First Report of Cylindrocarpon pauciseptatum Associated with Grapevine Decline from Castilla y León, Spain

Abstract

During a survey for grapevine decline, 10 young grapevines (cvs. Tempranillo and Verdejo) with low vigor and little foliage were collected between June 2008 and August 2009. Small pieces of vascular and brown wood were placed onto malt extract agar supplemented with 0.25 g/liter of chloramphenicol and incubated at 25°C. Fifteen resulting colonies were transferred to potato dextrose agar in petri dishes (90 mm). Colonies with white mycelium covered the dishes after 10 days of incubation at 25°C in darkness; mycelium gradually became yellowish with some brownish aspect. Macroconida were predominantly three septate (40 to 45 to 50 × 8.6 to 9 to 9.5 μm with a length and width ratio of 4.7 to 5 to 5.4), straight, and cylindrical with both ends broadly rounded. Chlamydospora and ovoidal microconidia were observed on synthetic nutrient-poor agar (1). Cylindrocarpon pauciseptatum was not easy to distinguish from other Cylindrocarpon species based on morphological characteristics. Ribosomal internal transcribed spacer region sequences of single-spore cultures confirmed the morphological identification and revealed 100% genetic identity with other isolates of C. pauciceptatum present in GenBank (EF607090), a sequence of the fragment was deposited with Accession No. EU983277. Pathogenicity tests were conducted with two isolates. The inoculations were done on 110R rootstock wood of four different young plants and 15 detached canes of current-season growth (cv. Tempranillo). Plants were inoculated with an agar plug containing C. pauciceptatum; controls were treated with agar only. Grapevines were maintained in a greenhouse at 20 to 25°C. After 3 to 4 months, C. pauciceptatum was reisolated from brown tissues and internal vascular lesions in 45% of inoculated samples, fulfilling Koch's postulates. Control plants were asymptomatic and C. pauciceptatum was not recovered. To our knowledge, this is the first report implicating C. pauciceptatum as a cause of grapevine black foot disease in Spain with potentially significant impact on grapevine nurseries. Reference: (1) H. J. Schroers et al. Mycol. Res. 112:82, 2008.

Effectiveness of Natural Antifungal Compounds in Controlling Infection by Grapevine Trunk Disease Pathogens through Pruning Wounds

“…The purpose of this treatment was to hasten the onset of symptoms since one of the biggest disadvantages in the analysis of these types of diseases is that the detection of symptoms under real field conditions is erratic and can vary significantly from year to year depending upon environmental conditions and other factors (47). Interestingly, there was a significant association between pathogen application on pruning wounds (line 1) and increased mortality compared to results in untreated control plants (line 10) [ 2 (1) ϭ 19.4, P Ͻ 0.001].…”

Real-Time PCR Detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

“…Our experience demonstrated that these fungi could be reisolated 1 cm over the inoculation point, and incipient wood symptoms could be observed. The effectiveness of the inoculation was evaluated using the conventional method of fungus isolation (22), consisting of cutting six wood chips (approximate diameter of 1 to 2 mm and approximate length of 0.5 to 1 cm) that were placed on malt extract agar (MEA) plates (Merck, Darmstadt, Germany) and incubated at 25°C in darkness until fungi grew to a size at which they could be isolated and placed on PDA plates. As de- scribed above, the PDA plates were incubated and the colonies that grew were morphologically and/or molecularly identified.…”

“…The occurrence of Phaeoacremonium spp. varies among grape-growing countries, with P. aleophilum the species most frequently found to affect grapevines worldwide (23,13,22). It was suggested that this pathogen disseminated from one host to another, such as kiwifruit and Prunus sp.…”

“…Petri disease and esca are grapevine diseases of worldwide importance. Outbreaks have been reported from France (20), California (31), Italy (27), Portugal (28), New Zealand (30), South Africa (19), Australia (11), and Spain (22).…”

“…These two species represent around 16 to 18% of the 30 species associated with grapevine decay in Castilla y León, Spain (22). Once the PCR protocols were established with purified DNA from isolated fungi, as is usually done, attempts were made to detect these fungi in spore suspensions without DNA purification.…”