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Summary The parasitic weed Orobanche cumana (sunflower broomrape) constrains sunflower production in eastern and southern Europe and in the Middle East. Although genetic resistance is the most effective control method, new parasite races evolve overcoming sunflower resistance. In this work, highly virulent populations of O. cumana were analysed for pathogenicity and genetic diversity. The virulence of 11 populations from Hungary, Romania, Spain and Turkey was assessed and compared after infection of sunflower inbred lines to differentiate races of the parasite under glasshouse conditions. Molecular diversity among and within 27 parasite populations was studied by RAPD‐PCR, UPGMA and amova analyses. Highly virulent race F was identified in Hungary, Spain and Turkey. The most virulent race (G) was also found in Turkey. The molecular analysis among highly virulent populations of O. cumana identified four molecular clusters, respectively, grouping populations from Central Spain, Hungary, South Spain and Turkey. The genetic homogeneity within parasite populations was confirmed, since no molecular divergences were found within them. This work constitutes the first geographical study of O. cumana together with pathogenicity and molecular traits inherent to each geographical group, and provides useful information for possible phylogenetic analyses of O. cumana. In addition, molecular markers associated with geographical origin could be developed and used as diagnostic tools to track new broomrape introductions into areas free of virulent races where they might represent a threat to sunflower production.

Verticillium wilt and leaf mottle of sunflower, caused by the fungus Verticillium dahliae (Vd) has become a major constraint to sunflower oil production in temperate European countries. Information about Vd from sunflower is very scarce despite genetics, molecular traits and pathogenic abilities of fungal strains affecting many other crops being widely known. Understanding and characterizing the diversity of Vd populations in those countries where sunflowers are frequent and severely affected by the fungus are essential for efficient breeding for resistance. In this study, we have analyzed genetic, molecular and pathogenic traits of Vd isolates affecting sunflower in European countries. When their genetics was investigated, almost all the isolates from France, Italy, Spain, Argentina, and Ukraine were assigned to vegetative compatibility group (VCG) 2B. In Bulgaria, Turkey, Romania, and Ukraine, some isolates were assigned to VCG6, but some others could not be assigned to any VCG. Genotyping markers used for Vd affecting crops other than sunflower showed that all the isolates were molecularly identified as race 2 and that markers of defoliating (D) and non-defoliating (ND) pathotypes distinguished two well-differentiated clusters, one (E) grouping those isolates from Eastern Europe and the other (W) all those from the Western Europe and Argentina. All the isolates in cluster W were VCG2B, while the isolates in cluster E belonged to an unknown VCG or to VCG6. When the host range was investigated in the greenhouse, the fungus was highly pathogenic to artichoke, showing the importance of farming alternatives in the management of Verticillium attacks. Sunflower genotypes were inoculated with a selection of isolates in two experiments. Two groups were identified, one including the isolates from Western Europe, Argentina, and Ukraine, and the other including isolates from Bulgaria, Romania, and Turkey. Three pathogenic races were differentiated: V1, V2-EE (Eastern Europe) and V2-WE (Western Europe). Similarly, three differentials are proposed for race identification: HA 458 (universal susceptible), HA 89 (resistant to V2-EE, susceptible to V2-WE) and INRA2603 (susceptible to V2-EE, resistant to V2-WE). The diversity found in Vd affecting sunflower must be taken into account in the search for resistance to the pathogen for European environments of sunflower production.

The increasing limitation of agrochemicals for disease control is a major challenge for European agriculture and a spur to developing environmentally friendly approaches such as biological control. Entomopathogenic fungi, which have been used in the control of insect pests for a long time, also have other uses, such as being antagonists of fungi, including plant pathogens. We determined the in vitro effect of three strains of Metarhizium brunneum and two of Beauveria bassiana against Verticillium dahliae and Cadophora helianthi, causal agents of sunflower wilts. Both M. brunneum and B. bassiana were able to inhibit the mycelial growth of the sunflower pathogens and, according to the dual culture and microscopy results, two types of antagonism were observed as being dependent on the strain: competition and/or antibiosis. Greenhouse experiments showed that, after soil treatments with entomopathogens and plant inoculation by root immersion in conidial suspensions of V. dahliae, the entomopathogens were able to efficiently persist in the soil, and three of the four strains even significantly reduced the severity of symptoms in sunflowers. Interestingly, molecular analysis showed that all the strains were able to establish themselves as endophytes in sunflowers in the absence of V. dahliae. When the plants were inoculated with V. dahliae, we detected the pathogen, but not the entomopathogen, in the sunflowers by molecular methods. The results of this work suggest that the protection conferred by M. brunneum and B. bassiana against verticillium wilt might not be plant‐located, but is probably the consequence of their competition with V. dahliae in the soil.

Sunflower (Helianthus annuus L.) plants with symptoms of interveinal chlorosis were observed in the summer of 2013 in one field in Cadiz (Spain) where the performance of 30 hybrids was assessed. Symptoms affected 80% of the hybrids with incidence as high as 90%. Chlorosis and yellowing near the leaf margin were visible at floral initiation, and they progressed from the lower to upper leaves. Mottled leaves were observed near the top of the plants. On severely affected leaves, chlorotic patches enlarged, coalesced, and large areas of the leaves became necrotic and dried. Cross sections of the lower stem showed a brown discoloration of the vascular system. The fungus that was consistently isolated from stem and petiole tissues of sunflower plants was morphologically identified as Verticillium dahliae Kleb. (Vd) (5) and molecularly confirmed by PCR amplification of the 526-bp band (4). The race of the isolates was determined in a greenhouse experiment at 18 to 28°C from February to April 2014. Isolates 1-13 and 2-13 of Vd, obtained from two of the hybrids in 2013, one of them being Transol, were independently inoculated to 1-month-old plants of each of three sunflower genotypes: the susceptible hybrid Transol and the inbred lines HA89 (carrying the V1 gene for resistance to Vd) (2) and HAR5 (resistant to other diseases but with unknown reaction to Vd). Plants were inoculated by immersing roots in a suspension of 106 conidia per ml for 30 min. Inoculated plants were individually transplanted to 1-liter pots filled with sand/silt. Roots of the control treatments were immersed in water. Six replications (pots) were established for each isolate × genotype combination, according to a complete randomized 3 × 3 factorial design. Five weeks after inoculation, symptoms developed in 100% of the plants in the three sunflower genotypes. Severity of symptoms (SS) in each plant was assessed as percentage of foliar tissue affected. Significantly higher SS occurred on inoculated plants as compared to non-inoculated plants, which did not develop symptoms. Mean disease severity on inoculated plants was 80% (averaged across isolates and genotypes). A significant effect of genotypes was obtained. Mean SS averaged across isolates were 98, 73, and 69% for HAR5, HA89, and Transol, respectively. When stem tissues from the three sunflower genotypes were sampled and incubated on potato dextrose agar at 25°C, the mycelial growth of Vd was confirmed for the inoculated plants but not for the control plants. Isolates of Vd infecting the resistant inbred line HA89 have only been identified in Argentina (1) and the United States (3). To our knowledge, this is the first report of a race overcoming the V1 gene in HA89 in Europe. This poses a risk to commercial sunflower breeding programs in European countries. References: (1) A. B. Bertero de Romano and A. Vázquez. Page 177 in: Proc. 10th Int. Sunf. Conf., Surfers Paradise, Australia, 1982. (2) G. N. Fick and D. E. Zimmer. Crop Sci. 14:895, 1974. (3) T. Gulya. Helia 30:115, 2007. (4) J. Mercado-Blanco et al. Plant Dis. 87:1487, 2003. (5) W. E. Sackston. Plant Dis. Rep. 41:885, 1957.