Abstract

Melon necrotic spot virus (MNSV) is one of the major viruses affecting cucurbit crops worldwide. However, very little information on the epidemiology of the disease caused by this virus is available, especially regarding seed transmission, seed disinfection, the presence and distribution of the virus and its fungal vector Olpidium bornovanus in the world, and the genetic diversity of both entities. Therefore, discovering the epidemiological aspects and variability in populations of these infectious agents is a key factor to manage the disease. Similarly, some aspects of the diagnostic methods of MNSV and O. bornovanus and of other Olpidium species, including O. brassicae and O. virulentus, may clearly improve. The aforementioned species transmit different viruses in various horticultural crops, a scenario which needs studying to better understand the interactions between viruses and fungal vectors, and to determine the host range of these entities. Seed transmission of MNSV was assessed in seedlings grown from commercial melon seeds. Seedlings at the cotyledon and adult stages were analyzed by DAS-ELISA and RT-PCR to detect the virus. No seedling group was positive for MNSV by DAS-ELISA. However, RT-PCR revealed that at least 7.0-8.0% of seedlings were infected, corresponding to seed-to-seedling transmission rates of 11.3-14.8%. Different seed-disinfection treatments were evaluated to prevent seed transmission of MNSV. The results suggest that a treatment of 144 h at 70°C could eradicate the virus in melon seeds without affecting germination. Given the importance of acquiring O. bornovanus in seed transmission of MNSV, a multiplex PCR method to detect this fungus was established. This method also detected with high sensitivity and differentiated other Olpidium species that infect the roots of different vegetable crops, O. brassicae and O. virulentus, to distinguish these species, which is not possible by a simple morphological observation of resting spores. This method also detected O. bornovanus in water samples. Additionally, the specificity and sensitivity of the method were evaluated, and the method was validated with samples of 9 different crops from 11 American, European and African countries. The geographic incidence and genetic diversity of MNSV and Olpidium in different cucurbits species from several Latin American (Guatemala, Honduras, Mexico and Panama) and European (Italy and Spain) countries have been studied. Twenty-nine MNSV isolates of these countries were selected to study the genetic diversity of all the proteins that compose the genome of this virus (p29, p89, p7A, p7b, p42) and to determine the phylogenetic relationships of the MNSV population. This population varies in geographical terms. The genetic diversity observed in the entire p7A and p7b proteins suggests that these coding regions are under strong selection pressure. We also analyzed the ITS regions of ribosomal DNA of 40 Olpidium isolates associated with each geographic region of this study. These isolates were related to the host from which they were obtained. Finally, we conducted two surveys (2006 and 2008) to determine the presence and distribution of MNSV and O. bornovanus in the main cucurbit-growing regions of Panama where these entities were identified in 12% and 45% of the samples analyzed, respectively, but only in melon. O. bornovanus was observed in all the regions where MNSV was detected. Neither MNSV nor its fungus vector has been cited in Panama before this thesis. Likewise, the MNSV and O. bornovanus isolates obtained in this study were molecularly analyzed to estimate their variability. Additionally, other viruses that infect cucurbits were analysed in 2008, including CMV, SqMV, PRSV, WMV-2 and ZYMV, because symptoms similar to those producing these viruses were reported that year. The presence and distribution of each virus varied according to the region and cucurbit species analyzed.