Cecília Rego

Involvement of "Phaeoacremonium" spp. and "Cylindrocarpon destructans" with Grapevine Decline in Portugal

In an attempt to determine the aetiology of young vine decline in Portugal a study was carried out in rootstock nurseries and in young vineyards during the last few years. Rootstock nurseries located in the most important production areas of Portugal (Ribatejo-Oeste and Beira Litoral) were surveyed. The fungi most frequently isolated from discoloured wood at the base of the stem were Cylindrocarpon destructans and Phaeoacremonium spp., although Acremonium sp., Phomopsis sp., Fusarium spp., Fusicoccum sp., Gliocladium sp., Phoma sp. and Sphaeropsis sp. were also present. Young vines (2- to 8-year-old) showing decline symptoms were collected from various vinegrowing regions of Portugal. Disease incidence was variable but decline symptoms were present in several wine and table grapevine cultivars grafted onto different rootstocks (99R, 110R, 1103P, 101-14, 140Ru, 5BB and 161-49). Isolations from symptomatic internal tissues revealed that also C. destructans and P. chlamydosporum were the predominant fungi isolated from the basal end of the rootstocks and grafting tissues. In the pathogenicity tests carried out with C. destructans isolates the pathogen was reisolated from inoculated plants showing black-foot symptoms but never from the controls. Our results point out that both C. destructans and P. chlamydosporum might be involved with young grapevine decline in Portugal.

Cylindrocarpon root rot: multi-gene analysis reveals novel species within the Ilyonectria radicicola species complex

Ilyonectria radicicola and its Cylindrocarpon-like anamorph represent a species complex that is commonly associated with root rot disease symptoms on a range of hosts. During the course of this study, several species could be distinguished from I. radicicola sensu stricto based on morphological and culture characteristics. DNA sequence analysis of the partial β-tubulin, histone H3, translation elongation factor 1-α and nuclear ribosomal RNA-Internal Transcribed Spacer (nrRNA-ITS) genes were employed to provide further support for the morphological species resolved among 68 isolates associated with root rot disease symptoms. Of the various loci screened, nrRNA-ITS sequences were the least informative, while histone H3 sequences were the most informative, resolving the same number of species as the combined dataset across the four genes. Within the Ilyonectria radicicola species complex, 12 new taxa are delineated occurring on a diverse range of hosts, the most common being Cyclamen, Lilium, Panax, Pseudotsuga, Quercus and Vitis.

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.

Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine.

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, (-)-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) (-)-4-chloro-terremutin hydrate (4) and(+)-4-hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-(-)-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)(-)-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with (-)-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxins.

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.

Draft Genome Sequence of Diplodia seriata F98.1, a Fungal Species Involved in Grapevine Trunk Diseases

ABSTRACT The ascomycete Diplodia seriata is a causal agent of grapevine trunk diseases. Here, we present the draft genome sequence of D. seriata isolate F98.1 (37.27 Mb, 512 contigs, 112 scaffolds, and 8,087 predicted protein-coding genes).