Genetic and Molecular Approach to Verticillium dahliae Infecting Sunflower

Abstract

Verticillium leaf mottle and wilt caused by the fungus Verticillium dahliae is a major disease of sunflower in Argentina and the USA. In the summer of 2013, an important outbreak of the disease occurred in one field in the Southwest of Spain. The determination of vegetative compatibility groups (VCGs) of V. dahliae is used in the genetic characterization of the fungus. Molecular markers have also been developed and applied for the analysis of V. dahliae, particularly for those isolates infecting artichoke, cotton and olive tree. The objective of this work was to determine the genetic and molecular features of V. dahliae of sunflower. Three V. dahliae isolates, one from Argentina collected in 2012 and two collected in Spain in 2013, were analysed. The VCGs were determined by complementation between nit mutants of the isolates from sunflower and the VCGs reference strains. Molecular characterization was conducted by polymerase chain reaction using primer pairs that are diagnostic of either V. dahliae species, defoliating or non-defoliating pathotypes, or VCGs. Complementation tests between nit mutants and reference strains clearly showed that the three isolates from sunflower belong to the VCG2B. The VCG2B has been identified in V. dahliae from crops as cotton, artichoke, eggplant, pepper and tomato among others. When molecularly analysed, the three V. dahliae isolates infecting sunflower had the same molecular pattern than the one found for non-defoliating isolates of V. dahliae pathogenic to artichoke or cotton. The results of this work show the closeness between non-defoliating isolates of V. dahliae infecting artichoke, cotton and sunflower and suggest that any of these three species can serve as carrier and source of inoculum for Verticillium outbreaks in them.

Genetics, Host Range, and Molecular and Pathogenic Characterization of Verticillium dahliae From Sunflower Reveal Two Differentiated Groups in Europe

“…The molecular characterization of all the isolates was performed using diagnostic primers of race 1 ( Usami et al, 2007 ; de Jonge et al, 2012 ) and race 2 ( Short et al, 2014 ), as well as with the diagnostic markers of D and ND pathotypes described for Vd infecting olive tree and artichoke ( Carder et al, 1994 ; Mercado-Blanco et al, 2001 , 2002 , 2003 ; Collado-Romero et al, 2009 ). Isolates of the D pathotype of Vd are pathogenic on sunflower among other crop species ( Jiménez-Díaz et al, 2017 ), but previous findings by our research group showed that those affecting sunflower are molecularly similar to ND isolates of the fungus that are pathogenic to artichoke or cotton ( García-Carneros et al, 2014 ). Besides, races of Vd from sunflower have been determined on the basis of the reaction of particular host genotypes (or differentials) carrying genes of resistance from different sources, but racial characterization by means of molecular markers for race has not been addressed so far.…”

“…The VCG of 38 monoconidial isolates, including those previously characterized by our research group ( García-Carneros et al, 2014 ), were determined by generation and characterization of nitrate non-utilizing ( nit ) mutants of each of them and determination of vegetative compatibility. Nit mutants were generated on Water Agar Chlorate medium as colonies presenting a faint growth on Czapek-Dox Agar (CDA) with no aerial mycelium ( Korolev and Katan, 1997 ) and phenotyped on CDA amended with hypoxanthine as described by Correll et al (1987) .…”

“…In contrast, these markers showed a high molecular diversity of ND pathotypes belonging to 2A, 2B, and 4B VCGs ( Pérez-Artés et al, 2000 ; Mercado-Blanco et al, 2001 , 2002 , 2003 ). Preliminary results from our research group showed that the molecular pattern of Vd isolates infecting sunflower in Argentina and Spain matched that of the ND pathotype of artichoke and/or cotton, pointing to the closeness between ND isolates affecting these three crops and, therefore, suggesting that any of them could serve as a carrier and source of inoculum for Verticillium outbreaks ( García-Carneros et al, 2014 ).…”

“…Isolates of Vd affecting sunflowers in Canada showed weak reactions with testers from VCG4A and 4B; one isolate was identified as VCG3 and another one was compatible with all VCG groups except VCG2A ( El-Bebany et al, 2013 ). In previous works by our research group, isolates from Argentina and Spain were adscribed to VCG2B ( García-Carneros et al, 2014 ). Since Vd reproduces asexually, isolates in the same VCG could be genetically distinct populations with a similarity in a number of physiological, ecological, pathogenic, and host-range traits ( Jiménez-Díaz et al, 2006 ; Collado-Romero et al, 2008 ; Korolev et al, 2009 ).…”

Evidence of soil‐located competition as the cause of the reduction of sunflower verticillium wilt by entomopathogenic fungi

“…All V . dahliae isolates used were previously characterized in a multidisciplinary study (Martin‐Sanz et al ., 2018b) and were selected on the basis of their different geographical origin and pathogenic race (Garcia‐Carneros et al ., 2014; Martin‐Sanz et al ., 2018b). C. helianthi isolates were selected from the plant pathogen culture collection in the Field Crop Disease Laboratory at the Institute for Sustainable Agriculture from CSIC (IAS‐CSIC) in Cordoba, Spain (Molinero‐Ruiz, 2019).…”