Assessment of Maternal Effects and Genetic Variability in Resistance to Verticillium dahliae in Olive Progenies.

The use of genetic resistance is likely the most efficient, economically convenient and environmentally friendly control method for plant diseases, as well as a fundamental piece in an integrated management strategy. This is particularly important for woody crops affected by diseases in which mainly horizontal resistance mechanisms are operative, such as Verticillium wilt, caused by Verticillium dahliae. In this study, we analyzed the variability in resistance to Verticillium wilt of olive trees in progenies from five crosses: 'Picual' × 'Frantoio', 'Arbosana' × 'Koroneiki', 'Sikitita' × 'Arbosana', 'Arbosana' × 'Frantoio' and 'Arbosana' × 'Arbequina' and their respective reciprocal crosses. Additionally, seedlings of 'Picual' and 'Frantoio' in open pollination were used as controls. In October 2016 and 2018, the fruits were harvested, and seeds germinated. Six-week-old seedlings were inoculated by dipping their bare roots in a conidial suspension of V. dahliae, and disease progress in terms of symptom severity and mortality was evaluated weekly. Additionally, seedling growth was evaluated every two weeks. At the end of the experiment, no significant differences were found for any of the assessed parameters when reciprocal crosses were compared. These results suggest that there is no maternal or paternal effect in regard to the heritability of resistance. In addition, this study identifies the best crosses for obtaining the highest number of resistant genotypes, highlighting the importance of the selection of specific cultivars to optimize the breeding process.

Lack of Evidence for Transmission of Verticillium dahliae by the Olive Bark Beetle Phloeotribus scarabaeoides in Olive Trees.

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is one of the most important diseases affecting olive crops in the Mediterranean area. With the aim to evaluate the role of Phloeotribus scarabaeoides (Bernard) (olive bark beetle) as a dispersal vector of V. dahliae, several experiments were conducted in semi-controlled conditions from May 2009 to April 2012. Groups of olive trees (2.5-year-old) certified free from V. dahliae were covered by a mosquito net and exposed to adults of P. scarabaeoides by three different ways: (1) branches or trunks collected in several olive orchards from trees severely affected by Verticillium wilt and showing apparent entry holes (mating galleries) of P. scarabaeoides; (2) adults of olive bark beetle extracted from damaged branches collected in the field; (3) adults from damaged branches that were superficially inoculated with V. dahliae. The fungus V. dahliae was not detected either by microbiological and molecular techniques from shoots of olive trees with galleries of the insect or from any of the tissues of the collected beetle adults from the galleries. Additionally, Verticillium wilt disease symptoms were not observed in olive trees exposed to the olive bark beetles. Moreover, the pathogen was never detected from any beetle adults that were recovered from the mating galleries of branches or trunks collected in several olive orchards from trees severely affected by Verticillium wilt. We conclude that P. scarabaeoides is not a vector of V. dahliae under the investigated experimental conditions.

Effect of Cultivar Resistance and Soil Management on Spatial-Temporal Development of Verticillium Wilt of Olive: A Long-Term Study.

Verticillium wilt, caused by Verticillium dahliae, challenges olive cultivation and an Integrated Disease Management (IDM) approach is the best-suited tool to combat it. Since 1998, an IDM strategy in an orchard (called Granon, Spain) of the susceptible cv. Picual was conducted by increasing planting density with moderately resistant cv. Frantoio, chemical weed control, and replanting of dead olives with cv. Frantoio following soil solarization. The Verticillium wilt epidemic in Granon orchard was compared to the epidemic in a non-IDM orchard (called Ancla, Spain) with plowed soil and dead Picual olives replanted with the same cultivar. Field evaluations (2012-2013) showed an incidence and severity of the disease as Picual-Ancla > Picual-Granon > Frantoio-Granon. The spatiotemporal dynamics of the Verticillium epidemics from 1998 to 2010 were monitored with digital images using SIG. The annual tree mortalities were 5.6% for Picual olives in Ancla orchard, and 3.1 and 0.7% for Picual and Frantoio olives in Granon orchard, respectively. There was a negative relationship between the mortality of olive trees (%) by the pathogen and the height (m) above sea level. The annual mortality of cv. Picual olives was positively correlated with spring rainfalls. The Index of Dispersion and beta-binomial distribution showed aggregation of Verticillium-dead olives. In conclusion, this IDM strategy considerably reduced the disease in comparison with traditional agronomic practices.

Starch Hydrolysis and Vessel Occlusion Related to Wilt Symptoms in Olive Stems of Susceptible Cultivars Infected by Verticillium dahliae.

This study investigated starch content, amount of pathogen DNA and density of occluded vessels in healthy and Verticillium dahliae infected olive shoots and stems. Starch hydrolysis is considered a mechanism to refill xylem vessels that suffered cavitation by either, drought conditions or pathogen infections. The main objective of this work was to evaluate this mechanism in olive plants subjected to V. dahliae infection or to drought conditions, in order to know the importance of cavitation in the development of wilting symptoms. In initial experiments starch content in the shoots was studied in trees of cultivars differing in the level of resistance growing in fields naturally infested with V. dahliae. The starch content, esteemed by microscopic observation of stem transversal sections stained with lugol, decreased with the level of symptom severity. Results were confirmed in a new experiment developed with young plants of cultivars 'Picual' (highly susceptible), 'Arbequina' (moderately susceptible) and 'Frantoio' (resistant), growing in pots under greenhouse conditions, either inoculated or not with V. dahliae. In this experiment, the pathogen DNA content, quantified by real-time PCR, and the density of occluded vessels, recorded by microscopic observations of transversal sections stained with toluidine blue, were related to the symptoms severity caused by the pathogen. Finally, a drought experiment was established with young plants of the cultivar 'Picual' grown in pots under greenhouse conditions in order to compare the effects caused by water deficit with those caused by the pathogen infection. In both cases, results show that starch hydrolysis occurred, what indirectly evidence the importance of xylem cavitation in the development of the symptoms caused by V. dahliae but in the water stressed plants no vessel occlusion was detected.

Characterization of a novel single-stranded RNA mycovirus related to invertebrate viruses from the plant pathogen Verticillium dahliae.

Fungal viruses, also known as mycoviruses, are widespread in all major groups of fungi. Mycoviruses from plant pathogens can reduce the virulence of their host fungus and have therefore potential as biological control agents. This has spurred the identification of novel mycoviruses in plant pathogens, research which is greatly contributing to our understanding of these organisms. In this work, we report the characterization of a novel monopartite mycovirus from Verticillium dahliae, the main causal agent of Verticillium wilt. This novel mycovirus, which we termed Verticillium dahliae RNA virus 1 (VdRV1), was identified in three different isolates of V. dahliae collected in olive growing areas of the Guadalquivir valley, southern Spain. We determined that the VdRV1 genome is a positive (+) single-stranded (ss) RNA, 2631 nucleotides in length, containing two open reading frames. VdRV1 showed few similarities with known mycoviruses, only with a group of unassigned (+) ssRNA mycoviruses which are related to plant viruses classified within the family Tombusviridae. However, phylogenetic analysis revealed that VdRV1 and the unassigned (+) ssRNA mycoviruses have a closer relationship with recently reported invertebrate viruses. This result indicates that as more viral sequences become available, the relationships of mycoviruses with viruses from other hosts should be reexamined. Additionally, the work supports the hypothesis of a heterogeneous origin for mycoviruses.