A Bipartite Geminivirus with a Highly Divergent Genomic Organization Identified in Olive Trees May Represent a Novel Evolutionary Direction in the Family Geminiviridae.

Olea europaea Geminivirus (OEGV) was recently identified in olive in Italy through HTS. In this work, we used HTS to show the presence of an OEGV isolate in Portuguese olive trees and suggest the evolution direction of OEGV. The bipartite genome (DNA-A and DNA-B) of the OEGV-PT is similar to Old World begomoviruses in length, but it lacks a pre-coat protein (AV2), which is a typical feature of New World begomoviruses (NW). DNA-A genome organization is closer to NW, containing four ORFs; three in complementary-sense AC1/Rep, AC2/TrAP, AC3/REn and one in virion-sense AV1/CP, but no AC4, typical of begomoviruses. DNA-B comprises two ORFs; MP in virion sense with higher similarity to the tyrosine phosphorylation site of NW, but in opposite sense to begomoviruses; BC1, with no known conserved domains in the complementary sense and no NSP typical of bipartite begomoviruses. Our results show that OEGV presents the longest common region among the begomoviruses, and the TATA box and four replication-associated iterons in a completely new arrangement. We propose two new putative conserved regions for the geminiviruses CP. Lastly, we highlight unique features that may represent a new evolutionary direction for geminiviruses and suggest that OEGV-PT evolution may have occurred from an ancient OW monopartite Begomovirus that lost V2 and C4, gaining functions on cell-to-cell movement by acquiring a DNA-B component.

Fungal Communities Associated with Peacock and Cercospora Leaf Spots in Olive.

Venturia oleaginea and Pseudocercospora cladosporioides are two of the most important olive fungal pathogens causing leaf spots: peacock spot, and cercosporiosis, respectively. In the present study, fungal communities associated with the presence of these pathogens were investigated. Overall, 300 symptomatic and asymptomatic trees from different cultivars were sampled from Alentejo, Portugal. A total of 788 fungal isolates were obtained and classified into 21 OTUs; Ascomycota was clearly the predominant phylum (96.6%). Trees from cultivar 'Galega vulgar' showed a significant higher fungal richness when compared to 'Cobrançosa', which in turn showed significant higher values than 'Picual'. Concerning plant health status, symptomatic plants showed significant higher fungal richness, mainly due to the high number of isolates of the pathogens V. oleaginea and P. cladosporioides. In terms of fungal diversity, there were two major groups: ca. 90% of the isolates found in symptomatic plants belonged to V. oleaginea, P. cladosporioides, Chalara sp., and Foliophoma sp. while ca. 90% of the isolates found in asymptomatic plants, belonged to Alternaria sp. and Epicoccum sp. This study highlights the existence of different fungal communities in olive trees, including potential antagonistic organisms that can have a significant impact on diseases and consequently on olive production.

Antagonistic activity of fungi of Olea europaea L. against Colletotrichum acutatum.

Fungi naturally present in olive trees were identified and tested for their antagonistic potential against Colletotrichum acutatum. A total of 14 isolates were identified, 12 belonged to genera Alternaria, Epicoccum, Fusarium, Aspergillus, Anthrinium, Chaetomium, Diaporthe, Nigrospora, one to family Xylariaceae and one was unclassified. All fungal isolates showed some inhibitory action over the growth of C. acutatum during dual culture growth, however, when agar-diffusible tests were performed only five fungal isolates caused C. acutatum growth inhibition: Alternaria sp. isolate 2 (26.8%), the fungus from Xylariaceae family (14.3%), Alternaria sp. isolate 1 (10.7%); Diaporthe sp. (10.7%), Nigrospora oryzae (3.5%). Volatile substances produced by these isolates were identified through gas-chromatography techniques, as phenylethyl alcohol, 4-methylquinazoline, benzothiazole, benzyl alcohol, lilial, galaxolide, among others. These inhibitory volatiles could play a significant role in reduction of C. acutatum expansion in olive and their study as potential biocontrol agents should be further explored.

Detection of sweet potato virus C, sweet potato virus 2 and sweet potato feathery mottle virus in Portugal.

Field sweet potato plants showing virus-like symptoms, as stunting, leaf distortion, mosaic and chlorosis, were collected in southwest Portugal and tested for the presence of four potyviruses, sweet potato virus C (SPVC), sweet potato virus 2 (SPV2), sweet potato feathery mottle virus (SPFMV), sweet potato virus G (SPVG), and the crinivirus sweet potato chlorotic stunt virus (SPCSV). DsRNA fractions were extracted from symptomatic leaves and used as templates in single and multiplex RT-PCR assays using previously described specific primers for each analyzed virus. The amplified reaction products for SPVC, SPV2 and SPFMV were of expected size, and direct sequencing of PCR products revealed that they correspond to the coat protein gene (CP) and showed 98%, 99% and 99% identity, respectively, to those viruses. Comparison of the CP genomic and amino acid sequences of the Portuguese viral isolates recovered here with those of ten other sequences of isolates obtained in different countries retrieved from the GenBank showed very few differences. The application of the RT-PCR assays revealed for the first time the presence of SPVC and SPFMV in the sweet potato crop in Portugal, the absence of SPVG and SPCSV in tested plants, as well as the occurrence of triple virus infections under field conditions.

Genetic diversity of the coat protein of Olive mild mosaic virus (OMMV) and Tobacco necrosis virus D (TNV-D) isolates and its structural implications.

The genetic variability among 13 isolates of Olive mild mosaic virus (OMMV) and of 11 isolates of Tobacco necrosis virus D (TNV-D) recovered from Olea europaea L. samples from various sites in Portugal, was assessed through the analysis of the coat protein (CP) gene sequences. This gene was amplified through reverse transcriptase polymerase chain reaction (RT-PCR), cloned, and 5 clone sequences of each virus isolate, were analysed and compared, including sequences from OMMV and TNV-D isolates originally recovered from different hosts and countries and available in the GenBank, totalling 131 sequences. The encoded CP sequences consisted of 269 amino acids (aa) in OMMV and 268 in TNV-D. Comparison of the CP genomic and amino acid sequences of the isolates showed a very low variability among OMMV isolates, 0.005 and 0.007, respectively, as well as among TNV-D isolates, 0.006 and 0.008. The maximum nucleotide distances of OMMV and TNV-D sequences within isolates were also low, 0.013 and 0.031, respectively, and close to that found between isolates, 0.018 and 0.034, respectively. In some cases, less variability was found in clone sequences between isolates than in clone sequences within isolates, as also shown through phylogenetic analysis. CP aa sequence identities among OMMV and TNV-D isolates ranged from 84.3% to 85.8%. Comparison between the CP genomic sequences of the two viruses, showed a relatively low variability, 0.199, and a maximum nucleotide distance between isolates of 0.411. Analysis of comparative models of OMMV and TNV-D CPs, showed that naturally occurring substitutions in their respective sequences do not seem to cause significant alterations in the virion structure. This is consistent with a high selective pressure to preserve the structure of viral capsid proteins.