Leire Molinero-Ruiz

Diagnosis of the infection of sunflower by Orobanche cumana using multicolour fluorescence imaging

Abstract Orobanche cumana is an holoparasite and thus totally dependent on sunflower for fixed carbon. Initial stages of the infection occur in the first weeks after sowing and are critical for the establishment of a continuum between the host and the parasite vascular system. From that moment the parasite obtains its supply of water, mineral nutrients, and assimilates from the host plant. Alterations of plant metabolism can be detected using remote sensing techniques for detection of fluorescence emitted by plants. One of these indirect techniques is multicolour fluorescence imaging. In this work, we assessed the early infection of sunflower by O. cumana using multicolour fluorescence imaging and we inferred physiological processes affected in sunflower plants infected by the parasite. Ten germinated seeds of the inbred line NR5 were inoculated with population LP2013 of O. cumana. The same number of not inoculated seeds was used as control. Sunflower was planted in pots with soil mixture and grown in greenhouse at 12–22°C for 6 weeks. Multicolour fluorescence imaging was conducted 3, 4, and 5 weeks after inoculation. The two first pairs of fully expanded leaves of each sunflower plant were imaged, and, for each measure date, five fluorescence variables in inoculated plants were compared to those in the control. Three weeks after inoculation, when symptoms of infection were still not observed, decreased levels of blue and green fluorescence and increased far-red fluorescence were observed in leaves of the inoculated plants. At 4 and 5 weeks after inoculation, when inoculated plants displayed symptoms of infection by O. cumana, differences in fluorescence between inoculated plants and the controls were the same and statistically supported. These results are consistent with an increase in total chlorophyll content of sunflower plants infected by O. cumana, and a decrease in the accumulation of secondary metabolites, both related to the need of higher photosynthetic activity to supply the parasite with photosynthate. Biochemical mechanisms underlying alterations in photosynthesis must be further investigated. The results obtained showed that multicolour fluorescence imaging can be used to detect fluorescence differences in inoculated sunflower as early as 3 weeks after inoculation. Therefore, this technique can be used as a diagnostic tool for early detection of genotypes of sunflower which are susceptible or resistant to O. cumana.

Fluorescence Imaging in the Red and Far-Red Region during Growth of Sunflower Plantlets. Diagnosis of the Early Infection by the Parasite Orobanche cumana

Broomrape, caused by the root holoparasite Orobanche cumana, is the main biotic constraint to sunflower oil production worldwide. By the time broomrape emerges, most of the metabolic imbalance has been produced by O. cumana to sunflower plants. UV-induced multicolor fluorescence imaging (MCFI) provides information on the fluorescence emitted by chlorophyll (Chl) a of plants in the spectral bands with peaks near 680 nm (red, F680) and 740 nm (far-red, F740). In this work MCFI was extensively applied to sunflowers, either healthy or parasitized plants, for the first time. The distribution of red and far-red fluorescence was analyzed in healthy sunflower grown in pots under greenhouse conditions. Fluorescence patterns were analyzed across the leaf surface and throughout the plant by comparing the first four leaf pairs (LPs) between the second and fifth week of growth. Similar fluorescence patterns, with a delay of 3 or 4 days between them, were obtained for LPs of healthy sunflower, showing that red and far-red fluorescence varied with the developmental stage of the leaf. The use of F680 and F740 as indicators of sunflower infection by O. cumana during underground development stages of the parasite was also evaluated under similar experimental conditions. Early increases in F680 and F740 as well as decreases in F680/F740 were detected upon infection by O. cumana. Significant differences between inoculated and control plants depended on the LP that was considered at any time. Measurements of Chl contents and final total Chl content supported the results of MCFI, but they were less sensitive in differentiating healthy from inoculated plants. Sunflower infection was confirmed by the presence of broomrape nodules in the roots at the end of the experiment. The potential of MCFI in the red and far-red region for an early detection of O. cumana infection in sunflower was revealed. This technique might have a particular interest for early phenotyping in sunflower breeding programs. To our knowledge, this is the first work where the effect of a parasitic plant in its host is analyzed by means of fluorescence imaging in the red and far-red spectral regions.

A New Race of Verticillium dahliae Causing Leaf Mottle of Sunflower in Europe

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.

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

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.

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.

Cadophora malorum: a new pathogen of sunflower causing wilting, yellowing and leaf necrosis in Russia

Russia devotes more than six million hectares to sunflower (Helianthus annuus L.) crop every year being, together with Ukraine, the most important producer countries worldwide. From 2014 an increase of wilt diseases is being observed in the country. Plants with symptoms of wilting and leaf yellowing and necrosis were observed in two commercial fields and one demonstrative field trial in Orenburg region in August 2016, and in one commercial field in Saratov region in August 2015. Disease incidence was estimated between 15-30% depending on the field and yield from infected plants was near to zero. Four symptomatic plants per location were collected for observation and pathogen isolation. From each sample, the basal part of the stem was surface sterilized for 5 min in 10% sodium hypochlorite solution, rinsed in water, cut longitudinally and sections of the cortex with brownish appearance indicative of possible fungal colonization were incubated in water-agar medium at 25oC in darkness. Fungal colonies were c...

Use of Blue-Green Fluorescence and Thermal Imaging in the Early Detection of Sunflower Infection by the Root Parasitic Weed Orobanche cumana Wallr.

Although the impact of Orobanche cumana Wallr. on sunflower (Helianthus annuus L.) becomes evident with emergence of broomrape shoots aboveground, infection occurs early after sowing, the host physiology being altered during underground parasite stages. Genetic resistance is the most effective control method and one of the main goals of sunflower breeding programmes. Blue-green fluorescence (BGF) and thermal imaging allow non-destructive monitoring of plant diseases, since they are sensitive to physiological disorders in plants. We analyzed the BGF emission by leaves of healthy sunflower plantlets, and we implemented BGF and thermal imaging in the detection of the infection by O. cumana during underground parasite development. Increases in BGF emission were observed in leaf pairs of healthy sunflowers during their development. Lower BGF was consistently detected in parasitized plants throughout leaf expansion and low pigment concentration was detected at final time, supporting the interpretation of a decrease in secondary metabolites upon infection. Parasite-induced stomatal closure and transpiration reduction were suggested by warmer leaves of inoculated sunflowers throughout the experiment. BGF imaging and thermography could be implemented for fast screening of sunflower breeding material. Both techniques are valuable approaches to assess the processes by which O. cumana alters physiology (secondary metabolism and photosynthesis) of sunflower.

La marchitez tardía del maíz (Zea mays L.) causada por Cephalosporium maydis en la Península Ibérica, y otros hongos asociados

The soil-borne fungal species associated with Cephalosporium maydis, as the causal agent of late wilt of corn at the Iberian Peninsula, were identified by monitoring 19 fields showing wilt symptoms in the major crop areas between 2011 and 2012. In 47% of the fields, C. maydis was not identified, but Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani and Trichoderma harzianum were found to infect corn plants. In the remaining fields, besides C. maydis, other soil-borne fungi were identified at considerable percentages: F. verticillioides (19%), F. proliferatum (19%), F.equiseti (9%), F. oxysporum (9%) and Pythium oligandrum (9%). The vascular growth of C. maydis and other fungal species in corn plants was confirmed by means of analyses with plants showing wilt from three different fields. Both C. maydis and F. graminearum, F. equiseti, F. proliferatum and T. harzianum were isolated at the insertion between the root and the stem and at 10cm height on the stem of plants. The effect of infection by C. maydis on corn plant production was quantified in pots under semi-natural conditions in 2011. In inoculated plants, there was a 54% reduction in the weight of cobs, in addition to a reduction in the weight of roots and shoot (leaves and stem) which were significantly smaller compared to the non-inoculated control; this suggests the great economic impact that late wilt may have under natural conditions. Similarly, this study highlights the complexity degree of the etiology of late wilt, which should be studied by confirming the pathogenesis of the soil-borne fungi identified in the corn with the aim of determining the role of each species on the development of the disease and/or the severity of symptoms.Las especies de hongos de suelo asociadas a Cephalosporium maydis como agente causal de la marchitez tardia del maiz en la Peninsula Iberica se identificaron muestreando 19 campos con sintomas de marchitez en las principales zonas de cultivo entre 2011 y 2012. En el 47% de los campos no se identifico C. maydis, pero si Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani y Trichoderma harzianum infectando las plantas de maiz. En los campos restantes, junto a C. maydis se identificaron otros hongos de suelo en porcentajes apreciables: F. verticillioides (19%), F. proliferatum (19%), F.equiseti (9%), F. oxysporum (9%) y Pythium oligandrum (9%). El crecimiento vascular de C. maydis y de otras especies fungicas en plantas de maiz se confirmo analizando plantas con marchitez procedentes de tres campos diferentes. Tanto C. maydis como F. graminearum, F. equiseti, F. proliferatum y T. harzianum se aislaron de la insercion entre la raiz y tallo y a 10 cm de altura en el tallo de las plantas. El efecto de la infeccion por C. maydis sobre la produccion de las plantas de maiz se cuantifico en macetas y condiciones seminaturales en el 2011. En plantas inoculadas se obtuvo una reduccion del peso de las mazorcas del 54%, ademas de pesos de raiz y de parte aerea (tallo y hojas) significativamente menores en comparacion con el control no inoculado, lo que sugiere el gran impacto economico que puede tener la marchitez tardia en condiciones naturales. Asimismo este trabajo pone de manifiesto el grado de complejidad de la etiologia de la marchitez tardia, que deberia ser estudiado mediante la confirmacion de la patogenicidad de los hongos de suelo identificados en maiz, con el fin de determinar el papel que puede jugar cada una de estas especies en el desarrollo de la enfermedad y/o severidad de los sintomas.

Harpophora maydis affecting maize in Southern Europe: different growth media, long-term storage and in vitro effect of extracts of Lycium europaeum L.

Trabajo presentado en el 15th Congress of the Mediterranean Phytopathological Union, celebrado en cordoba (Espana) del 20 al 23 de junio de 2017.Comunicacion oral presentada en el 15th Congress of the Mediterranean Phytopathological Union, June 20–23, 2017, Cordoba, Spain.Early assessment of late wilt of maize (Harpophora maydis) and the control effect of Lycium europaeum extracts. C. RODRÍGUEZ-MALLOL1, R. TEJ1,2, L. MOLINERO-RUIZ1. 1Department of Crop Protection, Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Alameda del Obispo s/n, 14004 Córdoba, Spain. 2Physiology and Biochemistry of Plant Response to Abiotic Stresses Unit, Faculty of Sciences of Tunis, University of Tunis El Manar, 1060 Tunis El Manar, Tunisia. E-mail: lmolinero@ias.csic.es

Genetic and phenotypic diversity of Verticillium dahliae populations from sunflower in Europe

Trabajo presentado en el 15th Congress of the Mediterranean Phytopathological Union, celebrado en cordoba (Espana) del 20 al 23 de junio de 2017.Comunicacion oral presentada en el 15th Congress of the Mediterranean Phytopathological Union, June 20–23, 2017, Cordoba, Spain.Early assessment of late wilt of maize (Harpophora maydis) and the control effect of Lycium europaeum extracts. C. RODRÍGUEZ-MALLOL1, R. TEJ1,2, L. MOLINERO-RUIZ1. 1Department of Crop Protection, Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Alameda del Obispo s/n, 14004 Córdoba, Spain. 2Physiology and Biochemistry of Plant Response to Abiotic Stresses Unit, Faculty of Sciences of Tunis, University of Tunis El Manar, 1060 Tunis El Manar, Tunisia. E-mail: lmolinero@ias.csic.es