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

Mosca S, Li Destri Nicosia MG, Cacciola SO, Schena L - PLoS ONE (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Agraria, Università Mediterranea, Reggio Calabria, Italy.

ABSTRACT
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.

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Genotype networks based on ITS sequences of Colletotrichum acutatum sensu lato (A), C. gloeosporioides s.l. (B) and C. boninense s.l. (C), detected in different olive tissues in 3 different phenological phases (June, October and December).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. Table 2). 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. Fig. 1) and named using the initials of the corresponding species as follows: C. godetiae (Glo), C. acutatum s.s. (Acu), C. gloeosporioides s.s. (Glo), C. kahawae (Kah), C. acutatum s.l. (Acusl), C. gloesporioides s.l. (Glosl) and C. boninense s.l. (Bonsl).
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pone-0114031-g002: Genotype networks based on ITS sequences of Colletotrichum acutatum sensu lato (A), C. gloeosporioides s.l. (B) and C. boninense s.l. (C), detected in different olive tissues in 3 different phenological phases (June, October and December).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. Table 2). 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. Fig. 1) and named using the initials of the corresponding species as follows: C. godetiae (Glo), C. acutatum s.s. (Acu), C. gloeosporioides s.s. (Glo), C. kahawae (Kah), C. acutatum s.l. (Acusl), C. gloesporioides s.l. (Glosl) and C. boninense s.l. (Bonsl).

Mentions: The cloning of 37 positive PCR fragments from different olive tissues and the subsequent sequencing of 20 clones per sample yielded 740 reliable DNA sequences. The analysis of the complete panel of sequences enabled the identification of 15 different genotypes represented by at least two different sequences (Fig. 1; Fig. 2). All detected genotypes belonged to species of the genus Colletotrichum. Seven out of 15 genotypes (God1, God7, Acu1, Acu2, Glo, Kah, and Bonsl) were identical to reference sequences while the remaining 8 genotypes showed one or few polymorphic bases compared to currently available GenBank deposited sequences. However, genotypes perfectly matching reference sequences were much more diffused, accounting for the great majority of positive samples (Fig. 2).


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

Mosca S, Li Destri Nicosia MG, Cacciola SO, Schena L - PLoS ONE (2014)

Genotype networks based on ITS sequences of Colletotrichum acutatum sensu lato (A), C. gloeosporioides s.l. (B) and C. boninense s.l. (C), detected in different olive tissues in 3 different phenological phases (June, October and December).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. Table 2). 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. Fig. 1) and named using the initials of the corresponding species as follows: C. godetiae (Glo), C. acutatum s.s. (Acu), C. gloeosporioides s.s. (Glo), C. kahawae (Kah), C. acutatum s.l. (Acusl), C. gloesporioides s.l. (Glosl) and C. boninense s.l. (Bonsl).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263604&req=5

pone-0114031-g002: Genotype networks based on ITS sequences of Colletotrichum acutatum sensu lato (A), C. gloeosporioides s.l. (B) and C. boninense s.l. (C), detected in different olive tissues in 3 different phenological phases (June, October and December).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. Table 2). 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. Fig. 1) and named using the initials of the corresponding species as follows: C. godetiae (Glo), C. acutatum s.s. (Acu), C. gloeosporioides s.s. (Glo), C. kahawae (Kah), C. acutatum s.l. (Acusl), C. gloesporioides s.l. (Glosl) and C. boninense s.l. (Bonsl).
Mentions: The cloning of 37 positive PCR fragments from different olive tissues and the subsequent sequencing of 20 clones per sample yielded 740 reliable DNA sequences. The analysis of the complete panel of sequences enabled the identification of 15 different genotypes represented by at least two different sequences (Fig. 1; Fig. 2). All detected genotypes belonged to species of the genus Colletotrichum. Seven out of 15 genotypes (God1, God7, Acu1, Acu2, Glo, Kah, and Bonsl) were identical to reference sequences while the remaining 8 genotypes showed one or few polymorphic bases compared to currently available GenBank deposited sequences. However, genotypes perfectly matching reference sequences were much more diffused, accounting for the great majority of positive samples (Fig. 2).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Agraria, Università Mediterranea, Reggio Calabria, Italy.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus