DNA-Dependent Detection of the Grapevine Fungal Endophytes Aureobasidium pullulans and Epicoccum nigrum

Abstract

Aureobasidium pullulans and Epicoccum nigrum are frequently reported as endophytes of various crops, including grapevine (Vitis vinifera). Because of their potential role as biological control agents against grapevine pathogens, we examined the occurrence of A. pullulans and E. nigrum in two grapevine varieties (Merlot and Prosecco) in Italian vineyards where spontaneous recovery from phytoplasma disease is recurrent. Species-specific primers for A. pullulans and two genetically distinct strains of E. nigrum were designed in variable regions of ITS1 and ITS2. Primer specificity was confirmed by polymerase chain reaction using purified DNA from other fungal endophytes that are usually encountered during isolation attempts from grapevine tissues and from several other strains of A. pullulans and E. nigrum isolated from other sources. In order to determine the occurrence of the two endophytes in grapevine plants, DNA was extracted from shoots of 44 grapevines collected in six vineyards from different localities of northeast Italy. Both endophytes were detected and their identity was confirmed by restriction fragment length polymorphism (RFLP) patterns obtained from reference strains. RFLP analyses confirmed the presence of two E. nigrum strains belonging to different RFLP groups in grapevine. The molecular methods described allowed a sensitive, specific, and reliable identification of the two endophytes in grapevine.

Fungal endophytic communities associated to the phyllosphere of grapevine cultivars under different types of management

“…This classical endophytic genus has also been reported to possess several antifungal properties against grapevine pathogens such as P. viticola or B. cinerea (Fowler et al 1999). Epicoccum nigrum is being developed commercially due to its capability to produce secondary metabolites with antibiotic activity (Martini et al 2009). In summary, 66 % of the isolates obtained in this study belonged either to Alternaria or to Epicoccum species, which means that endophytic communities may constitute a source of biocontrol agents useful to control important vine diseases.…”

“…In addition, Aureobasidium pullulans was not isolated from grapevines in this study. This fungus has shown to be very frequent in grapevine plants (Martini et al 2009;Gonzalez & Tello 2011;Grisan et al 2011;Schmid et al 2011;Pancher et al 2012) and its role as antagonist against several pathogens has led to its commercial development to control B. cinerea. Differences found may be due to the cultivars and environmental dissimilarities, which have shown to have a very important impact in the fungi present (N unez-Trujillo et al 2012;Pancher et al 2012).…”

“…Endophytic fungal communities have been studied less, but have showed some very interesting and important results (Martini et al 2009;Gonzalez & Tello 2011). Their presence has shown to interfere in wine quality through the production of toxic metabolites that some fungi produce, such as ochratoxin A (OTA) produced by Aspergillus spp.…”

Guazuma ulmifolia (Sterculiaceae), a new natural host of 16SrXV phytoplasma in Costa Rica

“…The pellet was re-suspended in 2 mL of CTAB 2% and two aliquots (about 600 µL) were collected and incubated at 60ºC for 30 min, with sporadic agitation. The next steps of DNA extraction were carried out according to Martini et al (2009). The extracted DNA was quantified by Nanodrop ND1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and diluted in sterile water to obtain a concentration of about 20 ng/μL.…”

“…The DNA extraction protocol using ethylene glycol monomethyl ether in the initial steps permitted to obtain good quality DNAs for successful PCR amplifications, which were not achieved using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) and Martini et al (2009) protocol without modified initial steps. This plant species produces a gummy product during the DNA extraction process and that may be the cause of unsuccessful amplification.…”