Molecular analysis of Colletotrichum species in the carposphere and phyllosphere of olive.

A metagenomic approach based on the use of genus specific primers was developed and utilized to characterize Colletotrichum species associated with the olive phyllosphere and carposphere. Selected markers enabled the specific amplification of almost the entire ITS1-5.8S-ITS2 region of the rDNA and its use as barcode gene. The analysis of different olive samples (green and senescent leaves, floral residues, symptomatic and asymptomatic fruits, and litter leaves and mummies) in three different phenological phases (June, October and December) enabled the detection of 12 genotypes associated with 4 phylotypes identified as C. godetiae, C. acutatum s.s., C. gloeosporioides s.s. and C. kahawae. Another three genotypes were not identified at the level of species but were associated with the species complexes of C. acutatum, C. gloeosporioides and C. boninense sensu lato. Colletotrichum godetiae and C. acutatum s.s. were by far the most abundant while C. gloeosporioides s.s. was detected in a limited number of samples whereas ther phylotypes were rarely found. The high incidence of C. acutatum s.s. represents a novelty for Italy and more generally for the Mediterranean basin since it had been previously reported only in Portugal. As regards to the phenological phase, Colletotrichum species were found in a few samples in June and were diffused on all assessed samples in December. According to data new infections on olive tissues mainly occur in the late fall. Furthermore, Colletotrichum species seem to have a saprophytic behavior on floral olive residues. The method developed in the present study proved to be valuable and its future application may contribute to the study of cycle and aetiology of diseases caused by Colletotrichum species in many different pathosystems

Use of quantitative PCR detection methods to study biocontrol agents and phytopathogenic fungi and oomycetes in environmental samples

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. © 2013 Blackwell Verlag GmbH

Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes

In recent years quantitative PCR (qPCR) detection methods have been widely utilised to detect phytopathogenic fungi and oomycetes and have greatly contributed to the advancement of knowledge in plant pathology. However, major drawbacks and common errors, most typical of earlier reports, still affect many methods currently available in the literature. Errors can be made throughout the entire process for the development of qPCR methods, at the level of selection of appropriate DNA extraction and purification protocols, identification of suitable target regions, choice of the chemistry, design and validation of specific primers and probes, analysis of sensitivity, choice of an absolute and/or relative quantification approach and analysis of the risk of detecting target DNA from dead sources. In the present review the above mentioned steps are analysed, highlighting their critical aspects and providing a practical guide for the users

List of species and isolates utilized to evaluate the specificity of <i>Colletotrichum</i>-genus-specific primers and corresponding positive (+) or negative (-) amplification results obtained in PCR reactions with pure culture DNA samples.

<p>List of species and isolates utilized to evaluate the specificity of <i>Colletotrichum</i>-genus-specific primers and corresponding positive (+) or negative (-) amplification results obtained in PCR reactions with pure culture DNA samples.</p

Genotype networks based on ITS sequences of <i>Colletotrichum acutatum sensu lato</i> (A), <i>C. gloeosporioides s.l.</i> (B) and <i>C. boninense s.l.</i> (C), detected in different olive tissues in 3 different phenological phases (June, October and December).

<p>According to the caption (bottom right of the figure) different colors were used to connect detected genotypes and analyzed olive samples. Empty white boxes in the caption indicate analyzed samples that did not produce any positive amplification, while white boxes containing “na” indicate non-analyzed samples. The letters “T1”, “T2” and “A1” inside the circles were used to indicate sampling fields where genotypes were detected (Cfr. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114031#pone-0114031-t002" target="_blank">Table 2</a>). The size of each circle represents the relative frequency of genotypes in terms of number of samples in which they were detected. Genotypes were identified according to their phylogenetic collocation (Cfr. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114031#pone-0114031-g002" target="_blank">Fig. 1</a>) and named using the initials of the corresponding species as follows: <i>C. godetiae</i> (Glo), <i>C. acutatum s.s.</i> (Acu), <i>C. gloeosporioides s.s.</i> (Glo), <i>C. kahawae</i> (Kah), <i>C. acutatum s.l.</i> (Acusl), <i>C. gloesporioides s.l.</i> (Glosl) and <i>C. boninense s.l.</i> (Bonsl).</p

List of <i>Colletotrichum</i> species and ITS genotypes identified in different olive tissues collected in three olive orchards on the Gioia Tauro plain (southern Italy).

<p>*Number of samples in which each genotype was detected</p><p>**Accession numbers</p><p>The number of samples and the orchards (Cfr. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114031#pone-0114031-t002" target="_blank">Table 2</a>) in which each genotype was detected is reported together with GenBank accession numbers for sequences. Genotypes were grouped according to their phylogenetic identification (Cfr. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114031#pone-0114031-g001" target="_blank">Fig. 1</a>).</p><p>List of <i>Colletotrichum</i> species and ITS genotypes identified in different olive tissues collected in three olive orchards on the Gioia Tauro plain (southern Italy).</p

Summary of results of field surveys conducted with different olive tissues collected in 3 phenological phases from 8 different plants located in three fields (T1, T2, A1).

<p>*GPS coordinates: T1 (38°22'53.0"N, 15°56'27.5"E), T2 (38°22'15.1"N 15°55'38.3"E) and A1 (38°24'44.6"N, 15°56'23.1"E). (nd)  =  not analyzed samples; (-) analyzed samples that did not produce positive amplifications.</p><p>Detected phylotypes were associated with <i>Colletotrichum godetiae</i> (Cgo), <i>C. acutatum sensu stricto</i> (Ca), <i>C. gloeosporioides s.s.</i> (Cgl), <i>C. kahawae</i> (Ck), and non well-defined species of <i>C. acutatum</i> s.l (Casl), <i>C. gloeosporioides s.l.</i> (Cgsl) and <i>C. boninense s.l.</i> (Cbsl). Numbers in brackets represent the percentage of sequences associated with different phylotypes in each cloned PCR fragment.</p><p>Summary of results of field surveys conducted with different olive tissues collected in 3 phenological phases from 8 different plants located in three fields (T1, T2, A1).</p

Qualitative and quantitative impacts of Bactrocera oleae on the fungal microbiota of ripe drupes

Trabajo presentado en el 15th Congress of the Mediterranean Phytopathological Union (Plant health sistaining Mediterranean ecosystems), celebrado en Córdoba (España) del 20 al 23 de junio de 2017.The olive fly, Bactrocera oleae, is a major key pest of olive drupes, greatly affecting quality and quantity of olive oil production. Fungus species associated with olive drupes can also have important impacts on olive production. However, little is currently known about the interaction between olive fly and fungi. Ripe olive drupes of three olive varieties, either with or without olive fly infestations, were collected in southern Italy.This research was supported by the Italian Ministry of Education, University and Research (MIUR) with the grant “Modelli sostenibili e nuove tecnologie per la valorizzazione delle olive e dell’olio extravergine di oliva prodotto in Calabria - PON Ricerca e competitività 2007–2013 (PON03PE_00090_02).Peer reviewe