogene soorten die in verband gebracht worden met sudden decline disease, bladverwelking en bladvlekken op dadelpalmen, waarbij zowel fytopathologische als voedselveiligheids kwesties aan de orde komen. De gevoeligheid van de isolaten voor zeven fungiciden werd geëvalueerd zowel in in vitro proeven als in het veld. In vitro bleken de contactfungiciden uitstekend te werken. Van de doordringende fungiciden vertoonde Luna Experience een hogere werkzaamheid tegen Fusarium soorten om de myceliumgroei te remmen. Ook vertoonde het een vergelijkbaar effect tegen Alternaria en Curvula-ria soorten. Wat contactfungiciden betreft, werd de beste remming gerapporteerd voor Biobac WP. En het derde fungicide dat een uitstekende werking tegen Alternaria vertoonde was Carial Top. Een veldproef met deze drie beste fungiciden tegen dadelpalm bladziekte veroorzaakt door A. arbores-cens, bevestigde hun werkzaamheidDate palm (Phoenix dactylifera L.), is widely cultivated across North Africa. In Tunisia, with about 300 thousand tons of fruits produced per year, date palm occupies a strategic place in the socio-economic stability of semiarid and arid regions. Several biotic and abiotic factors can compromise date palm cultivation. In 2017–2018, extensive symptoms of sudden decline, leaf wilt symptoms were and leaf spots observed on date palms in southern Tunisia. Based on morphological identification, Fusarium was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative Fusarium strains at species level and investigate their phylogenetic relationships. The main species detected was F. proliferatum, and at a much lesser extent, F. brachygibbosum, F. caatingaense, F. clavum, F. incarnatum, and F. solani. Pathogenicity on the ‘Deglet nour’ variety plantlets and the capability to produce mycotoxins were also assessed. All Fusarium species were pathogenic complying Koch’s pos-tulates. F. proliferatum strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All F. brachygibbosum strains pro-duced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. Fusarium caatingaense, F. clavum, F. incar-natum produced only BEA. F. solani strains produced MON, BEA, and ENNs

Phylogeny and mycotoxin profile of pathogenic Fusarium species isolated from sudden decline syndrome and leaf wilt symptoms on date palms (Phoenix dactylifera) in Tunisia

In 2017-2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, Fusarium was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative Fusarium strains at species level and investigate their phylogenetic relationships. The main species detected was Fusarium proliferatum, and at a much lesser extent, Fusarium brachygibbosum, Fusarium caatingaense, Fusarium clavum, Fusarium incarnatum, and Fusarium solani. Pathogenicity on the Deglet Nour variety plantlets and the capability to produce mycotoxins were also assessed. All Fusarium species were pathogenic complying Koch's postulates. Fusarium proliferatum strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All F. brachygibbosum strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. Fusarium caatingaense, F. clavum, F. incarnatum produced only BEA. Fusarium solani strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of Fusarium species on date palms, concerning both phytopathological and food safety issues

Table_1_Phylogeny and mycotoxin profiles of pathogenic Alternaria and Curvularia species isolated from date palm in southern Tunisia.docx

Date palm (Phoenix dactylifera L.), is a widely cultivated crop across North Africa, with about 300 thousand tons of fruits produced per year, in Tunisia. A wide range of fungal pathogens has been associated with leaf spots of date palm, Alternaria species being the most frequently reported. Symptomatic leaves of Deglet Nour variety were randomly collected in six localities in Tunisia. We used a polyphasic approach to identify 45 Alternaria and five Curvularia strains isolated from date palm, confirming their pathogenicity. Sequencing of allergen Alt-a1, glyceraldehyde-3-phosphate dehydrogenase (gpd) and calmodulin genes allowed us to group 35 strains in Alternaria Section, and 10 strains in Ulocladioides section. Based on sequencing analyses of Internal Transcribed Spacer, gpd and elongation factor genomic regions, all Curvularia strains were identified as Curvularia spicifera. All Alternaria and Curvularia species tested on date palm plantlets proved to be pathogenic, fulfilling Koch’s postulates. Although no significant differences were observed among the species, the highest mean disease severity index was observed in A. arborescens, while the lowest corresponded to C. spicifera. The capability of these strains to produce mycotoxins in vitro was evaluated. None of the A. consortialis strains produced any known Alternaria mycotoxin, whereas more than 80% of the strains included in Alternaria section Alternaria produced variable amounts of multiple mycotoxins such as alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid and tentoxin. Curvularia spicifera strains produced detectable traces of fumonisins B. This work reports a first comprehensive multidisciplinary study of mycotoxigenic Alternaria species and C. spicifera associated with leaf spot disease on date palm.</p

Image_1_Phylogeny and mycotoxin profiles of pathogenic Alternaria and Curvularia species isolated from date palm in southern Tunisia.TIF

Date palm (Phoenix dactylifera L.), is a widely cultivated crop across North Africa, with about 300 thousand tons of fruits produced per year, in Tunisia. A wide range of fungal pathogens has been associated with leaf spots of date palm, Alternaria species being the most frequently reported. Symptomatic leaves of Deglet Nour variety were randomly collected in six localities in Tunisia. We used a polyphasic approach to identify 45 Alternaria and five Curvularia strains isolated from date palm, confirming their pathogenicity. Sequencing of allergen Alt-a1, glyceraldehyde-3-phosphate dehydrogenase (gpd) and calmodulin genes allowed us to group 35 strains in Alternaria Section, and 10 strains in Ulocladioides section. Based on sequencing analyses of Internal Transcribed Spacer, gpd and elongation factor genomic regions, all Curvularia strains were identified as Curvularia spicifera. All Alternaria and Curvularia species tested on date palm plantlets proved to be pathogenic, fulfilling Koch’s postulates. Although no significant differences were observed among the species, the highest mean disease severity index was observed in A. arborescens, while the lowest corresponded to C. spicifera. The capability of these strains to produce mycotoxins in vitro was evaluated. None of the A. consortialis strains produced any known Alternaria mycotoxin, whereas more than 80% of the strains included in Alternaria section Alternaria produced variable amounts of multiple mycotoxins such as alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid and tentoxin. Curvularia spicifera strains produced detectable traces of fumonisins B. This work reports a first comprehensive multidisciplinary study of mycotoxigenic Alternaria species and C. spicifera associated with leaf spot disease on date palm.</p