Use of beneficial bacteria and their secondary metabolites to control grapevine pathogen diseases

Grapevine is one of the most important economic crops yielding berries, wine products as well as derivates. However, due to the large array of pathogens inducing diseases on this plant, considerable amounts of pesticides—with possible negative impact on the environment and health—have been used and are currently used in viticulture. To avoid negative impacts of such products and to ensure product quality, a substantial fraction of pesticides needs to be replaced in the near future. One solution can be related to the use of beneficial bacteria inhabiting the rhizo- and/or the endosphere of plants. These biocontrol bacteria and their secondary metabolites can reduce directly or indirectly pathogen diseases by affecting pathogen performance by antibiosis, competition for niches and nutrients, interference with pathogen signaling or by stimulation of host plant defenses. Due to the large demand for biocontrol of grapevine diseases, such biopesticides, their modes of actions and putative consequences of their uses need to be described. Moreover, the current knowledge on new strains from the rhizo- and endosphere and their metabolites that can be used on grapevine plants to counteract pathogen attack needs to be discussed. This is in particular with regard to the control of root rot, grey mould, trunk diseases, powdery and downy mildews, pierce’s disease, grapevine yellows as well as crown gall. Future prospects on specific beneficial microbes and their secondary metabolites that can be used as elicitors of plant defenses and/or as biocontrol agents with potential use in a more sustainable viticulture will be further discussed

Fungal endophytes from arid areas of Andalusia: high potential sources for antifungal and antitumoral agents

Native plant communities from arid areas present distinctive characteristics to survive in extreme conditions. The large number of poorly studied endemic plants represents a unique potential source for the discovery of novel fungal symbionts as well as host-specific endophytes not yet described. The addition of adsorptive polymeric resins in fungal fermentations has been seen to promote the production of new secondary metabolites and is a tool used consistently to generate new compounds with potential biological activities. A total of 349 fungal strains isolated from 63 selected plant species from arid ecosystems located in the southeast of the Iberian Peninsula, were characterized morphologically as well as based on their ITS/28S ribosomal gene sequences. The fungal community isolated was distributed among 19 orders including Basidiomycetes and Ascomycetes, being Pleosporales the most abundant order. In total, 107 different genera were identified being Neocamarosporium the genus most frequently isolated from these plants, followed by Preussia and Alternaria. Strains were grown in four different media in presence and absence of selected resins to promote chemical diversity generation of new secondary metabolites. Fermentation extracts were evaluated, looking for new antifungal activities against plant and human fungal pathogens, as well as, cytotoxic activities against the human liver cancer cell line HepG2. From the 349 isolates tested, 126 (36%) exhibited significant bioactivities including 58 strains with exclusive antifungal properties and 33 strains with exclusive activity against the HepG2 hepatocellular carcinoma cell line. After LCMS analysis, 68 known bioactive secondary metabolites could be identified as produced by 96 strains, and 12 likely unknown compounds were found in a subset of 14 fungal endophytes. The chemical profiles of the differential expression of induced activities were compared. As proof of concept, ten active secondary metabolites only produced in the presence of resins were purified and identified. The structures of three of these compounds were new and herein are elucidated.This work was supported by Fundación MEDINA and the Andalusian Government grant RNM-7987 ‘Sustainable use of plants and their fungal parasites from arid regions of Andalucía for new molecules useful for antifungals and neuroprotectors’

Taxonomy and multi-locus phylogeny of cylindrocarpon-like species associated with diseased roots of grapevine and other fruit and nut crops in California

Black foot disease is a common and destructive root disease of grapevine caused by a multitude of cylindrocarpon-like fungi in many viticultural areas of the world. This study identified 12 cylindrocarpon-like fungal species across five genera associated with black foot disease of grapevine and other diverse root diseases of fruit and nut crops in the Central Valley Region of California. Morphological observations paired with multi-locus sequence typing of four loci, internal transcribed spacer region of nuclear rDNA ITS1-5.8S-ITS2 (ITS), beta-tubulin (TUB2), translation elongation factor 1-alpha (TEF1), and histone (HIS), revealed 10 previously described species; Campylocarpon fasciculare, Dactylonectria alcacerensis, D. ecuadoriensis, D. macrodidyma, D. novozelandica, D. torresensis, D. valentina, Ilyonectria capensis, I. liriodendri, I. robusta, and two new species, Neonectria californica sp. nov., and Thelonectria aurea sp. nov. Phylogenetic analyses of the ITS+TUB2+TEF1 combined dataset, a commonly employed dataset used to identify filamentous ascomycete fungi, was unable to assign some species, with significant support, in the genus Dactylonectria, while all other species in other genera were confidently identified. The HIS marker was essential either singly or in conjunction with the aforementioned genes for accurate identification of most Dactylonectria species. Results from isolations of diseased plant tissues revealed potential new host associations for almost all fungi recovered in this study. This work is the basis for future studies on the epidemiology and biology of these important and destructive plant pathogens

Taxonomic revision and multi-locus phylogeny of the North American clade of Ceratocystis

The North American clade (NAC) of Ceratocystis includes pathogenic species that infect a wide range of woody hosts. Previous phylogenetic analyses have suggested that this clade includes cryptic species and a paraphyletic C. variospora. In this study, we used morphological data and phylogenetic analyses to characterize NAC taxa, including Ceratocystis isolates causing a serious disease of almond trees in California. Phylogenetic analyses based on six gene regions supported two new species of Ceratocystis. Ceratocystis destructans is introduced as the species causing severe damage to almond trees in California, and it has also been isolated from wounds on Populus and Quercus in Iowa. It is morphologically similar to C. tiliae, a pathogen on Tilia and the most recently characterized species in the NAC. Ceratocystis betulina collected from Betula platyphylla in Japan is also newly described and is the sister taxon to C. variospora. Our six-locus phylogenetic analyses and morphological characterization resolved several cryptic species in the NAC

Morphological and molecular identification of Eutypa lata on grapevine in Serbia

During a survey from 2004 to 2016, symptoms of grapevine dieback were observed in six vineyards in Serbia. Symptoms initially appeared as small, chlorotic, and necrotic spots along the rim of the leaves, deformation of leafs, and the appearance of shortened shoots, often with the so-called zigzag internodes. Over time, partial or complete dying of the vines developed. Symptomatic samples were collected and submitted to laboratory analysis. Based on the morphological characterization, the isolated fungus was initially identified as belonging to Eutypa species. Pathogenicity tests showed that 47 selected isolates caused tissue necrosis around the site of inoculation, chlorosis, and deformation of the leaves, along with the appearance of tiny, necrotic spots on the periphery of the leaf that fall off over time. Inoculated cuttings were stunted, dwarf-like with zigzag internodes. Molecular identification was done with sequence and phylogenetic analysis of ITS, TUB, and RPB2 genomic regions. Based on the phylogenetic analysis, all isolated fungi were determined as Eutypa lata

Efficacy of fungicides on mycelial growth of diatrypaceous fungi associated with grapevine trunk disease

Several species of Diatrypaceae have been recently isolated from the wood of cankered grapevines in several regions of the world and shown to be pathogenic with varying degrees of virulence when inoculated in stems of potted vines. Existing management strategies have focussed on the evelopment of procedures and products to prevent or reduce Eutypa lata infection. The best method to prevent infection is by applying fungicides to wounds, but there are few registered chemicals for any of the diatrypaceous fungi in Australia. Six selected fungicides were evaluated in vitro for their efficacy in reducing mycelial growth of Eutypa lata, Cryptovalsa ampelina, Diatrypella vulgaris, Eutypa leptoplaca, Eutypella citricola and Eutypella microtheca. Carbendazim, fluazinam, tebuconazole, and prothioconazole + tebuconazole were effective at inhibiting mycelial growth of all Diatrypaceae spp. tested. Pyraclostrobin reduced colony diameter of most of the fungal species by 50% or more. Pyrimethanil was ineffective at reducing mycelial growth of these pathogens. Five of the six fungicides evaluated in this study have shown efficacy in vitro against Diatrypaceae spp. and require further evaluation in the field. This study represents the first approach for fungicide evaluation against mycelial growth of diatrypaceous fungi, other than Ea. lata. It contributes to the development of integrated management strategies for grapevine trunk diseases. © 2011 Australasian Plant Pathology Society Inc.Peer Reviewe