The taxonomic status of Colletotrichum gloeosporioides sensu lato (s.l.) associated with olive anthracnose is still undetermined and the pathogenic ability of this species complex is controversial. In the present study, isolates obtained from olive and provisionally identified as C. gloeosporioides s.l. on the basis of morphological and cultural features were reclassified using ITS and TUB2 as DNA barcode markers and referred to seven distinct species, recently separated within C. gloeosporioides (C. aenigma, C. gloeosporioides sensu stricto (s.s.), C. kahawae, C. queenslandicum, C. siamense and C. theobromicola) and C. boninense (C. karstii) species complexes. Furthermore, isolates of C. kahawae were ascribed to the subspecies ciggaro by analysing the GS gene. A single isolate, not in either of these two species complexes, was not identified at the species level. In pathogenicity tests on detached olive drupes some of these species, including C. aenigma, C. kahawae subsp. ciggaro, C. queenslandicum, C. siamense and C. karstii, were shown to be weakly pathogenic. Moreover, they were found very sporadically on olive. In contrast, some isolates of C. gloeosporioides s.s. and isolates of C. theobromicola proved to be virulent on both green and ripening olives. This study gives a better insight into both the aetiology and the epidemiology of olive anthracnose and might have implications for biosecurity and quarantine because C. theobromicola has never been reported in major European olive-producing countries.

Quantitative polymerase chain reaction (qPCR) is a versatile technique for the accurate, sensitive, reliable and high‐throughput detection and quantification of target DNA in various environmental samples, and in recent years, it has greatly contributed to the advancement of knowledge in the plant pathology field. Indeed, this technique is ideal to evaluate inoculum threshold levels and to study the epidemiology, biology and ecology of phytopathogenic fungi and oomycetes, thus opening up new research opportunities to investigate host–pathogen interactions and to address tasks related to quarantine, eradication and biosecurity. Moreover, it can be a useful tool in breeding programs. The present review analyses the most relevant applications of qPCR for the detection and quantification of filamentous fungi and oomycetes within host tissues and in soil, air and water, along with brief paragraphs focusing on new application fields such as the detection and quantification of mycotoxigenic fungi and biocontrol agents. The high potentiality of qPCR for present and future applications is highlighted together with a critical analysis of major drawbacks that need to be corrected to definitively confirm it as a preferential routine quantitative detection method.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.transfection | plant pathogen adaptation | host species jump

: The aim of this study was to investigate the occurrence, diversity, and distribution of Phytophthora species in Protected Natural Areas (PNAs), including forest stands, rivers, and riparian ecosystems, in Sicily (Italy), and assessing correlations with natural vegetation and host plants. Fifteen forest stands and 14 rivers in 10 Sicilian PNAs were studied. Phytophthora isolations from soil and stream water were performed using leaf baitings. Isolates were identified using both morphological characters and sequence analysis of the internal transcribed spacer (ITS) region. A rich community of 20 Phytophthora species from eight phylogenetic clades, including three new Phytophthora taxa, was recovered (17 species in rhizosphere soil from forest stands and 12 species in rivers). New knowledge about the distribution, host associations, and ecology of several Phytophthora species was provided.

Two distinct Phytophthora taxa were found to be associated with brown rot of pomelo (Citrus grandis), a new disease of this ancestral Citrus species, in the Vinh Long province, Mekong River Delta area, southern Vietnam. On the basis of morphological characters and using the ITS1-5.8S-ITS2 region of the rDNA and the cytochrome oxidase subunit 1 (COI) as barcode genes, one of the two taxa was provisionally named as Phytophthora sp. prodigiosa, being closely related to but distinct from P. insolita, a species in Phytophthora Clade 9, while the other one, was closely related to but distinct from the Clade 2 species P. meadii and was informally designated as Phytophthora sp. mekongensis. Isolates of P. sp. prodigiosa and P. sp. mekongensis were also obtained from necrotic fibrous roots of Volkamer lemon (C. volkameriana) rootstocks grafted with ‘King’ mandarin (Citrus nobilis) and from trees of pomelo, respectively, in other provinces of the Mekong River Delta, indicating a widespread occurrence of both Phytophthora species in this citrus-growing area. Koch’s postulates were fulfilled via pathogenicity tests on fruits of various Citrus species, including pomelo, grapefruit (Citrus x paradisi), sweet orange (Citrus x sinensis) and bergamot (Citrus x bergamia) as well as on the rootstock of 2-year-old trees of pomelo and sweet orange on ‘Carrizo’ citrange (C. sinensis ‘Washington Navel’ x Poncirus trifoliata). This is the first report of a Phytophthora species from Clade 2 other than P. citricola and P. citrophthora as causal agent of fruit brown rot of Citrus worldwide and the first report of P. insolita complex in Vietnam. Results indicate that likely Vietnam is still an unexplored reservoir of Phytophthora diversity.