Teresa Nascimento

Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines

Black foot is an important disease of grapevines, which has in recent years been recorded with increased incidence and severity throughout the world, affecting grapevines both in nurseries and young vineyards. In the past the disease has been associated with infections by Ilyonectria macrodidyma, Ilyonectria liriodendri, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare. Based on published data, a high level of genetic diversity was detected among isolates of I. macrodidyma. To resolve this issue, we employed a multigene analysis strategy (based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers on both sides of the 5.8S nuclear ribosomal RNA gene) along with morphological characterisation to study a collection of 81 I. macrodidyma-like isolates from grapevine and other hosts. Morphological characters (particularly conidial size) and molecular data (highest resolution achieved with histone H3 nucleotide sequence) enabled the distinction of six monophyletic species within the I. macrodidyma complex, four of which (Ilyonectria alcacerensis, Ilyonectria estremocensis, Ilyonectria novozelandica, and Ilyonectria torresensis) are described here. This work forms part of an effort by the International Council on Grapevine Trunk Diseases to resolve the species associated with black foot disease, which we believe will clarify their taxonomy, and therefore help researchers to devise control strategies to reduce the devastating impact of this disease.

Transcriptome and metabolome reprogramming in Vitis vinifera cv. Trincadeira berries upon infection with Botrytis cinerea

Vitis vinifera berries are sensitive towards infection by the necrotrophic pathogen Botrytis cinerea, leading to important economic losses worldwide. The combined analysis of the transcriptome and metabolome associated with fungal infection has not been performed previously in grapes or in another fleshy fruit. In an attempt to identify the molecular and metabolic mechanisms associated with the infection, peppercorn-sized fruits were infected in the field. Green and veraison berries were collected following infection for microarray analysis complemented with metabolic profiling of primary and other soluble metabolites and of volatile emissions. The results provided evidence of a reprogramming of carbohydrate and lipid metabolisms towards increased synthesis of secondary metabolites involved in plant defence, such as trans-resveratrol and gallic acid. This response was already activated in infected green berries with the putative involvement of jasmonic acid, ethylene, polyamines, and auxins, whereas salicylic acid did not seem to be involved. Genes encoding WRKY transcription factors, pathogenesis-related proteins, glutathione S-transferase, stilbene synthase, and phenylalanine ammonia-lyase were upregulated in infected berries. However, salicylic acid signalling was activated in healthy ripening berries along with the expression of proteins of the NBS-LRR superfamily and protein kinases, suggesting that the pathogen is able to shut down defences existing in healthy ripening berries. Furthermore, this study provided metabolic biomarkers of infection such as azelaic acid, a substance known to prime plant defence responses, arabitol, ribitol, 4-amino butanoic acid, 1-O-methyl- glucopyranoside, and several fatty acids that alone or in combination can be used to monitor Botrytis infection early in the vineyard.

Black foot of grapevine: sensitivity of Cylindrocarpon destructans to fungicides

Black foot disease of grapevine is caused by Cylindrocarpon spp., with C. destructans being the main pathogen isolated from vine cuttings and young vineyards in Portugal. Few recommendations for black foot disease control are presently available, and they are not easy to implement within commercial nurseries. In this study, 14 fungicides were evaluated for their effect on the mycelial growth and conidium germination of four field isolates of C. destructans. Mycelial growth of the pathogen was inhibited by DMI fungicides, prochloraz (EC50 values <0.09 mg l-1) and to a lesser extent by difenoconazole (EC50 values <2.25 mg l-1), by the benzimidazole fungicide benomyl (EC50 values <0.35 mg l-1), and by the mixtures cyprodinil + fludioxonil and carbendazim + flusilazole, which gave EC50 values <0.75 mg l-1. Among these, only cyprodinil + fludioxonil (EC50 values <0.15 mg l-1), the strobilurin fungicides, azoxystrobin and trifloxystrobin (EC50 values <2.27 mg l-1) and the phenylsulphamide fungicide tolylfluanid (EC50 < 0.54 mg l-1) were effective in reducing conidium germination. Results from in vivo studies, carried out on potted grapevine plants (cultivar Castelao) showed that benomyl, tebuconazole and the mixtures carbendazim + flusilazole and cyprodinil + fludioxonil significantly (α=0.05) improved plant growth (plant height and number of roots) and decreased disease incidence compared with non-treated plants.