First report of wilt of almond caused by Verticillium dahliae in Tunisia

Almond (Prunus dulcis) cultivation in Tunisia dates back to ancient times and has been known since the Carthaginian era (Gouta et al., 2011). In some orchards, intercropping with olive and vegetables is a common practice. Environmental conditions are typically Mediterranean, and many fungal diseases affect almond trees in the region. During a routine survey in June 2010, almond trees (cv. Masetto) grafted onto the rootstock 'Garnem' showed symptoms of yellowing, leaf fall and twig and branch dieback.  Discoloration of vascular tissue was visible after cutting open affected stems (Fig. 1). These symptoms were observed in intensively managed orchards in the Northern part of Tunisia.

Samples were collected from branches and twigs showing the characteristic symptoms, and were kept on cold packs before bringing to the laboratory for isolations. Portions of symptom-bearing branches were surface sterilised with 2% sodium hypochlorite, then rinsed with sterile distilled water and allowed to air dry. Isolations from these tissues consistently yielded a Verticillium sp. on potato dextrose agar (PDA) amended with 25 mg/l of streptomycin sulphate. All isolates obtained produced dark colonies with small black microsclerotia on PDA, a defining feature that distinguishes V. dahliae from V. albo-atrum (Smith, 1965). Based on morphological characteristics (Hawksworth & Talboys, 1970) the fungus was tentatively identified as Verticillium dahliae Kleb. The identification was further confirmed by comparison of internal transcribed spacer (ITS) sequence data with reference isolates. The ITS region of rDNA was amplified by polymerase chain reaction (PCR) with primers ITS1F/ITS4 (Gardes & Bruns, 1993) and sequenced using primers ITS5/ITS4. Sequences from almond isolates in this study (GenBank Accession Nos. JQ902034 and JQ902035) were identical to other sequences in the GenBank, and matched the sequence of V. dahliae from tomato (GU060637).

A pathogenicity test was performed on ten grafted almond plants (cv. Mazetto, two years old). Trees were inoculated by root immersion in a suspension of 10⁷ conidia/ml of V. dahliae (isolate for sequence JQ902034) while a set of control trees was similarly submerged in sterile water. The inoculated trees were maintained in a glasshouse at daily average temperatures between 25° and 28°C and relative humidity between 50 and 80%. Symptoms developed 60 days post inoculation and were typical of the original symptoms observed on diseased trees in the field. The symptoms consisted of chlorosis, wilting and necrosis of apical leaves that progressed downwards on the tree. V. dahliae was consistently re-isolated from infected vascular tissues of symptomatic trees as described above, completing Koch’s postulates. Non-inoculated plants remained healthy.

Although V. dahliae has been previously reported on artichoke (Jabnoun-Khiareddine et al., 2008) and olive trees (Triki et al., 2006) in Tunisia, this is the first report of Verticillium wilting on almond trees in Tunisia. At this time, the economic importance of Verticillium wilt on almond cultivation in Tunisia is limited. However, Verticillium wilt might become an important economic problem for almond farmers in the future, since almond cultivation is expanding in many agricultural areas previously dedicated to tomato crops.