Evaluation of a Pomegranate Peel Extract as an Alternative Means to Control Olive Anthracnose

Olive anthracnose is caused by different species of Colletotrichum spp. and may be regarded as the most damaging disease of olive fruit worldwide, greatly affecting quality and quantity of the productions. A pomegranate peel extract (PGE) proved very effective in controlling the disease. The extract had a strong in vitro fungicidal activity against Colletotrichum acutatum sensu stricto, was very effective in both preventive and curative trials with artificially inoculated fruit, and induced resistance in treated olive tissues. In field trials, PGE was significantly more effective than copper, which is traditionally used to control the disease. The highest level of protection was achieved by applying the extract in the early ascending phase of the disease outbreaks because natural rots were completely inhibited with PGE at 12 g/liter and were reduced by 98.6 and by 93.0% on plants treated with PGE at 6 and 3 g/liter, respectively. Two treatments carried out 30 and 15 days before the expected epidemic outbreak reduced the incidence of the disease by 77.6, 57.0, and 51.8%, depending on the PGE concentration. The analysis of epiphytic populations showed a strong antimicrobial activity of PGE, which sharply reduced both fungal and bacterial populations. Because PGE was obtained from a natural matrix using safe chemicals and did not have any apparent phytotoxic effect on treated olive fruit, it may be regarded as a safe and effective natural antifungal preparation to control olive anthracnose and improve olive productions

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 and Application of a Quantitative PCR Detection Method to Quantify Venturia oleaginea in Asymptomatic Olive (Olea europaea) Leaves.

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important for the development of effective control strategies. Current knowledge is incomplete owing to a lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the internal transcribed spacer ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming the appropriateness of the target region for developing a quantitative PCR (qPCR) assay. Furthermore, high-throughput sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive, enabling the detection of approximately 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results and enables detection of V. oleaginea in all olive organs, including those in which OLS cannot be studied using previously available methods

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

Phylogenetic trees built using unique sequences representative of all detected genotypes (♦) together with sequences of reference isolates of <i>Colletotrichum acutatum sensu lato</i>[1], <i>C. gloeosporioides s.l.</i>[2] and <i>C. boninense s.l.</i>[3].

<p>Genotypes were identified as <i>C. godetiae</i> (A), <i>C. acutatum s.s.</i> (C), <i>C. gloeosporioides s.s.</i> (D) and <i>C. kahawae</i> (F). Three additional genotypes were associated with 2 (B), 6 (D) and 3 (F) species within <i>C. acutatum s.l.</i>, <i>C. gloeosporioides s.l</i> and <i>C. boninense s.l.</i>, respectively. Separate analyses were conducted for each species complex. Numbers on nodes represent the posterior probabilities for the maximum likelihood method.</p

Pomegranate Peel Extracts as Safe Natural Treatments to Control Plant Diseases and Increase the Shelf-Life and Safety of Fresh Fruits and Vegetables

Although the Green Revolution was a milestone in agriculture, it was accompanied by intensive use of synthetic pesticides, which has raised serious concerns due to their impact on human and environmental health. This is increasingly stimulating the search for safer and more eco-friendly alternative means to control plant diseases and prevent food spoilage. Among the proposed alternatives, pomegranate peel extracts (PPEs) are very promising because of their high efficacy. In the present review, we discuss the complex mechanisms of action that include direct antimicrobial activity and induction of resistance in treated plant tissues and highlight the importance of PPE composition in determining their activity. The broad spectrum of activity, wide range of application and high efficiency of PPEs against bacterial, fungal and viral plant pathogens suggest a potential market not only restricted to organic production but also integrated farming systems. Considering that PPEs are non-chemical by-products of the pomegranate industry, they are perceived as safe by the public and may be integrated in circular economy strategies. This will likely encourage agro-pharmaceutical industries to develop commercial formulations and speed up the costly process of registration